Annals of Biomedical Engineering

, 36:1281

Measuring Mechanical Properties, Including Isotonic Fatigue, of Fast and Slow MLC/mIgf-1 Transgenic Skeletal Muscle

Authors

    • Department of Mechanical and Aeronautical EngineeringUniversity of Rome “La Sapienza”
  • Antonio Musarò
    • Department of Histology and Medical Embryology, IIMUniversity of Rome “La Sapienza”
  • Emanuele Rizzuto
    • Department of Histology and Medical Embryology, IIMUniversity of Rome “La Sapienza”
Article

DOI: 10.1007/s10439-008-9496-x

Cite this article as:
Del Prete, Z., Musarò, A. & Rizzuto, E. Ann Biomed Eng (2008) 36: 1281. doi:10.1007/s10439-008-9496-x

Abstract

Contractile properties of fast-twitch (EDL) and slow-twitch (soleus) skeletal muscles were measured in MLC/mIgf-1 transgenic and wild-type mice. MLC/mIgf-1 mice express the local factor mIgf-1 under the transcriptional control of MLC promoter, selectively activated in fast-twitch muscle fibers. Isolated muscles were studied in vitro in both isometric and isotonic conditions. We used a rapid “ad hoc” testing protocol that measured, in a single procedure, contraction time, tetanic force, Hill’s (Fv) curve, power curve and isotonic muscle fatigue. Transgenic soleus muscles did not differ from wild-type with regard to any measured variable. In contrast, transgenic EDL muscles displayed a hypertrophic phenotype, with a mass increase of 29.2% compared to wild-type. Absolute tetanic force increased by 21.5% and absolute maximum power by 34.1%. However, when normalized to muscle cross-sectional area and mass, specific force and normalized power were the same in transgenic and wild-type EDL muscles, revealing that mIgf-1 expression induces a functional hypertrophy without altering fibrotic tissue accumulation. Isotonic fatigue behavior did not differ between transgenic and wild-type muscles, suggesting that the ability of mIgf-1 transgenic muscle to generate a considerable higher absolute power did not affect its resistance to fatigue.

Keywords

Transgenic muscleTwitch forceIsometric and isotonic forceHill’s equationMuscle power measurementMuscle isotonic fatigue measurement

Introduction

MLC/mIgf-1 transgenic mice were generated for the first time in 2001 using a tissue-restricted transgene, encoding a locally acting isoform of insulin-like growth factor-1 (mIgf-1) under the transcriptional control of Myosin Light Chain (MLC) promoter.38 Expression of the mIgf-1 transgene was shown to be selectively restricted to skeletal muscle, and to be predominantly expressed in muscles with a high ratio of fast-twitch fibers, such as extensor digitorus longus (EDL: 94.2% fast-twitch fibers).38 In contrast, it was activated at very low levels in slow-twitch muscles such as the soleus (60% slow-twitch fibers).4,38 Transgenic EDL muscles exhibited an increase in muscle mass and maximum force, while no significant changes of muscle mass and maximum force were observed in transgenic soleus.38 Moreover, the increase of muscle mass in transgenic EDL was proportional to the increase of muscle absolute force, while specific force was about the same as for wild-type EDL; this outcome indicates that the increase in muscle mass promoted by mIgf-1 expression is a “functional hypertrophy” without accumulation of fibrotic tissue in the muscle.38 Finally, localized mIgf-1 transgene expression sustains hypertrophy and regeneration in senescent skeletal muscles38 without the adverse side effects seen in other Igf-1 transgenic models20,41; thus the MLC/mIgf-1 transgenic mouse represents an excellent animal model of persistent muscle hypertrophy, which is already utilized for research on muscle dystrophy and cachexia.9,15

Only isometric force of MLC/mIgf-1 muscles has already been measured38; therefore, in this paper we wished to fully characterize MLC/mIgf-1 transgenic skeletal muscles in mice, determining their functional parameters in both isometric and in isotonic conditions. To do so we carried out in vitro experiments, on isolated EDL and soleus muscles from transgenic and wild-type mice.

Since the mIgf-1 overexpression is restricted to fast-twitch fibers, no significant differences were expected between slow-twitch muscle (soleus) functional parameters of transgenic and control mice, while an increase in maximum absolute force and power was expected for EDL transgenic muscles. Even though nothing could be hypothesized about the resistance to fatigue of transgenic EDL and soleus muscles, we extended previous studies analyzing the resistance to fatigue of EDL and soleus muscles in isotonic conditions, for both wild-type and MLC/mIgf-1 transgenic mice.

Many experimental protocols have been proposed, to study in vitro the functional properties of muscle tissue at all structural levels, from single fibres10,1618,22 to the whole muscle.2,5,9,13,21,2831,39 Muscle shortening and force are two important parameters associated with the mechanical properties of muscles, and are the fundamental measurements used to study muscle specimens in vitro, either in isometric or isotonic conditions. Isometric contraction is a situation where the muscle is stimulated and develops a force at a constant length; it is a useful test to determine parameters such as twitch force, tetanic force and contraction time (the time to reach the peak force after delivery of single electrical pulses). However, body movements are generated with dynamic muscle actions. For this reason, the force–velocity relationship of a muscle represents a fundamental property that should be studied. Furthermore, fatigue during dynamic contractions may be more relevant than during repetitive isometric contractions.

All the parameters mentioned above have been widely investigated for several muscle types3,6,8,23,32,34,35,45 and for different animal models.1,19,25,33,43 Isotonic fatigue, however, has been described in only a few studies.14,40,46 It results in a larger decline of maximum force than isometric fatigue tests,44 and is a model that better simulates the fatigue conditions occurring in vivo. Furthermore, by studying isotonic fatigue, it is possible to measure both the mechanical work done during muscle shortening, and the related power changes.

Therefore, to fully characterize the functional properties of MLC/mIgf-1 skeletal muscle, we developed an experimental protocol to quickly and repetitively evaluate all the parameters introduced above, including an estimation of isotonic fatigue. The procedure is based on the electrical stimulation of an isolated muscle, kept in a buffered bathing solution, and on the simultaneous acquisition of muscle displacement, velocity of shortening, and force. This experimental protocol should be easily usable with every traditional experimental set-up to study muscle in vitro. The instrumentation has to have only one specific feature: the system used to actuate the muscle should be able to switch continuously and smoothly between force and position control modes, in order to allow for the experimental protocol to run without interruptions.

Materials And Methods

Experimental Procedure

Both male and female wild-type (FVB) and MLC/mIgf-1 mice were used in this study. The mice were maintained in a temperature controlled room (22 ± 1 °C) with a 12 h light–dark cycle and were sacrificed at 14–16 weeks of age. Each mouse was sacrificed by cervical dislocation before the experiment. All experiments were conducted in accordance with the Italian Law which governs the use of animals in experimentation and the procedure was approved by the local ethics review board.

The two EDL and the two soleus muscles were immediately excised from each mouse and put in a storage solution. This solution was obtained adding potassium phosphate (1.2 mM), magnesium sulfate (0.57 mM), calcium chloride (2.00 mM), and HEPES (10.0 mM) to the Krebs-Ringer bicarbonate buffer (Sigma K4002), and was gassed with a mixture of 95% O2 and 5% CO2 at room temperature. The osmolarity and the pH values were inside the standard deviation values of the Krebs-Ringer bicarbonate buffer (pH: 7.3 ± 0.3; Osmolarity: 267 ± 5% mOsm/L).32,43 The muscle to be tested was mounted vertically in a temperature controlled (30 °C) chamber where it was immersed in the Krebs-Ringer bicarbonate buffer solution, with 10 mM Glucose, also continuously gassed with a mixture of 95% O2 and 5% CO2.36 One end of the muscle was linked to a fixed clamp while the other end was connected to the lever-arm of an Aurora Scientific Instruments 300B actuator/transducer system, using a nylon thread, as shown in Fig. 1. Nylon thread compliance was equal to 0.187 ± 0.004 μm/mN, while muscle compliance resulted equal to 2.000 ± 0.082 μm/mN. The lever-arm could be controlled either in force or in position mode, and allowed for switching between isometric and isotonic measurements without removing the specimen from the bath.
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Figure 1

Experimental setup: the isolated muscle is mounted vertically in a temperature controlled chamber (30 °C) filled with Krebs-Ringer bicarbonate buffer solution, continuously gassed with a mixture of 95% O2 and 5% CO2. One end is linked to a fixed clamp while the other end is connected to the lever-arm of the actuator/transducer with a nylon thread. Electrical stimulation is provided with two platinum electrodes which deliver constant current spikes of 200 mA

The isolated muscle was electrically stimulated by means of two platinum electrodes, located 2 mm from each side of the muscle, with 200 mA controlled current pulses (which resulted in a pulse voltage of around 10 V). For each experiment, the initial muscle length was adjusted to the length (L0) which produced the highest twitch force. The optimal fiber length (Lf) was determined by multiplying that value of L0 for the fiber length to the muscle length ratio (0.44 for the EDL and 0.71 for soleus) indicated in the literature.11,32 The muscle cross-sectional area (CSA) was determined, either for EDL and soleus, dividing the muscle mass m by the product of Lf and the density of mammalian skeletal muscle11 (1.06 mg/mm3):
$$ {\text{CSA}}{\left( {{\text{mm}}^{{\text{2}}} } \right)}{\text{ = }}\frac{{{\text{m}}{\left( {{\text{mg}}} \right)}}} {{L_{{\text{f}}} {\left( {{\text{mm}}} \right)}{\text{ $ \times $ 1}}{\text{.06}}{\left( {{{\text{mg}}} \mathord{\left/ {\vphantom {{{\text{mg}}} {{\text{mm}}^{{\text{3}}} }}} \right. \kern-\nulldelimiterspace} {{\text{mm}}^{{\text{3}}} }} \right)}}}. $$

Experimental Protocol

The rationale of the experimental protocol was to measure isometric and isotonic parameters inducing as little fatigue as possible in the isolated muscle. Therefore, the protocol had to be as short as possible. The phases of the protocol are shown in Fig. 2 and were as follows: the muscle was initially held isometric, and was stimulated with two 0.5 ms single pulses: during the first twitch, isometric twitch force and contraction time were measured, while the second pulse was added to check the consistency of the values obtained with the first twitch and to increase the period of muscle equilibration before applying tetanic stimulation. Contraction time is the time interval the isolated muscle needs to reach the peak force from the delivery of a single electrical pulse.
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Figure 2

Experimental protocol: force values evoked for one MLC/mIgf-1 EDL muscle. Isometric contractions are activated by two single twitches, two pulse trains to produce one un-fused and one fused tetanus. Isotonic contractions are activated by eight pulse trains applied in a pseudo-random sequence with the after-load technique. Isotonic fatigue is elicited by repetitive pulse trains with the muscle shortening against the Fref = Fmax/3. An intertrial interval of 180 s is provided between single twitches and tetanic contractions. An intertrial interval of 150 s is provided between isotonic contractions. During isotonic fatigue, pulse trains are applied every 1 s

The muscle was then maintained isometric and stimulated with two trains of 0.1 ms pulses: the first train (0.6 s at 120 Hz for EDL muscles; 1 s at 30 Hz for soleus muscles) used a frequency that induced unfused tetanus, while the second one (0.6 s at 180 Hz for EDL muscles; 1 s at 60 Hz for soleus muscles) produced a fused tetanus and evoked the maximal tetanic force. One measurement of maximum isometric force is shown in Fig. 3a. To minimize muscle fatigue during this phase of the protocol, a rest period of 3 min was allowed between pulse train stimulations.
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Figure 3

Contraction measurements for one MLC/mIgf-1 EDL muscle: (a) isometric force measured during a fused tetanus stimulation; (b) isotonic force, velocity and muscle shortening measured during one pulse train of the after-load technique. Muscle force increases till it reaches the programmed after-load applied by the actuator/transducer lever arm: at this point the muscle starts to shorten. Force amplitude (line) is mN, shortening amplitude (dashed) is mm × 50, velocity (dot) is mm/s × 3, spike amplitude is V × 2.5

The actuator was then switched to force control, and the “after-load” technique was applied to the muscle specimen to measure the force–velocity relationship in isotonic conditions. The muscle was stimulated with 8 pulse trains at its tetanic frequency. The duration of the trains was 0.4 s for EDL muscles and 0.6 s for soleus muscles. During each pulse train the muscle was allowed to shorten against 8 different constant loads in the following order: 20, 65, 30, 80, 35, 10, 50, 15% of its maximum force. Intertrial interval was 150 s for EDL and 180 s for Soleus. Displacement and velocity of shortening were measured during each stimulation. An example of isotonic force and muscle contraction is shown in Fig. 3b.

Measurement of fatigue concluded the protocol. The muscle was repeatedly stimulated in isotonic conditions with a series of 0.1 ms pulses, delivered in trains of 0.4 s duration, at a frequency of 120 Hz for EDL and 60 Hz for soleus. In this phase, the intertrial interval was only 1 s. The muscle shortened against a load equal to one-third of its maximum force (which we term reference force). This value was chosen because it has been shown that un-fatigued muscles generate their maximum power at that force level.27,40,44 The fatigue test was considered ended when the isolated muscle was no longer able to shorten against the reference force. The muscle specimens were blotted (not dry) and were weighed with tendons removed immediately after each experiment so that their mass m could be used to compute the specific force \({F}/{{{\text{CSA}}}} \) (kN/m2), where CSA is the muscle cross-sectional area described previously.

To evaluate whether the isotonic fatigue section of our experimental protocol could have induced anoxia in the central region of the isolated muscle,7 two additional tests were carried out in several separate experiments. The aim of these experiments was to determine whether muscle performance (twitch force and maximum isometric force) changed after fatigue. Thus those variables were measured again after the fatigue runs in EDL and soleus, both for wild-type and MLC/mIgf-1 transgenic mice. In order to determine whether the fatigue run could induce anoxia and tissue necrosis during our experiments, we stained muscles with Evans Blue Dye12,24 and evaluated the possible extent of fiber damage by histochemical analysis. After the fatigue test the muscles were rapidly removed and incubated for 60 min in 0.1% Evans Blue/oxygenated Krebs Ringer solution. Muscles were then washed 2 × 10–15 min in oxygenated Krebs Ringer, embedded in a O.C.T. compound and immediately frozen in melting isopentane for histological analysis. Frozen sections (7 μm) were fixed in cold acetone, rinsed in PBS, and covered with aqueous mounting media containing DAPI nuclear stain (Vectashield; Vector Laboratories). Fluorescent fibers were viewed under an inverted microscope (Axioskop 2 plus; Carl Zeiss Microimaging, Inc.) and images were processed using Axiovision 3.1. Twelve fields within the areas suspected of damage were analyzed and the Evans Blue positive fibers were identified for each field using Scion Image 4.0.3.2. software. Several EDL and soleus un-fatigued muscles were treated with Evans Blue Dye as negative control. A tibialis anterior (TA) muscle was purposely injured by mechanical pinching or by cardiotoxin (CTX) injection and treated with Evans Blue Dye as a positive control of the marker.

Parameter Calculation

Muscle displacement, velocity of shortening and force were acquired at 1080 Hz during the pulse train stimulation phases with a National Instruments NI-6014 data acquisition board. Contraction time and evoked forces were determined immediately, while Hill’s equation and fatigue parameters were computed during muscle recovery periods. Hill’s equation expresses the relationship between muscle force F and muscle shortening velocity v, for the entire range of F.26 The F–v relationship was calculated fitting the eight experimental points obtained with the after-load technique using a least-squares best-fit method to the Hill’s equation: \( {\left( {F + a} \right)} \times {\left( {v + b} \right)} = c, \) as depicted in Fig. 5. The F–v curves were extrapolated to \( v = 0 \) and \( F = F_{{\max }}. \) At any other given force F, the shortening velocity is v; while a, b, and c are positive constants. The maximum velocity of shortening vmax of the muscle is also determined by extrapolation, as the value of v when F = 0.

During the isotonic fatigue run, the muscle repeatedly shortened against a constant load equal to its reference force value (1/3 × Fmax).27 By multiplying the constant load both for the displacement and for the highest shortening velocity measured during each contraction (see Fig. 3b), mechanical work and mechanical power were calculated for each muscle.

Results

A total of 13 MLC/mIgf-1 transgenic (TG) and 17 wild-type (WT) mice were used to obtain 18 TG EDL, 19 TG soleus, 24 WT EDL, and 26 WT soleus muscles.

Body and muscle mass were measured for each TG and WT animal. Muscle length was also measured and the CSA was calculated with the equation indicated in the experimental procedure. Mean values of the main morphological parameters are reported with their uncertainties in Table 1, where we marked with * the values the Student’s t-test indicated to be significantly different between TG and WT. TG EDL muscles were 29.2% heavier but only 3.0% longer than the corresponding WT ones. The TG EDL CSA was 25.2% greater than the WT one.
Table 1

Morphological characteristics of animal models and muscles: TG = MLC/mIgf-1 transgenic; WT = wild-type; CSA = cross-sectional area

 

TG

WT

Whole body mass (g)

31.5 ± 0.61*

29.0 ± 0.47*

EDL weight (mg)

15.5 ± 0.7*

12.0 ± 0.6*

EDL length (mm)

10.30 ± 0.78

10.00 ± 0.65

EDL CSA (mm2)

3.23 ± 0.21*

2.58 ± 0.12*

Soleus weight (mg)

8.8 ± 0.4

7.9 ± 0.4

Soleus length (mm)

7.56 ± 0.39

7.46 ± 0.29

Soleus CSA (mm2)

1.57 ± 0.10

1.43 ± 0.11

Numbers are mean value ± SEM

p < 0.05

Results of the isometric test section are summarized in Fig. 4. Bars indicate the specific force mean value (F/CSA)mean ± the standard error of the mean (SEM) obtained during single pulse stimulation (twitch force), unfused tetanus, and fused tetanus stimulation. A comparison has been made between MLC/mIgf-1 transgenic and wild-type animal models, both for EDL (fast-twitch) and soleus (slow-twitch) muscles.
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Figure 4

Comparison of specific forces (F/cross-sectional area) measured for EDL (a) and soleus (b) in isometric conditions. Single pulse elicited a twitch-force. The indicated frequencies are for un-fused and fused tetanus contractions. TG are MLC/mIgf-1 transgenic muscles and WT are wild-type. Values are mean ± SEM

When stimulating the MLC/mIgf-1 EDL muscle with a 120 Hz pulse train, the absolute force was 19% (p < 0.05) greater than for the corresponding WT muscle; when stimulating at the tetanic frequency of 180 Hz, the absolute force increase for the transgenic EDL was 21.5% (p < 0.05). However, when we computed the specific force, for each isometric force data set, there were no significant differences between TG and WT animal models with regard to any of the measured variables.

Furthermore, EDL and soleus twitch contraction time did not differ significantly between TG and WT groups (see Table 2 further on).
Table 2

Contractile features of MLC/mIgf-1 transgenic muscles (TG) compared to wild-type muscles (WT)

 

EDL–TG

EDL–WT

SOL–TG

SOL–WT

Contraction time (ms)

11.8 ± 0.3

12.5 ± 0.3

31.6 ± 1.5

29.3 ± 1.3

Vmax/L0 (mm s−1/mm)

4.47 ± 0.36

4.41 ± 0.31

2.09 ± 0.11

2.14 ± 0.13

Wmax/m (mW/g)

114.59 ± 7.03

110.22 ± 6.29

20.08 ± 1.48

22.26 ± 1.63

EDL = extensor digitorus longus; SOL = soleus. Numbers are mean value ± SEM

For the isotonic tests, for each muscle, the force and velocity values measured during the eight isotonic stimulations were fitted to the F–v equation. Figure 5 shows the average F–v curve for EDL muscles along with data points and uncertainties (±SEM) superimposed. The measured points and the corresponding values from F–v equation were highly correlated (R > 0.99) for every data set.
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Figure 5

Average Hill’s F–v curves for transgenic (TG) and wild-type (WT) EDLs. The data points deriving from the eight isotonic stimulations with the after-loads are also indicated on the graph together with their uncertainties. Values are mean ± SEM

Mean values of curvatures (a/Fmax) from the F–v function42 were 0.425 ± 0.039 (TG) and 0.364 ± 0.024 (WT) for EDL muscles, and were 0.335 ± 0.019 (TG) and 0.350 ± 0.029 (WT) for soleus muscles. The normalized maximum shortening velocity vmax/L0 was computed for each muscle and is reported in Table 2. As shown in Fig. 5, shortening velocity v measured at each load F < Fmax did not vary significantly between WT and TG muscles.

The power (W = F × vmean) generated by the muscle as a function of the normalized load (F/Fmax) was computed using the average shortening velocity vmean measured during each isotonic contraction; the normalized power curve (W/m) for EDL muscles is shown in Fig. 6. Again, the highest absolute power generated by TG EDL muscles (1.77 ± 0.11 mW) was 34.1% higher than that generated by WT (1.32 ± 0.09 mW), while the mean values of the normalized power did not differ significantly among TG and WT groups. The values of maximum normalized power were attained at a force which was around one-third of the corresponding maximum force: Fref = (33.65 ± 2.2)% of Fmax for WT; and Fref = (34.9 ± 3.1)% of Fmax for TG.
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Figure 6

Normalized mechanical power (F × vmean/muscle mass) for transgenic (TG) and wild-type (WT) EDLs. Maximum power is generated when the muscle contracts against a force (reference force Fref) around one-third of its maximum force (Fmax), both for TG and WT models. Velocity vmean is the average shortening velocity measured during each contraction. Values are mean ± SEM

Finally, mechanical work (Fref × shortening) and power (Fref × vmean) generated by the muscles during the isotonic fatigue runs were computed for each trial in which the muscles were able to shorten against the constant load Fref = Fmax/3. The work and power curves measured while fatiguing the muscles are shown in Fig. 7a for EDL and 7-B for soleus. In the first seconds of the fatigue stimulation, WT EDL produced about 8% more work than TG EDL, and both muscle types stopped shortening at the same time (after about 25 s). In contrast, TG EDL generated a higher absolute power than WT, although both produced lower values than those measured in the preceding isotonic after-load section. Nevertheless, both TG and WT EDL showed a potentiation during the initial few seconds. Fatigue appeared to start a bit earlier for WT EDL than for TG EDL.
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Figure 7

Mechanical work (Fref × shortening) and power (Fref × vmean) measured during the development of isotonic fatigue for transgenic and wild-type EDL (a), and for transgenic and wild-type soleus (b). Muscles repeatedly contract against a resistant load, equal to the reference force (Fref = 1/3 × Fmax). TG is transgenic and WT is wild-type. Values are mean ± SEM

For soleus muscles, as can be seen in Fig. 7b, the isotonic fatigue behavior was basically the same for TG and WT muscles. This observation is consistent with the finding that no significant differences emerged from the F–v curve and the normalized power between TG and WT soleus.

The adequacy of diffusive O2 supply to the isolated muscle during the isotonic fatigue runs was tested both by additional isometric force tests, carried out after the fatigue section, and by Evans Blue Dye labeling. Nine minutes after the isotonic fatigue run, the maximum isometric force for EDL muscles was approximately the same as that measured before fatigue. Six minutes after the isotonic fatigue run, soleus muscles generated the same isometric force measured before fatigue. The outcome of the Evans Blue Dye labeling tests is shown in Fig. 8 and revealed no evidence of necrotic or damaged fibers.
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Figure 8

Evans Blue Dye labeling test to evaluate whether isotonic fatigue induced anoxia and fiber necrosis in the central region of MLC/mIgf-1 transgenic muscles. Florescence images of un-fatigued EDL (a) and un-fatigued soleus (b) reported as negative controls. Florescence images of EDL (c) and soleus (d) after being subjected to fatigue in the experimental protocol. Florescence images of a tibialis anterior injured by mechanically pinching (e) and cardiotoxin injection (f), reported as a positive control of the marker. Damaged and necrotic fibers are in red

Discussion

Localized mIgf-1 transgene expression sustains hypertrophy and regeneration in senescent skeletal muscle38 and counteracts muscle decline associated with muscular dystrophy,9 activating satellite cells and enhancing the recruitment of circulating stem cells.37 Thus, this experimental model offers promising advantages to design specific therapeutic approaches for human diseases. Within this context, we investigated the contractile behavior of MLC/mIgf-1 transgenic mice skeletal muscle both in isometric and isotonic conditions, including in a single experimental protocol the measurement of isotonic muscle fatigue. Most of the functional parameters studied here were previously unknown for MLC/mIgf-1 skeletal muscle.

The experimental results confirmed that the overexpression of mIgf-1 did not affect the functional properties of an almost completely slow muscle as the soleus. There were no significant differences between TG and WT for any measured parameter in soleus. MLC/mIgf-1 soleus showed an insignificantly (p > 0.50) smaller mean value of isometric specific force (see Fig. 4b) than wild-type.

Transgenic EDL, in contrast, showed an important increase in the absolute tetanic force (on average +21.5%) but no significant differences emerged when calculating the specific force, indicating that MLC/mIgf-1 mice are a good model of a completely functional muscle hypertrophy. This result is consistent with the histological analysis,38 where it was shown there is no additional fibrotic tissue in the increased MLC/mIgf-1 skeletal muscle mass. The constancy of the F–v curve between TG and WT EDL muscles (see Fig. 5) is another acknowledgment of this result.

No differences were found between the contraction time of TG and WT EDL muscles, and the measured values were consistent with published data,4,11,29,32 suggesting again that the over-expression of mIgf-1 did not affect fiber composition. The curvature of the F–v curve, the maximum shortening velocity and the normalized power curve was not significantly different between transgenic and wild-type muscles. For each load value, the shortening velocity did not differ significantly between the two groups.

With regard to muscle fatigue curves, wild-type EDL produced a statistically insignificant higher mean mechanical work in the first seconds of the tests, while transgenic EDLs produced a higher mean power (Fref × vmean) and showed a tendency to undergo fatigue slightly later than wild-type muscles. However, it should be noted that the work and the power measured in the fatigue section were obtained with the after-load technique, where the isolated muscles had to shorten against the reference force, a force equal to one-third of their maximal force. Therefore, transgenic EDLs were exerting a force 21.5% higher than wild-type.

In the first few seconds of the fatigue run of the protocol, before the muscle shows signs of fatigue (smaller shortening displacements), the muscle contracts against a force which is around one-third of Fmax. However, it is important to note that as soon as fatigue begins, the maximum isometric force is reduced and the force–velocity relationship changes its shape; at this point, this load is no longer optimal. To keep an isolated muscle to contract against its optimal force one should measure the maximum isometric force during the progress of fatigue and continuously adjust the external resistant load. For this reason, the fatigue curves presented in Fig. 7 can only be considered to compare the fatigue behavior of WT and TG muscles subject to the same experimental conditions.

The finding that a lower absolute power was measured for both TG and WT muscles during the fatigue run than during the isotonic after-load run, can be due to the short rest interval (3 min) between the two sections of the experiment. This rest interval should have been at least 6 min for soleus and 9 min for EDL.

A further concern about the fatigue section of the protocol was the adequacy of diffusive O2 supply to the central regions of the isolated muscle.7 The functional tests done with TG and WT muscles after the fatigue run revealed that, after an adequate resting time, the isolated muscles completely recover their original tetanic force levels. This observation indicates that isolated muscles subjected to our isotonic fatigue protocol were not subjected to irrecoverable damages due to anoxia. The florescence analysis, done by Evans Blue Dye fiber labeling, confirmed that no tissue necrosis due to anoxia was induced by the fatigue test in the central region of the muscle. Un-fatigued (control) and fatigued muscles had only a few necrotic fibers on the edge of the muscle. This was probably due to mechanical damages occurred when excising the muscles or most probably to unspecific stain. On the contrary, injured TA muscles displayed necrotic fibers not only on the edge but also in the center of the muscle (see Fig. 8e–f).

It needs to be highlighted that both tests discussed here rule out severe anoxia and central fiber necrosis, but do not exclude central fiber hypoxia, possibly occurred during the final seconds of the fatigue runs. Because hypoxia driven fatigue is related to P(O2) in the central fibers of the muscle, the only effective way to resolve this issue would be to measure P(O2) in the central part of the muscles during the fatigue run.

However, there are aspects of our current data that can be interpreted to suggest that tissue hypoxia is not the main factor causing fatigue in these experiments: if fiber hypoxia had remarkably affected the isotonic fatigue behavior of our isolated muscles, then one would expect that transgenic EDLs (whose CSA is 25.2% bigger than WT) would have a faster decay of contractile responses during the final seconds of the fatigue run. Instead, the mean work and power curves reported in Fig. 7a, indicate that fatigue still develops at the same time for both WT and TG EDL. Although we stress that this is suggestive that anoxia is not causing fatigue, and not proof, we are still confident that the data from our experimental protocol reporting the fatigue behavior of wild type and MLC/mIGF-1 muscles can be used, at least, for comparison purposes.

In conclusion, a complete characterization of the functional properties of MLC/mIgf-1 mice skeletal muscle has been carried out, with an in-depth analysis of the isotonic features, done for the first time on this transgenic animal model. We developed an experimental protocol that allowed us to determine quickly and repetitively a number of parameters which describe the muscle functionality, both in isometric and in isotonic conditions. Although, this experimental protocol was developed for use with MLC/mIgf-1 transgenic muscles, it can be utilized with muscles from any other animal models, or to study the changes of muscle functionality after a possible pharmacological or surgical treatment.

Acknowledgments

The authors wish to thank professor Peter Grigg for invaluable discussions about muscle physiology and for polishing the English of the manuscript, and professor Mario Molinaro for his continuous encouragements during the experiments. This research was partially supported by Telethon (grant n. GSP030543) and MDA (grant no. 3986).

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© Biomedical Engineering Society 2008