Investigating In Vivo Myocardial and Coronary Molecular Pathophysiology in Mice with X-Ray Radiation Imaging Approaches
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While suitable imaging techniques and biophysical sensors are available for regional evaluation of myocardial function and local function in large coronary vessels in large animals and humans, small animals still remain a challenge for studies of regional cardiac and coronary function in vivo. This review briefly discusses the importance of the coronary microvascular in cardiovascular disease states, including heart failure. We then focus on the approaches that have been developed that provide new insights into local myocardial function at the myofilament level and high resolution cine-angiography with synchrotron radiation for functional imaging of coronary micro-and macrovessels in vivo in rodents.
KeywordsMicrovessel Cine-angiography Small-angle X-ray scattering Myofilament dysfunction
15.1 Translating Imaging of Cardiac Function to Small Animals
Our current approaches to the study of cardiac function and the mechanisms that regulate it in both healthy and disease states rely heavily on biochemical assays of the expression of genes and proteins, extracted from some part of the myocardium. However, we know from studies of the anatomy and physiology of the heart over the past two centuries that in many regards the heart is far from uniform across different regions in its function from the cell to tissue levels. It is easy to forget, when considering molecular analyses, that heart disease too is more often than not, non-uniform in the way it affects the coronary vessels and cardiac muscle. The assessment of myocardial contractile-relaxation properties and coronary vascular function in specific regions of the heart in humans and large experimental animals are two areas in fundamental cardiovascular science and clinical diagnosis that have been greatly aided by the application of various invasive biophysical sensors and non-invasive or less invasive imaging techniques. However, rodents and in particular transgenic mice, are the most common subjects utilized for basic cardiovascular science to discover the mechanisms of disease development and progression and the efficacy of pharmacological and regenerative interventions. Nonetheless, the small physical size of the rodent heart and limitations in the spatial and or temporal resolution of many of the traditional techniques employed to study cardiac and coronary function has previously limited such assessments to global measures. Frequently, it has not been possible to make repeated measurements in the same animals in the past.
Herein, we describe our recent progress in the development of in vivo imaging approaches that permit repeated measurements of coronary vascular function utilizing microangiography and local cardiac actin-myosin cross-bridge dynamics utilizing synchrotron radiation (SR) based small angle X-ray scattering (SAXS). This combination of techniques is being used to investigate the important role of the coronary microcirculation in the origins of myocardial disease associated with diabetes, hypertension, obesity and heart failure. For detailed overviews of the cardiovascular application of these techniques, their merits and limitations, the reader is referred to our reviews on synchrotron imaging applications (Shirai et al. 2009, 2013). The high intensity X-ray radiation obtainable at third and fourth generation synchrotron facilities around the world is essential for in situ SAXS recordings from the beating heart in anaesthetised rodents. Lab X-ray sources, routinely used for protein crystallography and SAXS studies of ex vivo samples do not produce sufficient photon flux to enable in vivo recordings with the resolution of several milliseconds. While microangiography performed with SR tuned to energies selective for the elements of contrast imaging agents (such as iodine) facilitates the highest possible temporal and spatial resolution for vascular imaging (see Sect. 15.2.1), the current capabilities of optimized microfocus X-ray systems now permits in vivo imaging of microvessels and has some applications for coronary imaging. Our progress in this area is briefly presented in this chapter (see Sect. 15.3). Before describing how we can apply SAXS and microangiography in our ongoing studies in rats and now mice, we first discuss the motivation for utilizing these techniques.
Synchrotron facilities with the capability of biomedical imaging, including the techniques described in this chapter, are available in many continents. The Lightsources.org webpages (http://www.lightsources.org/) is a useful community based entry point for explanations of synchrotron radiation principles, recent applications and descriptions of the synchrotron facilities available worldwide for non-commercial and commercial research. These webpages also describe access programs for each of these facilities.
15.2 The Importance of the Microvessels in Sustaining Cardiac Function
Coronary regulation of the larger microvessels acts to adjust local blood flow to meet metabolic demands. An increase in cardiac work during stress and exercise is facilitated by an increase in coronary flow through the opening of side branches and more distal segments of the vascular tree (vasodilation). The ratio or the maximal increase in coronary flow above resting level is referred to as coronary flow reserve and is a useful index of the health of the coronary circulation. Coronary flow reserve can be determined by positron emission tomography, myocardial contrast echocardiography or invasively with electromagnetic flow probes, cine-angiography or the thermodilution technique. Regardless of the technique used, coronary flow reserve is an integrated measure of blood flow changes across the whole myocardium, including the large coronary arteries and the microvessels, and while very informative, it does not inform on how blood flow is distributed between vessel types or regions. However, direct visualization of coronary vascular responses during cine-angiography does permit analysis of regional changes in blood flow in terms of flow velocity, vessel calibre and visible vessel number. Hence, cine-angiography provides opportunities to investigate changes in coronary function in healthy and disease states.
Various studies now reveal that microvascular function impairment is an important contributor to the progression of many forms of heart failure, including hereditary and non-hereditary forms of hypertrophic cardiomyopathy (Olivotto et al. 2011; Camici et al. 2012), and the increasingly prevalent forms of heart failure with preserved ejection fraction (HFpEF) and angina (Franssen et al. 2016; Crea et al. 2017). There is growing evidence that endothelial dysfunction and ongoing vascular inflammation contribute not only to progressive rarefaction of coronary microvessels (Mohammed et al. 2015), but also increased stiffness and impaired relaxation of cardiac muscle (Franssen et al. 2016; Crea et al. 2017). Reduced nitric oxide bioavailability in the cardiomyocytes surrounding the microvessels is associated with diminished protein kinase G activity and reduced phosphorylation of the giant sarcomeric protein titin, and thus, increased passive tension (one component of muscle stiffening) of the sarcomeres and promotion of ventricular hypertrophy (Borbely et al. 2009; van Heerebeek et al. 2012; Paulus and Tschöpe 2013; Franssen et al. 2016). However, the roles of other vasodilators have not been considered in the current paradigm that links microvessel endothelial inflammation to the origins of HFpEF. It is well established that endothelium derived hyperpolarization factors (EDHF) are important for the regulation of microvessel flow and coronary flow reserve during exercise. Diabetes, obesity, hypertension and aging all impair microvessel EDHF production and or transmission of the hyperpolarization signal through the vessel wall (Park et al. 2008; Luksha et al. 2009; Jenkins et al. 2012; Behringer et al. 2013; Feher et al. 2014; Beyer et al. 2016; Chen et al. 2016; Garland and Dora 2016). Moreover, an imbalance of vasoconstrictor factors, including endothelin-1, rho-kinase and serotonin also contribute to vascular dysfunction in the same comorbidities, which in combination drive the development of HFpEF. Therefore, the roles of other vasodilators and vasoconstrictors in the deterioration of coronary flow reserve, myocardial contractile reserve and diastolic dysfunction remain to be investigated.
15.2.1 The Challenges Associated with Investigating Coronary Microvascular Function
We utilize direct arterial injection of bolus amounts of commercial iodinated contrast agents. The increased viscosity of pure contrast agent requires that contrast be injected with a fast syringe pump via an arterial catheter that is as short as possible to reduce resistance to bolus delivery, and placed as close to target vessels as possible. Typically, paediatric radiography catheters can be used for adult and adolescent rats and hamsters, but for studies utilizing mice then tapered or step-down catheters are essential. Polyethylene or polyurethane catheters with an outer diameter of 1.2F (0.4 mm), such as a cut down version of the FunnelCath™, can successfully deliver contrast agent boluses in mice in ~1 s if the distal narrow tube is shortened (a simple manual test can confirm the ideal length for bolus injection) (Pearson et al. 2017). Furthermore, in the case of coronary angiography in rodents, ideal coronary cine-angiograms can only be obtained if the tip of the catheter is inserted retrograde via the right carotid artery (most direct route) up to a position immediately behind the aortic valve, otherwise left ventricle pumping greatly dilutes contrast concentration and entry into the coronary arteries. When inserting narrow gauge catheters in mice the use of a guidewire is sometimes needed to assist catheter insertion when vascular remodeling makes the artery wall increasingly fragile.
15.2.2 Protocols for Assessment of Coronary Endothelial Function
As demonstrated in Fig. 15.3 with current X-ray detectors it is possible to acquire coronary angiograms at physiological heart rates (300–700 bpm) free from motion artefacts, even at video frame rates, utilizing shutters placed in the X-ray beam to reduce single frame exposure times to 1–5 ms. High speed detectors operating at 50–100 Hz with high sensitivity phosphor plates eliminate the need for shutters for imaging acquisition, but such shutters serve to reduce surface entry radiation dose when image acquisitions are repeated many times in the same animals. Furthermore, motion-induced artefacts during coronary imaging are nearly eliminated by briefly sustaining lung volume at end inspiration (a breath hold 3–4 s), without significantly altering haemodynamics.
15.3 Progress in Vascular Imaging of Small Animals with Lab Systems
Preclinical microCT systems are commercially available that enable CT angiography in animals up to the size of rabbits. However, effective pixel size for single projection images on the CMOS flat-panel detectors with a field of view of several cm3 is typically 100–200 μm. With such systems 2D angiography can at best reveal small arteries. Higher resolution acquisitions are only possible for ex vivo 3D acquisitions due to the prolonged scanning times (>5 min). Nonetheless, microfocus X-ray imaging systems are available that are suitable for real time imaging of the microvessels in vivo, but the challenge has been achieving fast imaging with sufficient absorption contrast to visualize vessels in organs that move, the heart and lungs. One such system that we have utilized for investigations of the hindlimb vasculature in peripheral arterial disease associated with diabetes is the Hitex system described in our recent study (Sonobe et al. 2015).
15.4 Application of In Vivo SAXS to the Study of Myocardial Function in Mice
Various research groups currently employ SAXS for muscle diffraction studies of contraction-relaxation mechanisms in cardiac muscle ex vivo, which is complemented by cell approaches to investigate excitation-contraction coupling in isolated cardiomyocytes. This can be routinely achieved with dedicated lab X-ray SAXS systems, but real-time analysis of myofilament function is achieved with SR as an X-ray source. We and others have developed approaches for SAXS investigations in the beating heart in situ utilizing rats and more recently mice (Pearson et al. 2004, 2007; Yagi et al. 2004; Toh et al. 2006; Shirai et al. 2013).
The authors gratefully acknowledge support from the Uehara Memorial Foundation. All experiments performed at the Japan Synchrotron Radiation Research Institute with approval of the SPring-8 Animal Experiment Review Committee (proposals 2012A1674, 2013B1767, 2015A1354, 2015B1533, 2017A1324).
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