Literature Review

Abstract

In this chapter, stretching is defined with reference made to various stretching techniques. An understanding of how force (generated by stretching) affects soft and connective tissue is looked at both macroscopically (muscle, tendon, myotendon junction) and microscopically (sarcomere, myofibrillar proteins, ECM). By referring to these levels, an appreciation is gained of how the magnitude and rate of force generated during stretching affects the body as a whole. In order to elucidate this further, reference is made to inflammation and exercise, both non-damaging and damaging. With stretching considered as a load on tissue, a comprehensive overview is focused on damaging exercise, neutrophils, macrophages, and cytokines and their importance with regard to acute inflammation. The idea is proposed that stretching intensity be considered as a mechanotransduction mechanism. Mechanotransduction refers to the process by which the cells and tissues of the body respond to their environment, with the conversion of a mechanical energy into biochemical signals. Tensegrity is the main tenant of mechanotransduction, concerned with the essential maintenance of mechanical stability, which may be influenced by stretching intensity. Depending on the stretching intensity, this may or may not prompt an inflammatory response.

Introduction

The human body is an “open” system exchanging matter, energy, and information with its immediate environment. This exchange often results in the manifestation of internal processes and transformations, defined by such concepts as homeostasis, self-regulation, allostasis, and autopoieses. These processes allude to the body’s innate ability to monitor its mechanical usage and its subsequent response through use of several mechanisms, based on the exchange of information between the external and internal environments. According to Hannan and Freeman (Hannan & Freeman, 1977), homeostasis is based on information exchanged between the system and the external environment allowing the system to maintain a state of equilibrium over time. Self-regulation is an adaptive mechanism by which the system maintains a balanced condition within its structural limits and through the exchange of information with its external environment (Beer, 1975), while allostasis references a transformational concept, the idea that maintenance of stability occurs through change (Mcewen & Wingfield, 2003). It suggests that through this process the organism actively adjusts to both predictable and unpredictable events (Mcewen & Wingfield, 2003). With autopoieses, the predominant feature is the self-organisation of a system’s ability to stimulate a selective mechanism designed to align its internal complexity with the complex external environment (Maturana & Varela, 1975). Although each of these transformational processes appear to be different in defining the relationship(s) between the internal and external environments, a common thread is the exchange and processing of information, the formative response of the body to either a physical or a mental stress. Within this current work, the focus is on the exchange of information between a physical process (i.e. stretching), specifically the body’s response(s) to the magnitude, and rate of such a stimulus. This information exchange alludes to the concept of mechanotransduction, “the process of converting physical forces into biochemical signals and integrating these signals into (a) cellular response” (Huang, Kamm, & Lee, 2004). More will be discussed later.

The practice of stretching is widespread in the arts (i.e. dancing), the general fitness population (i.e. stretching programmes), as well as sports (Taylor, Dalton, Seaber, & Garrett, 1990). Stretching is viewed as a physical strategy aimed at improving flexibility (Weerapong, Hume, & Kolt, 2004), performance (Handel, Horstmann, Dickhuth, & Gulch, 1997; Wilson, Elliot, & Wood, 1992), pain relief, and recovery from injury (Chandler et al., 1990; Jackson et al., 1978; Maffulli, King, & Helms, 1994). In addition, it has become the universal strategy for injury prevention in sports (Thacker, Gilchrist, Stroup, & Kimsey, 2004). Athletes, coaches, trainers, and medical professionals (physicians, physiotherapists, etc.) often recommend stretching to enhance performance and prevent injury and thus regard it as an essential component of numerous physical training programmes (Caplan, Rogers, Parr, & Hayes, 2009) and rehabilitation protocols (Feland, Myrer, & Merrill, 2001; Malliaropoulos, Papalexandris, Papalada, & Papacostas, 2004). A review by Prentice has indicated that four stretching methods are commonly used for sport activities—static, ballistic, dynamic, and proprioceptive neuromuscular facilitation (PNF) (Prentice, 1985).

The Definition of Stretching and Its Measurement

Stretching, a movement applied by an external and/or internal force stressing connective and muscle tissue mechanically, is used to increase muscle flexibility and/or joint ROM (Martins et al., 2013; Weerapong et al., 2004). It depends on the active and passive tension of the muscle, the MTU, and the proprioceptors of the musculoskeletal system, the muscle spindles, and the Golgi tendon organs (GTO) (Abdel-Aziem, Draz, Mosaad, & Abdelraou, 2013; Guissard & Duchateau, 2006; Knudson, 2006; Nikolaou, Macdonald, Glisson, Seaber, & Garrett, 1987). In addition, non-muscle elements such as ligaments, tendons, and bones provide physiological constraints for maximal muscle stretch in skeletal muscle (Agarkova, Ehler, Lange, Schoenuer, & Perriard, 2003). Active tension is the interaction of the actin and myosin filaments of the muscle , with passive being the elongation of the connective tissue beyond its resting length (Knudson, 2006). Both of these define the length-dependent properties of the muscle, which is strongly related to stretching, for the interaction of each implies that exercise interventions, like stretching, may have a complex effect on skeletal muscle, dependent on the interaction of the tissues and the nature of the training stimulus (Knudson, 2006). In other words, when muscle is stretched by various stretching techniques, this may account for changes in the active and passive tension of the muscle (Apostolopoulos, Metsios, Flouris, & Koutedakis, 2015). An understanding of the molecular pathways governing the ability of the muscle to respond to a functional demand presents a better comprehension of the influence of stretch intensity on the musculoskeletal tissue(s), since the cells are influenced by physical force(s).

The force generated during stretching is a load, with load referring to the magnitude and the rate of force developed (Elliot, 2006). An understanding of how force affects soft and connective tissue at both the macroscopic (muscle, tendons, and myotendon unit) and microscopic (sarcomere, myofibrillar proteins, ECM) level provides a better insight as to how the magnitude and rate of force generated during stretching affects the body as a whole. This knowledge is pivotal in designing a proper stretching protocol aimed at increasing athletic performance or rehabilitation from musculoskeletal pain and injury.

At the macroscopic level, change due to stretching is primarily functional, while at the microscopic level, it is reflected in the cells and the ECM of the muscles, the tendons, and the MTU. A reduction in reflex sensitivity of the skeletal muscle with repeated stretching alters its mechanical tension resulting in a functional change (Avela, Kyrolainen, & Komi, 1999; Proske, Morgan, & Gregory, 1993). With tendons, stretching may change their dynamic behaviour, confirmed in findings from animal studies demonstrating that the elasticity of tendons is changeable through stretching (Viidik, 1969; Witvrouw, Mahieu, Roosen, & Mcnair, 2007). Regardless of the functional or structural transformation associated with stretching, an understanding of how the body adapts to this force, both macroscopically and microscopically, is essential and will be discussed further.

The cytoskeletal organisation of the cells, in other words, the contractile forces of the microfilaments and the compression resistance of microtubules, is responsible for the cell response to any static and physical force sensed. This response is defined by the varying levels of resistance influenced by the cell’s attachment and adherence to the ECM and its relationship to adjacent cells. In addition, the cell itself also generates force influencing both its function and shape. This highlights the influence directed at the responsiveness of individual cells to any external force(s) (i.e. fluid flow, stretching), as well as the cellular responses to cell exerted forces connected to and influenced by the extracellular microenvironment (Discher, Janmey, & Wang, 2005).

By referring to both the macroscopic and microscopic levels, in particular the integration and transference of information through the numerous levels from the external environment to the cytoskeleton of the cell, an understanding is achieved regarding the degree of force (intensity) developed during stretching and how this may cause a structural and mechanical change to the tissue. The sensing of force by cells is critical for their proper function, being fundamental in mediating a load transfer, as well as sustaining an interaction between the cells and tissue critical for the interface of function and homeostasis (Benjamin, Evans, & Copp, 1986; Lu & Jiang, 2006; Woo et al., 1988). Movement from the macro to the micro represents a continuum, an intracellular transfer of information explaining the body’s ability to adapt to the external environment. This information is particularly important if one considers that the magnitude of force generated during stretching may determine the effectiveness of stretching regarding the structural adjustment of the connective and muscle tissue to this externally applied force(s). An effectiveness may help in resolving trauma to the connective and muscle tissue or create and exacerbate the damage to these structures.

The increase in ROM about a joint with stretching is facilitated by the synchronised interaction(s) of the many types of soft tissue (muscle, tendons, etc.) and their relation to bone (joints) (Shellock & Prentice, 1985). In addition to this physiological relationship, a functional/behavioural one exists, the placement of the body in a proper position facilitating a more effective stretch. This allows for a better isolation of a muscle group (i.e. hamstring, quadriceps, etc.) and their particular joint(s) (i.e. knee, ankle, hips, etc.), ensuring the ability to control and apply the appropriate amount of a mechanical stress (i.e. stretching) aimed at increasing the ROM while limiting tissue damage (Abdel-aziem et al., 2013; Apostolopoulos et al., 2015). In other words, the position assumed during stretching may influence the magnitude of the force generated prior to and during the stretch potentially altering the response of the muscle and tendon tissue (Abdel-aziem et al., 2013; Apostolopoulos et al., 2015).

Range of motion is an important factor in human athletic performance and in many traumatic or orthopaedic conditions (Maffulli et al., 1994). The degree and distribution of ROM in individuals may be essential in maximising physical performance as well as the genesis and presentation of injuries (Jackson et al., 1978). It can be specific to each side of the body even in the same joint (Chandler et al., 1990; Maffulli et al., 1994).

Flexibility is typically measured in degrees of full ROM to the point of mild discomfort from a starting position (Luttgens & Hamilton, 1997). Goniometers and inclinometers have been used to measure it, respectively. The former is widely used in clinical settings measuring any limitations while documenting the effectiveness of the intervention (Gajdosik & Bohannon, 1987). The two-arm goniometer is most commonly used for the evaluation of ROM (Lea & Gerhardt, 1995). Its usefulness, however, has been questioned since the starting position, the long axis of the limb, the centre of rotation, and the vertical and horizontal positions can only be visually estimated (Nussbaumer et al., 2010). Nevertheless, its reliability is high, confirming its continued use in clinical settings (Mayerson & Milano, 1984; Rothstein, Miller, & Roettger, 1983).

Similar to the goniometer, an inclinometer is portable and lightweight requiring little training to assess joint ROM (Kolber, Fuller, Marshall, Wright, & Hanney, 2012). Two types of inclinometers exist: gravity-based and digital. The former makes use of a liquid compartment or magnet establishing zero degrees when positioned in the vertical. With constant gravity as a reference point, this inclinometer can be used to assess joint mobility and ROM (Dejong, Nieuwboer, & Aufdemkampe, 2007; Kolber, Vega, Widmayer, & Cheng, 2011). Digital inclinometers are costly, with a major disadvantage being the requirement of the clinician or examiner to establish the zero point accurately in order to minimise the risk of measurement error prior to use (Kolber et al., 2012). Overall, the inclinometer also possesses good reliability when strict measurement protocols are adhered too (Kolber et al., 2011, 2012).

Classification of Stretches

Stretching is classified as static, dynamic, and pre-contraction (Fig. 2.1). Within these categories there are active (self-stretch) and passive (with or without a partner) for static stretching, active and ballistic for dynamic stretching, and PNF for pre-contraction stretching (Page, 2012; Weerapong et al., 2004). These categories are not universal with some authors suggesting the existence of only four types of stretches: static, ballistic, dynamic, and PNF (Behm & Chaouachi, 2011; Shellock & Prentice, 1985). Regardless of the classification, the common denominator for each technique is the placement of a certain load(s) on the muscles and the joint(s) being targeted.

Fig. 2.1

Classification of stretching. (Published with kind permission of copyright © Nikos C. Apostolopoulos 2018. All rights reserved)

Static

Static stretching is commonly employed to improve joint mobility and prevent contracture (Nakamura, Ikezoe, Takeno, & Ichihashi, 2012). It usually involves moving the limb to the end of its ROM and holding this position for 15–60 s at a point of discomfort or pain (Bandy, Irion, & Briggler, 1997; Behm & Chaouachi, 2011; Feland, Myrer, & Merrill, 2001; Sady, Wortman, & Blanke, 1982; Smith, 1994). It is performed passively with use of a partner or therapist or, actively, with the individual self-stretching. The primary use of static stretching has been for increasing the ROM about a joint (Bandy et al., 1997; Feland, Myrer, & Merrill, 2001), attributed to changes in the length and stiffness of the MTU of the limb being stretched (Behm & Chaouachi, 2011; Power, Behm, Farrel, Carroll, & Young, 2004). However, a controversy exists whether this change is due to a decrease in MTU stiffness (Wilson, Wood, & Elliot, 1991) or is it primarily an increase in the tolerance to stretching (Law et al., 2009; Magnusson, Simonsen, Aagaard, Sorensen, & Kjaer, 1996).

Static stretching takes advantage of the inverse myotatic reflex promoting muscle relaxation, thereby causing a further stretch and an increase in ROM (Feland, Myrer, Schulthies, Fellingham, & Measom, 2001). Other benefits have been the reduction of pain and injury (Safran, Seaber, & Garrett, 1989; Smith, 1994), a decrease in soreness (High, Howley, & Franks, 1989), as well as improved performance (Young & Behm, 2002). However, systematic reviews looking at the impact of static stretching for prevention of sports injury risk concluded that static stretching does not reduce injury rates (Bo Lauersen, Bertelsen, & Bo Andersen, 2014; Small, Mcnaughton, & Matthews, 2008) or change the muscle-tendon mechanical properties (Freitas et al., 2017). In addition, static stretching may affect the development of explosive force, jumping, as well as sprint performance (Winchester, Nelson, Landin, Young, & Schexnayder, 2008; Young & Behm, 2003). Nevertheless, a study has observed that static stretching prior to competition(s) does not negatively impact the vertical jump performance in trained women (Unick, Kieffer, Cheesman, & Feeney, 2005). These controversies, whether static stretching improves performance or not and reduces pain and soreness or whether the increase in ROM is due to stretch tolerance or a change in MTU stiffness, reflect the lack of our current understanding and thus the need for further research.

Ballistic

Similar to static stretching, with “ballistic” stretching, the joint is passively moved to its maximum ROM, whereupon a dynamic “ballistic” movement is applied on the stretched structure at the end of the ROM (Konrad & Tilp, 2014). This is in the form of a bouncing rhythmic motion using the momentum of a swinging body segment to lengthen the muscle (Bandy et al., 1997; Mahieu et al., 2007), which activates the stretch reflex resulting in a contraction of the muscle under stretch. This rapid change in tension, increasing with the magnitude and rate of the stretch, may produce a strain or rupture of the tissue making this form of stretching disadvantageous for improving ROM (ACSM, 2006; Page, 2012; Shrier & Gossal, 2000). It is noteworthy that articles referring to ballistic stretching as being disadvantageous consisted of a clinical commentary (Page, 2012), a nonsystematic literature review (Shrier & Gossal, 2000), and guidelines as suggested by the American College of Sports Medicine (ACSM, 2006). The views expressed in these articles represent findings associated with the notion that the rapid alternating “bouncing” at the end ROM increases risk of injury, although relevant good quality studies are currently lacking. In contrast, articles supporting ballistic stretching are all randomised clinical trials concluding that ballistic stretching is beneficial for increasing hamstring flexibility (Kumar & Chakrabarty, 2010; Morcelli, Oliveira, & Navega, 2013) or in combination with other techniques presents a more appropriate approach with regard to training and rehabilitation (Mahieu et al., 2007). Similar to static stretching, the aforementioned discrepancy illustrates the need for more robust research to provide better and relevant information and create a better understanding of the various stretching techniques.

Dynamic

Dynamic stretching involves moving a limb through its full ROM to the end ranges for several times (Page, 2012). This method has been introduced as a better alternative to static stretching for increasing ROM (Murphy, 1994). With dynamic stretching, movement begins from the neutral position of the joint, performed slowly and deliberately (Bandy et al., 1997). Murphy suggests that if the limbs are moved too quickly, a tendency to swing the limb exists which may cause the stretch reflex to be elicited at the endpoint of the movement during the lengthening of the muscle. The slow deliberate movement of the limb back to the neutral position is executed with use of an eccentric contraction of the muscle. This contraction by the antagonist muscle results in the relaxation of the lengthening muscle due to the principle of reciprocal inhibition. In other words, the muscle is reflexively inhibited (Murphy, 1994).

Proprioceptive Neuromuscular Facilitation (PNF)

This stretching technique developed to improve muscle elasticity to treat neurological dysfunctions (Knott & Voss, 1968) has been shown to have a positive effect on both active and passive ROMs (Hindle, Whitcomb, Briggs, & Hong, 2012). Currently two techniques, collectively referred to as PNF procedures (Etnyre & Lee, 1987) are used, the contract-relax and the contract-relax-antagonist-contract (Condon & Hutton, 1987; Hindle et al., 2012; Page, 2012), with each using a volitional contraction in order to increase ROM. The rationale behind the contract-relax technique is that the successive maximal excitations of the motor neurons during the volitional contractions will reflexively promote their subsequent inhibition (Kabat, 1950). With contract-relax-antagonist-contract, contraction and relaxation of the muscle to be stretched (i.e. the stretching of the target muscle) is followed by a concentric contraction of the opposing muscle. Activation of the opposing muscle is believed to reduce activation of the target muscle through reciprocal innervation (Hindle et al., 2012; Kabat, 1950; Knott & Voss, 1968; Voss, 1967). An example of a paired target and opposing muscle is the quadriceps and hamstring muscles, respectively. Although, it has been reported that PNF results in a greater increase in ROM compared to static stretching (Magnusson, Simonsen, Aagaard, et al., 1996; Magnusson, Simonsen, Dyhre-Poulsen, et al., 1996; Sady et al., 1982), with the effects lasting 90 min or more (Funk, Swank, Mikla, Fagan, & Farr, 2003), the mechanism(s) thought to facilitate this increase in muscle length has been questioned (Chalmers, 2004; Shrier & Gossal, 2000). In general, the literature suggests that activation of the opposing muscle motoneurones results in the simultaneous excitation of the Ia-inhibitory interneurons which synapse the target muscle motoneurones, resulting in a decrease in the neural activity of the target muscle (Hultborn, Illert, & Santini, 1976; Sharman, Cresswell, & Riek, 2006). The inhibition of the proprioceptive structures in the target muscle (i.e. Golgi tendon organ), in response to the contraction, or shortening of the opposing muscle, is responsible for the lengthening of the target muscle fibres (Hindle et al., 2012; Sharman et al., 2006). However, instead of observing a decrease in electromyography activity as suggested by reciprocal inhibition, inferring a reduction in active force production which would resist the lengthening of the muscle (Chalmers, 2004), the muscle exhibits both an active electromyography and a subsequent increase in muscle stiffness (Moore & Hutton, 1980; Shrier & Gossal, 2000). Therefore, increases in ROM of the target muscle (i.e. quadriceps) occurred despite the opposing muscle (i.e. hamstring) being under considerable tension suggesting that the PNF techniques (contract-relax and contract-relax-antagonist-contract) failed to evoke sufficient relaxation in the muscle to overcome tension generated by the stretch (Osternig, Robertson, Troxel, & Hansen, 1987). Regardless of this controversy, PNF procedures have consistently been observed to enhance ROM compared to either static or ballistic stretching (Etnyre & Abraham, 1986; Feland, Myrer, Schulthies, et al., 2001; Funk et al., 2003; O’hara, Cartwright, Wade, Hough, & Shum, 2011; Sady et al., 1982; Wallin, Ekblom, Grahn, & Nordenborg, 1985).

Macroscopic and Microscopic Information Processing Levels: Macroscopic—Muscle, Tendon, and Myotendon Unit (MTU)

Introduction

Since the time of the French philosopher and mathematician Rene Descartes (1596–1650) and the British natural philosopher Sir Isaac Newton (1643–1727), biologists have adopted the approach of studying the mechanism(s) of the body in response to the environment from both an ontological and epistemological level (Ahn, Tewari, Poon, & Phillips, 2006; Mazzochi, 2008). The former refers to the concept of the organism and how it is made with the latter focused on understanding those things that define it. To accomplish this, scientists embraced the concepts of simplification and reductionism, suggesting that the complexity of the body can be resolved by both an analysis and a reduction of everything to its simplest components. In other words, biological systems can be explained largely by referring to the physical and chemical properties of their individual components (Westerhoff & Palsson, 2004).

In vitro studies have been instrumental in discovering, observing, and determining individual components (cells), providing merit to the reductionist approach, albeit that the procedures are conducted in a controlled environment outside of the living organism. They were important in elucidating the assembly of myofibrils, a dynamic interaction dependent on the coordinated integration of myosin, actin, associated proteins, and other cytoskeletal elements. However, we need to be cognisant that these protein interactions frequently include an unmanageable number of binary interactions with uncertain functional significance (Ayalon et al., 2011). The whole organism is a dynamic, adaptive, and interactive network, a study of the relations between components possessing the ability for the development of new collective properties in order to maintain allostasis and stability through change (Mcewen & Wingfield, 2003).

An understanding of complex systems is accessible through in vivo studies. Unlike in vitro, they refer to an experimentation using a whole living organism as opposed to a partial or dead organism. They enable the observation of the various degrees of interactions of components at multiple levels, disclosing the behaviour of the system as a whole. The exchange between all levels provides the system with the plasticity to rapidly adapt to the changing environmental situations (Coffey, 1998), suggesting that the whole is far from equilibrium. It is an interplay between order and randomness absorbing as well as dissipating energy in the quest to maintain itself (Biebricher, Nicolis, & Schuster, 1995). For instance, compared to the study of individual muscle fibres through in vitro, a muscle in vivo is subjected to substantial transverse and bending loads that would not be present in isolation (Burkholder, 2007). These loads are imposed through pressures in contact with and attachment to bone, tendons, fascial structures, connective tissue adhesions to adjacent tissues, as well as other muscles (Burkholder, 2007; Huijing & Baan, 2001).

The ultimate aim of biological research is to understand the importance of information, specifically the inter- and intracommunication of cells and molecules, and their role in the development of and modulation of structure. Signals are forms of information bridging the external and internal environment(s) of a living organism, for although individual units (cells, molecules) feature prominently in the construction of the whole (tissues, organs, organisms), it is the sum of these units plus the local interactions (information) suggesting that the whole is greater than the sum of its parts.

Biological systems express a degree of randomness. The inherent positive and negative feedback loops are mechanisms of signal transduction defining adaptation. Adaptation governs the function of the cells considered as a form of a change or a level detector, with the former being sensitive to permutations in the magnitude of the stimulus and the latter to stimulus intensity (Mcburney & Balaban, 2009). The change detector is a high-pass filter, sensitive to a high frequency, and a rapid stimulus fluctuation, with the level detector being a low-pass filter, sensitive to a steady low-frequency stimulus (Mcburney & Balaban, 2009). Common amongst both filters is the measuring and processing of information from the internal and external environments decoded through in vitro and in vivo studies.

The magnitude , intensity, and rate of the transduced signal are core mechanisms of cellular information processing mediated by the concentration of molecules, themselves serving as signals (Uda & Kuroda, 2016). The signal generated holds the potential of explaining any observed dynamic behaviour of living systems. Understanding how this signal (information) is generated, stored, and used at the various levels, from the macro to the micro, and vice versa, explains the complex feedback and feedforward patterns of biological networks. These are a reflection of underlying processes characterised by the information garnered by the low and high frequency filters , providing a better understanding of the staggering complexity, robustness, and versatility of the structure of living systems and their function (Oltvai & Barabasi, 2002) in short, a manifestation of a specific integration of both molecular and cellular information and its influence on form and function. Therefore, the mechanical feedback or factors generated within and by the system is an important determinant of organismic form and an expression of information generated by the stiffness, stretchiness, and viscoelastic response of cells and tissues to a mechanical load (Wainwright, 1988).

One can start from the analysis of elementary components of a phenomenon, such as often studied by in vitro studies; however, to comprehend the reality of what actually occurs, we need to comprehend the phenomenon in its entirety. According to von Bertalanffy (Von Bertalanffy, 1968), one needs to observe from a higher level and from a holistic perspective. The human body is a system, a coherent whole consisting of a boundary distinguishing it from internal and external elements (Ng, Maull, & Yip, 2009). Studying the specificity of individual biological molecules involved in a structure or an activity is important, for complex structures and activities cannot be explained in isolation, since their components are frequently involved in many different processes (Van Regenmortel, 2004). Therefore, interest in this project is concerned with how the magnitude of a signal (information) in the form of a stretch (external force) influences the macroscopic and microscopic elements of the body. An understanding of the interrelationship(s) of the various components, from the tissues to the muscle cell, provides a better understanding of how stretching may influence the structure and function of the body and its relevance to inflammation and the inflammatory response.

Muscles, Tendons, and MTU

Muscle

Skeletal muscle is a composite of connective tissue, nerves, blood vessels, and primarily contractile material, classified as either smooth and striated (cardiac and skeletal) (Gillies & Lieber, 2011). This dynamic tissue that undergoes a significant degree of mechanical strain and cellular deformation with each contraction is a study of the interplay between chemical and mechanical inter-tissue signalling (Gumerson & Michele, 2011). To preserve its normal function, the skeletal muscle, which generates force and routinely undergoes cell shortening required for movement, limits mechanical cellular injury by adapting to changing workloads, thus ensuring a balance between degeneration and regeneration (Gumerson & Michele, 2011).

Skeletal muscle consists of bundles of cells anatomically and mechanically arranged in parallel, with each cell functioning independently and the total force generated being a sum of the forces of each cell (Goldstein, Schroeter, & Michael, 1991). It is a highly specialised structure responsible for transforming chemically stored energy into mechanical work, with the energy provided being in the form of adenosine triphosphate (ATP) (Adams & Schwartz, 1980). This function is dependent on the efficient coordination and integration of several subcellular biological networks responsible for the contraction, shortening, and development of tension (Smith, Meyer, & Lieber, 2013).

All skeletal muscles have adaptive potential, capable of modifying their structure in response to environmental changes, such as altered patterns of activity, pathological processes, metabolic conditions, and ageing (Bruton, 2002). When skeletal muscle is not recruited to generate force and movement, it is normally found in a relaxed state (Goldstein et al., 1991). The muscle possesses a remarkable ability of repair or regeneration following damage via an effective cellular repair system (Philippou, Maridaki, Theos, & Koutsilieris, 2012). This is stimulated in order to recover normal structure and function while preventing loss of mass since skeletal muscle is considered to be an irreversible postmitotic tissue lacking an ongoing cell replacement (Decary et al., 1997; Goldspink, 2005; Philippou et al., 2012). Skeletal muscle has two specialised junctional regions, the MTU and neuromuscular, with the former referring to the attachment of the muscle to the tendons and the latter to the innervation of the muscle by motor neurons.

Isometric, concentric, and eccentric activity are the three major muscular actions resulting from both the muscle’s active and passive components (Knudson, 2007). Isometric refers to an activity where the muscle length does not change, with both concentric and eccentric describing the shortening and lengthening of the muscle, respectively. Active and passive tension defines the length-dependent properties of the muscle strongly related to stretching (Knudson, 2006). The interaction of each suggests that exercise interventions, such as stretching, has a complex effect on skeletal muscle dependent on the interaction of the tissues and the nature of the training stimulus (Knudson, 2006).

Although much has been learned by observing muscle in response to various activities (i.e. weightlifting, running, sprinting) and forces (i.e. compression, tension, bending, rotation, shearing, and gravity), to gain a better understanding and insight into and about the mechanism(s) involved during force generation or its response to stretching, it is necessary that we take the system apart. This reductionist approach, with use of in vitro studies and assays, continues to contribute to the study of the interaction of the filament systems of striated skeletal muscle (endosarcomere) and its relationship and integration with the multitude of myofibrillar proteins (Ackermann et al., 2011; Batters, Veigel, Homsher, & Sellers, 2014). As discussed above an in vitro approach enables the simplification of complex objects making them easier to understand.

Muscles are composed of tubular cells called myocytes containing many chains of myofibrils and cylindrical structures, extending the complete length of the myocyte. The streamlined cylindrical shape of the myofibril is important for transmission of forces, for cylinders are designed to provide a passage for the flow of information from one place to another (Oiwa & Manstein, 2007; Volk, 1995). This structural design ensures a fundamental requirement of all cells, their ability to interpret, convert, and convey information gathered from their immediate environment. To accomplish such a task, several systems have been designed in nature. The G-protein family, responsible for processing and converting information about membrane shear stress (Frangos, Mcintire, & Eskin, 1988) and the integrin receptors, sensing the surface tension of the cell through ligand binding (Ruoslahti & Pierschbacher, 1988). In addition, there are the strain activated ion channels which alter conductance when deformed (Guharay & Sachs, 1984), and the cytoskeletal network (microfilaments, microtubules, intermediate filaments), responsible for causing a conformational change in proteins and a deformation of the nucleus, altering biological activity (Maniotis, Chen, & Ingber, 1997; Palmisano et al., 2015). All of these will be discussed further below.

The myofibril provides a scaffold for the spatial distribution of proteins responsible for integrating force production and transmission (Sanger et al., 2005). The smallest repeated segment of the myofibril, the sarcomere, is the fundamental unit of muscle contraction dominating the anatomy of both skeletal and cardiac striated muscle (Batters et al., 2014; Lange, Ehler, & Gautel, 2006; Sanger & Sanger, 2001) (Figs. 2.2 and 2.3). A wide range of sarcomere lengths exist suggesting that no single sarcomere length can be used to explain all muscles; however, what is known to determine sarcomere length is the function of the muscle itself (Lieber, Roberts, Blemker, Lee, & Herzog, 2017). Despite the fact that sarcomeric lengths have a preferred operating range, published work by William and Goldspink suggesting that sarcomeres adapt to a new length by adjusting the serial sarcomere numbers (i.e. increase or decrease) has been questioned (Williams & Goldspink, 1978a, 1978b). A study by Takahashi et al. demonstrated that although serial sarcomere numbers increased initially at the MTU 1-week postsurgical tensioning procedures, when the MTU was stretched beyond its “physiological point”, this was followed by a rapid decrease in sarcomere numbers over the remaining 8-week period, despite sarcomere length being constant (Takahasi, Wand, Marchuk, Frank, & Lieber, 2010), suggesting that adaptation of the sarcomere of skeletal muscle is extremely plastic.

Fig. 2.2

The sarcomere. (Published with kind permission of copyright © Nikos C. Apostolopoulos 2018. All rights reserved)

Fig. 2.3

The sarcomere depicting the four filaments (in red). (Published with kind permission of copyright © Nikos C. Apostolopoulos 2018. All rights reserved)

The sarcomere is the largest macromolecular complex known, assembled from many protein subunits organised in a highly specific way into filamentous (myosin, actin, titin, and nebulin) and anchoring (Z-disc, M-band) structures responsible for the contractile process (Gautel, Mues, & Young, 1999), and of cytoskeletal elements and their relative proteins (Fig. 2.3). Although the sarcomere specialises in force generation, its fundamental activity and response to a mechanical force (i.e. stretching) is dependent upon the correct positioning of hundreds of proteins located within, as well as on its periphery. These cytoskeletal elements consist of both dynamic and passive components involved in the transference of force but, more importantly, the relaying of information from one sarcomere to another. These proteins play both structural and regulatory roles and are responsible for connecting the sarcomere to the plasma membrane and from there to the ECM. At this cellular and organelle level, the exosarcomeric network, the intermediate filament lattice of the cytoskeleton, envelopes the sarcomere linking the Z-disc and M-lines to the membrane skeleton (costamere), mitochondria, nuclei, and sarcoplasmic reticulum (Barral & Epstein, 1999; Sanger, Sanger, & Franzini-Armstrong, 2004; Wang, Mccarter, Wright, Beverly, & Ramirez-Mitchell, 1993) (more discussed in sections below). It is believed that in muscle cells, this intermediate filament network is force bearing (Lazarides, 1980; Wang et al., 1993).

In addition to the exosarcomeric, the endosarcomeric network exists consisting of four filaments: the thin and thick comprised mainly of the actin and myosin proteins, respectively, as well as the third and fourth filaments, represented by the large myofibrillar extensible titin and the inextensible nebulin proteins, respectively (Kruger, Wright, & Wang, 1991; Tskhovrebova & Trinick, 2003) (Fig. 2.3). Both the exo- and endosarcomeric network systems, and their associated proteins, are responsible for the synchronous response and conversion of the microscopic contractile process at the sarcomere leading to a macroscopic action.

The unique cross-striated appearance of striated muscle, first reported in 1840 by Bowman (Bowman, 1840), arises as a result of the transverse alignment of sarcomeric striations of neighbouring myofibrils (Wang & Ramirez-Mitchell, 1983). The maintenance of this alignment is attributed to a filamentous bridge between the M-line and Z-disc and the intermediate filament (Lazarides, 1980, 1982; Wang & Ramirez-Mitchell, 1983). The alternating light (isotropic) and dark (anisotropic) bands are direct and precise alignments of the four filament systems, the lateral boundaries of the sarcomere defined by the Z-disc, and the intermediate filament of the exosarcomere (Clark, Mcelhinny, Beckerle, & Gregorio, 2002; Rui, Bai, & Perrimon, 2010).

The Filament Systems of Skeletal Muscle

Since the groundbreaking work of Hugh Huxley (Huxley, 1969; Huxley & Hanson, 1954), detailed investigations have been conducted of the properties of myofibrillar proteins exploring the molecular basis for muscle contraction. What has been uncovered is that the dynamic interaction responsible for the assembly of myofibrils is dependent upon the coordinated integration of myosin, actin, and associated proteins, as well as other cytoskeletal elements.

As mentioned above, four filaments comprise the sarcomere. The parallel arrays of the thin filaments spanning the I band (light region), overlapping with the thick filament in the A-band (dark region), and their affiliated proteins and protein complexes, all responsible for the generation of active force and the subsequent performance of mechanical work (Rui et al., 2010).

Situated in between two I-bands is the Z-disc, with the M-line located in the middle of the A-band (Fig. 2.2). Both these transverse structures are important regions, for the thin and thick filaments cross-link in the Z-disc and M-line, respectively. This biological design serves an important structural and functional role maintaining the integrity of the sarcomere during contraction and stretching by establishing a precise arrangement of the filaments (Katzemich et al., 2012). The amount of overlap is determined by the respective length of these filaments, in particular the thin filament. Research has uncovered that the length of the thick filament is constant (1.6 μm); however the length of the thin filament fluctuates from~1.0 to 1.3 μm (Burkholder, Fingado, Baron, & Lieber, 1994; Littlefield & Fowler, 2008). The extent of overlap between the thin and thick filament determines the sarcomere’s force-generating capacity as well as the physiological requirement of the muscle (Burkholder et al., 1994). For instance, short thin filaments are associated with a reduction in force due to a decrease in the overlap with the thick filament (Granzier, Akster, & Ter Keurs, 1991; Ottenheijm & Granzier, 2010). In addition, it has been observed that the M-line is more prone to distortion under stress compared to the Z-disc (Agarkova & Perriard, 2005; Gautel, 2011; Linke, 2008).

Titin and nebulin form the third and fourth filaments, with titin spanning half the sarcomere and nebulin traversing the full length of the thin filament, with both playing an important role in sarcomere organisation, strength, and development (Clark et al., 2002; Meyer & Wright, 2013) (Fig. 2.3). For a detailed review of the filament system(s), their associated proteins, and the Z-discs and M-line of the sarcomere, and cytoskeletal proteins, the reader is directed to the end of the chapter to the suggested “Further Reading” section. The material in this section provides a brief overview of the structure and important components of the sarcomere in order to provide an understanding of how these structures relate to one another and how stretching, specifically the magnitude of stretching, may be responsible for causing an inflammatory response.

Thin Filament (Actin, Troponin, Tropomyosin, and Tropomodulin) (Fig. 2.3)

The thin filament consists of actin comprised of two twisted α helices associated and affiliated with the regulatory protein complex consisting of tropomyosin and troponin (Kostyukova, 2008; Rui et al., 2010; von der Ecken et al., 2015; Wakabayashi, 2015). In addition, tropomodulin, which requires tropomyosin for optimal function, is a pointed end thin filament capping protein associated with the thin filament and nebulin (discussed below), responsible for maintaining the final length of the thin filament, preventing it from elongating or shortening (Mcelhinny, Kolmerer, Fowler, Labeit, & Gregorio, 2001; Ottenheijm & Granzier, 2010). It also inhibits the depolymerisation and polymerisation of the actin monomers (Kostyukova, 2008).

Tropomyosin and troponin are attached to the actin backbone at regular intervals and are important for regulating contraction and the giant protein nebulin (Gordon, Honmsher, & Regnier, 2000; Horowits, Luo, Zhang, & Herrera, 1996; Wang & Wright, 1988; Zot & Potter, 1987). In skeletal muscle, tropomyosin forms a continuous ropelike structure over a head-to-tail association strengthened by troponin (Schutt & Lindberg, 1992). Troponin, which binds to the tropomyosin molecule is responsible for regulating the motion of tropomyosin on the thin filament (White, Cohen, & Phillips, 1987). This relationship between tropomyosin and troponin is very important for the generation of maximal force imparted on the Z-disc, for tropomyosin relative to actin is an inextensible parallel component responsible for summing up all the individual forces developed over the length of the actin filament in the overlap zone between actin and myosin (Schutt & Lindberg, 1992).

The tension developed at the sarcomere is a manifestation of the helical segment of the actin filament determined by its attachment to both the tropomyosin at one end and to the myosin head on the other, with contraction occurring when the actin segments pull on tropomyosin while being anchored via cross bridges to the thick filaments (Schutt & Lindberg, 1992). The actin-tropomyosin-troponin filament protein complex is responsible for (a) the calcium (Ca²⁺)-dependent movement of troponin and tropomyosin on actin filament, since these two proteins are essential for Ca²⁺ regulation (Gordon et al., 2000; Szczesna & Potter, 2002; Wakabayashi, 2015), and (b) the interaction site(s) with myosin (Barua, Winkelmann, White, & Hitchcock-Degregori, 2012; Borovikov & Gusev, 1983; Kad, Kim, Warshaw, Vanburren, & Baker, 2005). This complex is anchored in the Z-disc extending towards the middle of the sarcomere interdigitating with the thick filaments in the A-band region (Fig. 2.2).

Thick Filaments (Myosin)

Myosin is the principle protein of the bipolar thick filament and, in association with several other proteins, forms a remarkable and precise network for the purpose of contraction in skeletal muscle (Adelstein, 1983). The associated proteins of the thick filament are critical in mediating sarcomeric protein interaction, such as thick filament assembly and regulation of muscle contraction (Barral & Epstein, 1999; Clark et al., 2002; Kenny, Liston, & Higgins, 1999). Myosin provides the force necessary to drive muscle contraction but is also important in the structural formation of the thick filament (Vikstrom et al., 1997). It is one of the three major classes of molecular motors, the other two being kinesin and dyneins, with all three driven by the hydrolyses of ATP (Llinas, 2015). Unlike kinesin and dyneins which interact with the microtubule cellular tracks, myosin networks with actin filament tracks play both a structural and enzymatic role in muscle contraction and intracellular motility (Llinas, 2015; Lowey & Trybus, 1995; Oiwa & Manstein, 2007). Myosin consists of 18 subfamilies, with myosin II, predominantly associated with skeletal muscle (Lutz & Lieber, 1999; Oiwa & Manstein, 2007).

The highly asymmetric myosin II protein is comprised of two dimerised myosin heavy chain (MHC) molecules with each forming a head and neck domain, intertwining with its neighbour to form an extended α-helical coiled-coil tail (Clark, Langeslag, Figdor, & Van Leeuwen, 2007; Craig & Padron, 2004; Rayment et al., 1993). Each myosin heavy chain consists of two subunits, the heavy and light meromyosin, with the head and neck being part of the former and the tail part of the light meromyosin (Clark et al., 2007; Lutz & Lieber, 1999) (Fig. 2.3). Both the head and neck are referred to as catalytic and regulatory domains (Borejdo, Ushakov, & Akopova, 2002), with the former consisting of the NH₂ terminal responsible for binding to actin exhibiting an actin-activated ATPase activity (Schutt & Lindberg, 1992). The neck consists of the two myosin light chain molecules, the essential and regulatory (Borejdo et al., 2002; Trybus, 1994), with these molecules functioning as a lever arm for force production (Clark et al., 2002; Craig & Padron, 2004; Krendel & Mooseker, 2005).

The ordered arrangement of myosin heads consists of an inherent periodicity of approximately 14.3 nm intervals on a helical array that repeats at every 42.9 nm (Trinick, 1996; Xu et al., 1999). The structure of the backbone of the thick filament consists mainly of staggered, closely packed myosin tails running parallel to the filament axis and is responsible for assembling into a bipolar filament (Adelstein, 1983). Assembly of the thick filament is very much dependent upon the interaction between the rod segments of the myosin (Craig & Padron, 2004). According to Crick’s “knobs-into-holes” model, stability for two α helices into a coiled coil is exhibited by an electrostatic arrangement by the strong attraction of the hydrophobic residues in the core of the coil with hydrophilic residues lying on the surface (Chew & Squire, 1995; Crick, 1953; Mclachlan & Karn, 1982). This charge distribution within the myosin tail accounts for the fundamental periodicities associated with the myosin filaments (Craig & Padron, 2004; Offer & Sessions, 1995).

Third and Fourth Filaments and Obscurin

Within the myofibril are found three of the largest proteins in the body, titin, nebulin, and obscurin, with titin and nebulin comprising the third and fourth filaments of the endosarcomere. Both titin and nebulin are integral components of the sarcomere, with obscurin intimately surrounding them, primarily at the level of the Z-disc and M-line during myofibrogenesis and the M-line in adult myocytes (Bang et al., 2001; Kontrogianni-Konstantopoulos, Jones, Van Rossum, & Bloch, 2003; Young, Ehler, & Gautel, 2001).

Mechanical stability of the muscle is maintained by the presence of the extensible titin and the inextensible nebulin filaments, with both located in the gap region between the thick and thin filaments (I- and M-band) and from the ends of the isolated thick filament (Figs. 2.2 and 2.3). Besides mechanical stability, these endosarcomeric filaments are responsible for maintaining the assembly characteristic of the sarcomere (Funatsu, Higuchi, & Ishiwata, 1990; Granzier & Labeit, 2005).

Titin (Connectin) (Third Filament): The Titan Amongst Proteins

Titin , a force-bearing protein, is the largest polypeptide in the body, a giant myofilament, whose name is derived from the Titan gods of Greek mythology, who possessed great strength and importance (Luft, 2006). Titin, once considered to play only a structural role by acting as a scaffold, ensuring the continuity of the myofibrils of striated muscle, has now emerged as a candidate for a controller of filament assembly, since it traverses from the Z-disc to M-lines (Furst, Osborn, Nave, & Weber, 1988; Maruyama, Kimura, Ohashi, & Kuwano, 1981; Trinick, 1996). Titin’s dynamic nature suggests it has an important role in intracellular signalling and the passive mechanical properties of the sarcomere (Granzier & Labeit, 2004; Labeit et al., 2003; Miller et al., 2003; Nishikawa et al., 2012; Tskhovrebova et al., 2010). Its incorporation into the M-line network suggests a role as a stress sensor since the M-line is believed to limit longitudinal misalignments of the thick filament during contraction, responsible for making the position of the thick filaments unstable (Agarkova & Perriard, 2005).

Titin is responsible for the assembly of myofibres with its deletion from the sarcomere resulting in a total loss and disruption of the myofibril assembly, despite the presence of other sarcomeric proteins (Van Der Ven, Bartsch, & Gautel, 2000; Young et al., 2001). Spanning half the sarcomere, with its NH₂ and COOH termini in both the Z-disc and M-lines (the middle of the A band), respectively (Furst et al., 1988; Labeit, Gautel, Lakey, & Trinick, 1992; Squire, 1997; Tskhovrebova & Trinick, 2001), the NH₂ terminal forms an elastic connection between the end of the thick filament and the Z-disc in the I-band region (Luther, 2009).

As an elastic connector, this assures adaptation to I-band length changes during contraction but more importantly contributes to the generation of passive shortening/tension of the sarcomere during a relaxed state (no active force generated by myosin cross-bridges) (Furst et al., 1988; Kontrogianni-Konstantopoulos, Ackermann, Bowman, Yap, & Bloch, 2009; Labeit & Kolmerer, 1995; Maruyama et al., 1981; Tskhovrebova, Houmeida, & Trinick, 2006) (Fig. 2.4). The I-band domain is the only region exhibiting elastic stretch dependence, observed to approximately double in length during normal activity (Trinick, 1996; Wang, Wright, & Ramirez-Mitchell, 1985). In addition, since titin-containing filaments are centrally symmetric to the M-line, this is responsible for keeping the A band at the centre ensuring balanced myosin-cross-bridge forces in both thick filament halves during active contraction (Horowits, Kempner, Bisher, & Podolsky, 1986).

Fig. 2.4

Titin relationship to Z-disc and M-line. (Published with kind permission of copyright © Nikos C. Apostolopoulos 2018. All rights reserved)

The titin protein is primarily composed of 244 repetitive and 17 unique non-repetitive domains, with the former consisting of 112 immunoglobulin and 132 fibronectin-III superfamily domains (~90% of its mass) (Gautel, 1996; Labeit et al., 1992). The 17 distinct motifs, accounting for ~10% of titin’s mass, are comprised of phosphorylation sites, a 28–30 residue proline-glutamate-valine-lysine (PEVK) motif found in the I-region and a serine/threonine kinase domain located at the COOH terminal in the M-line (Fig. 2.4) (Granzier & Labeit, 2005, 2006; Greaser, 2001; Sebestyen, Fritz, Wolff, & Greaser, 1996). The physiological force levels responsible for maintaining the structural integrity of the sarcomere, specifically the passive muscle force, are determined by the PEVK (Linke, Ivemeyer, Mundel, Stockmeier, & Kolmerer, 1998; Trombitas et al., 1998). The repetitive domains (immunoglobulin and fibronectin-III) provide binding sites for many diverse proteins within the myofibre, including myofibrillar and membrane components, signalling molecules responsible for integrating mechanical and contractile activity with regulatory mechanisms controlling both metabolism and gene expression (Kontrogianni-Konstantopoulos et al., 2009).

Nebulin (Fourth Filament): The Uncertain Protein

Nebulin , another myofibrillar protein, received its name from the word nebulous because of its unknown function which was eventually revealed in part by the generation of nebulin knockout mice models (Ottenheijm & Granzier, 2010). These models exposed the important role that nebulin played in regulating contraction and the overlap of the thin-thick filaments (Ottenheijm & Granzier, 2010). During the stretching of the sarcomere, nebulin, located parallel to the actin filaments, remains relatively fixed to these filaments (Horowits et al., 1996). This adherence is due to nebulin being composed of ~185 α-helical modular repeats that are each ~35 amino acids in size (McElhinny et al., 2001), with each repeat being a potential actin-binding motif (Witt et al., 2006).

Nebulin functions as a “molecular ruler”, defining the length of the thin filament, since a single nebulin polypeptide spans the length of actin (Horowits et al., 1996; Pfuhl, Winder, & Pastore, 1994). Two studies with nebulin knockout mice observed the constant length of the thin filament with nebulin present, with the length decreasing in mice deficient of nebulin (Bang et al., 2006; Witt et al., 2006). This relationship suggests that when sarcomeres are subjected to overstretch, both filaments, actin and nebulin, act in a coextensive relationship in situ, since no change in length is observed of the actin filament (Kruger et al., 1991). The length of the thin filament is important for determining the amount of force generated by a muscle based on its physiological requirement (Burkholder et al., 1994).

The NH₂ terminus of nebulin is found at the pointed end of the thin filament interacting with the capping protein tropomodulin (H-zone) (Labeit, Ottenheijm, & Granzier, 2011; McElhinny et al., 2001), while the COOH-terminus is anchored to the Z-disc via α-actinin (Horowits et al., 1996). Localised at the Z-disc, α-actinin, a cytoskeletal actin-binding protein, forms a latticelike structure stabilising the muscle contractile apparatus, as well as playing an important role in muscle contraction (Sjoblom, Salmazo, & Djinovic-Carugo, 2008), for muscle fibres deficient in nebulin develop less force (Labeit et al., 2011). Nebulin in conjunction with titin is an important regulator of Z-disc structure, specifically in its assembly and width (Gautel, Goulding, Bullard, Weber, & Furst, 1996). It plays a role in the transverse linking of the myofibrils limiting the degree to which they can move transversely during contraction or passive stretching, thereby preventing damage to the intermyofibrillar membrane systems (i.e. T-tubules and sarcoplasm reticulum) (Ottenheijm & Granzier, 2010). In addition, nebulin is required to connect desmin, the most abundant intermediate filament protein (discussed further below) in striated adult muscle to the Z-disc preventing a greater displacement of the Z-disc which occurs during the stretch of the myofibre (Ottenheijm & Granzier, 2010; Shah et al., 2002). In short, nebulin contributes to isometric force production, width specification of Z-disc, and myofibrillar connectivity (Labeit et al., 2011).

Obscurin: The Concealed Protein

Obscurin (obscure) and nebulin (nebulous) share similar synonyms, vague and ambiguous, for similar to nebulin, this novel titin-interacting protein was difficult to characterise because of its somewhat low abundance, large size, and complexity (Young et al., 2001). Obscurin , a multidomain protein concentrated on the peripheries of the Z-disc and M-line of the sarcomere is composed of signalling domains and adhesion modules arranged in tandem (Kontrogianni-Konstantopoulos & Bloch, 2005). Both obscurin’s NH₂ and COOH-terminal sequences are exposed at the sarcomere surface, located to and restricted to the Z-disc and M-lines, an exposure consistent with the distribution of the sarcoplasmic reticulum and T-tubules, also located on the periphery of the sarcomere, suggesting that obscurin is the anchoring mechanism for the specific localisation of this membrane system (sarcoplasmic reticulum and T-tubules) (Kontrogianni-Konstantopoulos et al., 2003; Kontrogianni-Konstantopoulos & Bloch, 2005). In addition, since obscurin binds to titin and the sarcomeric myosin (Young et al., 2001), its termini are more accessible to segments of the sarcoplasmic reticulum, the myofibrillar cytoskeleton, and the surrounding myoplasm (Franzini-Armstrong, 1994; Kontrogianni-Konstantopoulos et al., 2003; Kontrogianni-Konstantopoulos & Bloch, 2005; Young et al., 2001).

Obscurin, similar to titin and nebulin, fulfills the role of a “molecular ruler” as suggested by its relationship with the sarcomere and the cytoskeletal components (McElhinny et al., 2001). However, instead of determining the longitudinal dimensions, continuous reticular labelling of obscurin in cross sections suggests that it is associated with the diameter of the sarcomere enveloping the myofibrils at the Z-disc and the M-line (Kontrogianni-Konstantopoulos et al., 2003). In animal studies, removal of obscurin was associated with changes in the longitudinal architecture of the sarcoplasmic reticulum and associated proteins, suggesting a prominent role in aligning the sarcoplasmic reticulum with the myofibrils of striated muscle cells. Besides affecting the integrity of the sarcolemma, obscurin is responsible for reduced muscle exercise tolerance and with disturbances in the organisation of the ECM during skeletal muscle development (Lange, Perera, Teh, & Chen, 2012; Randazzo et al., 2013).

In conclusion, the role for titin and nebulin is the passive elasticity of the sarcomere, both longitudinally and laterally (Horowits et al., 1986), and the maintenance of a static and dynamic stability during relaxation and contraction, by primarily ensuring that the thick filaments are kept at symmetrical positions (Horowits et al., 1986; Horowits & Podolsky, 1987). Both serve as scaffolds or length-regulating templates of the thick and thin filaments, important for contraction (Kruger et al., 1991). In addition, obscurin , an anchoring mechanism for the specific localisation of the membrane systems (sarcoplasmic reticulum and T-tubules), is an integral and important component for both the assembly of the membrane systems and the contractile apparatus affiliated with and required for Ca²⁺ homeostasis (Kontrogianni-Konstantopoulos et al., 2003, 2006; Young et al., 2001).

Z-Disc, Intermediate Filaments, and Costameres

The primary function of the proteins/structures of the cytoskeleton of the muscle cell is to link, anchor, as well as tether structural components inside the cell, ensuring a cooperative movement between the contractile and elastic components of the muscle (Goldstein et al., 1991; Stromer, 1998). The Z-disc and affiliated proteins continue this enigmatic role, with novel proteins and interactions continuously being discovered (Faulkner, Lanfranchi, & Valle, 2001; Gontier et al., 2005; Sanger & Sanger, 2008). The Z-disc is formed by the overlap of the thin filaments from opposing half-sarcomeres, cross-linked to a stable network consisting of four pairs of Z-filaments each comprised of α-actinin (Z-disc protein), associated with stable cross-connections of thin filaments (Sorimachi et al., 1997; Stromer & Goll, 1972) (Figs. 2.2 and 2.3).

Z-Disc

The Z-disc lies perpendicular to the myofibrils marking the end of the sarcomere, and is considered a supramolecular structure linked to both muscle injuries and atrophies (Faulkner et al., 2000; Gontier et al., 2005). It serves as an anchoring site for the actin, titin, and nebulin protein filaments (Fig. 2.3) being the primary conduit for the generation of force by contraction (Clark et al., 2002).

The intricate molecular architecture of the Z-disc, linking the contractile units of the sarcomere in series, is central to collecting information of the mechanical force generated by the interaction between the myosin and actin within the sarcomere (Faulkner et al., 2001; Sanger & Sanger, 2008). In the past, the Z-disc was considered as a passive structure of the sarcomere linking actin filaments of opposite polarity (antiparallel) in subsequent sarcomeres for the successive transmission of force generated in the myofilaments (Frank, Kuhn, Katus, & Frey, 2006; Squire, Al-Khayat, Knupp, & Luther, 2005). However, changes in Z-disc structure associated with development of active tension in skeletal muscle suggest that it is a dynamic structure involved in determining the mechanical properties of the muscle (Goldstein et al., 1991).

Depending on their location, proteins associated with the Z-disc can be divided into those partially located in the Z-disc and extending into sarcomere (i.e. titin, actin, nebulin, MLP, myopodin, etc.) (Barash, Mathew, Lahey, Greaser, & Lieber, 2005; Barral, Hutagalung, Brinker, Hartl, & Epstein, 2002; Horowits et al., 1986; Hutagalung, Landsverk, Price, & Epstein, 2002; Kruger et al., 1991; Linnemann et al., 2010; Liu, Srikakulam, & Winkelmann, 2008; Mcelhinny, Kazmierski, Labeit, & Gregorio, 2003; Powers, Nishikawa, Joumaa, & Herzog, 2016; Schutt & Lindberg, 1992; Witt et al., 2006) and those located entirely within the Z-disc (i.e. α-actinin, telethonin, CapZ, etc.) (Caldwell, Heiss, Mermall, & Cooper, 1989; de Almeida Ribeiro et al., 2014; Faulkner et al., 2000; Miller et al., 2003; Rubtsov & Lopina, 2000; Torgan & Daniels, 2000, 2001; Wette, Smith, Lamb, & Murphy, 2017; Yamashita, Maeda, & Maeda, 2003; Zhou et al., 2001) (Figs. 2.5 and 2.6). The numerous proteins of this highly organised 3-D structure form multiple protein complexes interacting with both extramyofibrillar and intramyofibrillar domains (Stromer, 1995) (Fig. 2.7). The extramyofibrillar consisting of costameres and intermediate filaments will be discussed further below. The intramyofibrillar consists of the actin, titin, and nebulin filaments responsible for interfacing the thin and thick filaments between the Z-discs and the M-line . The relationship and interaction of these protein complexes and domains are responsible for two important attributes of the muscle cell cytoskeleton, its strength and its flexibility, accommodating changes in cell geometry in response to muscle contraction or a mechanical stress (Stromer, 1998).

Fig. 2.5

Z-disc proteins partially located in Z-disc (associations and function). (Published with kind permission of copyright © Nikos C. Apostolopoulos 2018. All rights reserved)

Fig. 2.6

Z-disc proteins fully located in Z-disc (associations and function). (Published with kind permission of copyright © Nikos C. Apostolopoulos 2018. All rights reserved)

Fig. 2.7

Z-disc proteins, intra- and extramyofibril. (Published with kind permission of copyright © Nikos C. Apostolopoulos 2018. All rights reserved)

The architectural organisation of the Z-disc is responsible for actively coordinating various cellular signalling pathways (Gontier et al., 2005). For instance, the width of the Z-disc depends upon a variety of factors such as muscle and fibre type and the difference in actin filament overlap responsible for structural transitions (Stromer, 1995). These transitions place a load on the mechanical properties of skeletal muscle, observed during the response of the sarcomere to mechanical stimuli in the form of either a contraction or a stretch (Goldstein, Schroeter, & Sass, 1982; Stromer, 1995). The Z-disc maintains a certain form and spacing regardless of an increase in load suggesting its ability to resist deformation with a passive stretch (Goldstein et al., 1991). This structural criterion allows for the ease of transmission of tension generated within the sarcomere to bones via the tendons, for the Z-disc functions as an anchor, adjoining sets of thin filaments end-on-end in myofibrils (Goldstein et al., 1991).

Maintenance of form and spacing provides stability within the Z-disc lattice over a long range of sarcomere lengths, an important feature in skeletal muscle, since it operates over a range of sarcomere lengths, during extension and contraction (Goldstein et al., 1991). In short, the muscle is better able to conform to change by adjusting and adapting to any mechanical stress. Therefore, understanding the complexity of the Z-disc proteins and their interactions is relevant to comprehending the influence of a mechanical force (i.e. stretching) on the sarcomere and the subsequent biochemical response (i.e. mechanotransduction).

No sarcomeric protein is an “island” onto its own, for most have at least one if not more binding partners forming a network to support, stabilise, and provide cohesion to the myofibre (Faulkner et al., 2001). In addition, this network, in association with other proteins and protein complexes found within the extramyofibril and the sarcolemma, forms a fundamental unit of response, a feedback-signalling machine. This process allows for the proper and significant relay of information from a mechanical stimulus (i.e. stretching) to the myofibre via the ECM, and depending on its magnitude, this may influence the skeletal muscle tissue aversely or favourably. An example of a negative disruption is eccentric contraction, an elongation of the skeletal muscle in an activated state, responsible for damaging the sarcomeres and impairing the excitation-contraction coupling (Agarkova et al., 2003). Ultrastructural studies observed a characteristic change in the sarcomere patterns associated with localised regions of overstretched sarcomeres exhibiting irregular and distorted Z-discs (Yeung, Belnave, Ballard, Bourreau, & Allen, 2002). The stress on the sarcomere results in a breakdown to structures involved in the excitation-contraction coupling (i.e. sarcoplasmic reticulum, T-tubules), itself linked to a leakage of ions confirmed by measurement of intracellular Ca²⁺, resulting in inflammation (discussed further below) (Balnave & Allen, 1995). For instance, passive stretching of mice skeletal muscle >50% of strain disrupted the sarcomere, suggesting work done to stretch muscle is an indicator of the magnitude of injury (Brooks, Zerba, & Faulkner, 1995). This response to the magnitude of a mechanical stimulus (i.e. stretching) suggests that the intensity of the activity can alter the morphology of the intra- and extramyofibrillar components initiating an inflammatory response.

Besides the actin-myosin complexes, effective propagation of a mechanical signal (information) from the environment to the cytoskeleton, and the ensuing chemical response, is dependent upon several subcellular complexes. Apart from the Z-disc and affiliated proteins, the other complexes consist of the cytoplasmic intermediate filament (i.e. desmin) and the two major costamere complexes (dystrophin-glycoprotein and the integrin-vinculin-talin).

Intermediate Filaments: The Mechanical Relay System

The depletion of thin and thick filaments from the myofibril with use of a high-salt buffer results in a cytoskeleton composed predominantly of intermediate filaments (Lazarides, 1980) (Fig. 2.8). These filaments form a dynamic cytoskeletal infrastructure connecting the peripheries of each successive Z-disc as well as forming a sleeve surrounding each myofibril (Wang & Ramirez-Mitchell, 1983). In cells subjected to mechanical stress they play important roles as force mechanosensors and mechanotransducers (Conover & Gregorio, 2011).

Fig. 2.8

Intermediate filaments and costameres. (Published with kind permission of copyright © Nikos C. Apostolopoulos 2018. All rights reserved)

The intermediate filaments form a continuous network linking the cell membrane to the cytoplasmic organelles and nuclear envelope thereby functioning and acting as a mechanical relay system for the transmission of muscle tension (Anderson, Li, & Goubel, 2001; Conover & Gregorio, 2011). This network is responsible for diverse functions such as force transmission, mechanochemical signalling, cellular integrity, and the mechanical integration of the contractile actions of the muscle fibre (Capetanaki, Bloch, Kouloumenta, Mavroidis, & Psarras, 2007). The skeletal muscle intermediate filaments associate with the sarcolemma at structures termed “costameres”, which are present at the membrane overlying the Z-discs of the sarcomere (Capetanaki et al., 2007).

Costameres: The Achilles Heel

The relationship between the costameres and muscle is likened to the Greek heroes, Hercules and Achilles, with the muscle responsible for performing impressive feats of strength (Hercules) and the costamere, its Achilles heel (Ervasti, 2003). Costameres, transverse elements of the sarcolemmal lattice, resemble riblike structures (“costa”, Latin for “rib”, and Greek “meros”, part) consisting of a doublet band of densely clustered patches of vinculin (a cytoskeletal protein involved with cell-cell and cell-matrix junctions) segregated into two rows flanking the Z-disc as well as overlying the I band of the underlying sarcomere (Pardo, D’angelo Siliciano, & Craig, 1983) (Fig. 2.8). The costameres circumferentially align in register with the periphery of the Z-disc of the force generating myofibrils, physically coupling the Z-disc to the sarcolemma (Ervasti, 2007). Costameric proteins identified and associated with the costamere/Z-disc axis are responsible for converting a mechanical stimulus to a change in both cell signalling and gene expression (Ervasti, 2003). Whether the muscle is contracted or stretched, the periodicity of the costamere is identical to the underlying sarcomere, with this relationship observed whether a bundle of superficial myofibrils is staggered with respect to an adjacent bundle, displaced, or discontinued (Pardo et al., 1983).

Costameres are composed of large complexes of integral and peripheral membrane proteins linked to the contractile apparatus of the sarcomere by intermediate filaments and to the ECM (Pardo et al., 1983). Besides playing a mechanical and functional role for the transmission of force from the contractile apparatus to the ECM, costameres protect the sarcolemma against contraction-induced damage (Anastasi et al., 2008).

Although it is beyond the scope of this manuscript to describe the numerous relationships and nuances formed between the sarcomere, the sarcolemmal structures (intermediate filament and costamere complexes), and the ECM, in response to a mechanical signal, what is presented is a simple hierarchy of interaction. This level of interaction is descriptive of a dynamic relationship and integration of numerous proteins, and protein complexes, responsible for a signal transduction cascade, specifically the transference and processing of information. Movement of this communication is accomplished by a cell-wide system, a highway, composed of key inter- and extramyofibrillar proteins responsible for the connectivity spanning the nuclear matrix to the ECM and beyond. This continuous network ensures transduction of a mechanical signal from the environment, traversing and influencing the response(s) of the structures affected at both the macroscopic (i.e. muscles, MTU, tendon) and microscopic (ECM, myofibrils, nucleus) levels. The result is a biochemical response, a feedback to the mechanical signal, and a mechanotransduction process.

Communication with cells is made possible through a tensegrity architecture for structural organisation (Ingber, 1997), the integrity of an object under tension, a term coined by Buckminster Fuller, based on an object created by his student, Kenneth Snelson (Connelly & Back, 1998). According to tensegrity, objects maintain their structural integrity by achieving a balance between compression and tension. Use of tensegrity to describe the relationship between the various levels of tissues and cells in response to a dynamic external load (i.e. stretching) provides an explanation for the hierarchy of interaction mentioned above. As the mechanical signal passes from the macroscopic to the cellular environment, the cells sense and transduce the force, accomplished by protein structures responsible for creating a tensional force balanced by interconnected structural elements resisting compression within the cells of musculoskeletal tissues (Anastasi et al., 2006; Connelly & Back, 1998). The result is the transduction of the force into changes of cellular biochemistry and gene expression, mechanotransduction (discussed below).

The structural and regulatory proteins associated with this cell-wide system are responsible for both active and passive load bearing within muscle sarcomeres (Shah et al., 2004). Of the Z-disc proteins, titin, actin, and α-actinin play a prominent role in establishing a connection with the ECM, with disruption of the interaction between titin and α-actinin translating into an interruption within structure of the Z-disc, since the amount of titin overlap coincides with the width of the Z-disc (Gregorio et al., 1998). Titin is a Z-disc linking protein acting to specifically determine attachment of the α-actinin within the Z-disc creating sites for the cross-linking of the thin filaments (Sorimachi et al., 1997). Α-actinin functions as a ligand for titin enabling the cross-link of the antiparallel titin and thin filaments from opposing sarcomere (Sorimachi et al., 1997) (Fig. 2.8), With the interaction of α-actinin, titin, and actin contributing to the structural continuity of the sarcomere.

The establishment of a connection between the myofibril and the ECM is manifested by a system of intermediary components acting between the Z-disc. The structure and function of these intermediate filament proteins connect the contractile apparatus to the sarcolemmal cytoskeleton. The most predominant intermediate protein is desmin, essential for maintaining the integrity of the myofibrils upon stress (Li et al., 1997). Desmin is co-expressed with the proteins synemin and paranemin, forming copolymers typically localised around α-actinin-rich Z-discs (Costa, Escaleira, Cataldo, Oliveira, & Mermelstein, 2004). This interrelationship suggests formation of large intermediate filament protein networks creating a greater opportunity for interaction with other structures (Capetanaki et al., 2007; Schweitzer et al., 2001). Synemin which consists of binding sites for desmin, a-actinin, and vinculin (quintessential costameric protein) plays an important function directly linking intermediate filaments to both the Z-disc and costameres (Bellin, Huiatt, Critchley, & Robson, 2001).

Lack of desmin in skeletal muscle is associated with irregularities in the organisation of myofibres (i.e. loss of alignment of myofibrils), Z-disc streaming, focal degeneration, but more importantly a reduction of myofibril anchorage to the plasma membrane at the costameres (Agbulut et al., 2001). Studies in mice lacking desmin have observed that desmin-based intermediate filaments are important in linking the Z-discs of superficial myofibrils to costameres at the sarcolemma (Stone, O’Neill, Catino, & Bloch, 2005). In addition, desmin is essential for the nucleus to withstand any mechanical load associated with contraction, for an extensive loss of its position occurs in absence of desmin during stretching of a passive fibre (Shah et al., 2004). Through its high-affinity association with nebulin, desmin attaches to the sarcomeres (Conover & Gregorio, 2011). Since desmin contains multiple binding sites for nebulin, this allows for the preservation of the interfibrillar connectivity at the Z-discs, the proper assembly of desmin intermediate filaments at the Z-disc, influencing the architecture of the actin thin filament in myocytes (Conover & Gregorio, 2011). This relationship between desmin and nebulin is crucial for the stability and proper spacing of adjacent thin filaments required for the proper transmission of the lateral force from Z-disc to Z-disc (Conover & Gregorio, 2011).

Costameres, concentrations of ECM receptor complexes, refer to a connection site made between the contractile apparatus (myofibrils) and the sarcolemma. They are involved in the transmission of and sensing of mechanical stress associated with muscles and are considered a “proteic” machinery associated with the sarcolemma, signalling, transmembrane, and ECM proteins (Anastasi et al., 2008; Gumerson & Michele, 2011) (Fig. 2.9). Costameres are considered bands of vinculin, encircling muscle fibres, repeating along its length with a periodicity corresponding to subjacent sarcomeres (Craig & Pardo, 1983). They have many characteristics in common with the cell-ECM type of adherens junction, in particular focal adhesion complexes, since they function as a cellular communication unit, a signalling highway tethered between the Z-disc, a hotspot for muscle cell signalling and the ECM (Anastasi et al., 2009; Burridge & Guilluy, 2016; Peter, Cheng, Ross, Knowlton, & Chen, 2011). In addition, they are associated with the protection of the sarcolemma against contractile damage, for the membrane-associated costameres are coupled physically to underlying sarcomeres, widening and narrowing in concert with underlying I band in stretched and contracted muscle (Craig & Pardo, 1983; Peter et al., 2011).

Fig. 2.9

Costamere associations. (Published with kind permission of copyright © Nikos C. Apostolopoulos 2018. All rights reserved)

Two protein complexes concentrated at the costamere and registered to the Z-disc of the sarcomere are, the dystrophin-glycoprotein and the integrin-vinculin-talin complex. Both are responsible for regulating the interaction between the cytoskeleton and the ECM (Anastasi et al., 2004). The former complex, consisting of dystrophin (an elongated cytoskeletal protein), dystroglycan, and the sarcoglycan transmembrane subcomplex, plays a critical role between the ECM-cell interaction (Anastasi et al., 2003). Dystrophin, linking the submembrane actin and the intermediate filament is important for the transmission of the force of contraction across the sarcolemma to the extracellular structures and maintenance of the architecture of skeletal muscle (Ervasti & Campbell, 1993; Peter et al., 2011). The dystroglycan and sarcoglycan complexes link the signal transducing units of the ECM to the myofibrillar contractile elements (Anastasi et al., 2004; Ohlendieck, 1996).

Dystyroglycan, generated from a single gene is cleaved into two proteins, the peripheral α-dystroglycan and the transmembrane β-dystroglycan (Michele & Campbell, 2003), with the former binding to α-laminin, a component of the ECM, and the latter, connecting the cytoskeleton to the ECM by binding to dystrophin (Campbell, 1995), completing a pathway responsible for transferring a mechanical signal between the ECM and cytoskeleton. Closely associated to the dystroglycan complex is the multi-member sarcoglycan complex, consisting of six genes, α-, β-, γ-, δ-, ε-, and ζ-sarcoglycan, of which the α- and γ- are associated with skeletal muscle (Anastasi et al., 2009). This transmembrane protein stabilises the interactions of dystrophin and its associated proteins (dystroglycan with the ECM) (Hack, Groh, & Mcnally, 2000). A marked reduction in dystrophin and the associated glycoproteins (dystroglycans and sarcoglycans) disrupts a critical linkage between the sarcolemmal cytoskeleton actin and the ECM, compromising the integrity and flexibility of the sarcolemma, affecting its mechanical stability during cycles of contraction and relaxation (Campbell, 1995).

The other complex, the vinculin-talin-integrin, regulates interactions between the cytoskeleton and the ECM, by the sensing of forces (compression, tensile, shear, etc.) from both an “outside-in” and “inside-out” signalling process. It is an integral part of a myriad of tissues (muscle, tendons, bones, etc.) exposed to an ever-changing mechanical stimulus generated by environmental forces and movement (Atherton, Stutchbury, Jethwa, & Ballestrem, 2016; Dufort, Paszek, & Weaver, 2011). In fact, the sustained disruption of the tensional homeostasis of the cell and tissues caused by a mechanical stimulus results in alterations in the ECM, serving as a catalyst for either repair or harm. A major player coordinating this mechanoresponsiveness is vinculin, a mechanosensitive protein involved in the adaptation of tissue to forces and the homeostasis of the actin cytoskeleton (Atherton et al., 2016). It is governed and regulated by intracellular forces generated during force transduction in relation to talin (Atherton et al., 2016). Talin, a ubiquitous large focal adhesion protein, is an essential structural link between integrin and the intracellular machinery, activating integrin and coupling it with the actin cytoskeleton (Atherton et al., 2015, 2016; Humphries et al., 2007). This process is reinforced by vinculin (Atherton et al., 2015, 2016; Humphries et al., 2007), for the force-induced mechanical unfolding of talin exposes recognition sites for vinculin, with its binding to talin being essential for the process of mechanotransduction at the cell-matrix adhesion site (Haining, Lieberthal, & Del Rio Hernandez, 2016). The importance of force in this whole process was demonstrated by both in vitro (Ciobanasu, Faivre, & Le Clainche, 2014) and in vivo (Margadant et al., 2011) studies. The former, composed of substrate-bound talin and actomyosin, demonstrated that the isometric contraction of actin exposed the vinculin-binding sites of talin increasing the affinity of vinculin to talin while decreasing talin refolding (Ciobanasu et al., 2014). Similarly, the latter study, with use of fluorescence microscopy, observed that cyclical stretching of talin by the contraction of actomyosin resulted in recruitment of vinculin (Margadant et al., 2011), highlighting the importance of the magnitude of the force. Therefore, the mechanotransduction response of talin, its unfolding, and the binding of vinculin, are important processes responsible for a greater stabilisation and adhesion between the cytoskeleton and the ECM (Haining et al., 2016).

As indicated above, talin and vinculin, which are tethered at the costamere, are localised at the site of the cell-matrix adhesion interacting with integrin (Peter et al., 2011). Integrin, a major force-bearing adhesion receptor protein, plays a dynamic critical role in the cell’s ability to sense and respond to the mechanics of its surroundings (Puklin-Faucher & Sheetz, 2009). A key player in both cell signalling and adhesion, it is an ideal candidate for mechanotransduction, since the application of force stimulates its recruitment to the cell-matrix adhesion site (Galbraith, Yamada, & Sheetz, 2002), with vinculin stabilising the integrin-talin-actin complex, reducing focal adhesion turnover (Critchley, 2005). This binding of vinculin to talin in response to the magnitude of a stretching force is pivotal for coupling integrin to the actin cytoskeleton and the transmission of force from the matrix to the actin cytoskeleton (Critchley, 2005; del Rio et al., 2009).

Integrins, allosteric proteins, are transmembrane heterodimeric receptors of α/β subunits (Green et al., 2011; Mayer, 2003), integrating the cells’ exterior (ECM) with its interior (cytoskeleton), maintaining the integrity of the cytoskeletal-ECM linkage (Barczyk, Carracedo, & Gulberg, 2010; Campbell & Humphries, 2011; Van Der Flier & Sonnenberg, 2001). They play a crucial role as direct mechanotransducers, transmitters of force to other elements (Ross et al., 2013). To date 18α and 8β chains have been discovered paired in a manner forming at least 24 receptors for cell adhesion molecules (Mayer, 2003; Plow, Haas, Zhang, Loftus, & Smith, 2000). Molecules involved in cell adhesion are responsible for binding cells together into multicellular organisms and tissues, enabling the communication of cells and tissues with their surroundings (Van Der Flier & Sonnenberg, 2001).

Integrins have evolved a highly responsive receptor mechanism, enabling the bidirectional transmission of signals (“inside-out” and “outside-in”) between the ECM and intercellular interactions (Anastasi et al., 2004; Burkin & Kaufman, 1999), which explains their involvement in various biological phenomena (i.e. tissue repair and remodelling and cell migration) (Belkin et al., 1996). As receptors for cell adhesion molecules, integrins create appropriate opportunities for mechanical connectivity by bringing cells together to form tissues. Tissues are the instrument by which cells communicate with the environment in response to a wide range of physical forces (Schwartz, 2010). How this is accomplished is a hallmark function of integrins. Through the α/β pairing, a recognition for multiple extracellular ligands occurs, with the primary function and purpose being cell adhesion, permitting transmission of signal and cell-cell interactions.

Ligands are large multi-adhesive ECM molecules binding to integrins (Barczyk et al., 2010). The α/β pairings of the integrin determine the ligand-binding specificities for the various ECM proteins (Anastasi et al., 2004). Most α subunits have been paired with one or two β subunits. The β subunit, composed of three subfamilies (β1, β2, and β3), is used to group the integrins, with β1 and β3 mediating cell-matrix adhesions expressed by all cell types and β2 restricted to leukocytes (Green, Mould, & Humphries, 1998). The β1 subfamily forms the largest group of receptors for ECM proteins (Mayer, 2003). It is expressed in mammalian focal contacts, costameres, the myotendinous junction, and the sarcolemmal membrane (Mayer, 2003). The β1 subfamily features prominently in the binding of an integrin receptor to the ligands of muscle of the ECM, the fibronectin (α5β1), laminin (α6β1 and α7β1), and collagen (α1β1 and α2β1). The dynamic formation between the integrin and its specific ligand is a requirement for the process of mechanotransduction. Because of this integrin-ligand interaction, cell adhesion and its engagement with the ECM induce integrin activation and intracellular signalling; nonetheless, with time, this adhesion-induced integrin activation subsides with the cell reaching a new state of equilibrium (Jalali et al., 2001). Since it is beyond the scope of the present manuscript to describe the numerous combinations of α/β pairings and variations and their ligand molecule(s), the reader is directed to articles at the end of the chapter.

In summary, every component of the mechanical linkage from the external environment to the cell-matrix (ECM ligands, integrins, costameres, intermediate filaments, etc.) and the cytoskeleton (sarcomeric proteins, Z-disc proteins, etc.) is responsible for the mechanical properties of the muscle and its response to a mechanical load. The associations developed between these proteins and protein networks establish a hierarchy of interaction, an information-processing pathway by which the cell communicates with the environment via cell adhesion. This dynamic connection between the specific ECM ligands and the integrin-mediated adhesions is critical for the transmission of a signal into the cell consisting of physical (rigidity, composition) and biochemical (extracellular protein ligands) information, information generated by the ECM protein networks [ligands (collagens, laminins, and fibronectin)]. This signal, a response to a physical displacement of the protein networks by exogenous and physiological forces, influences the mechanical connectivity within each tissue and cell (Paluch et al., 2015). This process, a key inherent design of complex organisms, allows for the modulation of the mechanical strength of tissues to match the force encountered (Schwartz, 2010). For instance, physical stress, such as the stretching of a single force-bearing cytoplasmic molecule in response to cyclical stretching alters the proteins’ unbinding kinetics, exposing binding sites for other proteins (i.e. talin for vinculin) (del Rio et al., 2009; Margadant et al., 2011). This response influences the cell-matrix interface in an attempt for the cell to adapt and establish a new equilibrium (Margadant et al., 2011). When cells and tissues are subjected to forces that are too high, the established regulatory mechanisms involved in maintaining their integrity breaks down. This loss in integrity is responsible for the release of cytoplasmic contents into the surrounding tissue sending chemotactic signals (movement of a cell in response to a chemical stimulus) into the tissue prompting the inflammatory response and the recruitment of inflammatory cells to the site of damage (Clarkson & Hubal, 2002; Elmore, 2007; Robertson, Maley, Grounds, & Papadimitriou, 1993).

Muscle Architecture

Although the microscopic structures of the sarcomere, the four myofilaments, the Z-disc, and the affiliated proteins form the functional unit of the muscle, with sarcomere length strongly influencing force generation or elongation, the best predictor of force generation of the muscle macroscopically, is muscle architecture (Lieber & Friden, 2000). Relative to the axis of force generation, the arrangement of muscle fibres within a muscle determines its force-velocity properties (Lieber, 1992; Moreau, Simpson, Teefey, & Diamano, 2010).

Behaviour of muscle contraction in humans has been evaluated based on the assumed in vivo function associated with joint(s) action. Since direct observation is impossible, the assumption has been that action at the joint is a reflection of the intrinsic characteristic of muscle fibres and how they are affected by anatomical factors within the muscle and joint system (Bobbert, Ettema, & Huijing, 1990; Powell, Roy, Kanim, Bello, & Edgerton, 1984; Trotter, 1993; Wickiewicz, Roy, Powell, & Edgerton, 1983; Wickiewicz, Roy, Powell, Petrine, & Edgerton, 1984). In short, the force-velocity relationship reported for the muscle is similar to the muscle fibres (Fukunaga, Kawakami, Kuno, Funato, & Fukashiro, 1997). This assumption is controversial for one cannot obtain information about the amount of shortening or lengthening occurring during the movement of the joint (Fukunaga et al., 1997). For instance, pennate muscles (unipennate, bipennate, multipennate), where muscle fascicles are arranged diagonally, running at several angles relative to the muscle’s force generating axis, make it very difficult to determine the architecture of the muscle during joint movement (Bobbert et al., 1990; Roy & Edgerton, 1992; Trotter, 1993) not to mention that it is almost impossible to describe the behaviour of the MTU from joint movement (Fukunaga et al., 1997).

Regardless of this controversy, developments in imaging techniques (i.e. ultrasonography and MRI) have made it possible to determine function of the muscle-tendon complex based on the structural alignment of the muscle fibres (Fukunaga et al., 1992; Hensriksson-Larsen, Wretling, Lorentzen, & Oberg, 1992; Kawakami, Abe, & Fukunaga, 1993; Kawakami, Abe, Kuno, & Fukunaga, 1995). These observations propose that orientation of the muscle fascicle suggests direction of the muscle’s pull and its strength (Lieber & Friden, 2000). If fibres extend parallel to the muscle’s force-generating axis, they possess a longitudinal architecture (i.e. biceps brachii). If they are running at a fixed angle (0–30°), they are classified as unipennate (i.e. vastus lateralis) and multipennate referring to fibres running at several angles relative to the muscle’s force-generating axis (i.e. gluteus medius). This structure-function relationship defines force production and movement providing a plausible explanation of the mechanical basis of muscle injury during movement (Lieber & Friden, 2000). According to Gans et al., the architecture of the fibre(s) and how they are attached to each other influences force generation, performance, and function (Gans & Abbot, 1991).

Tendon

As suggested by sections above, a complex dynamic interaction exists between the cell and its physical microenvironment. This interaction involves a set of pathways between its surface (i.e. focal adhesions, integrins, intermediate filaments, cytoskeleton) and the nucleus, culpable of a biochemical response to a mechanical perturbation (Goldmann, 2012). This pathway, integral to the cell’s ability to adapt in response to an externally applied force (tension, compression, shear stress, stretching), results in the stimulation of biochemical pathways fundamental for tissue development, homeostasis, maintenance, and repair (Banes et al., 1995).

Tendons are rich ECM connective tissue structures consisting of strong collagen fibril arrays and a tenocyte (a fibroblast), found within the ECM (Subramanian & Schilling, 2015). In response to a mechanical force, collagen and prostaglandins, released by the tenocytes, characteristic cells in tendons and ligaments, modify the composition and elastic properties of the ECM (Milz, Ockert, & Putz, 2009; Valdivia, Vega-Macaya, & Olguin, 2017). According to Kjaer, these changes to the ECM are responsible for the tendon’s ability to resist the mechanical load generated during muscle contraction as well as form a functional attachment to the bone (Kjaer, 2004; Milz et al., 2009).

Tendons, are a specialised load-bearing tissue whose cells are sensitive to mechanical stimuli during loading. They adapt their ECM in an anabolic or catabolic manner, influenced by the magnitude, frequency, direction, and duration of the applied load(s) (Lavagnino et al., 2015). Tendons primarily function by transmitting tensile load to the bone from the muscle, ensuring stability and a greater efficiency in the movement of the musculoskeletal system (Gelberman, Goldberg, & An, 1988). It has been suggested that during eccentric contractions they act as mechanical buffers protecting the muscle from damage (Latash & Zatsiorsky, 1993).

Structurally, the tendon is attached to the muscle at the MTU and to the bone via the teno-osseous junction. This viscoelastic material, of limited elongation, consists of dense wavy (zigzag) parallel fibres that change as the load increases, in a time-dependent manner. The limited elongation of the tendon is critical for its non-linear viscoelastic response to applied loads (Summers & Koob, 2002; Woo & Buckwalter, 1988). As with all biological systems exposed to mechanical stress, the properties of the tendon are highly dependent on their structure (tendon fibres) and cellular organisation, particularly the orientation and composition of its ECM (PG, glycoproteins, elastin, and water) (Duenwald, Vanderby, & Lakes, 2009; Wren, Beaupre, & Carter, 2000). Primarily composed of water, the ECM consists of type I collagen (~65–80% dry weight) and elastin (1–2% of dry weight) (Lavagnino et al., 2015).

Tendons are arranged in a structural hierarchy, with each level consisting of inherent tensile properties progressing from type I collagen (microscopic) molecules to collagen fibrils, that are assembled into fascicles, characteristic of the crimped pattern, and finally into the tendon fibre (macroscopic) (Kastelic, Galeski, & Baer, 1978; Summers & Koob, 2002). The structural arrangement and the inherent mechanical property of collagen is responsible for the main physical characteristics of the tendon (Lavagnino et al., 2015). As the magnitude of force or load is increased, the collagen’s ability to form covalent intermolecular and intramolecular cross-links, inhibiting sliding between adjacent fibres and fibrils, allows the tendon to withstand high tensile stress (~50 to 150 MPa) (Lavagnino et al., 2015; Woo & Buckwalter, 1988). The linear region of the stress-strain curve of a tendon is associated with the extension of the collagen fibres in the direction of traction, combined with a sliding of the fibrils in their ECM (Goulam Houssen, Gusachenko, Schanne-Klein, & Allain, 2011). Once the load is removed, the majority of changes caused are recoverable, with the time required for recovery after unloading being greater than the time required during the loading period of the tendon (Gelberman et al., 1988).

The structure of the tendon is poorly suited to withstand compressive loading (Wren et al., 2000), with chronic stress loads or overuse injuries causing multiple micro-traumatic events, disrupting its internal structure, and is responsible for the degeneration of the tendon cells and matrix (Kraushaar & Nirschl, 1999). An in vitro study examining the effects of stretch on a rabbit tendon fibroblast cell revealed that the mechanical load (stretch), and the subsequent biochemical response (pro-inflammatory cytokine IL-1β), was responsible for degenerative changes (Archambault, Tsuzaki, Heerzog, & Banes, 2002).

Myotendon Unit (MTU) (Fig. 2.10)

Referring to the function and architecture of the muscle, the tendon, and the MTU, an appreciation is gained for the role each plays in relation to the stress or strain developed both extrinsically and intrinsically. During intrinsic loading, the force developed by the contractile filaments of skeletal muscle progresses through the MTU to the tendon transforming the force from the muscle to the bone, creating joint movement (Huxley, 1969; Mccall, Byrnes, Dickinson, Pattany, & Fleck, 1996). Throughout this transition, the ECM is responsible for a functional link amongst the three tissues, playing a fundamental role in the transmission of force and the maintenance of tissue structure of tendons, ligaments, bone, and muscle (Kjaer, 2004). According to a comprehensive review, the ECM of both the tendons and intracellular connective tissue is a dynamic structure adapting structurally and functionally to mechanical load (Kjaer, 2004). In addition, the magnitude, frequency, and duration of forces on these tissues depend on their prior loading history (exercise, overuse, disuse) and the composition of the ECM (age, disease, microdamage) (Tidball & Quan, 1992).

Fig. 2.10

Myotendon unit. (Published with kind permission of copyright © Nikos C. Apostolopoulos 2018. All rights reserved)

The MTU found at either end of long cylindrical skeletal muscle fibres, is considered a significant component of the tension transmitting mechanism. It is characterised by four separate ultrastructural domains connecting the actin filaments and the associated cross-linking structures of the terminal sarcomere, with the matrix occupying the space between the collagen fibres of the tendon (Tidball, 1984; Trotter, Eberhard, & Samora, 1983). Of the sarcomeric forces generated and transmitted onto a tendon, a significant part of the force created is oriented parallel to the direction of the force ensuring that the junction is loaded in shear (Huijing & Baan, 2001; Trotter et al., 1983). This force is generated by transmembrane proteins, and not by the continuous bilayer of lipids (phospholipid, glycolipids, and cholesterol) separating the myofilaments from the connective tissue filaments (Trotter, Corbett, & Avner, 1981).

To facilitate this transmission by shear force, the MTU presents a complex geometry, with the plasma membrane at each end of the muscle cell being highly folded and invaginated, forming branch fingerlike cylindrical-shaped cell processes (Law, 1993; Trotter, Hsi, Samora, & Wofsy, 1985). Cylindrical shapes used in nature are synonymous with a structural connection allowing for the transmission of forces, a passage from one place to another (Volk, 1995). The cylindrical geometry of the digit-like extensions greatly amplifies the interfacial area between the muscle and connective tissue resulting in a decrease in stress at the muscle-tendon interface (Tidball, 1984; Trotter, Samora, & Baca, 1985). A reduction in junctional surface area is often accompanied by an increase in junction failure, suggesting that stresses in pathological conditions are higher than normal resulting in a mechanical failure, although the cells are less capable of force production (Tidball, 1984). Observations of this interfacial folding by investigators have interpreted this folding as an extension of the muscle cell into the tendon, with a complimentary extension of the tendon into the muscle cell (Mackay, Harrop, & Muir, 1969; Tidball, 1983). In addition to the folded interface, transmission of force from one sarcomere to its serial neighbour and eventually onto the MTU supports the viewpoint that the MTU is a specialised tissue designed for force transmission (Huijing & Baan, 2001).

During development of the muscle, tendon cells attach to the muscle through the ECM forming the MTU. This formation is a communication between the interaction of the integrins and the ECM molecules secreted by the muscle and tendons, as well as the contribution of dystroglycan, responsible for muscle binding to the ECM (Valdivia et al., 2017). The MTU is one of the two structures responsible for the transmission of a mechanical force between the outside and inside of muscle cells, based on the structural relationship between the cytoskeleton proteins, talin and vinculin, and components of the ECM, with the other structure being the costamere (Bozyczko, Decker, Muschler, & Horwitz, 1989; Pardo et al., 1983). Vinculin and talin function to connect the actin filaments to the muscle sarcolemma with regard to the MTU, as well as maintaining the internal myofibrillar structure and the transmission of tension during contraction with regard to the costameres (Bozyczko et al., 1989).

The fingerlike projections are interdigitations of the sarcolemma, where actin filaments extending from the last Z-disc are connected to the subsarcolemmal proteins indirectly interacting with the tendinous ECM proteins (Charvet, Ruggiero, & Le Guellec, 2012). Two separate transmembrane linkage systems are present in the MTU, each structurally linked by laminin, specifically laminin 211 (Charvet et al., 2012). Laminin is a multifunctional macromolecule found in the basement membrane and is the most abundant structural non-collagenous glycoprotein in the ECM (Aumailley & Smyth, 1998). The systems linked by laminin are the dystrophin-glycoprotein complex and the α7β1 integrin, as discussed above.

Suggestion that the “chain is no stronger than its weakest link” exemplifies the importance of the relationship of the sarcolemma to the basement membrane, a thin highly specialised sheet of ECM surrounding muscle. To insure a mechanical reinforcement of the sarcolemma during the cycle of contraction and relaxation, it is necessary that an intact link exists between the intracellular cytoskeleton and the surrounding basement membrane (Holmberg & Durbeej, 2013). This membrane consists of two layers, the basal and reticular lamina, with the latter being composed mainly of collagen and connected to the endomysium (Holmberg & Durbeej, 2013). The basal lamina, directly linked to the sarcolemma, consists of the lamina lucida and densa. Since it is beyond the scope of this manuscript to go into detail regarding laminin, the basement membrane, and the MTU, the reader is directed to material at the end of the chapter. What is presented is a pathway connecting the muscle to the MTU and to the tendon, the transmembrane systems, the structures of the ECM, and the cytoskeletal proteins.

Strongly present in the MTU, the biological functions of the laminin 211 are dependent on both the dystrophin-glycoprotein complex and the α7β1 integrin linkage system. As mentioned in the costamere section, the majority of integrins involved in cell-ECM adhesion share a common β1 subunit, with the main integrin in adult skeletal muscle being α7β1 (Song, Wang, Foster, Biesler, & Kaufman, 1992). This is localised peripherally around fibres enriched at the MTU, as well as the neuromuscular junctions (Bao, Lakonishok, Kaufman, & Horwitz, 1993). The α7 subunit, highly expressed at the MTU, exhibits a weaker presence in other regions of the plasma membrane, and is also a determinant of junctional specificity, serving to distinguish the MTU from other adherens junctions (Bao et al., 1993). The appearance of α7β1 integrin at the MTU correlates with insertion of myofibrils into subsarcolemmal densities and folding of the junctional membrane, playing a role in both the formation and integrity of the MTU, as well as force generation (Bao et al., 1993). The connection between the ECM and the α7β1 integrin is important for the transmission of mechanical forces and maintenance of skeletal muscle fibres (Grounds, Sorokin, & White, 2005).

Of the dystrophin-glycoprotein complex, α- and β-dystroglycans are major non-integrin cell surface receptors necessary for binding to components of the surrounding basement membrane (Holmberg & Durbeej, 2013; Michele & Campbell, 2003). The extracellular α-dystroglycan protein binds to laminin 211, a component of the ECM, and to β-dystroglycan, with β-dystroglycan connecting the cytoskeleton to the ECM by binding to dystrophin, which in turn combines to the actin cytoskeleton, completing a pathway responsible for the transfer of a mechanical signal between the ECM and cytoskeleton (Campbell, 1995).

In summary, the MTU is a highly specialised region between the muscle and tendon. It forms an integrated mechanical unit with the last sarcomere of the muscle and its associated intracellular contractile proteins, and the connective tissue proteins of the tendinous-enriched type I collagen molecules of the tendon ECM matrix (Charvet et al., 2012; De Palma, Marinelli, Pavan, & Bertoni-Freddari, 2011). The collagen fibrils of the tendon insert into deep recesses formed between the cylindrically folded fingerlike processes of muscle cells increasing the contact area between the muscle and tendon collagen fibres. This connection, in shear, is similar to the magnitude and character of a shear stress at focal adhesions. It has been suggested that since these structures express similar protein compositions, they are analogous, for cells adhere tightly to and exert force upon their respective ECM structures (Tidball, O’Halloran, & Burridge, 1986). The cytoskeletal proteins prominently localised at focal adhesions and the MTU are vinculin, talin, and α-actinin, key players in cell signalling and adhesion. As discussed above, these proteins are ideal candidates for mechanotransduction, since the application of force stimulates recruitment of vinculin to bind to the exposed sites of the unfolded talin protein (Haining et al., 2016). Vinculin is found in high concentrations at both focal adhesion complexes (Galbraith et al., 2002) and the MTU. The expression of talin at the MTU is similar to costameres and, likewise, regulated by mechanical loading. At the MTU, it possesses a specialised function in force coupling, important in maintaining the MTU under mechanical stress (Valdivia et al., 2017). It is important to note that the MTU is a dynamic structure where new sarcomeres are added in this junction in response to stretch-induced hypertrophy (Garrett, Nikolaou, Rtibbeck, Glisson, & Seaber, 1988; Williams & Goldspink, 1971). Passive stretch, within a physiological range, results in a 2% strain on the tendon but an 8% strain in the MTU demonstrating the differences in the viscoelastic properties of the various regions of the MTU (Magnusson, 1998).

Macroscopic and Microscopic Information Processing Levels: Microscopic—Force, Cells, Tissue, and the Extracellular Matrix (ECM)

Force, Cells, and Tissue

The impact of mechanical forces on tissue has been known for over a century, when Julius Wolf postulated that bone tissue adapts its structure to the mechanical environment (Eyckmans, Boudou, Yu, & Chen, 2011; Ingber, 2004). He observed that the trabeculae of the bone matched the principal stress lines caused by physical loading (Eyckmans et al., 2011), such that the coordinated growth of the tissue was guided by the mechanical forces imparted on it (Orr, Helmke, Blackman, & Schwartz, 2006). Mechanical force influences and plays a role in the development and evolution of the tissues (Silver, 2006), with tissues altering their structure to best meet the mechanical demands placed on them (Mueller & Maluf, 2002). The adaptive quality of the cell and ultimately the tissue is determined by force, in particular its response to the temporal, rate, and magnitude characteristics of a mechanical stimulus responsible for activating distinct signalling pathways affecting their state and function. This ability to adapt suggests that altering and manipulating the magnitude of the force(s) influences the outcome of the intervention. In addition, the magnitude, frequency, and duration of force on tissues and cells are dependent upon the prior loading history (exercise, disuse, overuse) and the composition of the ECM (tissue type, age, sex, disease, microdamage) (Lavagnino et al., 2015).

Forces interact with biological structures at various and multiple length scales influencing their forms and functions. In essence, the processing of information in units of individual molecules is via spatial-, conformational-, force-sensitive-, and temporal operations such as binding, unbinding, catalysis, degradation, and cross-linking (Xia & Kanchanawong, 2017). This ability of cells and tissues to sense and respond to a mechanical force regardless of its origin is central to many aspects of biology. The common denominator for such a response is cell shape, more precisely the deviation from “normal” shape, since all cells have a unique morphology (size and shape). This change in morphology has been associated with changes in cellular function since the maintenance of homeostasis is a catalyst of adjusting to the various stresses imparted on the cell. Biomechanical signals sensed by cells in vivo, are associated with cellular deformation, mechanical stress, as well as deformation of the nucleus in response to elongation (Freytes, Wan, & Vunjak-Novakovic, 2009). In addition, any diffusible factors directing cellular function are either secreted by the cells themselves (autocrine), produced by other cell types (paracrine), or are transported through the bloodstream (endocrine) (Freytes et al., 2009). With all biological tissues and their cells, the ECM and molecular factors incorporated into the ECM are responsible for altering cell function. Residual tensile “prestress” in the cytoskeleton, tensile strengths, and elastic stiffness (deformability) is influenced by the ECM, through attachment of the cell to the matrix (matrix ligand-integrin linkage), the cell-cell connections, and contractility. As suggested above, force(s) can be applied by the cell’s own cytoskeleton, or from external sources, influencing the adhesion(s) mediated by the transmembrane integrins responsible for triggering intracellular signals remodelling the ECM (Schwartz, 2010).

Physical force(s) (i.e. tension, compression, rotation, bend, shear, and gravity) (Fig. 2.11) place different structural and functional demands on tissues at the cellular level (Ingber, 1997; Vandenburgh, Hatfaludy, Karlisch, & Shansky, 1991). Force is both static and dynamic in nature (Watson, 1991), with the level of exposure over a given area defined by the magnitude, time, and direction of the stress application (Mueller & Maluf, 2002). For instance, if a cell protein undergoes a structural change from an extended to a contracted state, the application of the stretching force is responsible for changing its energy state (Khan & Sheetz, 1997). The direct response of the adhesions to the force is responsible for the recruitment of the ECM proteins.

Fig. 2.11

Forces. (Published with kind permission of copyright © Nikos C. Apostolopoulos 2018. All rights reserved)

Forces have been increasingly recognised as major regulators of cell structure and function (Jamney & Mcculloch, 2007; Khan & Sheetz, 1997) modulating almost all aspects of cell function, including growth, differentiation, migration, gene expression, protein synthesis, and apoptosis (Alenghat & Ingber, 2002). Actively contracting and innervated muscle fibres, subjected to active and passive tensions, can regulate the growth of the myofibre both longitudinally (Williams & Goldspink, 1971) and cross-sectionally (Goldberg, Etlingger, Goldspink, & Jablecki, 1975). Mechanical forces affect the form and function of tissues directly, with the effects of compression on the bone and cartilage and tension on the muscle being several examples (Alenghat & Ingber, 2002).

Each organism is constantly subjected to either an external mechanical stress (e.g. gravity, movements) or internal (contractile and hemodynamic) forces generated by both muscle and non-muscle cells (Chiquet, 1999). In processes ranging from the contraction of muscles to the alignment of the chromosomes at the metaphase plate, forces must be adjusted to appropriate levels by cells in order for them to function properly (Khan & Sheetz, 1997), suggesting that the ability for muscle tissue to respond to mechanical force is central to and relevant to the restoration of its structure and function (Orr et al., 2006).

Since tissue structures are dynamic and change in response to the physical demands placed on them, their growth and development are direct responses to the force (Frost, 1990). Passive stretch in young animals has been observed to cause an increase in muscle tension, stimulating skeletal muscle growth, by inducing an increase in protein accumulation in the muscle tissue (Goldspink, 1977; Vandenburgh, Hatfaludy, Sohar, & Shansky, 1990). It has been observed that mechanical tension in the form of a “passive” stretch is associated with hypertrophy of the skeletal muscle following a tenotomy of the gastrocnemius muscle (Schiaffino & Hanzlikova, 1970). This mechanical “tension” is one of the basic “trophic” factors acting on skeletal muscle, exerting a fundamental regulatory function, adapting the muscle to variable physiological demands (Schiaffino & Hanzlikova, 1970). Therefore, “continuous integration” of the internal responses to a mechanical force is integral to the homeostasis of the tissue, a biological adaptation and regulation of the internal milieu (Schulkin, 2004).

The Extracellular Matrix (ECM)

The relationship of the human body to the environment is not a linear cause-effect for the body is a highly intermeshed open energetic system. Its response to change, more importantly its behaviour to change, is informed by non-linear paradigms, most notably chaos theory and complex adaptive systems, a quantum rather than a linear event. By referring to the chaos theory, we are stating that the response of the body is sensitive to initial conditions that are highly variable and often difficult to predict, since many systems are involved in coherent responses to environmental cues (i.e. mechanical stress). The complex adaptive system involves multiple component parts interacting in a non-linear manner. The information processed from both the micro- and macroenvironments are important to maintain and organise the structure and function of the body. At the micro level, a large part of the information is based on a reciprocal relationship between the cells and the ECM, which was recognised over 30 years ago and is still a central concept in cell biology (Bissell & Aggeler, 1987).

The ECM in the muscle, is dynamically remodelled, according to the loads imposed on it during muscle growth, exercise, and as a response to damage (Purslow, 2008). It is a complex amalgamation of macromolecules (collagens, non-collagenous glycoproteins, elastin, and proteoglycans) capable of self-assembly, predominantly via non-covalent bonds (Kresse & Schonherr, 2001). At one time it was thought of as an inert, supportive scaffold, stabilising the physical structure of tissue; however, evidence from cell culture experiments suggests that it is a dynamic and complex environment characterised by biophysical, mechanical, and biochemical properties specific to each tissue (i.e. bone, muscle tendon) (Bowen, Jenkins, & Fraser, 2013; Lee & Nelson, 2012). The ECM is important in developmental and regenerative processes, transmembrane signalling, and force transmission (Badylak, 2002), serving as a reservoir for growth factors and cytokines, modulating their activation and turnover (Kresse & Schonherr, 2001). This dynamic structure adapts to physiological demands placed upon it, changing and remodelling, decreasing susceptibility to stress, modifying mechanical and viscoelastic properties, and increasing load resistance (Voermans et al., 2008). A study examining myoblast proliferation and differentiation on solubilised and 3D decellularised quadriceps muscle matrices of mice, suggests that in vivo muscle ECM is responsible for guiding cell positioning (Charturvedi et al., 2015).

A hierarchical organisation of ECM zones, corresponding to the endo-, peri-, and epimysium, the basement membrane of skeletal muscle, and the MTU exists, with evidence suggesting that the perimysium is continuous with the tendon since both consist of type I collagen (Gillies & Lieber, 2011). Since these zones overlap, classification of the components of the skeletal muscle consists of several features defined by histological, ultrastructural, and molecular appearances (Fig. 2.12). A symbiotic and reciprocal relationship exists between the cells and the ECM of each area, influenced by the biological relevance of external cues essential for their function and maintenance. For instance, cells encapsulated in biomimetic hydrogels, have been observed to remodel their surrounding matrix, producing new ECM molecules, with this remodelling behaviour influenced by several external cues such as the type of hydrogel and the absence or presence of mechanical and biochemical stimuli (Ahearne, 2014).

Fig. 2.12

Classification of skeletal muscle ECM. (Published with kind permission of copyright © Nikos C. Apostolopoulos 2018. All rights reserved)

To date, three areas of biological relevance exist: the physical properties (stiffness, viscoelasticity, tensile, compressive, shear), the spatial organisation (size, shape, morphology of adhesion surface), and the biochemical complexity of matrix molecules (collagens, laminins, fibronectin) (Akhmanova, Osidak, Domogatsky, Rodin, & Domogatskaya, 2015). These are fundamental to the form and function of the connective tissue, regulating the behaviour of cells, as well as determining both the development and maintenance of tissue homeostasis in response to force, both internally and externally (Mammoto & Ingber, 2010). By either a direct or indirect action, the ECM provides the structural foundation for mechanical integrity and tissue function, influencing and regulating the availability of growth factors and cytokines (Hynes, 2009; Pickup, Mouw, & Weaver, 2014). Regulating the production, degradation, and remodelling of its components, the ECM supports tissue development, function, and repair (Lu, Takai, Weaver, & Werb, 2011), for modulation of cell binding to the ECM is what drives cell growth and adaptation. These components, their relative amounts and organisation, and the ECM molecular scaffold are unique for each tissue, reflecting the specific functions required for the cells present in the tissue (Gattazzo, Urciuolo, & Bonaldo, 2014).

A substantial part of the volume of tissue is extracellular space, with their cells being joined, supported, and surrounded by an intricate network of macromolecules forming the ECM (Alberts et al., 2002). All cells at one time or another make contact with the ECM, either continuously or during important phases (Hynes, 2009). Cells have a unique relationship with their environment, in particular to mechanical stress, and coupled with the ECM proteins, are responsible for maintaining their shape, while providing structural support for tissues (Hynes, 2009). The magnitude of an external force can affect the response of the cell, altering the geometry of the ECM, for cells within connective tissue establish and maintain the ECM during development, remodelling it during adaptation, and repairing it in response to injury (Humphrey, Dufresne, & Schwartz, 2014). In previous sections, the importance of the communication between the ECM ligand and its specific cell integrin (i.e. vinculin-talin-integrins, costameres) was presented. This interactive relationship is responsible for the cell’s direct reaction to an externally applied or internally created force. The transmission of information to the cell provides the cues to process any changes to the topography of the surrounding ECM, such as its rigidity, deformability, and its anisotropy (Bershadsky, Balaban, & Geiger, 2003; Chen, 2008; Geiger & Bershadsky, 2002). This allows the cell to actively modify its microenvironment by synthesising or degrading the ECM, secreting cytokines, and communicating with other cells (Freytes et al., 2009).

The stiffness (rigidity) or elasticity of the ECM is an important material property of connective tissue. Stiffness allows cells to sense the external forces, and respond to the environment in an appropriate manner, via the process of mechanotransduction (Mammoto & Ingber, 2010). This property is a measure of the relationship between the applied force and its compliancy (i.e. stretchability), determining its ability to adapt to stress, as defined by the magnitude and rate of application of an external or internal stimulus. As previously discussed, cell adhesions formed by integrin receptors, are key and important transmembrane structures that process diverse sources of signals, and are the sites of the transmission of forces between the cytoskeleton and the ECM, with the ECM playing a central role in transducing the effects of force to regulate cell functions (Ross et al., 2013). These external and internal inputs influence cell-cell and cell-matrix contact, since any exposure to a mechanical perturbation is felt with and influenced by the ECM (Discher, Mooney, & Zandstra, 2009; Geiger, Spatz, & Bershadsky, 2009). In addition to magnitude, the frequency and duration of the mechanical stimuli on cells are also determined by their prior loading history (exercise, disuse, overuse) (Tidball & Quan, 1992).

The ECM is more plentiful than the cells it surrounds, composed of various proteins and polysaccharides secreted locally by the cells themselves, and assembled in an organised meshwork (Sapir & Tzlil, 2017). The hallmark of the ECM, is that it can be dynamically remodelled, tailored to both the structure and function of each tissue, reflecting the tissues inherent physiological state (Egebald, Rasch, & Weaver, 2010; Hynes, 2009). The mechanical properties of a tissue are determined by the ECM composition and orientation, in particular by the mechanical coupling of the cell matrix (Qi et al., 2006). An example of the importance of this coupling was provided by an experiment which observed that the elasticity of the matrix was responsible for specifying the lineage of a cell towards a neuron, a myoblast, or an osteoblast, by influencing the differentiation of a mesenchymal stem cell into a cell type matching the elastic properties of the substrate (Engler, Sen, Sweeney, & Discher, 2006). In most soft tissues (skin, muscle, brain), the ECM and the function of adherent cells contribute to and establish this elastic microenvironment (Discher et al., 2005). The ability to adapt to the substrate and its inherent functional requirements, is responsible for the diverse forms of tissues, such as becoming calcified to form the rock-hard structures of the bone or teeth, or adopt the ropelike organisation that gives tendons their enormous tensile strength (Alberts et al., 2002).

When referring to the relationship between the cells, the ECM, and the mechanical load, what unfolds is a simplified comprehensive summary of key components, the substrates, the effectors, and the sensors (Humphrey et al., 2014). The ECM is composed of a number of molecules belonging to three major families of matrix components, the macromolecules (the substrate), regulated by diverse biochemical factors, including growth factors, cytokines, and hormones: elastin(s) and fibrillar collagens, and the proteoglycans (PGs), with related glycosaminoglycans (GAGs) (Alberts et al., 2002; Bowen et al., 2013; Humphrey et al., 2014; Hynes & Naba, 2012; Lee & Nelson, 2012). The fibroblasts (the effector) are the primary cells in connective tissue responsible for building, maintaining, and organising the mechanical homeostasis of the matrix, producing and secreting the matrix macromolecules (Humphrey et al., 2014). And finally, the integrins (the sensors), are the main cellular components mediating the sensing and regulation of ECM mechanics (Humphrey et al., 2014). Presented below and summarised in Fig. 2.13, are the fundamental and key players responsible for governing the cell-ECM interactions. For a more in-depth information, the reader is directed to further readings at the end of the chapter.

Fig. 2.13

Key players in mechanical homeostasis. (Published with kind permission of copyright © Nikos C. Apostolopoulos 2018. All rights reserved)

The Substrate

The elastic fibres of tissues are composed mainly of elastin, a significant structural component of the ECM. Elastin, a chemically inert insoluble polymer, is one of the most stable constituents of the ECM of connective tissue, endowing them with extensibility and resilience, responsible for mechanical memory (ability to recoil back to their homeostatic state) (Baldwin, Simpson, Steer, Cain, & Kielty, 2013). It is very important in connective tissue subjected to repetitive distension and physical stress (i.e. elastic arteries). Expression and the synthesis of elastin typically occurs in fibroblasts and chondrocytes, with elevated levels of elastin synthesis observed following injuries, and as a consequence of pathogenic conditions (Rodgers & Weiss, 2005).

Fibrillar collagens are the most abundant in vertebrates, playing a structural role by contributing to the shape, molecular architecture, and mechanical properties of tissue (i.e. resistance to traction in ligaments) (Ricard-Blum, 2011). They are vital for transmission of force during muscular contraction (Kjaer, 2004). Consisting of five members synthesised by connective tissue cells, in particular fibroblasts, osteoblasts, and chondrocytes, the major and abundant types are collagens I, II, and III, with the minor and sparse types being collagens V and XI (Ricard-Blum & Ruggiero, 2005). In contrast to elastin, fibrillar collagens bestow stiffness and strength to connective tissues (Humphrey et al., 2014). It has been observed that similar to contractile proteins, human tendon and muscle collagen are highly responsive to exercise, due to the sensing and chemical transduction of a mechanical stress (Miller et al., 2005). This mechanotransduction process via the ECM, that is, the increased synthesis of collagen from the fibroblasts, is dependent upon an interplay between a mechanical load and the integrin(s) (Chiquet, Renedo, Huber, & Fluck, 2003; Ingber et al., 1994).

Proteoglycans, well-known components of the ECM, include cartilage, connective tissue, and cell surface molecules (Selkirk, 2000). These sugar-modified proteins consist of a protein core, with one or more long unbranched sugar polymers (glycosaminoglycans) attached through the process of glycosylation. Proteoglycans are responsible for providing a key stabilising force for proteins within microenvironments (Arey, 2012). Similar to ECM molecules and adhesion receptors, proteoglycans are quite diverse, with some being essential for the function and development of the skeleton, immune, and central nervous systems (Couchman, 2003). The various combinations of proteins (matrix, cell surface transmembrane) and glycosaminoglycan chains [hyaluronan (HA), chondroitin (CS), keratin (KS), dermatan (DS), and heparan (HS) sulphate] found in vertebrates account for the diverse proteoglycan molecules (Perrimon & Bernfield, 2001; Yanagishita, 1993). This structural diversity is responsible for various functions attributed to glycoproteins. For instance, syndecan, a family of transmembrane proteoglycans via their heparan chain, binds growth factors, ECM components, protease inhibitors, chemokines, and enzymes, and plays an active role in signal transduction (Perrimon & Bernfield, 2001). All cellular processes involving interactions at the cell surface (i.e. cell-matrix, cell-cell, ligand-receptor) involve proteoglycans, for these molecules avidly bind proteins and are abundant at the cell surface (Yanagishita, 1993).

The Effectors

The fibroblasts, metabolically active cells, are the primary mononuclear cell types responsible for the formation of the majority of ECM components including, collagen, fibronectin, proteoglycans, tenascin, and laminin (Gillies & Lieber, 2011; McNulty, 2006). Although skeletal muscle myoblasts are associated with production of collagens, fibroblasts are crucial for the assembly of collagen into a functional ECM (Chapman, Meza, & Lieber, 2016). Lying within the interstitial space between muscle fibres, fibroblasts are responsible for the production of the majority of skeletal muscle ECM (Gillies & Lieber, 2011). Coordinating the macromolecules, they regulate the synthetic and mechanical machinery, providing the overall structural organisation and mechanical properties of the tissue (Humphrey et al., 2014). With fibroblasts continually synthesising ECM proteins, a major function is the production and the homeostatic maintenance of the ECM . The skeletal muscle fibroblasts secrete and assemble fibrillar collagen isoforms composed primarily of collagens I and III (Light & Champion, 1984). These fibrillar molecules bear tremendous stress within the muscle, providing the ECM with the ability to transmit both longitudinal and lateral force from the muscle to tendons (Chapman et al., 2016; Kjaer, 2004; Tidball, 1991).

The Sensors

The integrins, mentioned above, are the main cellular components mediating the sensing and regulation of the ECM mechanics (Humphrey et al., 2014). They are essential for binding matrix proteins, the associated cytoskeletal and signalling proteins of focal adhesions, and the actomyosin cytoskeleton (Humphrey et al., 2014). Focal adhesions, are clusters of integrin transmembrane receptors, coupling the ECM to the actin cytoskeleton via actin-binding proteins, with sensing, signalling, and force transmission being informed by the actomyosin-integrin-actin-binding protein-ECM pathway (Ciobanasu, Faivre, & Le Clainche, 2013). The relationship between the actin-binding proteins, talin and vinculin was discussed earlier with regard to their importance for force transmission. In particular with vinculin, its association with focal adhesions, is correlated with the force applied at the site, with its recruitment to cells being abolished in the absence of talin (Ciobanasu et al., 2013).

Conclusion

Tissues and cells are exposed to diverse types of extrinsic mechanical forces including mechanical stretch (tension), compression, and shear stress (Carver & Goldsmith, 2013). Their behaviour is largely determined and influenced by interactions with neighbouring cells, systemic mechanical cues, and the skeletal muscle ECM. The structural integration between the individual muscle fibres and the ECM, with its collagen content linking tissues together, determines force transmission and absorption of loading energy, tissue structure maintenance, and repair for tendons, ligaments, bone, and muscle (Alexander, 1991; Alexander & Bennett-Clark, 1977; Gillies & Lieber, 2011; Kjaer, 2004). The altered expression of specific ECM proteins is part of the adaptive response to various types of mechanical stress (Chiquet, 1999). For instance, during diseased or injured states, the cell and the ECM adapt by altering muscle function accordingly (Gillies & Lieber, 2011). The mechanical stress caused by these states regulates production of ECM proteins directly, with the triggering of an intracellular signalling pathway activating the gene, or indirectly, with release of a paracrine growth factor (Chiquet et al., 2003). It is interesting to note that the environment surrounding the cell, determines its pattern of gene expression and phenotype, despite the genome of the cell remaining the same (Nelson & Bissell, 2006).

An in vitro study observed tension as a requirement for maintaining the phenotype typical of tendon fibroblasts, for which collagen I amounts to more than 20% of their total protein synthesis (Chiquet, 1999; Quinones, Neblock, & Berg, 1986). Interestingly, the same cells (collagen I) at sites of compression “transdifferentiate” into fibrocartilage (Benjamin & Ralphs, 1998; Chiquet, 1999), suggesting that the type of mechanical stress is a basic requirement for proper function. In this manuscript, the concern is with tension and tensile strength (i.e. resistance to pull) of the muscle and tendon, in the form of stretching. The tensile strength of the tissue, specifically the matrix, is based on intra- and intermolecular cross-links and the orientation and length of the collagen fibrils and fibres (Kjaer, 2004). The connective tissue of skeletal muscle and tendon is a “plastic” structure with a dynamic protein turnover possessing the capacity to adapt to changes in the external environment, such as mechanical loading or inactivity and disuse (Kjaer, 2004). The realisation that mechanical stimuli (load, force) contribute to the health and pathogenesis of the tissue, illustrates the need to research the magnitude and rate of stretching that best contributes to the overall health and recovery of the tissue(s) of the musculoskeletal system: the muscles, the tendons, and the MTU.

The Strain Factors of Stretching: Intensity, Duration, and Frequency

Stretching initiates a stress/strain response on the connective tissue dependent on its magnitude (intensity) and rate, which is influenced by the biochemical complexity of the matrix molecules (i.e. collagen, elastin, PGs, and GAGs) (Ransone, Geissler, Wilson, & Adams, 2012). As mentioned above, these fibrillar molecules bear tremendous stress within the muscle, providing the ECM with the ability to transmit both longitudinal and lateral force from the muscle to tendons, in response to a time-varying mechanical stimuli (Chapman et al., 2016; Hoffman, Grashoff, & Schwartz, 2011; Kjaer, 2004; Tidball, 1991); This stress/strain response is responsible for cellular changes as observed in an in vitro study, where the concentration in tenascin C and collagen XII, two ECM components of fibroblasts, increased by an eightfold during stretching (Chiquet, 1999).

According to DeDeyne (2001), the mechanical stimulus of stretching influences the ECM, detected by and transmitted via the integrin into the cell interior activating a series of nuclear proteins which modify gene transcription and regulate sarcomeregenesis. Sarcomeregenesis is the synthesis of contractile proteins produced by specific muscle genes by mechanotransduction (Martins et al., 2013). Mechanotransduction, the process by which mechanical energy is converted into biochemical signals, is critical for mediating adaptations to mechanical load in connective tissues which precipitates changes in intracellular biochemistry and gene expression (Hamill, 1997; Ingber, 2008b; Ko & Mcculloch, 2001). More on mechanotransduction will be presented below.

With cellular response to mechanical force coupled to the organisation of the ECM (Chen, Tan, & Tien, 2004), the ECM, similar to hormones and growth factors, plays an important role in cell growth and behaviour (Daniels & Solursh, 1991; Shimizu & Shaw, 1991). As discussed above, this interaction between the ECM and cell is largely mediated by the integrins, bridging the cell-ECM interactions (Hynes, 1992; Meredith, Fazeli, & Schwartz, 1993). The adhesion facilitated by the integrins ensures a mechanical connection between the ECM fibrils with the intracellular actin cytoskeleton, facilitating the response and adaptation to mechanical stimuli from the environment (Schwartz, 2010). This adhesion is not only essential for binding matrix proteins and the associated cytoskeletal and signalling proteins (Humphrey et al., 2014)., but provides the understanding of how the force generated by stretching is transmitted to the cytoskeleton.

Regardless of the stretching methods used to facilitate increases in ROM about a joint, what is common amongst all is the parameters of training: intensity, duration, and frequency (Marschall, 1999; Mujika et al., 1995). Prescriptions of intensity (how hard the stretch is, referencing the elongation of the muscle for a given rate of stretch), duration (time spent), and frequency (how often), are responsible for the adaptive response of the tissue to stretching or training (Mujika et al., 1995; Seiler, 2010; Young, Elias, & Power, 2006). In addition, the position assumed during stretching may influence directly or indirectly the intensity of the stretch, for the force generated prior to and/or during the stretch (passive or active), potentially alters the response of the muscle and tendon tissue and their components (i.e. collagen) (Apostolopoulos et al., 2015), which respond to altered levels of activity (Kjaer, 2004). A study comparing a standing to a supine hamstring stretch observed that the latter isolated the hamstring muscle better, was more comfortable, but more importantly facilitated a better relaxation response during stretching (Abdel-aziem et al., 2013).

Although duration and frequency are important in stretching programmes, with bouts of stretching (30–60 s) improving ROM and flexibility in the human muscle (Bandy et al., 1997), the intensity, the magnitude of force applied during stretching (Jacobs & Sciacia, 2011), and the rate of force may be of a greater significance. Too much force during stretching is associated with an inflammatory response and fibrosis (Brand, 1984; Mcclure, Blackburn, & Dusold, 1994). However, unlike duration and frequency, which are easier to quantify, with numerous articles referring to them in relation to stretching (Bradley, Olsen, & Portas, 2007; Brandenburg, 2006; Cornwell, Nelson, & Sidaway, 2002; Feland, Myrer, & Merrill, 2001; Kokkonen, Nelson, & Cornwell, 1998), intensity is more difficult. In relation to stretching, intensity is defined as a feeling, a perception associated with a certain amount of exertion. This relationship between perceived intensity and stimulus strength is a psychosocial response, an internal representation associated with the magnitude of the stimulus, evoking a feedback, essentially a relative measure exacting a conscious interpretation within the mind (MacKay, 1963). According to psychophysical laws, this perception or feeling represents a concept referred to as a “magnitude production” (Stevens, 1971). In other words, the value associated with the intensity of the stretch conveyed by the individual, is an adjustment in their response, producing a match to the stimulus. In short, intensity is a qualitative response unique to individuals. This qualitative nature explains why intensity, with respect to stretching, remains under researched, accounting for the lack of relevant articles.

Therefore, the aim of the current project is to look at stretch intensity and its influence on the musculoskeletal tissues, with a particular focus placed on the relationship of the magnitude of the force generated during stretching and how this may cause inflammation and the inflammatory response. Stretching imparts a mechanical energy on the body, with stretching intensity (i.e. low vs. high) potentially influencing the mechanical equilibrium between the tension-generating muscle and tension-resisting tendons. In short, the manipulation of stretch intensity may provide answers for the proper recovery of the musculoskeletal tissue, thereby improving its function and athletic performance, as well as for the treatment of various musculoskeletal disorders, by influencing inflammation and the inflammatory response .

Inflammation

Inflammation is an essential immune response that enables survival during infection or injury and maintains tissue homeostasis under a variety of noxious conditions. Inflammation comes at the cost of a transient decline in tissue function, which can in turn contribute to the pathogenesis of diseases of altered homeostasis.

… Medzhitov ( 2010 )

Introduction

The history of Inflammation and its description can be traced back to both ancient Egypt and Greece (Granger & Senchenkova, 2010). Hippocrates, the father of medicine, coined the term oedema to describe inflammation, referring to it as a necessary process of healing following tissue damage (Granger & Senchenkova, 2010). Based on a visual observation, the Roman medical writer, Celsus, is credited with describing the four cardinal signs of acute inflammation: rubor et tumor cum calore et dolore (redness and swelling, with heat and pain) (Marmelzat, 1977; Medzhitov, 2010). The fifth sign, functio laesa (loss of function), was added a century and a half later, by the Greco-Roman physician/physiologist, Galen, being a characteristic sign for both acute and chronic inflammation (Lawrence, Willoughby, & Gilroy, 2002; Rather, 1971; West, 2014). In the eighteenth century, the Scottish surgeon John Hunter wrote, “inflammation in itself is not to be considered as a disease, but as a salutary operation consequent to some violence or some disease” (Palmer, 1835), further reinforcing the importance of inflammation with regard to homeostasis of the body.

Inflammation is an important and useful host defence mechanism in response to a foreign body challenge or tissue injury. It is a necessary information process of our tissues and their ability to function and survive; however, it can also cause further damage (Slauson & Cooper, 2002). In 1972, Thomas Lewis wrote, “Our arsenal for fighting off bacteria is so powerful, and involves so many different defense mechanisms, that we are more in danger from them than the invaders. We live in the midst of explosive devices; we are mined” (Thomas, 1972).

Inflammation attempts to restore the damaged tissue to its pre-injury state. It consists of an innate system of cellular and humoral responses (Rock & Kono, 2008; Ward, 2010). It is either acute or chronic, with acute defining a series of responses beginning within hours and lasting for several days, characterised by the influx of neutrophils and macrophages, cells of the immune response (Ward, 2010). The adaptive value of the acute inflammatory response is removal of cellular debris, necrotic tissue, and the repair of damaged blood vessels, myofibres, and the ECM (Cannon & St. Pierre, 1998). However, if the injury is persistent or a foreign material in the wound cannot be eliminated, this progresses to chronic inflammation, with a preponderance of lymphocytes and macrophages (Lucke et al., 2015), suggesting that chronic inflammation is not defined by the duration of the response (Ward, 2010). In order for tissue to return to a normal state, what is needed is a moderation of the four components of a typical inflammatory response: the inducing stimulus, the sensors, the inflammatory mediators, and the target tissues (Medzhitov, 2010). The inducing stimulus (i.e. muscle or tissue damage, injury, infections, etc.) initiates the inflammatory response, with the outcome (i.e. apoptotic or damaged cells) being detected by the sensors of the inflammatory pathway (i.e. resident macrophages, toll-like receptors, etc.) at the site of damage. These sensors induce production of the inflammatory mediators [i.e. cytokines (IL-1β, IL-6, and TNF-α)], which act locally (site of damage) and systemically (other parts of the body), effecting various target tissues (i.e. blood vessels, endothelial cells, liver, etc.) of the inflammatory response (Haslett, 1992; Medzhitov, 2010).

The disturbance of the microcirculation (i.e. infection, tissue injury) begins the inflammatory response, with leukocytes and serum proteins moving from the blood to the extravascular tissue (Lawrence et al., 2002; Medhitov, 2008). This is controlled and regulated by the release of vasoactive and chemotactic mediators (Lawrence et al., 2002). The nature of the inflammatory trigger (i.e. infection, tissue damage) is responsible for the path preceding the inflammatory response (i.e. bacterial, viral infections, tissue damage, etc.) (Medzhitov, 2010). With this present work, the concern is the response to a muscle disturbance caused by a mechanical stimulus (i.e. stretching), since this occurs in the absence of an infection.

Several mechanisms are responsible for inflammation: the injurious stimulation of mast cells and nerves, bleeding caused by tissue damage, cell death due to injury, contusions, and eccentric exercise (Rock & Kono, 2008; Smith, 1991, 1994). Mechanical injury to the myofibre activates an immediate necrosis along the entire length of the myofibre exposing the sarcoplasm (Li, Cummins, & Huard, 2001). The myofibres nearest the damage undergo a hypercontraction, tearing apart into a series of irregular dense masses of myofilaments in the form of contraction clots (Warren, Lowe, & Armstrong, 1993). These clots activate an extracellular protein kinase responsible for initiating degeneration of muscle tissue and local inflammation (Aronson et al., 1998).

Eccentric exercise and acute stretch injuries are examples of mechanical stimuli, given that the force generated causes an excessive overload of the contractile elements of the skeletal muscle exceeding its habitual requirements (Toumi & Best, 2003). Structurally, there is a disarrangement of the myofilament in the sarcomeres, damage to the sarcolemma, loss of fibre integrity, and the subsequent leakage of the muscle proteins into the blood (Jones, Newham, Round, & Tolfree, 1986). This functional change results in a decrease or loss in muscle force, changes in mechanical (Brockett, Morgan, & Proske, 2001) as well as proprioceptive properties (Walsh, Allen, Gandevia, & Proske, 2006), and is responsible for triggering an acute response (Hellsten, Frandsen, Orthenblad, & Sjodin, 1997). According to Tidball (2005), the extent of the inflammatory response is determined by the degree of muscle damage, the magnitude of the inflammation, and the injury-specific interaction between the invading inflammatory cells and the damaged muscle. Muscle inflammation is defined by several phases, destruction, repair, and remodelling (Jarvinen, Jarvinen, Kaariainen, Kalimo, & Jarvinen, 2005), with each characterised by the appearance of predominant inflammatory cell types (Philippou et al., 2012).

Neutrophils, Macrophages, Cytokines, and Acute Phase Response

Neutrophils and Macrophages

The release of reactive oxygen species by necrotic cells caused by damage to muscle tissue triggers the inflammatory response (inducing stimulus) (Cheung, Hume, & Maxwell, 2003). Once within the interstitium, the inflammatory cells activate the resident satellite cells (the sensors) releasing a chemotactic agent, resulting in the infiltration of circulating inflammatory cells (Clarkson & Hubal, 2002; Robertson et al., 1993). These inflammatory cells migrate into the site of inflammation through the endothelial wall of the skeletal muscle cells, via the process of rolling, adhesion, and transmigration (Ley, Laudanna, Cybulsky, & Noursharhg, 2007).

In the early stages of acute inflammation, the first responders are the neutrophils and polymorphonuclear leukocytes, recruited to the inflammatory site. Their activation increases their longevity several fold, which ensures their presence enabling them to carry out complex activities contributing to the resolution of inflammation or shaping the adaptive immune response (Kolaczkowska & Kubes, 2013). Neutrophils are detected within the first hour, peaking between 24 and 48 h and remaining for up to 5 days (Smith, Kruger, Smith, & Myburgh, 2008; Tidball, 2005). In addition to their phagocytic activity, they release proteases aiding in the degradation and removal of the cellular debris (Tidball, 2005). Neutrophils are gradually replaced by macrophages, appearing approximately 2 days post damage, further contributing to the degradation of the damaged tissue and the removal of necrotic cellular debris (Lawrence et al., 2002; Philippou et al., 2012). In addition, macrophages play a key role in repair (Cannon & St. Pierre, 1998; McLennan, 1993). Unlike neutrophils, macrophages consist of two phenotypes, the phagocytic pro-inflammatory, responsible for removal of cellular debris and necrotic tissue (neutrophils), and the non-phagocytic anti-inflammatory , involved in muscle repair, which peaks 4 days post damage and remains elevated for many days afterwards (Malm et al., 2000; Smith et al., 2008; Tidball, 2005; Tidball & Villalta, 2010). Both neutrophils and macrophages are responsible for the release of cytokines. More will be discussed below.

Cytokines

Cytokines , are small glycoproteins released by activated immune cells regulating inflammation. They are produced by a number of cell types (i.e. leukocytes) (Khan, 2008). In response to tissue damage and infection, cytokines function as intercellular messengers responsible for the movement of immune cells towards the site of inflammation (Kushner, 1993; Zhang & An, 2007). These soluble mediators are multifunctional, such that more than one cytokine is capable of acting on the same target cell negotiating similar functions (Akira, Hirano, Taga, & Kishimoto, 1990).

The term cytokine is a general term with specific terms referring to their origin. Cytokines are subdivided into either pro- or anti-inflammatory. They act locally in an autocrine (a hormone action binding to receptors on and affecting the function of the same cell that produced it) (The Free Dictionary, 2016), or a paracrine (a communication between cells producing a signal-inducing changes to nearby cells) manner, and are capable of an endocrine communication, responsible for cell-to-cell messaging over a long distance affecting a target organ (i.e. liver). Cytokines are characterised by pleiotropy (multiple biological actions), redundancy (shared biological actions), synergy (two or more cytokines’ additive effect), and antagonism (two or more cytokines’ inhibitory effects), with these characteristics responsible for creating a complex and intricate network (Nicola, 1994; Veskler, 2005). Although muscle damage stimulates a local cytokine cascade, initiated by various cell types (fibroblasts, neutrophils, and macrophages), a clear-cut and specific contribution of each cell type and the subsequent specific roles of each cytokine is very difficult to determine (Smith et al., 2008). Types of molecules included under the cytokine umbrella are the tumour necrosis factors (TNF-x), the growth factors (x-GF), and the interleukins (IL-x) (secreted by some leukocytes acting on other leukocytes) (Fig. 2.14).

Fig. 2.14

Summary of cytokines . (Published with kind permission of copyright © Nikos C. Apostolopoulos 2018. All rights reserved)

Neutrophils , the first responders of the immune system migrate to the affected sites in response to injury (Kim & Luster, 2015). They are a source of chemokines produced in the bone marrow (Furze & Rankin, 2008), being a group of cytokines specifically released to recruit discrete leukocyte subpopulations (Scapini et al., 2000; Von Vietinghoff & Ley, 2008). One such chemokine is the pro-inflammatory cytokine IL-1β, a potent chemoattractant (a chemical agent inducing a cell to migrate towards it) for macrophages (Smith et al., 2008), with the neutrophils and macrophages responsible for the release of another cytokine, TNF-α. Both IL-1β and TNF-α, besides initiating the degradation of the damaged muscle tissue (Peake, Nosaka, & Suzuki, 2005), are responsible for the expression of a third pro-inflammatory cytokine, IL-6 (Frost & Lang, 2005). IL-6, a known pleiotropic (affects the activity of multiple cell types) cytokine is released into circulation in response to multiple homeostatic perturbations including injury, trauma, and acute infections (Streetz, Wustefeld, Klein, Manns, & Trautwein, 2001). These three cytokines form a complex intricate network with the expression of each influencing the other, with IL-1 and TNF-α being potent inducers of IL-6, and IL-6 regulating the expression of TNF-α (Akira et al., 1990; Akira, Taga, & Kishimoto, 1993; Mcgee, Bamberg, Vitukus, & Mcghee, 1995). This synergistic relationship is an attempt to regulate the immune response and the inflammatory reaction (Akira et al., 1990, 1993; Streetz et al., 2001). In addition, IL-6 production in contrast to IL-1β and TNF-α has been linked to a contraction-induced response by skeletal muscle (Smith et al., 2008). The release of these cytokines locally is responsible for initiating a systemic response to tissue damage, the acute phase response (Streetz et al., 2001).

Acute Phase Response

The acute phase response is activated as a feedback to disturbances of the homeostasis of the organism (Kushner, 1982) (Fig. 2.15). It is linked to both a major adaptive and defensive role regarding tissue injury and infection. The acute phase response, an intrinsic response, is initiated after the first few days following the inducing stimulus and is associated with a vast number of changes, both systemic and metabolic (Kushner, 1982). Its objective is the containment and destruction of infectious agents, removal of damaged tissue, and assisting in repair (Kushner, 1982). As mentioned above, IL-1β, TNF-α, and IL-6 are responsible for its activation; however, although IL-1 and TNF-α are considered mediators with regard to initiating the acute phase response, they are not responsible for inducing a full acute phase response (Akira et al., 1990); that role primarily belongs to IL-6, initially referred to as a hepatocyte-stimulating factor (Fuller & Grenett, 1989; Gruys, Toussaint, Niewold, & Koopmans, 2005; Ramadori, Van Damme, Rieder, & Meyer Zum Buschenfelde, 1988; Streetz et al., 2001) (Fig. 2.5).

Fig. 2.15

Acute phase response. (Published with kind permission of copyright © Nikos C. Apostolopoulos 2018. All rights reserved)

The acute phase response refers to changes in the concentrations of numerous acute phase proteins produced in the liver (Kushner, 1993; Streetz et al., 2001). It is primarily induced by IL-6, a messenger between the hepatocytes synthesising the acute phase proteins and the local site of damage (Baumann & Gauldie, 1990; Whicher & Westacott, 1992). The acute phase proteins are influential in one or more stages of inflammation, healing, or an adaptation to a noxious stimulus (Gabay & Kushner, 1999). In humans, C-reactive protein, fibrinogen, and serum amyloid alpha are acute phase proteins (Jain, Gautam, & Naseem, 2011). Normally present in trace amounts in the plasma, an averse stimulus initiates a dramatic increase in their rate of synthesis and concentration by the liver (Kushner, Ganapathi, & Schultz, 1989).

Interest in the present work is with C-reactive protein, in particular high-sensitivity C-reactive protein (hsCRP), as a measure of inflammation in response to various stretching intensities. C-reactive protein is present at the very onset of any infection or tissue injury (Castell et al., 1990; DuClos, 2000; Heinrich, Castell, & Andus, 1990). With its synthesis not specific to any disease, increases or decreases in its concentration are proportional to the inflammatory stimulus (Libby, Ridker, & Maseri, 2002). The expression of C-reactive protein is considered a more accurate reflection of the acute phase response compared to any other biomarkers (Kilicarslan, Uysal, & Roach, 2013; Libby et al., 2002). It has a long biological half-life of 19–20 h (Marino & Giotta, 2008; Pepys & Hirschfield, 2003; Vigushin, Pepys, & Hawkins, 1993). This allows for the stability of concentration levels that are not subject to a circadian variation such as IL-6 (Meier-Ewert et al., 2001), consisting of a diurnal variation, being low in the morning with higher concentrations measured both in vivo or ex vivo before bedtime (Meier-Ewert et al., 2001; Sothern et al., 1995; Vgontzas et al., 1999).

C-reactive protein, which represents the downstream integration of overall cytokine activation, is credited with playing contradictory roles (Libby et al., 2002). The synthesis of pro-inflammatory cytokines (IL-1α and β, TNF, and IL-6) by C-reactive protein suggests a magnification of the inflammatory response; however, its synthesis of interleukin-1 receptor antagonist (IL-1ra), in circulating monocytes, suggests an anti-inflammatory role, with the subsequent suppression of pro-inflammatory cytokines in tissue macrophages in the presence of IL-6 (Tilg, Trehu, Atkins, Dinaello, & Meir, 1994). This anti-inflammatory role is partially explained by its relationship with IL-6 (Samols, Agrawal, & Kushner, 2002), since IL-6 is responsible for synthesis of IL-1ra and the TNF-a antagonists, directly suppressing synthesis of the pro-inflammatory cytokines (IL-1β and TNFα) (Tilg et al., 1994). Interestingly, with chronic inflammation, IL-6 expresses a pro-inflammatory role (Srirangan & Choy, 2010), contradicting its beneficial protective role in resolving acute inflammation (Gabay, 2006). In this capacity, IL-6 favours the accumulation of mononuclear cells through the continuous secretion of monocyte chemoattractant protein-1 and anti-apoptotic functions on T-cells at the site of injury (Atreya et al., 2000). According to Gabay, the expressed synthesis and increased concentration of IL-6 serves as a transition from acute to chronic inflammation (Gabay, 2006). Therefore, IL-6 is a pivotal keystone cytokine , for if tissue damage is not resolved during the acute inflammatory response, its role shifts to perpetuating inflammation as seen in a condition such as rheumatoid arthritis (Hashizume & Mihara, 2011; Metsios, Stavropoulos-Kalinoglou, & Kitas, 2015; Srirangan & Choy, 2010; Yoshida & Tanaka, 2014).

Inflammation and Exercise

In the literature, inflammation and the inflammatory response have been examined relative to either non-damaging (e.g. concentric) or damaging (e.g. eccentric) exercise, with the study by Canon et al. being the first to suggest that cytokines are released in response to exercise (Cannon & Kluger, 1983). As stretching is considered a form of physical activity used by athletes and rehabilitation patients (Page, 2012; Weerapong et al., 2004), by investigating physical activity and its relationship to the inflammatory response, this provides a plausible explanation of the relationship of stretching to inflammation. Similar to exercise (non-damaging and damaging), efficacy of stretching is determined by the parameters of training: intensity, duration, and frequency (Marschall, 1999; Mujika et al., 1995; Seiler, 2010). In this section, both non-damaging and damaging exercise relevant to the inflammatory response(s) will be discussed.

Non-damaging Exercise and the Inflammatory Response

Since the study by Cannon and Kluger (1983), exercise has been observed to induce the cytokine cascade characterised by the release of the following specific cytokines, the pro-inflammatory cytokines (TNF-α, IL-1β, and IL-6) (Drenth et al., 1995; Sprenger et al., 1992), and the anti-inflammatory cytokines [IL-1ra, TNF-α antagonists, and IL-10 (cytokine inhibitors)] (Drenth et al., 1995; Ostrowski, Rohde, Asp, Schjerling, & Pedersen, 1999; Pedersen & Hoffman-Goetz, 2000). With habitual (non-damaging) exercise, a marked increase in the concentration of plasma IL-6 compared to other cytokines occurs (Pedersen & Hoffman-Goetz, 2000), contradicting a study that suggests this increase is due to muscle damage (Bruunsgaard et al., 1997). Support for the former study was provided by an investigation that observed the return of IL-6 to resting level postexercise, despite the continued expression of the two markers for muscle damage, namely, creatine kinase and myoglobin (Peake et al., 2005). With the immune response dependent on the ability of leukocytes to transmigrate from circulation to the site of inflammation through the endothelium, increases in the concentration of adhesion molecules on the surface of leukocytes and endothelial cells occur in response to muscle damage (Akimoto et al., 2002; Alberts et al., 2002; Carlos & Harlan, 1994; Chen et al., 2006; Kaplanski, Marin, Montenero-Julian, Mantovani, & Farnarier, 2003; Reihmane & Dela, 2013; Springer, 1994; Vestweber, 2007). However, during habitual exercise, this increase in adhesion molecules does not occur, remaining relatively unchanged postexercise or during recovery (Chaar et al., 2011; Smith et al., 2000). Considering that numerous cells from a variety of tissues can produce pro-inflammatory cytokines (i.e. IL-6, TNF-α), alteration in the concentration levels of these cytokines is not necessarily reflective of changes in their production by circulating leukocytes (Reihmane & Dela, 2013; Starkie, Rolland, Angus, Anderson, & Febraio, 2001). Therefore, since the rise in the concentration of plasma IL-6 during habitual exercise is not due to the circulating leukocytes, this rise is believed to be associated with muscle contraction (Steensberg et al., 2000, 2001, 2002).

The release and rise of IL-6 from skeletal muscle during exercise (eccentric and concentric) (Hiscock, Chan, Bisucci, Darby, & Febbraio, 2004) was measured during sustained marathon running (Ostrowski, Rohde, Zacho, Asp, & Pedersen, 1998), the contraction of knee extensor muscles of a single leg during a 5h concentric exercise (Steensberg et al., 2000), and during cycling (Ullum et al., 1994). With no changes observed for pre-mRNA IL-6, IL-1α, IL-1β, and TNF-α in the blood mononuclear cells due to habitual exercise, increases in plasma IL-6 was less likely related to the increased production of cytokines (Ullum et al., 1994). Unlike eccentric exercise, which is associated with micro-injuries to both the muscle and connective tissue, concentric exercise provides an opportunity to observe the expression of IL-6 during non-damaging exercise (Macintyre, Reid, & Mckenzie, 1995). The amount of plasma IL-6 released by the knee extensor muscles was ~100-fold greater compared to pre-exercise, with levels remaining relatively unchanged in the non-exercised leg (Steensberg et al., 2000). This several fold rise in the concentration of IL-6 has been detected in clinical studies concerned with serious infection (Bruunsgaard, Skinhoj, Qvist, & Pedersen, 1999; Damas et al., 1992; Hack et al., 1989) and observed in a study investigating prolonged running (i.e. a marathon competition) (Starkie et al., 2001). Unlike the aforementioned studies which had participants perform a concentric exercise (i.e. bicycling, knee extension exercise) (Steensberg et al., 2000; Ullum et al., 1994), this study employed an eccentric exercise in the form of running (Starkie et al., 2001). Interestingly, the concentration of IL-6 associated with the eccentric exercise was not much greater than the concentric exercise, demonstrating that damage was not a requisite for its release during habitual exercise (Pedersen & Febbraio, 2008) (Fig. 2.16).

Fig. 2.16

Relationship of IL-6 relative to concentric and eccentric non-damaging exercise. (Modified from Pedersen and Febbraio 2008)

An investigation in which the calf muscles of male Wistar rats were electrically stimulated concentrically or eccentrically, observed a local production of IL-6 in the exercising leg, with levels for the non-stimulated remaining unchanged (Jonsdottir et al., 2000), supporting the view that expression of IL-6 is closely related to muscle contraction . In addition, significant differences in muscle fibre type were not observed with similar levels of IL-6 measured in both the red and white fibres of the soleus and gastrocnemius and the gastrocnemius, respectively (Jonsdottir et al., 2000). Analogous to the studies by Ullum et al. (1994) and Steensberg et al. (2000), this study confirmed that muscle damage was not a requisite for increases in plasma IL-6 during habitual exercise, but released in response to muscle contraction (Jonsdottir et al., 2000).

Myokines

Myokines are cytokines produced, released, and expressed by muscle fibres exhibiting a paracrine or endocrine effect (Pedersen, Akerstrom, Nielsen, & Fischer, 2007). They account for exercise-associated immune changes, playing a role in resolving both exercise-associated metabolic changes following training adaptation (Pedersen et al., 2007). The myokines in response to habitual exercise are responsible for expressing IL-6, IL-1ra, IL-8, IL-10, IL-15, and TNF-α into circulation, with TNF-α appearing in response to very intense long duration exercise (e.g. marathon running) (Febbraio & Pedersen, 2002, 2005; Nielsen et al., 2007; Starkie et al., 2001; Ullum et al., 1994). This late release of TNF-α does not precede the expression of IL-6 as occurs with sepsis or muscle-damaging exercise (Pedersen & Febbraio, 2008) (Fig. 2.17).

Fig. 2.17

Comparison of muscle damage (a) vs. habitual exercise (b) induced cytokines (note: during muscle damage IL-6 release is preceded by TNF-α). (Modified from Pedersen and Febbraio 2008)

Interleukin-6, IL-8, and IL-15 are classified as myokines since their expression is regulated by the type of muscular contraction (Marino, Scuderi, Provenzano, & Bartoccioni, 2011; Pedersen & Febbraio, 2008). Both IL-6 and IL-8 are regulated by concentric contraction at the protein and mRNA level (Akerstrom et al., 2005; Chan, Carey, Watt, & Febbraio, 2004; Febbraio & Pedersen, 2002, 2005; Pedersen et al., 2007), with IL-15 regulated by resistance training (Nielsen et al., 2007). Production of IL-6 in response to muscle activity has been linked to muscle repair (Steensberg et al., 2000), with an increased expression observed by regenerating mice myofibres exposed to a crush trauma (Kurek, Nouri, Kannourakis, Murphy, & Austin, 1996). Interleukin-8, a chemoattractant cytokine, is produced by a variety of blood and tissue cells, with a distinct specificity for neutrophils, attracting and activating them at the site of inflammation and regulating the neutrophil-endothelial interaction (Bickel, 1993). Interleukin-15, a pleiotropic cytokine expressed at the mRNA level of numerous normal human tissues (i.e. activated monocytes, dendritic cells, fibroblasts, and osteoclasts), is involved in both adaptive and innate immune responses inducing the synthesis of TNF-α (Itsumi, Yoshikai, & Yamada, 2009; Liew & Mcinnes, 2002).

In order to determine the source of IL-6 in contracting muscle, the reverse transcription-polymerase chain reaction technique was used to detect and quantify gene expression (i.e. mRNA) (Keller et al., 2001). Levels of IL-6 transcription and mRNA on the nuclei and total RNA were measured from muscle biopsies obtained pre-, during, and post non-damaging exercise. Participants performed a two-legged dynamic knee extensor exercise (50–60% of maximal workload) for 180 min, as well as being on a diet regimen eliciting either normal (control) or low (~60% of normal) muscle glycogen levels (Keller et al., 2001). Muscle biopsies prior to exercise observed that the nuclear transcription gene for IL-6 was nearly undetectable, with a rapid and pronounced increase in the transcription rate of IL-6 observed post exercise at the nuclear level by the reverse transcription-polymerase chain reaction technique (Keller et al., 2001; Malm et al., 2000). In response to a reduction in muscle glycogen concentration, a greater rise in the transcription for the IL-6 gene after 90 min (~40-fold) and 180 min (~60-fold) of exercise was observed (Keller et al., 2001). In addition, a significant increase in IL-6 mRNA (>100-fold) was observed after 180 min of exercise vs. control (~30-fold) suggesting that besides the type of muscular contraction (i.e. eccentric or concentric), concentration of muscle glycogen is also critical in the release of IL-6 during habitual exercise (Keller et al., 2001; Steensberg et al., 2001).

Muscle as an Endogenous Glucose-Producing Organ

Skeletal muscle is considered an endogenous glucose-producing organ influencing the disposal of glucose, with a greater increase in glucose uptake occurring during contraction compared to the maximal stimulation by insulin (Febbraio, Hiscock, Sacchetti, Fischer, & Pedersen, 2004). Considering that IL-6 is produced in the absence of inflammatory markers, and that expression of intramuscular IL-6 mRNA is exacerbated in response to compromised glycogen concentration, expression of IL-6 due to glycogen content suggests that skeletal muscle plays a metabolic role as well (Pedersen & Febbraio, 2008). A study evaluating the metabolic effects of IL-6 for treatment of metastatic renal cell cancer observed that IL-6 produced an endocrine response in patient’s administered recombinant human IL-6 (Stouthard et al., 1995), with a greater increase in hepatic glucose occurring on the recombinant human IL-6 infusion vs. the control (Stouthard et al., 1995). In agreement with the previous study, a study that administered IL-6 subcutaneously to healthy volunteers noticed an increase in glucose (Tsigos et al., 1997). The release of glucagon induced by IL-6 increased hepatic glycogenolysis contributing to elevations of blood glucose (Tsigos et al., 1997). An in vitro study designed to investigate whether radioactive glucose released from cultured hepatocytes consisted of radioactive pre-labelled glycogen pools observed that IL-6 was a strong glucoregulatory cytokine stimulating the release of hepatic glucose (Ritchie, 1990). Interestingly, in the past, IL-6 was once referred to as a hepatocyte-stimulating factor (Heinrich et al., 1998).

According to Gleeson (2000), the release of IL-6 in lieu of glucose concentration and contracting muscle suggests that this myokine acts as a hormone mediating the hepatic glucose output necessary for maintaining the homeostasis of glucose during non-damaging exercise. Therefore, since skeletal muscle is considered an endocrine organ, responsible for the production, release, and expression of several cytokines, an important link has been established between skeletal muscle , metabolic changes, and the modification of cytokine production in tissue and organs in response to habitual exercise (Pedersen, 2006; Pedersen et al., 2001).

Intensity, Duration, and Mode of Exercise

The extent to which the muscle is activated depends on the intensity, duration, and the mode of exercise (i.e. the amount of muscle mass recruited) (Pedersen & Febbraio, 2008). A study investigating the importance of intensity and the release of IL-6 had seven untrained healthy male participants perform a 45-min knee extension exercise with both legs kicking at a frequency of 60 kicks per minute at 25% W_max, on two independent parallel one-leg knee extension ergometers, simultaneously (Helge et al., 2003). The blood was collected from both the femoral artery and veins at 15, 30, and 40 min. After 45 min, the exercise was stopped and the load was adjusted. When exercise resumed for another 35 min, one leg performed a knee extension exercise kicking at 65% W_max (moderate intensity), with the other kicking at 85% W_max (high intensity). The blood was sampled at 15, 30, and 35 min, with a muscle biopsy taken pre- and post-80 min of exercise. The main finding of the study was that the release of IL-6 by the working muscle was related to both the intensity of the exercise and the glucose uptake (Helge et al., 2003), confirming that the release of IL-6 by the contracting muscle is important for maintaining glucose homeostasis during habitual exercise (Gleeson, 2000). Interestingly, although the release of IL-6 by the contracting muscle is prompted by the need to supply fuel, this myokine also accommodates the need for fuel by stimulating lipolysis in adipose tissue in response to diminished muscular carbohydrate (Helge et al., 2003).

Besides intensity, duration of exercise is associated with release of IL-6. A study investigating the effect of a prolonged one-legged dynamic knee extensor exercise with six healthy males for 5 h at a power output of 25 W, representing 40% of their peak power output (W_max), observed an increase in production and high turnover of IL-6 (Steensberg et al., 2000). Participants moved the ankle over a range of ~60° (from 90 to 30° angle), with the blood collected before and after each hour of exercise from both the femoral artery and vein of the exercising leg and the femoral vein of the resting leg. Over the last 2 h of exercise, a release of IL-6 from the muscle was 17-fold higher than the arterial concentration (Steensberg et al., 2000). The effects of a prolonged exercise in the form of marathon running (Copenhagen Marathon) also measured an increase in IL-6 mRNA locally in activated skeletal muscle (Ostrowski et al., 1998). Blood samples were drawn from the antecubital vein of 16 male marathoners 1 week before, immediately after, and 2 h postrace, with similar time periods for muscle biopsies (vastus lateralis) taken from eight of the participants. The expression of mRNA was not observed in the circulating blood mononuclear cells suggesting that no contribution occurred from muscle damage. In addition, although increases in plasma IL-6 was observed postexercise, its decline thereafter coincided with an increase in IL-1ra (anti-inflammatory cytokine) concentration 2 h postexercise (Ostrowski et al., 1998), suggesting that acute exercise creates an IL-6 anti-inflammatory environment.

Another source for the release of IL-6 is the mode of exercise, with the mass of skeletal muscle recruited during exercise influencing the expression of the cytokines. A study in which ten experienced triathletes performed two cycling and running sessions spread out over a 4- to 6-week period, observed that running (eccentric exercise), which recruited more muscle mass, had a greater release in IL-6 versus cycling (concentric exercise) (Nieman et al., 1998). Similarly, this observation was supported by a study that observed a pronounced increase in systemic concentration of IL-6 occured during running vs. cycling (Febbraio & Pedersen, 2002). In conclusion, circulating levels of IL-6 in response to habitual exercise occur without muscle damage, with this rise in concentration influenced by the intensity, duration , and the amount of muscle mass recruited (Jonsdottir et al., 2000; Pedersen & Fischer, 2007; Steensberg et al., 2000; Ullum et al., 1994).

Damaging Exercise and the Inflammatory Response

Since the 1960s, sports scientists observed that small lesions in muscle structure expressing a small foci of inflammation and degenerative changes, including fibre necrosis, were characteristic of a single bout of exhaustive exercise – either short-lasting intensive or long-lasting moderate (Vihko, Rantamaki, & Salminen, 1978). This disruption is associated with crush and strain injuries, overloading, and eccentric exercise, responsible for initiating a sequence of cellular responses, expressing an inflammatory response (Tidball, 1995). Unlike the cytokine cascade observed for habitual exercise, the release of IL-6 is preceded by TNF-α, with the order of release occurring as follows: IL-1β, TNF-α, and IL-6. It should be noted that IL-6, classified as an “inflammation-responsive” cytokine, does not directly cause inflammation, even though its infusion has resulted in fever in humans (Mastorakos, Chrousos, & Weber, 1993). In addition, unlike IL-1β and TNF-α, it is not associated with capillary leakage or shock, the upregulation of nitric oxide or matrix metalloproteinase, which are all major inflammatory mediators stimulated by both IL-1 and TNF-a (Barton, 1997; Mastorakos et al., 1993). As mentioned above, IL-6 is a primary inducer of C-reactive protein and for the synthesis of cytokine inhibitors (IL-1ra, soluble TNF-α receptors, and IL-10).

With muscle damage, the sequence/time response of the cytokines of the local inflammatory response, is firstly pro-inflammatory in nature, with the release of IL-1β and TNF-α expressed in skeletal muscle (Cannon & St. Pierre, 1998), followed by the expression of the anti-inflammatory cytokines IL-6, IL-1ra, soluble TNF-α receptors, and IL-10 (Fig. 2.18). Although cytokines are classified as either pro- or anti-inflammatory, this classification is quite simplistic given that some cytokines (i.e. IL-6) may act as either (Cavaillon, 2001). According to Cavaillon (2001), the nature of the activating signal, the sequence and timing of action, and the nature of the target cell, greatly influences the properties of the cytokines and their subsequent expression as either pro- or anti-inflammatory in nature.

Fig. 2.18

Cytokine cascade related to muscle damage. (Modified from Pedersen 2006)

Stages Associated with Muscle Damage Exercise and Inflammation

The series of mind maps (Figs. 2.19, 2.20, 2.21, 2.22, 2.23, 2.24, 2.25, and 2.26) and Table 2.1 below suggest a road map concerning acute inflammation and muscle-damaging exercise. These “stages” and Table 2.1 were created to provide a logical sequence of events, in an attempt to simplify the complex processes associated with muscle-damaging exercises, acute inflammation, and muscle regeneration. These “stages” are not definitive in nature but rather suggestive (but evidence-informed), since the complex processes affiliated with acute inflammation [pro-inflammatory myocytes in circulation, transmigration and adhesion molecules, cytokines (pro- and/or anti-inflammatory, etc.)] overlap and are not isolated incidents. Based on collective research findings, four “stages” have been identified and mapped. The first stage refers to muscle damage, the cause responsible for the loss of the organisation of the sarcomere, with the second stage referring to the autolytic calpain , referencing the disruption of cellular Ca²⁺ by a mechanical stimulus, expressing the upregulation of calpain (calcium-activated proteases). This protease is involved in the autolysis of the muscle components. The third stage, adhesion and transendothelial migration (TEM) of leukocytes, refers to activation of the endothelium responsible for attracting the leukocytes (i.e. neutrophils) to the site of inflammation, and finally, the fourth stage, the inflammatory cells. This last stage references the neutrophils, as well as the pro- and anti-inflammatory macrophages activated at the site of inflammation.

Fig. 2.19

The four stages with regard to muscle-damaging exercise and inflammatory response. (Published with kind permission of copyright © Nikos C. Apostolopoulos 2018. All rights reserved)

Fig. 2.20

Stage1: Muscle damage. (Published with kind permission of Copyright © Nikos C. Apostolopoulos 2018. All rights reserved)

Fig. 2.21

Stage 2: Autolytic calpain. (Published with kind permission of Copyright © Nikos C. Apostolopoulos 2018. All rights reserved)

Fig. 2.22

Stage 3: Adhesion and transendothelial migration of leukocytes. (Published with kind permission of copyright © Nikos C. Apostolopoulos 2018. All rights reserved)

Fig. 2.23

Stage 4: Inflammatory cells. (Published with kind permission of copyright © Nikos C. Apostolopoulos 2018. All rights reserved)

Fig. 2.24

Stage 4: Inflammatory cells—neutrophils. (Published with kind permission of copyright © Nikos C. Apostolopoulos 2018. All rights reserved)

Fig. 2.25

Stage 4: Inflammatory cells—pro-inflammatory macrophages (phagocytic). (Published with kind permission of copyright © Nikos C. Apostolopoulos 2018. All rights reserved)

Fig. 2.26

Stage 4: Inflammatory cells—anti-inflammatory macrophages (non-phagocytic). (Published with kind permission of copyright © Nikos C. Apostolopoulos 2018. All rights reserved)

Table 2.1 Time scale of inflammatory cell response in relation to the phases of muscle regeneration post muscle damage (Published with kind permission of copyright © Nikos C. Apostolopoulos 2018. All rights reserved)

Stage 1: Muscle Damage (Fig. 2.20)

The catalyst for inflammation is the loss of normal sarcomeric organisation, due to the retraction of the myofibrils, in response to exercise-induced muscle damage (Tidball, 1995). The first study referring to morphological disruptions of the muscle in humans, had five healthy males rapidly run down ten flights of stairs for ten times, with the rest period consisting of taking the elevator to the tenth floor (Friden, Sjostrom, & Ekblom, 1981). Muscle biopsies obtained from the right and left soleus 2 weeks prior and on the second and seventh day postexercise, were divided into two halves, with one-half prepared for electron microscopy, and the other for enzyme histochemistry. At the cellular level, muscle fibres prior to and postexercise appeared normal, tightly packed, in well-organised fascicles, suggesting no signs of ischemic fibre necrosis or rupture in the sore muscles. However, at the subcellular level, focal disturbances of the characteristic cross-striated band pattern of the muscle was observed 2 days postexercise, with disturbances estimated to being three times greater comparable to sections from both control and 7 days postexercise. The Z-disc showed marked “broadening” and “streaming” (structural disruption of the material of Z-disc ripped out across a large portion of the sarcomere) with the myofilamentous material in the adjacent sarcomeres being either supercontracted or disorganised. In addition, the normally regular and complex fine structure of the Z-discs had gaps in their characteristic lattice pattern. The high myofibrillar tension developed during activation of the contractile material (interdigitating arrays of the thick and thin myofilaments) accounts for this mechanical disruption in the Z-discs, suggestive of a potential weak link in the contractile chain of the myofibres (Friden et al., 1981).

A study suggesting that muscle damage following eccentric work is mechanically induced, with further disruption caused by mechanical and biochemical factors, had four healthy normal participants perform a 20-min step test (Newham, Mcphail, Mills, & Edwards, 1983). The quadriceps group of one leg performed a concentric (stepping up) with the other an eccentric (stepping down) contraction, each lasting 1 s at a stepping frequency of 15 cycles/min (Newham, Mcphail, et al., 1983). Biopsies for three participants were taken immediately prior to exercise, with further biopsies taken immediately, and at 24 and 48 h postexercise, with the latter times coinciding with pain and tenderness of the eccentrically contracted quadriceps [delayed onset muscle soreness (DOMS)]. Biopsies prepared for electron microscopy observed areas of myofibrillar disruption. These disruptions were counted and classified as either “focal” (areas affecting one or two adjacent myofibrils and one or two adjacent sarcomeres), “extensive” (areas affecting more than two adjacent myofibrils and sarcomeres or a fibre containing more than ten focal areas), or “very extensive” (areas containing more than one extensive area of damage). No abnormalities were observed in the internal architecture of the concentrically activated quadriceps, either pre- or postexercise.

Abnormalities in the eccentric contracted quadriceps immediately postexercise continued, with changes observed 1–2 days later. Histologically 59% appeared “normal”, with 16% and 8% expressing “focal” and “extensive”, and “very extensive” disruptions, respectively. In samples taken approximately 30 h postexercise, 45% appeared “normal”, with 6%, 23%, and 28% revealing “focal”, “extensive”, and “very extensive” changes, respectively, disclosing that the myofilaments of the sarcomeres in the eccentric exercise quadriceps immediately and at 30 h postexercise were disorganised with the Z-disc material “streaming” across the sarcomere (Newham, Mcphail, et al., 1983).

With damage varying based on the time period, more sarcomeres exhibited greater and extensive damage at 30 h post-eccentric exercise (Fig. 2.27). This time course of morphological changes, with more damage observed considerably later in the postexercise period vs. immediately post, suggests the delayed release of creatine kinase (muscle damage biomarker) into circulation, peaking 4–5 days postexercise (Newham, Jones, & Edwards, 1983). Interestingly, a study investigating the effects of passive stretching in relation to an immobilised soleus muscle of male Wistar rats, observed that stretching was also responsible for morphological changes to the muscle fibre (Gomes, Cornachione, Salvini, & Mattiello-Sverzut, 2007). Ultrastructural examination observed a disruption of the sarcomere, with the myofibrils appearing fragmented at the Z-disc, prompting the conclusion that passive stretching needs to be applied carefully in order to prevent muscle damage (Gomes et al., 2007).

Fig. 2.27

Measurement of histological changes. (Published with kind permission of copyright © Nikos C. Apostolopoulos 2018. All rights reserved)

A rapid invasion of inflammatory cell populations lasting from days to weeks, initiated by muscle damage, is instrumental in promoting further disruption in muscle homeostasis or repair (Tidball, 2005). The dead or dying cells release inflammatory mediators, cytokines, as well as chemokines, inducing chemotaxis (movement of an organism or cell in response to a chemical stimulus) in nearby responsive cells (Shek & Shephard, 1998). The injury-specific interaction between the invading inflammatory cells and muscle, the previous history of muscle use (i.e. repetitive injuries vs. acute, eccentric vs. concentric exercise), and the magnitude of response, determines whether the overall inflammatory response will be beneficial or detrimental (Tidball, 2005). In other words, the size and nature of the disruption, and the sequencing, timing, and concentration levels of pro- and anti-inflammatory cytokines, are responsible for the continued disruption or recovery of the muscle.

Stage 2: Autolytic Calpain (Fig. 2.21)

Studies referring to humans, intact animals , isolated muscle, and single muscle fibres, have repeatedly shown that the primary site of damage is within the muscle fibre itself (Allen, 2001). Lesions, referred to as micro-injuries, that are usually subcellular, occur in small proportions in response to relatively intense, long duration, or eccentric exercise (Armstrong, Ogilvie, & Schwane, 1983). Regardless of the stimulus (i.e. crush injury, muscle strain, inflammation), this damage initiates a sequence of cellular responses caused by a mechanical disruption, preceding a biochemical response (Faulkner, Brooks, & Opiteck, 1993). Although mechanical disruption can explain a reduction in force production, the increased expression of inflammatory cells is responsible for an additional decrease in function (Faulkner et al., 1993; Macintyre, Reid, Lyster, Szasz, & Mckenzie, 1996; Tidball, 2002). The extensive disruption of the structural components of the muscle, expressed as a loss of normal sarcomere organisation with a retraction of the myofibrils from the injured site, results in a structurally intact sheath surrounding the damaged region, consisting of a basement membrane and endomysium proteins (Faulkner et al., 1993; Tidball, 1995). This disruption, a disturbance of the cross-striated band pattern affecting the myofibrillar Z-disc (streaming and broadening), involves all or a few of the sarcomeres, either in series or parallel (Friden, Sjostrom, & Ekblom, 1983). Ultrastructural damage involves the crumpling of the interface between the thin and thick filaments, since their overlap is an integral component of the myofilament structure (Brown & Hill, 1991; Higuchi, Yoshioka, & Maruyama, 1988; Horowits, 1992). Further, the disruption at the level of the Z-disc is associated with both mitochondrial and sarcoplasmic reticulum vacuolisation, an adaptive physiological response to numerous environmental changes limiting damage (Belcastro, Maclean, & Gilchrist, 1985; Friden et al., 1983). It has been suggested that vacuolisation, a distinct form of cell death, is a degeneration resulting in lytic responses (Henics & Wheatley, 1999).

Catabolic events associated with muscle damage involve the autolysis of components (Armstrong, 1990; Huijbregts, 2001), affiliated with the disruption of and the homeostasis of cellular Ca²⁺ (Armstrong, 1990; Belcastro, Shewchuk, & Raj, 1998), which is released from intercellular stores that gain entry from the extracellular space (Spencer, Lu, & Tidball, 1996). This disruption is characterised by a derangement of the sarcomere, fragmented or swollen sarcoplasmic reticulum elements and mitochondria, and lesions in the plasma membrane (Belcastro et al., 1998). The Ca²⁺ molecule is important for triggering muscle contraction, relaxation, and controlling the energetics of the muscle, by regulating the provision of ATP (Berchtold, Brinkmeier, & Muntener, 2000; Tate, Hyek, & Taffet, 1991). Within the myofibril, a variety of Ca²⁺-binding proteins (i.e. calmodulin, calpains), not involved directly in the process of contraction and relaxation exist, which are very important for muscle plasticity and performance (Berchtold et al., 2000; Suzuki, Hata, Kawabata, & Sorimachi, 2004). With the release and upregulation of Ca²⁺ occuring in response to the disruption of the sarcomere, and the muscle’s inability to buffer it, the “calcium-dependent protease” calpain is expressed and upregulated (Tidball, 1995). This Ca²⁺ binding protein, an inactive enzyme found within the cytosol of skeletal muscle (Kumamoto et al., 1992), is activated in the presence and increase in Ca²⁺ (Suzuki et al., 2004). When activated, selective proteolysis of various structural, metabolic, and/or contractile elements (Belcastro et al., 1998), hydrolysing enzymes (phosphatases and kinases), and cytoskeletal and membrane proteins occurs (Kunimatsu, Higashiyama, Sato, Ohkubo, & Sasaki, 1989). Although this proteolysis can occur at both the Z- and I-disc regions of the sarcomere, a higher propensity happens to occur at the Z-disc (Kumamoto et al., 1992), with prominent muscle proteins, including myofibrillar and major Z-disc proteins, and those involved with myofibril linkage to cell membranes (i.e. talin and vinculin) being cleaved (Takahasi, 1990). This calpain at the Z-disc suggests that this area of the muscle is the most susceptible to degradation (Dayton, Reville, Goll, & Stromer, 1976). The protease activity of calpain has been observed during myofibrillar degradation, with the loss of Z-disc proteins seen in 22% of the myofibrils isolated from the skeletal muscle of exercising rats (Belcastro, Parkhouse, Dobson, & Gilchrist, 1988; Goll, Dayton, Singh, & Robson, 1991). Calpain cleaves troponin, tropomyosin, α-actinin, nebulin, titin, desmin, the sarcolemmal-associated spectrin complex of proteins, and membrane adhesion molecules (integrin, cadherin, N-CAM) (Belcastro et al., 1998; Goll, Thompson, Li, Wei, & Cong, 2003). Since its action is rather disruptive, destabilising and altering the substrate proteins makes them more susceptible to various cellular proteases (Saido, Sorimachi, & Suzuki, 1994) (Fig. 2.28).

Fig. 2.28

Subsequent steps associated with the autolysis by calpain. (Published with kind permission of copyright © Nikos C. Apostolopoulos 2018. All rights reserved)

Eccentric exercise is associated with activation of calpain, since levels of intracellular Ca²⁺ remain slightly elevated above normal resting cytoplasmic levels for 24–48 h (Murphy, 2009). Following bouts of strenuous exercise, the proteolysis of skeletal muscle proteins by calpain (Belcastro et al., 1998) is linked to an increase in leukocyte populations (Camus et al., 1992; Field, Gougeon, & Marliss, 1991; Gabriel, Schwartz, Steffens, & Kindermann, 1992), for the peptides released are chemoattractive for neutrophils without producing any cytotoxic effects (Kunimatsu et al., 1989, 1993, 1995).

A study designed to investigate the relationship between calpain-like protease and neutrophil accumulation, measured accumulations of neutrophil through the activity of myeloperoxidase (MPO) (Raj, Booker, & Belcastro, 1998), an enzyme used to measure and determine neutrophil migration into muscles (Morozov, Tsyplenkov, Goldberg, & Kalinski, 2006). To measure this relationship, 15 male Wistar rats were randomly assigned to either a control (n = 5) or exercise (n = 5) group, with another exercise group (n = 5) investigating the extent of how calpain promotes neutrophil accumulation, with the latter group pre-injected with a cysteine protease inhibitor (reduces activity of calpain-like protease) 1 h prior to exercise. With speed of the motorised treadmill set at 25 m/min and at an 8% grade, the rats ran on the treadmill for 60 min or until voluntary termination. Postexercise rats, matched for control and exercise were euthanised with blood and muscle tissue (plantaris and ventricles) collected. Determination of calpain-like and MPO activity was performed on serial samples from the same muscle, with plasma creatine kinase also being measured to determine muscle damage. A major observation was the positive relationship between the activity of the calpain-like protease and MPO for all the studied tissues (plantaris and ventricles). More importantly, a link between the underlying processes with skeletal muscle calpain-like and MPO activity was observed showing a responsiveness to an exercise stimulus (Raj et al., 1998). Rats injected with the cysteine protease inhibitor were associated with a lower MPO, suggesting that neutrophil accumulation is dependent upon Ca²⁺-stimulated cysteine proteases (Raj et al., 1998).

Since the release of the peptides by the activity of calpain was chemoattractive for neutrophils (Kunimatsu et al., 1989), this signifies the shift from “stage 2” to “stage 3” (Fig. 2.19). With the aforementioned study by Raj et al. (1998), although the underlying mechanism regarding the enhanced responsiveness to exercise stimulus of the plantaris vs. cardiac muscle of calpain-like activity and MPO is unknown, they suggest that the synthesis of calpain is possibly related to the greater force production (intensity) generated by the plantaris muscle. Skeletal muscle contraction is associated with a greater metabolic demand dependent on the Ca²⁺-homeostasis, with eccentric exercise linked to a continued degradation of myofibrillar protein, a metabolic effect, affiliated with an extensive delay vs. immediate damage (Evans & Cannon, 1991). Interestingly, elevation of muscle Ca²⁺ observed in female Sprague-Dawley rats following stretching suggests that stretching may be a potential mechanism for disruption of the homeostasis of Ca²⁺ (Armstrong et al., 1993).

Stage 3: Adhesion and Transendothelial Migration (TEM) of Leukocytes (Fig. 2.22)

Recruitment of leukocytes to an injured site is a hallmark of the inflammatory response, for “no immune response”, either innate or adaptive, can occur unless leukocytes cross the blood vessels. This complex multistep process, with each step being a prerequisite for the next, consists of many adhesion molecules on both leukocyte and endothelial cell surfaces consisting of overlapping functions (Schenkel, Mamdouh, & Muller, 2004) (Fig. 2.29). When skeletal muscle is damaged, leukocytes, specifically neutrophils, are attracted to the site of injury through the process of adhesion and transendothelial migration. In vitro (cultured endothelium) and in vivo (animal intravital microscopy) studies suggest that transendothelial migration is a critical step in the regulation of the inflammatory response, contingent upon leukocytes crossing the endothelial lining of the blood vessels to enter the site of inflammation (Kim & Luster, 2015; Muller, 2013). It is important to note that regulation of the inflammatory response is very critical, for beneficial and collateral damage occurs once the leukocytes leave circulation.

Fig. 2.29

Transendothelial migration. (Published with kind permission of copyright © Nikos C. Apostolopoulos 2018. All rights reserved)

The largest pool of circulating leukocytes in the bloodstream, the neutrophils (40–80%), persist for hours to days until they reach senescence and are cleared from the body (Kim & Luster, 2015). Because of their sheer volume, they are often marginalised towards the vascular wall by collision with red blood cells, a process responsible for their adherence to the vascular endothelium (Sundd, Pospieszalska, & Ley, 2013). Since it is beyond the scope of this manuscript to describe the intricacies of adhesion and transmigration, readers are directed to the “Further Readings” section at the end of the chapter.

Before adhesion and transmigration can take place, leukocyte recruitment is initiated by the release of a chemoattractant agent, such as the release of the peptides by the activity of calpain, as a chemoattractant agent for neutrophils (Kunimatsu et al., 1989). The importance of the release of this agent was investigated by a study observing responses of leukocytes to skeletal muscle damage (i.e. crush injury) of the mid-region of the tibialis anterior of female inbred Swiss mice at zero (0), three (3), and 24 h post crush injury vs. non-injured muscle (Robertson et al., 1993). A chemotactic response occurred at both 3 and 24 h post damage, with migrating leukocytes accumulating in clumps near regions where muscle fragments were pronounced. Although an accumulation of neutrophils occurred at 3 h, a far stronger chemotactic response was observed for exudate vs. resident macrophages within 24 h (Minutti, Knipper, Allen, & Zaiss, 2017; Robertson et al., 1993). No chemoattractant response was present for macrophages in normal uninjured skeletal muscle, nor muscle removed immediately post crush (0 h) (Robertson et al., 1993). Considering that neutrophils and macrophages were expressed in injured muscle several hours post damage (3 and 24 h), and not immediately after, suggests the activation of resident fibroblasts and macrophages. Normally residing in a quiescent state in the endothelium, resident macrophages and the activation of the endothelium attract and activate inflammatory cells during the early stages of muscle injury (Tidball, 1995).

During homeostatic conditions, the endothelial cell layer lining the vascular lumen, serves as a barrier limiting the infiltration of leukocytes into tissues; however, during injury, the inflamed vessel wall selectively recruits leukocytes in response to various stimuli (Kim & Luster, 2015). In a sequence of adhesive steps, leukocytes attach to the endothelial wall of the blood vessel and roll along the wall to the endothelial borders traversing the endothelium, migrating through the interstitial tissue into the site of injury (Ley et al., 2007; Muller, 2013). Commencement of this process is reliant upon the release of the pro-inflammatory cytokines responsible for activating the endothelial cells. This initiates a well-orchestrated series of adhesive interactions, with the increased expression of adhesion molecules (E- and P-selectins) and integrin ligands, responsible for recruitment of neutrophils (Kim & Luster, 2015; Puri et al., 2005). The enhancement of the E- and P-selectin on the endothelial surface during inflammation act as receptors for the recruitment of leukocytes into the site of damage (Mitroulis et al., 2015), with expression of E-selectin inducing the synthesis of TNF-α (Cannon & St. Pierre, 1998; Weller, Isenmann, & Vestweber, 1992). Activation of the endothelium results in a release of IL-1β, IL-6, and IL-8 (Detmers et al., 1991; Liu & Spolarics, 2003), with induction of IL-8 by IL-1β promoting endothelial adhesion and the chemotaxis for neutrophils (Colditz, Zwahlen, Dewald, & Baggiolini, 1989; Willems, Joniau, Cinque, & Van Damme, 1989), with blocking of IL-1β activity using IL-1βra, reducing production of IL-8 by 85% (Porat, Poutsiaka, Miller, Granowitz, & Dinarello, 1992). Expression of IL-1β and TNF-α in response to active stretching during eccentric exercise, has been observed to augment leukocyte adhesion molecules on human endothelial cells (Bevilacqua, Pober, Mendrick, Cotran, & Gimbrone, 1987; Malm et al., 2000).

The primary step of capture, rolling, and slow rolling, which enables the adherence of leukocytes to the endothelium under conditions of blood flow, is dependent upon the rapid reversible bonds between the selectin family of adhesion molecules and the neutrophils (Ley et al., 2007). Interestingly, the shear stress of blood flow is a requisite step for adhesion, strengthening the bond between the selectins and the neutrophils, for when the flow is stopped, the cells detach. The E- and P-selectins are vital for capture and rolling, for mice lacking their expression exhibit more than a ten-fold elevated rolling velocity (Puri et al., 2005).

The processes of rolling and slow rolling bring the neutrophils into contact with the endothelial cells, whereupon, they can be activated further by pro-inflammatory (IL-1 and TNF-α) agents and chemokines on the surface of the endothelial cells (Muller, 2013). The chemokines, contacting the chemokine receptors on the leukocytes, activate the leukocyte integrins (Mitroulis et al., 2015). As mentioned above, integrins are heterodimeric adhesion receptors (α/β) with most of them binding to ECM proteins (β1 integrin) (Hynes, 1992). However, β2 integrin is associated with leukocytes whose ligands are the intracellular adhesion molecules 1 and 2 (ICAM-1 and ICAM -2) (Hynes, 1992; Muller, 2013). Their activation results in a conformational change favouring binding to their ligands expressed on the endothelial cells of the inflamed endothelium. Once activated, the integrins of the leukocytes bind tightly to their ligands on the endothelial cells, thereby allowing leukocytes to arrest on the endothelial surface (Muller, 2013). Mice who are deficient in β2 show an increased leukocyte rolling velocity suggesting that this integrin contributes significantly to the “slowing-down” of rolling neutrophils (Dunne, Ballantyne, Beaudet, & Ley, 2002; Muller, 2013).

Binding of integrins to ligands triggers the “outside-in signalling” pathway strengthening adhesion of leukocytes, but more importantly facilitating the formation of focal adhesion and further integrin-dependent steps (Mitroulis et al., 2015). Prior to the final step of transendothelial migration, leukocytes are engaged in the process of locomotion following firm arrest. This process is dependent upon the β2 integrin during which the leukocytes crawl on the surface layer in order to identify an appropriate site on the endothelium to migrate through its monolayer. Locomotion ensures that the leukocytes move efficiently to the interendothelial junctions for transendothelial migration to occur (Mitroulis et al., 2015; Phillipson et al., 2006).

The processes of leukocyte rolling, activation, adhesion, and locomotion are all reversible; however, transendothelial migration is not (Muller, 2013). Once the leukocyte squeezes through the endothelial cell borders in an amoeboid fashion, crossing the endothelial cells via the process of diapedesis, this is considered as the point of no return in the inflammatory response (Muller, 2013). By committing to diapedesis, the leukocyte cannot go back.

Stage 4: Inflammatory Cells (Fig. 2.23)

Neutrophils (Fig. 2.24)

Neutrophils and macrophages dominate the inflammatory response, with neutrophils being the first responders migrating into the affected tissue. Following eccentric exercise, they invade the injured skeletal muscle within several hours, remaining for several days (Karalaki, Filli, Philippou, & Koutsilieris, 2009; Tidball, 1995). By measuring the activity of the MPO enzyme, a large increase in neutrophil concentration in male Sprague-Dawley rat muscle was reported immediately after 1 h of treadmill running at 0% grade at 25 m/min until voluntary termination (Belcastro, Arthur, Albisser, & Raj, 1996). Neutrophil presence was confirmed in a study with nine healthy sedentary untrained men performing three sets of 15 min bouts of downhill treadmill running (negative 16% incline) at 75% HR_max, separated by 5 min rest periods (Fielding et al., 1993). Percutaneous needle biopsies of the vastus lateralis were taken pre-, 45 min, and 5 days postexercise , with blood samples obtained pre-, immediately post, and at 3 and 6 h and 1, 2, 5, and 12 days postexercise. A relationship between neutrophil and immunohistochemical staining for IL-1β was observed, with a significant positive correlation between neutrophil infiltration and the ratio of ultrastructural damage to total Z-discs (r = 0.66; P < 0.05). At 45 min, an accumulation of neutrophils occurred, remaining elevated for 5 days post, with immunohistochemical staining of muscle cross sections revealing a 135% increase in IL-1β immediately post and increasing to 250% 5 days postexercise. This association between neutrophils and IL-1 has been confirmed by other studies as well (Figarella-Branger, Civatte, Bartoli, & Pellissier, 2003; Pedersen, Ostrowski, Rohde, & Bruunsgaard, 1998; Philippou et al., 2012; Smith et al., 2008).

Increases in the concentration of neutrophils within 1–6 h post injury suggest that these circulating leukocytes play a prominent role in the early expression of the inflammatory response (Kanda et al., 2013; Orimo, Hiyamuta, Arahata, & Sugita, 1991; Papadimitriou, Robertson, Mitchell, & Grounds, 1990; Summers et al., 2010). They are important for removal of necrotic tissue by the process of phagocytosis, continuing the inflammatory response with the release of the pro-inflammatory cytokines (IL-1β and TNF-α) (Cannon & St. Pierre, 1998; Smith et al., 2008). Elevation in neutrophils has been observed with passive stretching in 4-month old adult mice as well (Pizza, Koh, Mcgregor, & Brooks, 2002).

Neutrophils are a source for the synthesis of IL-1β, TNF-α (Dubravec, Spriggs, Mannick, & Rodrick, 1990; Tiku, Tiku, & Skosey, 1986), and reactive oxygen species (i.e. superoxide or hydrogen peroxide), cytotoxic molecules which can lyse cell membranes leading to further muscle damage (Canon et al., 1991; Mittal, Siddiqui, Tran, Reddy, & Malik, 2014; Philippou et al., 2012). Reactive oxygen species are a double-edged sword, for in conjunction with other chemokines and growth factors, they also participate in muscle repair (Barbieri & Sestili, 2012). According to investigators, expression of these cytotoxins is dependent on the intensity and type of exercise responsible for muscle damage (Nieman, 1997; Tidball, 2005).

Pro- and Anti-inflammatory Macrophages (Figs. 2.25 and 2.26)

After the initial invasion by neutrophils, macrophages appear. These active inflammatory cells are a source for pro-inflammatory cytokines promoting the removal of cellular debris and muscle tissue remodelling (Kanda et al., 2013). They are comprised of several subtypes with distinct functions, with their phenotype characterised by the molecular environment of the damage (McLennan, 1996; Philippou et al., 2012). In other words, dependent on the environment, macrophages can adopt either a pro- or anti-inflammatory phenotype (Bystrom et al., 2008; Novak & Koh, 2013). For instance, the continued presence and accumulation of neutrophils at the inflammatory site are responsible for the presence of pro- rather than anti-inflammatory macrophages (Pizza, Petersen, Baas, & Koh, 2005) with the former producing reactive oxygen species, IL-1β, and TNF-α (Bencze et al., 2012) and the latter, when activated, producing IL-10, downregulating the production of IL-12, characteristic of the inhibition of inflammation (Bencze et al., 2012).

With the use of a specific panel of antibodies, based on the antigenicity of the various macrophage subtypes (ED1⁺ monocytes, ED1⁺ macrophages, ED2⁺Ox6⁻ and ED2⁺Ox6⁺, and Ox6⁺), McLennan investigated the arrival and departure time, as well as the specific location within the lesion of damaged muscle for the various macrophage subtypes (McLennan, 1996). The tibialis anterior muscle of adult male Wistar rats (n = 55) was exposed to a freeze injury using the blunt end of a stainless steel surgical probe (3 × 4 mm) cooled in liquid nitrogen. The rats were sacrificed at various hours (0.5, 1, 1.5, 2, 3, 5, 7, 9, 11, and 18) and days (1, 2, 3, 3.5, 4, 4.5, 5, 6, 7, 8, 11, 14, 19, and 21) post lesion of the muscle. Sections of the muscle were prepared for analysis using antihaemopoietic cell antibodies. Within 1 h of freezing, damage/dead fibres had swollen, differentiating them from healthy fibres. This initial event occurred uniformly throughout the lesion, with subsequent cellular events occurring where the lesion bordered the undamaged fibres, progressively moving from the periphery to the centre, creating a gradient of maturity. Events within the central core were usually delayed by 2–3 days. Cell infiltration on the periphery began by 3 h with some fibres being completely phagocytosed by 1 day. Myotube formation was observed to occur by 2 days, with the boundary between the damaged and undamaged portion becoming undiscernible by 3 weeks.

Circulating Macrophages (ED1⁺ Monocytes and ED1⁺ Pro-inflammatory)

Within an hour of damage and in response to inflammatory signals (cytokines and chemokines), the ED1⁺ monocyte infiltrated the epimysium penetrating the injury within 3 h. The primary role of this monocyte is to replenish the population of tissue-resident macrophages during homeostasis and inflammation (Murray & Wynn, 2011). Upon infiltration, the ED1⁺ monocyte is activated into ED1⁺ macrophages, preceding ED2⁺ macrophages and Ox6⁺ cells. According to Butterfield et al., ED1⁺ monocytes are the predominant macrophage phenotype in circulation transforming into the ED1⁺ macrophage upon activation (Butterfield, Best, & Merrick, 2006). The presence of ED1⁺ cells, associated with mature lesions, clearly indicates where damage has occured, for near the end of the degenerative phase, ED1⁺ macrophages are less apparent.

Resident Macrophages (Ox6⁺, ED2⁺Ox6⁻, ED2⁺Ox6⁺, and ED2⁺)

Resident macrophages, representing up to 10–15% of the total cell number during quiescent states in adult mammals, are versatile cells found in essentially all adult mammal tissues, with this number increasing in response to inflammation (Italiani & Boraschi, 2014). Concentrations of OX6⁺, a major subtype of resident macrophages, are completely absent early in the lesion, possibly in response to the death of the resident macrophages, as the damaged area contained some OX6⁺ cellular debris, with their numerical density behind the lesion being normal. Their appearance in the core of the lesion always occurred after the presence of ED1⁺ monocytes/macrophages with their numbers exceeding ED1⁺ cells. Increased accumulation of Ox6⁺ within the necrotic fibres, indicates early stages of degeneration, being less abundant in extensively phagocytosed fibres. As the lesion regenerated, the Ox6⁺ cells become more localised to the endomysium and perimysium, being initially more abundant than ED2⁺ macrophages in the endomysium, with the converse for the perimysium. They are normally located in both the endo- and perimysium, with their concentrations being greater during regeneration than in undamaged muscles. Three weeks post damage, their numerical density declined, making it difficult to delineate between damaged and undamaged tissue.

According to Honda et al. (Honda, Kimura, & Rostami, 1990) and McLennan (McLennan, 1993), ED2⁺Ox6⁻ is a major type of resident macrophage in skeletal muscles. Within lesioned areas, they appeared to die and rapidly regenerate. After 1 h post damage, the ED2⁺ macrophages were no longer detectable; however, they were still abundant in undamaged portions of the muscle. At 3 h a progressive increase in their numbers occur on the fringe, with a heightened accumulation formed on the fringes of the lesions and the perimysia behind them by 1–2 days (Honda et al., 1990; McLennan, 1993). It should be noted that the majority of ED2⁺ in the connective tissue, both behind and overlaying the lesions, were of the ED2⁺Ox6⁻ phenotype. ED2⁺ macrophages are not associated with necrotic fibres during early stages of phagocytosis (Honda et al., 1990).

Spatial Sequencing of Inflammatory Cells

Evidence exists suggesting that overload, overuse, and compression induce structural damage and inflammation to both muscle and tendon tissue (Kuipers, 1994; Messner, Wei, Andersson, Gillquist, & Rasanen, 1999; Sharma & Maffulli, 2006; Sorichter, Puschendorf, & Mair, 1999; Soslowsky et al., 2000). A study specifically referring to the time course of inflammatory cell accumulation in two animal models, with regard to structural damage and inflammation following a tendon injury (acute tendinopathy), provides a summary of the spatial sequencing of the various inflammatory cells, specifically the accumulation and decrease in the concentration of the neutrophils, ED1+ (phagocytic) and ED2+ (non-phagocytic) macrophages (Marsolais, Cote, & Frenette, 2001). Although this study referred to a tendon injury, regardless of the inciting factors, the events associated with inflammation and tissue damage are considered to be the same, with the kinetics and the magnitude of the response dependent on the extent of the damage as well as the muscle damaged (Butterfield et al., 2006; Carlson & Faulkner, 1983; Charge & Rudnicki, 2004; Lefaucheur & Sebille, 1995; Tidball, 1995).

This study by Marsolais et al. (2001) consisted of female Wistar rats, and comprised of several models. With the first model, a blunt dissection of the right isolated exposed Achilles tendon was performed, and injected with 30μl of crude collagenase dissolved in sterile phosphate buffered solution (PBS) near the osteo-tendinous junction of 24 rats, inducing Achilles tendinitis (Davidson et al., 1997). The procedure was repeated with sham-operated animals using the same volume of PBS without collagenase. Ambulatory controls not injected with either collagenase or PBS were allowed to roam freely in their cage. Since the sham group in the first model expressed a high number of inflammatory cells (neutrophils and ED1⁺ macrophages), following the exposed Achilles tendon procedure, a second model was setup to determine whether the surgical intervention and not the percutaneously injected collagenase and PBS was responsible for inflammation. Rats in this model were injected with the collagenase and PBS percutaneously without any surgical intervention, with the same volume of PBS without collagenase being injected in the sham animals, and with the ambulatory control rats not being injected with either collagenase or PBS. Rats in the first model (experimental, sham, and ambulatory control) were sacrificed at 1, 3, 7, 14 or 28 days post collagenase and PBS injection, with the rats in the second model sacrificed after 1 and 3 days. For both models, the sham animals were sacrificed at 1 and 3 days. A comparison of neutrophils and ED1⁺ and ED2⁺ macrophages between the two groups (experimental and sham) for both models was performed at 1 or 3 days post collagenase or PBS injections, with this time period being reflective of the time encompassing an extensive cell accumulation of the inflammatory cells.

The injured tendon expressed an accumulation of leukocytes with a rapid infiltration of neutrophils followed by ED1⁺ macrophages but to a lesser degree, with regard to post collagenase injection. There was a 46-fold increase in neutrophil concentration at 1 day post injury in the exposed Achilles tendon animals, decreasing more than 70% after 3 days, with a return to control values at 7, 14, and 28 days post injury. The time points used in this study (1, 3, 7, 14, and 28 days) are suggestive of the phases of inflammation and its resolution (Frenette, St-Pierre, Cote, Mylona, & Pizza, 2002) (Table 2.1). A comparison of immunohistochemistry results revealed a high concentration of neutrophils after 1 day of the injured tendon vs. being completely absent in the tendon from both the ambulatory controls and those that had recovered at 28 days. Concerning ED1⁺, an 18-fold increase occurred at 1 day, decreasing slightly at 3 days post injury, with the most significant decrease occurring by day 7, matching ambulatory control concentrations after 14 days. Comparing ED1⁺ to neutrophils, the latter had a twofold increase in numbers after 1 day, with ED2⁺ values augmented after 28 days (Table 2.2). Comparing the sham to the ambulatory control rats in the first model, a significant increase in both neutrophils and ED1⁺ concentrations was observed in the sham animals, which were exposed to the surgical procedure minus the collagenase and PBS. This result suggests that a mechanical stimulus is a major contributing factor for inducing muscle damage and inflammation (Frenette et al., 2002; Kuipers, 1994; MacIntyre et al., 1995).

Table 2.2 Sequence of inflammatory cells

Interestingly, the appearance of leukocytes was similar in both the nonexposed and exposed Achilles tendon groups at 1 and 3 days, confirming that regardless of the insult causing the damage, the sequencing of the cells of the inflammatory response is the same (Butterfield et al., 2006; Tidball, 1995). The nonexposed Achilles tendon procedure was associated with a decrease in the magnitude of the leukocyte cell accumulation, ~35% for neutrophils, and ~39% for ED1⁺ macrophages. Following collagenase injection, ED2⁺ macrophages increased in the nonexposed Achilles tendon group after 3 days. With both the ambulatory controls and sham animals, which underwent the nonexposed Achilles tendon procedure, no significant difference in the number of inflammatory cells was expressed. Based on the results of this study (Marsolais et al., 2001), as well as several other studies, neutrophils and ED1⁺ macrophages express a phagocytic phenotype responsible for the removal of cellular debris, with ED2⁺ macrophages associated mainly with the regeneration of damaged muscle (Al-Mokdad, Shibata, & Nakagawa, 1997; Massimino et al., 1997; McLennan, 1993, 1996; St. Pierre & Tidball, 1994).

By referring to Table 2.2, based on the Marsolais et al. (2001) study, we notice that in model one [mechanical insult (blunt dissection with injection of collagenase and PBS) to Achilles tendon] vs. model two [no exposure to mechanical stimulus (blunt dissection)], the ED2+ (non-phagocytic) macrophages associated with recovery accumulate earlier post collagenase injection (Table 2.2; yellow box). This observation suggests that the degree and magnitude of muscle damage are catalysts for the accumulation and phenotype of inflammatory proteins. As observed in studies referencing neutrophils, and pro- and anti-inflammatory macrophages, the neutrophils and the macrophages dominate the inflammatory response to muscle damage, with the ED1+ and ED2+ macrophages possessing diverse roles with regard to the inflammatory response and repair (Karalaki et al., 2009; Philippou et al., 2012; Tidball, 1995). The ED1+ macrophages feature prominently in the removal of necrotic tissue, with the ED2+ macrophages associated with muscle tissue repair typically observed at later stages of inflammation. The early appearance of the ED2+ macrophage suggests that the regeneration process occurs earlier with a less traumatic insult on the tendon tissue (model 2 nonmechanical stimulus) (Marsolais et al., 2001). Therefore, the degree of muscle damage, and the interaction and coordination of the various infiltrating inflammatory cells, are important for the outcome of the repair process of the muscle (Marsolais et al., 2001). This observation is significant when considering that the magnitude of stretching intensity may be responsible for causing inflammation as well as the recovery of the muscle from injury.

Neutrophils and macrophages play an important role in both the initial response to tissue damage and the subsequent resolution and regeneration of muscle fibre. At the site of tissue damage, these inflammatory cells coexist, with their existence dependent on their ability to perform distinct functions (Marsolais et al., 2001; McLennan, 1996). According to Tidball et.al (1999) macrophages do not contribute to membrane disruption during inflammation even though concentration of ED1⁺ (phagocytic) macrophages is observed to peak with muscle damage (Tidball et al., 1999). However, the ability of the macrophages, specifically ED2⁺, to repair the damage is quite limited in the absence of neutrophils, since the primary role of neutrophils is the removal of cellular debris through phagocytosis, prompting the macrophages for cellular regeneration and repair (Grounds, 1987; Wynn & Vannella, 2016). On the other hand, the continued presence and accumulation of neutrophils can impair tissue regeneration, since they can induce a secondary muscle injury by modifying skeletal muscle proteins oxidatively (Pizza et al., 2005). Elevated levels of neutrophils have also been associated with passive stretches, without any signs of injury (Pizza et al., 2002).

With regard to the macrophages, the microenvironment in which they find itself is essential for their expressed phenotype (ED1⁺, ED2⁺) (McLennan, 1996), with the polarisation signals being apoptotic cells (i.e. dead neutrophils), as well as cytokines released by other inflammatory cells (Butterfield et al., 2006; Duque & Descoteaux, 2014). For instance, a microenvironment populated predominately by necrotic tissue expresses ED1⁺ (Marsolais et al., 2001; McLennan, 1996). Similar to the neutrophils, ED1⁺ are activated by pro-inflammatory cytokines TNF-α and IL-1β (Hirani, Antonicelli, Strieter, Wiesener, & Ratcliffe, 2001). Once ED1⁺ monocytes are activated to the ED1⁺ macrophage phenotype, they contribute to the inflammatory response by releasing pro-inflammatory cytokines, such as prostaglandin-E2 and IL-1β, thereby recruiting more neutrophils, and thus heightening the inflammatory response (Scott, Khan, Cook, & Duronio, 2004). The activated ED1⁺ macrophages produce nitrogen and oxygen intermediaries [nitric oxide (NO) and superoxide] which are highly toxic and have the capacity to cause damage to neighbouring tissues (Nathan & Ding, 2010). They are believed to be involved in various chronic inflammatory diseases (Sindrilaru et al., 2011) making it very important to control their responses in order to prevent further collateral tissue damage (Murray & Wynn, 2011).

As observed by the studies above, the ED2⁺ macrophages appear during the latter stages of inflammation (Marsolais et al., 2001; McLennan, 1996). Similar to ED1⁺, ED2⁺ macrophages originate in the bone marrow from hematopoietic stem cells and are expressed in circulation as anti-inflammatory monocytes, becoming resident tissue macrophages (Bosurgi, Manfredi, & Rovere-Querini, 2011; Brigitte et al., 2010; Yang, Zhang, Yu, Yang, & Wang, 2014). These represent 10–15% of the total adult mammal cell population (Italiani & Boraschi, 2014). Unlike the preceding ED1⁺ macrophage, which is drawn to the necrotic tissue to phagocytose the cellular debris and apoptotic neutrophils, the primary role for ED2⁺ is that of tissue repair through cell signalling and cytokine production (Fernando, Reyes, Iannuzzi, Leung, & Mckay, 2014; Philippou et al., 2012). Interestingly, unlike the ED1⁺ macrophages, derived from the circulating monocytes and involved in severe inflammatory injuries, resident macrophages are involved in their repopulation after a mild injury (Italiani & Boraschi, 2014). These resident macrophages mediate tissue repair by releasing a number of growth-promoting factors and cytokines, such as fibroblast growth factor, insulin-like growth factor, and transforming growth factor (TGF)-β1 (Philippou et al., 2012; Robertson et al., 1993; Wahl et al., 1987), as well as IL-10, an anti-inflammatory cytokine attenuating the ED1⁺ macrophage (Tidball & Villalta, 2010). In addition, ED2⁺ macrophages produce matrix metalloproteinases and tissue inhibitors of metalloproteinases controlling the turnover of ECM (Wynn, 2008), as well as removing and digesting dead cells and debris, that would otherwise promote the responses of the ED1⁺ macrophage (Atabai et al., 2009; Baron & Wynn, 2011).

Interestingly, secretion of TGF-β1 by ED2⁺ signifies a shift from the ED1⁺ phenotype (inflammatory macrophage) towards the ED2⁺ (anti-inflammatory macrophage) in response to the phagocytosis of apoptotic neutrophils and necrotic muscle cells (Arnold et al., 2007; Ashcroft, 1999; Fadok et al., 1989; Tidball & Villalta, 2010). This shift serves to resolve muscle inflammation. Apoptosis (programmed cell death) is an energy-efficient means of removing the cellular debris, since the cells that phagocytose the debris (i.e. neutrophils and ED1⁺ macrophages) do not have to migrate and do not cause further inflammation (Brown, Kao, & Greenhalgh, 1992). More importantly, it is a necessary step in replacing cell populations in order to begin the next phase of healing (Greenhalgh, 1998). However, if the natural apoptotic events are tampered with, an exacerbation rather than a resolution of inflammation occurs, since the balance between the cellular numbers is lost leading to non-normal tissue repair (Greenhalgh, 1998). Further, the cytokines, fibroblast growth factor, insulin growth factor-1, and TGF-β1, are important for recruiting and activating fibroblasts which begin the repair process by secreting matrix molecules such as collagen (Butterfield et al., 2006). In both animals and humans, acute limited injury is accompanied by only a transient increase in TGF-β1, without fibrosis occurring (Border & Noble, 1994). However, the stimulus of repeated injuries increases the production of TGF-β1, sustaining its presence and levels, leading to the further deposition of ECM and the increased expression of tissue fibrosis (Chikenji et al., 2014; Zimkowska et al., 2009).

Therefore, infiltration of the leukocytes to the site of injury and inflammation, sequencing of the neutrophils and the macrophages (pro-inflammatory preceding anti-inflammatory), their coexistence and communication in response to the early removal of cellular debris, the apoptosis of the inflammatory cells, and the release of growth factors and cytokines, are essential for the resolution of tissue damage. Any delay in the sequencing and proper function of these processes slows down the recovery from tissue damage and is responsible for the disproportionate amount of tissue destruction and increased fibrotic tissue (i.e. scar tissue), affecting the function of the muscle. In addition, if the clearance of apoptotic cells is defective and there is a continued accumulation and persistence of leukocytes (i.e. neutrophils), this is responsible for the progression from acute to chronic inflammation (Lawrence et al., 2002). Since we are more concerned with the acute inflammatory response with regard to stretching intensity, it is beyond the scope of this manuscript to refer to chronic inflammation and the subsequent mechanisms related to its expression.

Concluding Remarks for Stage Four

Neutrophils and macrophages are responsible for the release of the pro-inflammatory cytokines IL-1β and TNF-α, with their production being responsible for the expression of IL-6 (Frost & Lang, 2005). These three cytokines form a complex network, with the expression of each influencing the other, in an attempt to regulate the inflammatory response (Akira et al., 1990). Interleukin-6, the mediator of the acute inflammatory response, is responsible for the release of C-reactive protein, which plays a prominent role as an anti-inflammatory mediator inducing the expression of IL-1ra, as well as sTNF-receptors (anti-inflammatory cytokines) (Fig. 2.30) (Tilg et al., 1994; Tilg, Dinaello, & Meir, 1997).

Fig. 2.30

Expression of anti-inflammatory cytokines. (Published with kind permission of copyright © Nikos C. Apostolopoulos 2018. All rights reserved)

As emphasised, the microenvironment of a given tissue is responsible for transformation of the macrophage monocyte into the macrophage phagocytic phenotype (Duque & Descoteaux, 2014). Many of the cytokines capable of biasing the phenotype of the circulating monocyte are provided by the endothelium as well as the surrounding lymphocytes, responsible for activating the ED1⁺ or ED2⁺ macrophages (Gordon & Taylor, 2005; Martinez, Helming, & Gordon, 2009; Martinez, Sica, Mantovani, & Locati, 2008). The ED1⁺ macrophages produce pro-inflammatory cytokines (IL-1β, TNF-α, IL-6, and IL-12) mediating destruction of pathogens and the removal of cellular debris, with ED2⁺-activated macrophages producing anti-inflammatory cytokines (IL-10, TGF-β1, and IL-6), associated with repair (Fernando et al., 2014; Flynn, Chan, & Lin, 2011). IL-6 possesses a Janus-like nature, for based on the surrounding microenvironment, it determines the macrophage phenotype (ED1⁺, ED2⁺), with its absence preventing recovery (Fernando et al., 2014; Kopf et al., 1994; McLennan, 1996). It is necessary for the resolution of inflammation as observed in mice deficient in IL-6 (IL-6^−/−) (Kopf et al., 1994). Although these mice (IL-6^−/−) did not express any developmental abnormalities, they were unable to generate an acute phase response (Kopf et al., 1994), suggesting that its induction is an important in vivo distress signal coordinating the activities of the hepatocytes, macrophages, and lymphocytes (Kopf et al., 1994). The relationship between IL-6 and C-reactive protein represents the body’s mechanism for resolving the acute inflammatory response.

Mechanotransduction

Conditions under which the muscle is loaded such as during eccentric and concentric exercise, or passive stretching, have been associated with the expression of cytokines (Helge et al., 2003; Ostrowski et al., 1998; Pedersen et al., 2001; Pedersen & Fischer, 2007; Steensberg et al., 2000, 2002; Ullum et al., 1994) and the elevation of neutrophils (Frenette et al., 2002; Koh, Petersen, Pizza, & Brooks, 2003a; Mcloughlin, Mylona, Hornberger, Esser, & Pizza, 2003; Pizza et al., 2002). The muscle’s response to load is influenced by the parameters of intensity, duration, and frequency (Marschall, 1999; Mujika et al., 1995), with a literature review suggesting that intensity may be of greater significance (Apostolopoulos et al., 2015). According to this review, intensity, in particular stretching intensity, is qualitative in nature compared to the quantifiable parameters of duration and frequency, accounting for why stretching intensity continues to remain relatively under researched (Apostolopoulos et al., 2015). It proposes that stretching intensity be considered as a force or load (Apostolopoulos et al., 2015), for too little force is associated with an elastic response with little or no gain in ROM, and too much force injuries tissue prompting an inflammatory response (Brand, 1984; Jacobs & Sciacia, 2011; McClure et al., 1994). The review recommends that stretching intensity, and body position during stretching, should be considered when investigating perceived muscle soreness, inflammation, and their influence on muscle health and performance (Apostolopoulos et al., 2015, p. 275). 

Exercise intensity influences the synthesis of cytokines with increases in IL-6 being observed (Ostrowski, Schjerling, & Pedersen, 2000; Peake, Suzuki, et al., 2005). A study investigating the effect of intense running observed significant increases in concentrations of leukocytes (neutrophils, monocytes) (Ostapiuk-karolczuk et al., 2012). With regard to stretching intensity and performance, a study suggests that to achieve maximum jump height athletes should perform a static stretching intensity <50% of the point of discomfort prior to performance (Behm & Kibele, 2007).

Many physiological functions depend on the ability of cells and tissues to sense and react to mechanical force or load (external and internal) with their response being essential for proper development and function (Hamill & Martinac, 2001; Kung, 2005). For instance, cells associated with baroreceptors, proprioceptors, spindle receptors, and Golgi tendon organs sense blood pressure, positions of limbs, as well as muscle stretch and tension, respectively (Kung, 2005). According to Goldspink, skeletal muscle possesses the ability to adapt its structure, function, and metabolism in response to a mechanical stimulus (i.e. stretching and overload) and that to a large extent this stimulus regulates the expression of individual myosin genes (biochemical response) (Goldspink, 1999). Goldspink is alluding to the concept of mechanotransduction, a process defining the response and relationship of the cells and tissues to their environment, translating any physical input or mechanical perturbation into a biochemical or biological signal (Goldspink, 1999; Hamill & Martinac, 2001; Kung, 2005; Previtera, 2004).

Mechanotransduction defines the conversion of mechanical energy (tension, compression, shear, etc.) into biochemical signals (Yavropoulou & Yovos, 2016). It is considered a key regulator of many physiological processes such as, the regulation of stem-cell differentiation (Engler et al., 2006), fibroblast migration (Lo, Wang, Dembo, & Wang, 2000), the initiation of inflammation, and the triggering of cytokine production (Makino et al., 2007; Previtera, 2004). With stretching defined as an external and/or internal force (Weerapong et al., 2004), and the magnitude of the force applied as the intensity of the stretch (Jacobs & Sciacia, 2011), stretching intensity may be considered as a mechanotransduction mechanism, thereby influencing the inflammatory response, aiding in the recovery or further damage of the muscle.

Mechanotransduction references the behaviour of collective interactions within complex networks adopting a “top-down” rather than a “bottom-up” approach (Ingber, 2008a). Rather than reverse engineer from the cellular level, it tries to understand mechanical behaviour by implying a higher-order architecture, and how this is influenced by physical forces (Huang, Sultan, & Ingber, 2006; Ingber, 2008a). The main tenet of mechanotransduction is the concept of tensegrity (Ingber, 2008a). Tensegrity is concerned with the essential maintenance of mechanical stability created when the compression-bearing rigid structures stretch or tense the flexible tension-bearing members. This imparts a compression on the rigid structures, which in turn creates a counteracting force, that is equilibrated throughout the structure, which itself is in a prestress state (Ingber, 1998, 2008a). In short, this neologism defines the structural shape of a tensegret, as a balance attained by the interaction between a set of members in tension and compression (Anastasi et al., 2006; Connelly & Back, 1998). With organisms constructed as a hierarchy of systems, tensegrity is observed at different levels from the macroscopic (bones, muscles, tendons, etc.) to the atomic scale (Anastasi et al., 2006). In other words, each system is comprised of its own tensional integrity, with the tensegrity at one level or size scale composed of smaller tension/compression elements at another level, with all levels being prestressed (Anastasi et al., 2006; Ingber, 2008a) (Fig. 2.31).

Fig. 2.31

Hierarchy of systems (macro to micro). (Published with kind permission of copyright © Nikos C. Apostolopoulos 2018. All rights reserved)

An increase in stiffness is a hallmark of prestressed structures and hierarchies, which is essential for the mechanical responsiveness to mechanical stress or load, itself being influenced by the intensity of the stress (McMahon, 1984). An example of a hierarchy of tensegrity levels is the relationship between the different layers of the musculoskeletal system, from the macroscopic to the cellular. At the macroscopic, the stable vertical form of the body relative to the force of gravity is maintained between the compression-bearing bones of the skeleton and the tensile pull of the muscles, tendons, and ligaments (Ingber, 1998). The prestress arises from the balance between the contractile forces generated by the muscle cells, countered by the bone matrix’s ability to resist (Ingber, 2006). Below this level, mechanical force is distributed through the myofascial and ECM connections between adjacent muscle bundles, blood vessels, and nerve tracts (Ingber, 2006). At the cellular level, balance between compression and tension of the individual muscle bundles and blood vessels is achieved between tractional forces at the parenchymal cells, resisting the forces exerted by the stiffened ECMs surrounding the connective tissue cells, ensuring stability (Ingber, 2006). At the Z-disc, a certain form and spacing is maintained regardless of an increase in load, suggesting its ability to resist deformation with a passive stretch (Goldstein et al., 1991). This structural criterion allows for the ease of transmission of tension generated within the sarcomere to bones via the tendons, for the Z-disc functions as an anchor, adjoining sets of thin filaments end-on-end in myofibrils (Goldstein et al., 1991). This rearranging at many size scales of the musculoskeletal system protects it against injury, since the molecular components comprising the tensed ECMs and interconnected cytoskeletal elements within adherent cells, adjust accordingly to the mechanical load imparted on the system as a whole (Ingber, 2006; Komulainen, Takala, Kuipers, & Hesselink, 1998; Ralphs, Waggett, & Benjamin, 2002). The importance of this architectural hierarchy is in its ability to provide a biochemical response to a mechanical perturbation, expressed in terms of the importance of the muscle and its ability to adapt to its immediate environment (Gans, 1991). In particular, muscle’s effectiveness (efficiency) in terms of energy consumption and the generation of force are facilitated by the placement of sarcomeres in fibres, and fibres into muscle, and the role muscle plays in relation of the organism to its environment (Gans & Abbot, 1991). This relationship of the fibres to one another, of the tendons to the aponeuroses, and skeletal elements (bones, joints, etc.) may influence the displacement of force in terms of tissue damage and response to stretching. In turn, feedback to the magnitude and rate of force by stretching intensity on tissue may influence its response to load and to the acute inflammatory response. In other words, since the process of mechanotransduction is concerned with the conversion of a mechanical perturbation into a biological signal, and stretching is a physical activity related to force development (Weerapong et al., 2004), the intensity of the stretch (i.e. magnitude of force) may be linked to either the muscle’s recovery from damage or not (Jacobs & Sciacia, 2011). Research needs to be conducted in order to investigate this relationship.

In skeletal muscle, the contractile filaments are maintained in a highly ordered structure by specialised proteins (i.e. actin, titin, desmin, etc.), with unaccustomed eccentric exercise or a mechanical stimulus inducing an adverse reaction causing a leakage of protein (creatine kinase) from skeletal muscle (Feasson et al., 2002). The most striking morphological feature of this reaction is the extensive sarcomeric disruption and Z-disc streaming (Friden et al., 1981, 1983; Friden, Kjorell, & Thornell, 1984). A loss of this sarcomeric order of the myofibrils initiates the autolysis of the damaged components in response to the loss of and upregulation of Ca² (Tidball, 1995). The severity of stretching, defined as muscle fibres stretched by 50% or greater of the optimum force generating length, is observed with a reduction in the maximum Ca²⁺-activated force (contraction), with 25% of the optimum force generating length not associated with a force deficit in skeletal muscle (Balnave, Davey, & Allen, 1997). In other words, sarcomere inhomogeneity was observed with severe stretching (Balnave et al., 1997), with an elevation of muscle Ca²⁺ via an influx of Ca²⁺ from the extracellular space observed during static stretching of rat soleus muscle, suggesting a disruption of the homeostasis of Ca²⁺ (Armstrong et al., 1993). This loss of and upregulation of Ca²⁺ is responsible for the activation of calpain (Armstrong, 1990; Belcastro et al., 1998). This calcium-activated protease cleaves a wide variety of myofibrillar and cytoskeletal proteins found in the muscle [α-actinin (Takahasi, 1990), vinculin (Evans, Robson, & Stromer, 1984), including talin (Fox, Goll, Reynolds, & Phillips, 1985), desmin and titin (Huang & Forsberg, 1998)], with these cleaved proteins acting as chemoattractants for neutrophils (Kunimatsu et al., 1989). A study investigating passive stretching in adult male mice observed elevated levels of neutrophils without overt signs of injury (Pizza et al., 2002).

Therefore, since the muscle is a highly plastic tissue adaptable to various situations (i.e. physical activity, injury, stretching) (Salvini, Durigan, Peviani, & Russo, 2012), mechanotransduction may be the mechanism by which stretching, in particular stretching intensity, influences adaptation. Considering that stretching intensity, defined as the magnitude of force or torque applied to the joint during a stretching exercise (Jacobs & Sciacia, 2011), is responsible for stressing connective and muscle tissue mechanically (Martins et al., 2013), we present the view that this is associated with a biochemical response. Muscle stretching expresses specific genes promoting sarcomeregenesis and remodelling of the ECM in shortened and atrophied muscles (Martins et al., 2013); it also influences the elastic and plastic responses of the MTU, affecting its flexibility and the generation of muscle strength (Gajdosik, 2001). In addition, stretching modifies the morphology, cellular orientation, and shape of fibroblasts (mechanical response), with the upregulation of genes encoding for cellular and extracellular components (β1 integrins, β-actin, and types I and III collagen) (biochemical response), described through the process of mechanotransduction (Kaneko et al., 2009). With the position of the cell maintained within the tissue architecture by external complexes (integrins, costameres), which act to set a “baseline” level of cell tension (tensegrity), changes in this tension result in the cell’s response to a mechanical load (Wu, Fannin, Rice, Wang, & Blough, 2011). The cell increases its stress state by contracting and applying both a mechanical load onto itself, with a subsequent biochemical response, directed to adjacent cells, in direct contact or through the extracellular matrix (Banes et al., 1995). This is a critical response designed to accommodate and acclimate functional loading in tissues (Orr et al., 2006). With mechanical force being a primary regulator of biological functions, mediating development of tissue (i.e. skeletal muscle, tendons etc.), and influencing diverse cellular processes (cell growth, differentiation, protein synthesis) (Alenghat & Ingber, 2002; Ingber, 2003; Wu et al., 2011), the propagation of a biochemical signal associated with stretching (Koh, Petersen, Pizza, & Brooks, 2003b; Pizza et al., 2002) alludes to its possible mechanoregulatory role. With the response of fibroblasts to mechanical load being dependent upon stretching magnitude, frequency, and duration (Wang, Thampatty, Lin, & Im, 2007; Wang, Yang, Li, & Shen, 2004), the loading of tissue based on stretching intensity (low, medium, high), through the process of mechanotransduction, may shed light on how this mechanical stimuli may influence the muscle and connective tissue.

Stretching and Inflammatory Cells

The expression of neutrophils relative to lengthening and isometric contractions and passive stretching was investigated in a study wherein 71 adult male mice were divided into lengthening and isometric contractions or passive stretching groups (Pizza et al., 2002). During lengthening, the extensor digitorum longus was stimulated and lengthened to 20% of its optimal fibre length, held at its optimal length (isometric), or lengthened without stimulation (passive stretching). Each exercise consisted of 75 repetitions lasting 5 min, with controls allowed to roam freely. Mice were sacrificed at 6 h or 3 days after initial in situ procedure, with immunohistochemistry for both neutrophils and macrophages conducted on 10 μm cross sections excised from the midbelly of the extensor digitorum longus. Inflammatory cells were counted and expressed as a number per cubic millimetre, with the number of fibres invaded by these cells counted and expressed as a percentage of the total number. At 3 days, passive stretching and isometric contractions accounted for a 5.5- and 3.7-fold increase in neutrophils, respectively, with lengthening contractions accounting for a 7.9-fold increase, compared to control. This rise in neutrophils occurred whether muscle activity resulted in injury or not, suggesting that passive stretching was a stimulus for one or more chemoattractants for neutrophils, without any overt impairments in function or histological disruption (Pizza et al., 2002). This result needs to be investigated further, for it may be related to the disturbed homeostasis and dysregulation of intracellular Ca²⁺ which is associated with the proteolytic activation of the ubiquitously expressed calpain expression in skeletal muscle, responsible for degrading contractile and other muscle proteins (Fatouros & Jamurtas, 2016; Tu, Levin, Hamilton, & Borodinsky, 2016). In addition, passive stretching was observed to promote protection of adult mice muscle following lengthening contractions , reducing the force deficit, the number of overtly injured fibres, and the accumulation of inflammatory cells (Koh et al., 2003a; Koh & Brooks, 2001).

Using complimentary approaches, an ex vivo model consisting of excised mouse subcutaneous tissue, and an in vivo model consisting of a subcutaneous microsurgical injury to the back of mice, a study investigated the effects of a brief tissue stretch on TGF-β1, and connective tissue matrix remodelling (Bouffard et al., 2008). Elevated extracellular levels of TGF-β1 is associated with the activation of fibroblasts, a major ECM effector, responsible for the increased synthesis of collagen, elastin, and proteoglycan (substrates of the ECM) (Bouffard et al., 2008). With changing levels of mechanical forces (i.e. immobilisation, exercise, and stretching) implicated in connective tissue remodelling (Bouffard et al., 2008), externally applied mechanical forces are believed to reduce collagen deposition during tissue repair and scar formation (Cummings & Tillman, 1992). In the ex vivo model, subcutaneous tissue, kept in organ culture for 4 days, was stretched (20% strain for 10 min, 1 day post excision) or not, with mice in the in vivo model randomised into a stretching (20–30% strain for 10 min twice a day for 7 days) or non-stretching group. The stretching protocol involved stretching of the trunk of the mouse while suspended by its tail, forcing it to extend its front and hind limbs towards a surface slightly inclined relative to vertical, with the distance measured, being the difference between the ipsilateral hip and shoulder joint during stretching and at rest (~20–30% greater during stretching). TGF-β1 protein was lower in the stretched tissue (ex vivo), with a significant rise in type 1 procollagen (in vivo) in the absence of stretch, suggesting that brief tissue stretching attenuates increases in soluble TGF-β1 and type 1 procollagen. These results hint that stretching is relevant in response to tissue injury (Bouffard et al., 2008).

A study by Smith et al. documented whether stretching (i.e. static or ballistic stretching) was responsible for DOMS (Smith et al., 1993). Most studies investigating DOMS and stretching were concerned whether stretching (i.e. static, passive, active, ballistic, dynamic, and PNF) can influence DOMS (Lund, Vestergaard-Poulsen, Kanstrup, & Sejrsen, 1998; Wessel & Wan, 1994). Based on eligible randomised clinical trials, a Cochrane Collaboration Review investigated whether stretching before, after, or before and after exercise was beneficial in treating or preventing DOMS (Herbert, De Noronha, & Kamper, 2011). This review concluded that regardless of when the muscle was stretched, no clinically important reductions in DOMS occur (Herbert et al., 2011). However, stretching intensity was not taken into account.

The primary outcome measured in the Smith et al. (1993) study, in response to static or ballistic stretching of a similar intensity and duration, was creatine kinase. Participants randomly assigned to either stretching group performed three identical sets of 17 stretching exercises, with the static group remaining stationary during each 60 s stretch and the ballistic group bouncing in time to a metronome (60 bounces/min). Blood samples were collected at pre- and 24, 48, 72, 96, and 120 h postexercise for creatine kinase, with rate perceived exertion scores recorded following each stretch using the Borg 6–20 scale. Although significant increases in DOMS were observed in both groups, static stretching was associated with more. Unfortunately, no mention was made of the stretching intensity (i.e. mild, discomfort, pain). Interestingly, the time course and extent of DOMS associated with static stretching is similar to that reported for eccentric exercise, with discomfort ensuing in the first 24–48 h, peaking between 24 and 72 h, and subsiding within 5–7 days (Ebbeling & Clarkson, 1989).

With stretching associated with morphological changes to muscle fibre (i.e. disruption of sarcomere) (Gomes et al., 2007), the disruption in the homeostasis of Ca²⁺ (Armstrong et al., 1993), increases in neutrophils (Koh et al., 2003a; Pizza et al., 2002), and a primary cause of DOMS (Smith et al., 1993), interest in this manuscript was concerned with stretching intensity. With stretching defined as a force (intensity) responsible for stressing connective and muscle tissue (Martins et al., 2013), the magnitude of force identified with stretching intensity (low, medium, high) may be responsible for stimulating a response of the interconnected layers from the macro (muscles and tendons) to the micro level (sarcomeres). In other words, the tissues and cells of each layer may respond differently to low- (i.e. no pain, gentle) versus high-intensity (i.e. pain, discomfort) stretching, conceivably responsible for inducing, increasing, maintaining, or alleviating the acute inflammatory response. The activation and mobilisation of immune cells mediated by the cytokines, released by the injured muscle, triggers inflammation (Fatouros & Jamurtas, 2016). To examine this assumption, three studies were designed. To our knowledge, studies one and two were the first to investigate stretching in humans with reference to blood biomarkers for inflammation and the acute inflammatory response. The first study investigated whether high-intensity passive static stretching (IS) (i.e. discomfort with some pain), used in many sports, in particular in aesthetic (i.e. gymnastics) and martial arts (i.e. taekwondo, judo, etc.), is responsible for an acute inflammatory response. High-sensitivity C-reactive protein and pro-inflammatory cytokines (IL-1β, TNF-α, and IL-6) were measured. The second study compared various passive static stretching intensities (low, medium, and high) to determine if a relationship exists between these different intensities and the expression of the acute inflammatory response. The blood biomarker measured was hsCRP. With study three, the applicability of passive static stretching intensity (low and high) in relation to recovery of the musculoskeletal tissue from an unaccustomed eccentric exercise was investigated. Unaccustomed eccentric exercise has been credited with the development of DOMS and strength loss (Fatouros & Jamurtas, 2016). Although perceived muscle soreness and muscle function are indirect measures, the totality of an injury to the muscle is a decrease in its ability to develop maximum force (Faulkner et al., 1993). DOMS is a form of acute inflammation, with the sensation of perceived muscle soreness representing inflammatory pain (Lieber & Friden, 1993; MacIntyre et al., 1995; Smith, 1991). Soreness and pain are considered contributing factors in regulating muscle activity, with muscular strength remaining depressed throughout the period of soreness (Talag, 1973). This interruption to the muscle tissue is associated with a disorganisation of the myofibrillar material, in particular, a focal disruption of the sarcomeres, with the Z-disc being the most vulnerable structure to an eccentric exercise-induced injury (Friden & Lieber, 2001). By specifically focusing on stretching intensity , what is presented is the concept that the magnitude of stretching intensity may be a contributing factor responsible for aiding in the proper recovery of the muscle. This has implications for exercise training and athletic performance, and may shorten the time of rehabilitation from musculoskeletal disorders and pain.