Dystrophic cardiomyopathy: role of TRPV2 channels in stretch-induced cell damage

Aims Duchenne muscular dystrophy (DMD), a degenerative pathology of skeletal muscle, also induces cardiac failure and arrhythmias due to a mutation leading to the lack of the protein dystrophin. In cardiac cells, the subsarcolemmal localization of dystrophin is thought to protect the membrane from mechanical stress. The absence of dystrophin results in an elevated stress-induced Ca2+ influx due to the inadequate functioning of several proteins, such as stretch-activated channels (SACs). Our aim was to investigate whether transient receptor potential vanilloid channels type 2 (TRPV2) form subunits of the dysregulated SACs in cardiac dystrophy. Methods and results We defined the role of TRPV2 channels in the abnormal Ca2+ influx of cardiomyocytes isolated from dystrophic mdx mice, an established animal model for DMD. In dystrophic cells, western blotting showed that TRPV2 was two-fold overexpressed. While normally localized intracellularly, in myocytes from mdx mice TRPV2 channels were translocated to the sarcolemma and were prominent along the T-tubules, as indicated by immunocytochemistry. Membrane localization was confirmed by biotinylation assays. Furthermore, in mdx myocytes pharmacological modulators suggested an abnormal activity of TRPV2, which has a unique pharmacological profile among TRP channels. Confocal imaging showed that these compounds protected the cells from stress-induced abnormal Ca2+ signals. The involvement of TRPV2 in these signals was confirmed by specific pore-blocking antibodies and by small-interfering RNA ablation of TRPV2. Conclusion Together, these results establish the involvement of TRPV2 in a stretch-activated calcium influx pathway in dystrophic cardiomyopathy, contributing to the defective cellular Ca2+ handling in this disease.

Rescue of neonatal cardiac dysfunction in mice by administration of cardiac progenitor cells in utero

Striated preferentially expressed gene (Speg) is a member of the myosin light chain kinase family. We previously showed that disruption of the Speg gene locus in mice leads to a dilated cardiomyopathy with immature-appearing cardiomyocytes. Here we show that cardiomyopathy of Speg(-/-) mice arises as a consequence of defects in cardiac progenitor cell (CPC) function, and that neonatal cardiac dysfunction can be rescued by in utero injections of wild-type CPCs into Speg(-/-) foetal hearts. CPCs harvested from Speg(-/-) mice display defects in clone formation, growth and differentiation into cardiomyocytes in vitro, which are associated with cardiac dysfunction in vivo. In utero administration of wild-type CPCs into the hearts of Speg(-/-) mice results in CPC engraftment, differentiation and myocardial maturation, which rescues Speg(-/-) mice from neonatal heart failure and increases the number of live births by fivefold. We propose that in utero administration of CPCs may have future implications for treatment of neonatal heart diseases.

Telomerase Is Essential for Zebrafish Heart Regeneration.

After myocardial infarction in humans, lost cardiomyocytes are replaced by an irreversible fibrotic scar. In contrast, zebrafish hearts efficiently regenerate after injury. Complete regeneration of the zebrafish heart is driven by the strong proliferation response of its cardiomyocytes to injury. Here we show that, after cardiac injury in zebrafish, telomerase becomes hyperactivated, and telomeres elongate transiently, preceding a peak of cardiomyocyte proliferation and full organ recovery. Using a telomerase-mutant zebrafish model, we found that telomerase loss drastically decreases cardiomyocyte proliferation and fibrotic tissue regression after cryoinjury and that cardiac function does not recover. The impaired cardiomyocyte proliferation response is accompanied by the absence of cardiomyocytes with long telomeres and an increased proportion of cardiomyocytes showing DNA damage and senescence characteristics. These findings demonstrate the importance of telomerase function in heart regeneration and highlight the potential of telomerase therapy as a means of stimulating cell proliferation upon myocardial infarction.

Pan-epicardial lineage tracing reveals that epicardium derived cells give rise to myofibroblasts and perivascular cells during zebrafish heart regeneration.

Myocardial infarction (MI) leads to a severe loss of cardiomyocytes, which in mammals are replaced by scar tissue. Epicardial derived cells (EPDCs) have been reported to differentiate into cardiomyocytes during development, and proposed to have cardiomyogenic potential in the adult heart. However, mouse MI models reveal little if any contribution of EPDCs to myocardium. In contrast to adult mammals, teleosts possess a high myocardial regenerative capacity. To test if this advantage relates to the properties of their epicardium, we studied the fate of EPDCs in cryoinjured zebrafish hearts. To avoid the limitations of genetic labelling, which might trace only a subpopulation of EPDCs, we used cell transplantation to track all EPDCs during regeneration. EPDCs migrated to the injured myocardium, where they differentiated into myofibroblasts and perivascular fibroblasts. However, we did not detect any differentiation of EPDCs nor any other non-cardiomyocyte population into cardiomyocytes, even in a context of impaired cardiomyocyte proliferation. Our results support a model in which the epicardium promotes myocardial regeneration by forming a cellular scaffold, and suggests that it might induce cardiomyocyte proliferation and contribute to neoangiogenesis in a paracrine manner.

Force-controlled patch clamp of beating cardiac cells.

From its invention in the 1970s, the patch clamp technique is the gold standard in electrophysiology research and drug screening because it is the only tool enabling accurate investigation of voltage-gated ion channels, which are responsible for action potentials. Because of its key role in drug screening, innovation efforts are being made to reduce its complexity toward more automated systems. While some of these new approaches are being adopted in pharmaceutical companies, conventional patch-clamp remains unmatched in fundamental research due to its versatility. Here, we merged the patch clamp and atomic force microscope (AFM) techniques, thus equipping the patch-clamp with the sensitive AFM force control. This was possible using the FluidFM, a force-controlled nanopipette based on microchanneled AFM cantilevers. First, the compatibility of the system with patch-clamp electronics and its ability to record the activity of voltage-gated ion channels in whole-cell configuration was demonstrated with sodium (NaV1.5) channels. Second, we showed the feasibility of simultaneous recording of membrane current and force development during contraction of isolated cardiomyocytes. Force feedback allowed for a gentle and stable contact between AFM tip and cell membrane enabling serial patch clamping and injection without apparent cell damage.

Regulation of the cardiac Na+ channel NaV1.5 by post-translational modifications.

The cardiac voltage-gated Na(+) channel, Na(V)1.5, is responsible for the upstroke of the action potential in cardiomyocytes and for efficient propagation of the electrical impulse in the myocardium. Even subtle alterations of Na(V)1.5 function, as caused by mutations in its gene SCN5A, may lead to many different arrhythmic phenotypes in carrier patients. In addition, acquired malfunctions of Na(V)1.5 that are secondary to cardiac disorders such as heart failure and cardiomyopathies, may also play significant roles in arrhythmogenesis. While it is clear that the regulation of Na(V)1.5 protein expression and function tightly depends on genetic mechanisms, recent studies have demonstrated that Na(V)1.5 is the target of various post-translational modifications that are pivotal not only in physiological conditions, but also in disease. In this review, we examine the recent literature demonstrating glycosylation, phosphorylation by Protein Kinases A and C, Ca(2+)/Calmodulin-dependent protein Kinase II, Phosphatidylinositol 3-Kinase, Serum- and Glucocorticoid-inducible Kinases, Fyn and Adenosine Monophosphate-activated Protein Kinase, methylation, acetylation, redox modifications, and ubiquitylation of Na(V)1.5. Modern and sensitive mass spectrometry approaches, applied directly to channel proteins that were purified from native cardiac tissues, have enabled the determination of the precise location of post-translational modification sites, thus providing essential information for understanding the mechanistic details of these regulations. The current challenge is first, to understand the roles of these modifications on the expression and the function of Na(V)1.5, and second, to further identify other chemical modifications. It is postulated that the diversity of phenotypes observed with Na(V)1.5-dependent disorders may partially arise from the complex post-translational modifications of channel protein components.

Interplay between cardiac function and heart development

Mechanotransduction refers to the conversion of mechanical forces into biochemical or electrical signals that initiate structural and functional remodeling in cells and tissues. The heart is a kinetic organ whose form changes considerably during development and disease. This requires cardiomyocytes to be mechanically durable and able to mount coordinated responses to a variety of environmental signals on different time scales, including cardiac pressure loading and electrical and hemodynamic forces. During physiological growth, myocytes, endocardial and epicardial cells have to adaptively remodel to these mechanical forces. Here we review some of the recent advances in the understanding of how mechanical forces influence cardiac development, with a focus on fluid flow forces. This article is part of a Special Issue entitled: Cardiomyocyte Biology: Integration of Develomental and Environmental Cues in the Heart edited by Marcus Schaub and Hughes Abriel.