By examining a healthy cardiac tissue sectioned in a plane parallel to the direction of its fibers, (Figure 3d), highly dense structures appear to connect two adjacent cardiomyocytes. These intercalated disks (ID) contain multiple adhesive components that ensure mechanical coupling and rapid coordinated electrical propagation, therefore are vital for the integrity of the myocardium and its ability to function as a whole191. Three types of adhesive junctions exist in the ID domain: fascia adherens and desmosomes, which are primarily mechanical; whereas gap junctions are the sites of intercellular electrical coupling.
Fascia adherens and desmosomes anchor actin filaments (described previously, involved in the contractile machinery) and intermediate filaments (a component of the cytoskeleton) at the plasma membrane of adjoining cells respectively, thereby provide physical and mechanical attachment between the cells191, 192. In fascia adherens, transmembrane protein N-‐cadherin connects sarcomeric actin filaments and maintains strong cell-‐cell adhesion. Desmosomes are small and discrete structures composed of three separate families of proteins: desmosomal cadherin, armadillo proteins and plakins193. In the normally working myocardium, both fascia adherens and desmosomes are involved in the sensing and regulation of mechanical stresses acting along the longitudinal axis. The area composita, a mixed type of junctional structure, is exclusive to the ID of mammalian species suggesting an evolutionary mechanism to strengthen mechanical coupling in the heart of higher vertebrates194. Their role is essential in organs subjected to high mechanical stresses, such as the skin and the heart. Mutations in genes coding for these junctional proteins are involved in cardio-‐cutaneous inherited pathologies195, such as Naxo’s disease196, 197.
Connexin43 (Cx43) is the major protein constituting gap junctions, which are made of two connexons (or hemichannels)198 and allow non-‐selective diffusion of ~1kDa molecules199, ions, and second messengers, thereby offering electro-‐metabolic coupling. 3 types of connexins exist in the heart: Cx40, 43 and 45 (the number refers to their molecular weights; 43 being the most abundant).
Saffitz et al. describe the spatial distribution of gap junctions as well suited for the heart’s functional requirements200, where large gap junctions at the ends of cells presumably facilitate the efficient intercellular current transfer and provide favorable current-‐to-‐load conditionsx 201. Gap junction specific proteins and density have a direct influence on the velocity of propagation. The measured CV in Purkinje fibers (~3m.s-‐1) is considerably higher than the one recorded in the ventricular bulk muscle (~0.5-‐0.7 m.s-‐1 longitudinally; 0.3-‐0.4 m.s-‐1 transversely)202. The fast propagation is partly due to the presence of different connexins in the gap junctions of these cells, where the amount of Cx40 is at least 3 folds higher in Purkinje fibers than regular cardiomyocytes203. On the other hand, high anisotropic ratio (AR) was also attributed to the shape of cells, the number and spatial orientation of cellular connections rather than the anisotropic distribution of gap junctions190. The
x Saffitz et al. defined current-‐to-‐load as the difference between the minimum amounts of current required in maintaining conduction and the actual amount delivered.
crista terminalis, a discrete bundle of atrial fibers that conducts impulses from the SAN to the AVN, is characterized by a relatively high AR compared to the bulk ventricular muscle (~10 vs. ~3 respectively), despite the relatively fewer gap junctions present201. It was suggested that because of the elongated shape of the cells found in the crista terminalis, wave fronts traveling in the transverse direction must traverse more intercellular junctions per unit distance traveled, and thus would encounter more resistance to their propagation than wave fronts traveling in the longitudinal direction190, 204.
In reality, mechanical stresses can induce changes in gap junction density at the ID, subsequently affecting the intercellular communication and propagation velocity on the macroscopic level. Saffitz et al. argue that the close proximity of gap junctions to mechanical adhesive junctions within the ID, and their ribbon like structures oriented perpendicular to the long axis of the cell is a revolutionary adaptation that would protect them from shear stress200. In favor of this point, pulsatile mechanical stretch was shown to markedly upregulate Cx43 in cultured neonatal cardiomyocytes, inducing remodeling and redistribution of gap junctions and possibly alterations in electrical conduction205. In addition, observations suggest that mechanical integrity is a requirement for a normal excitation, and formation of adhesion junctions is a prerequisite for gap junction formation206. On the other hand, Cx43 was not found to be a prerequisite for the organization of mechanical junctions192 and that electrical conduction was maintained in Cx43-‐deficient mice, suggesting an alternative mechanism of electrical propagation in the context of severely reduced gap junctions207. A growing line of evidence suggests that altered mechanical coupling or loss of cell-‐cell contact for any reason (chemical mediator, hypertrophy, dilatation, scarring… etc.) would directly modulate electrical pacing of the heart. Whereas, the opposite is not yet as tangible: impaired electrical coupling doesn’t affect mechanical coupling206.
Under normal physiological conditions, cardiac cells don’t show similar electrical coupling along the transverse axis, as they do along the longitudinal one14. The costamere and the t-‐tubules are the two major structures in the LM domain (Figure 3c). The term costamere refers to the rib-‐like bands that encircle the cardiomycoyte perpendicular to its long axis208. It consists of a complex protein network that not only forms a physical attachment of the underlying Z-‐line to its outer stress tolerant extracellular matrix (ECM), but also is the site where contractile forces within the cardiomyocyte are directly transmitted to the ECM209. In fact, externally applied strain on the ECM was sufficient through this protein complex to induce myofibril contraction, indicating that both externally applied and intrinsically stimulated mechanical forces were bidirectionally transmitted through the costameres209. The major consequence of muscular contraction is shortening and deformation. A process during which the contractile machinery of the sarcomere must remain connected to both the sarcolemma and the ECM, for the adequate transmission of force and the proper coordination of contraction within the three-‐dimensional muscular structure210. Cardiomyocytes, as load-‐bearing cells, not only generate and transmit mechanical contractions, but they are also equipped through the costameres to sense physical forces, to transduce them into biochemical signals, and generate appropriate responses leading to alterations in cellular structure and function211. This mechanotransduction is particularly complex in the heart, since individual cardiac cells should adapt to many simultaneously ongoing processes212: externally applied mechanical forces, internal loads (blood volume, arterial pressure), neurohormonal modulations, by either reacting instantaneously on the short-‐term or modifying gene expression for long-‐term responses212, then transmitting the same message to adjacent cells and their surrounding ECM for a synchronized contraction.
Two important macromolecular protein complexes play the regulatory role at the costamere: the dystrophin-‐syntrophin mutliprotein complex (DSMC) and the integrin complex14 (Figure 3c). Integrins
Anisotropy 23
are heterodimers composed of chains with a long extracellular domain, which binds extracellular laminin to the cytoskeletal actin213. The principal role of the integrin complex is mechanotransduction, anchoring at its cytoplasmic side many signaling molecules and kinases.
Inactivation of the cardiac integrin gene in mice resulted in an enhanced predisposition to stress-‐
induced cardiomyopathy214 and dilated ventricular chambers215. 1.2.2. The Dystrophin Molecule and the DSMC
The DSMC, similarly to the integrin complex, is composed of transmembrane, cytoplasmic and extracellular proteins216. In the cardiac cell, the transmembrane proteins are the sarcoglycans, the sarcospan and the dystroglycans that in turn bind to extracellular laminin. On the cytoplasmic side, dystrophin binds to syntrophin, dystrobrevin217, and neuronal nitric oxide synthase and attaches this multimolecular complex to cytoskeletal actin218. The DSMC is essential in stabilizing the sarcolemma upon physical stresses. Dystrophin (~430kDa) is mainly expressed in striated muscles, including cardiac muscle cells219 and its main function lies in linking membrane proteins to the actin cytoskeleton220 and maintaining the stability of the dystrophin-‐syntrophin multiprotein complex (DSMC) in the LM domain221. An interesting fact is that dystrophin isn’t restricted to muscles, but is also expressed in the central nervous system (such as the cerebellum, cortical neurons, hippocampus)222. Mutations of dystrophin lead to a severe progressive muscle wasting disease, Duchenne muscular dystrophy (DMD), or to a less severe form Becker’s dystrophy223.
DMD is a devastating X-‐linked degenerative muscle disease, with an incidence of 1:3300 live male births annually223. In patients with DMD, muscular biopsies characteristically demonstrate encoring or degenerating muscle fibers, often observed in clusters224. Small immature centrally nucleated cells are also observed and they correspond to the regenerative processes that take place in the early phases of the disease, creating a balance between necrotic and regenerating muscle tissue225. Later, the regenerative capacity of the muscle is exhausted and muscle fibers are continuously replaced with connective and adipose tissues225. The pathology becomes more prominently manifested with age and patients are usually wheelchair ridden in early adolescence224.
The responsible gene and its product, dystrophin, have been identified more than 30 years ago226, and the mouse model (mdx) harboring a spontaneous mutation similar to the one observed in man has been extensively studied, as a mouse model of the pathology227, 228. These experimental murine models are one of the best tools not only to investigate the primary defect, but also to delineate the causal chain of events leading to the observed pathological finding229, 230. In the case of DMD, despite major advancements done in the field, a comprehensive understanding of the mechanism leading from the absence of dystrophin to the muscular degeneration is still lacking231. Several hypotheses have been proposed to clarify the pathophysiology of the underlying deficits resulting from the absence of dystrophin232. Among the current hypotheses is the mechanical hypothesis, which suggests that membrane fragility in DMD patients allows the accumulation of proteins that are not normally present in the muscle fibers, indicating increased membrane permeability233. On the other hand, the calcium hypothesis proposes that disruptions to the DSMC can result in instability of the sarcolemma that permits calcium entry through membrane tears, when the sarcolemma is stretched during lengthening muscle contractions234. For a comprehensive review on the different available hypotheses involved in the pathology of DMD, refer to the review by Deconinck and Dan (2007)231.
The importance of dystrophin from a cardiac perspective becomes obvious when almost all DMD-‐
patientsxi develop severe cardiac manifestations of their disease, that culminate into dilated cardiomyopathy235, heart failure and increased propensity of sudden cardiac death224. Henceforth, understanding the mechanisms behind the induced arrhythmia in the dystrophic substrate requires the knowledge not only of the membrane effects due to the primary channel defect, but also of the intracellular signals, developmental effects on intercellular communication, integration of possible compensatory responses and other environmental factors that modulate the translation of the primary defect from the gene to the organ229. Despite known discrepancies in the electrophysiology of mouse and man, the mdx mouse has been continuously developed in the last 30 years to elucidate the role of dystrophic loss in the pathology of DMD.
Dystrophin has multiple protein-‐protein interaction domains, including a specific domain in its carboxylic terminus facilitating its interaction with syntrophin217. The latter has a PDZ domain xiithat mediates interactions with the carboxylic end of various ionic channels, including NaV1.5xiii, which has a PDZ-‐domain binding motif in its carboxylic-‐terminal tail. Interestingly, among the nine different NaV channels, only the ones expressed in striated muscles (i.e. NaV1.4 and NaV1.5) have such PDZ-‐binding motifs in their carboxylic ends236. This domain was found necessary for indirect interactions with dystrophin via syntrophin236. Gavillet et al. have initially shown that NaV1.5 expression was decreased in the mdx mouse, the functional consequence of which was a reduction in the whole cell INa,f current237. The authors ruled out other factors that could potentially lower the availability of NaV1.5, hence decrease the upstroke current (such as increased Vrmp, or alterations in activation-‐inactivation kinetics of NaV1.5 in the mdx heart). As dystrophin and syntrophin are virtually absent from IDs in normal condition, Petitprez et al. subsequently demonstrated that the interaction between NaV1.5 and dystrophin is exclusive to the LM of the myocytes238. All these findings combined, suggest that NaV1.5 is exclusively regulated by dystrophin on the LM of cardiomyocytes, the absence of which in the dystrophin-‐deficient mouse ensues the loss of NaV1.5 from the LM and reduction in the total INa,f at the upstroke. On the other hand, other studies have emphasized the interaction of SAP97 (another regulatory protein described in section 1.4.6) with cardiac ionic channels239, 240 including NaV1.5 at the IDs238, which also involves the PDZ-‐domain. In these studies, SAP97 enhanced INa,f without affecting the biophysical properties of the current or the intrinsic properties of the channels238. SAP97 has been also found not to have any major influence on conductance or opening probability nor time of the channels; rather it is thought to enhance the density of functional channels241. Silencing SAP97 lead however to a drastic deterioration of both INa,f and Ito,1 (the transient outward K+ current or early repolarization current)238. How SAP97 modulates functional NaV1.5 is not yet well understood.