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 diseas

IP 3 and Ca 2+ signals in the heart: boost them or bust them?

Ce mémoire décrit la synthèse ainsi que les propriétés complexantes et catalytiques de divers phosphites et phosphinites construits sur le bord inférieur de p-tert-butyl-calix[4]arènes régiosélectivement préfonctionnalisés. Les calix[4]arènes sont des composés macrocycliques résultant de la condensation de phénols p-substitués avec du formaldéhyde. Ils constituent d'excellentes plateformes de préorganisation pour la confection de ligands polytopiques. La première partie de ce mémoire est consacrée à la synthèse de calixarènes dans lesquels trois atomes d'oxygène "phénoxy" sont pontés par un atome de phosphore, le quatrième étant substitué par une fonctionnalité à atome d'oxygène donneur (éther, ester, oxyde de phosphine, amide). Ces phosphites, qui sont particulièrement robustes en milieu basique, présentent vis-à-vis de métaux de transition soit un comportement P-monodentate, soit P,Ochélatant. Associés à du rhodium, ils fournissent des catalyseurs très actifs en hydroformylation de l'octène. Une influence nette du pouvoir donneur de la fonction auxiliaire oxygénée a été mise en évidence, et ce tant au niveau de l'activité que de la sélectivité en aldéhyde. La seconde partie de ce travail décrit des calixarènes porteurs de deux groupes PX2 (X = OPh ou Ph) greffés sur des atomes d'oxygène occupant des positions distales de la matrice, les deux autres phénoxy ayant été substitués soit par une chaîne alkyle soit par un groupement porteur d'une fonction carbonyle. Ces ligands ont tendance à former facilement avec des ions transitionnels des complexes chélatés. Les complexes du type [Rh(diphos)(acac)] présentent une bonne activité en hydroformylation de l'octène. Ce sont les ligands phosphites qui conduisent aux meilleures sélectivités en nonanal, probablement en raison de leur aptitude à former autour du métal une poche étroite qui favorise la formation de l'intermédiaire "Rh(n-alkyle)" au détriment de l'intermédiaire "Rh(iso-alkyle)". La dernière partie de ce mémoire est consacrée à la diquinone formée lors de l'oxydation chimique ou électrochimique du p-tert-butyl-calix[4]-{OCH2P(O)Ph2}2-(OH)2. Une étude par diffraction des rayons X établit la conformation cône partiel du composé à l'état solide. Des études par RMN montrent que les noyaux quinoniques basculent rapidement à travers la cavité macrocyclique

Dystrophic cardiomyopathy: amplification of cellular damage by Ca2+ signalling and reactive oxygen species-generating pathways

Aims Cardiac myopathies are the second leading cause of death in patients with Duchenne and Becker muscular dystrophy, the two most common and severe forms of a disabling striated muscle disease. Although the genetic defect has been identified as mutations of the dystrophin gene, very little is known about the molecular and cellular events leading to progressive cardiac muscle damage. Dystrophin is a protein linking the cytoskeleton to a complex of transmembrane proteins that interact with the extracellular matrix. The fragility of the cell membrane resulting from the lack of dystrophin is thought to cause an excessive susceptibility to mechanical stress. Here, we examined cellular mechanisms linking the initial membrane damage to the dysfunction of dystrophic heart. Methods and results Cardiac ventricular myocytes were enzymatically isolated from 5- to 9-month-old dystrophic mdx and wild-type (WT) mice. Cells were exposed to mechanical stress, applied as osmotic shock. Stress-induced cytosolic and mitochondrial Ca2+ signals, production of reactive oxygen species (ROS), and mitochondrial membrane potential were monitored with confocal microscopy and fluorescent indicators. Pharmacological tools were used to scavenge ROS and to identify their possible sources. Osmotic shock triggered excessive cytosolic Ca2+ signals, often lasting for several minutes, in 82% of mdx cells. In contrast, only 47% of the WT cardiomyocytes responded with transient and moderate intracellular Ca2+ signals. On average, the reaction was 6-fold larger in mdx cells. Removal of extracellular Ca2+ abolished these responses, implicating Ca2+ influx as a trigger for abnormal Ca2+ signalling. Our further experiments revealed that osmotic stress in mdx cells produced an increase in ROS production and mitochondrial Ca2+ overload. The latter was followed by collapse of the mitochondrial membrane potential, an early sign of cell death. Conclusion Overall, our findings reveal that excessive intracellular Ca2+ signals and ROS generation link the initial sarcolemmal injury to mitochondrial dysfunctions. The latter possibly contribute to the loss of functional cardiac myocytes and heart failure in dystrophy. Understanding the sequence of events of dystrophic cell damage and the deleterious amplification systems involved, including several positive feed-back loops, may allow for a rational development of novel therapeutic strategie

Calcium waves driven by "sensitization” wave-fronts

Objective: Cellular Ca2+ waves are understood as reaction-diffusion systems sustained by Ca2+-induced Ca2+ release (CICR) from Ca2+ stores. Given the recently discovered sensitization of Ca2+ release channels (ryanodine receptors; RyRs) of the sarcoplasmic reticulum (SR) by luminal SR Ca2+, waves could also be driven by RyR sensitization, mediated by SR overloading via Ca2+ pump (SERCA), acting in tandem with CICR. Methods: Confocal imaging of the Ca2+ indicator fluo-3 was combined with UV-flash photolysis of caged compounds and the whole-cell configuration of the patch clamp technique to carry out these experiments in isolated guinea pig ventricular cardiomyocytes. Results: Upon sudden slowing of the SERCA in cardiomyocytes with a photoreleased inhibitor, waves indeed decelerated immediately. No secondary changes of Ca2+ signaling or SR Ca2+ content due to SERCA inhibition were observed in the short time-frame of these experiments. Conclusions: Our findings are consistent with Ca2+ loading resulting in a zone of RyR ‘sensitization' traveling within the SR, but inconsistent with CICR as the predominant mechanism driving the Ca2+ waves. This alternative mode of RyR activation is essential to fully conceptualize cardiac arrhythmias triggered by spontaneous Ca2+ releas

Cardiac phenotype of Duchenne Muscular Dystrophy: Insights from cellular studies

Dilated cardiomyopathy is a serious and almost inevitable complication of Duchenne Muscular Dystrophy, a devastating and fatal disease of skeletal muscle resulting from the lack of functional dystrophin, a protein linking the cytoskeleton to the extracellular matrix. Ultimately, it leads to congestive heart failure and arrhythmias resulting from both cardiac muscle fibrosis and impaired function of the remaining cardiomyocytes. Here we summarize findings obtained in several laboratories, focusing on cellular mechanisms that result in degradation of cardiac functions in dystrophy. This article is part of a Special Issue entitled "Calcium Signaling in Heart"

NO-dependent CaMKII activation during β-adrenergic stimulation of cardiac muscle

Aims During β-adrenergic receptor (β-AR) stimulation, phosphorylation of cardiomyocyte ryanodine receptors by protein kinases may contribute to an increased diastolic Ca2+ spark frequency. Regardless of prompt activation of protein kinase A during β-AR stimulation, this appears to rely more on activation of Ca2+/calmodulin-dependent protein kinase II (CaMKII), by a not yet identified signalling pathway. The goal of the present study was to identify and characterize the mechanisms which lead to CaMKII activation and elevated Ca2+ spark frequencies during β-AR stimulation in single cardiomyocytes in diastolic conditions. Methods and results Confocal imaging revealed that β-AR stimulation increases endogenous NO production in cardiomyocytes, resulting in NO-dependent activation of CaMKII and a subsequent increase in diastolic Ca2+ spark frequency. These changes of spark frequency could be mimicked by exposure to the NO donor GSNO and were sensitive to the CaMKII inhibitors KN-93 and AIP. In vitro, CaMKII became nitrosated and its activity remained increased independent of Ca2+ in the presence of GSNO, as assessed with biochemical assays. Conclusions β-AR stimulation of cardiomyocytes may activate CaMKII by a novel direct pathway involving NO, without requiring Ca2+ transients. This crosstalk between two established signalling pathways may contribute to arrhythmogenic diastolic Ca2+ release and Ca2+ waves during adrenergic stress, particularly in combination with cardiac diseases. In addition, NO-dependent activation of CaMKII is likely to have repercussions in many cellular signalling systems and cell type

Burkitt's lymphoma with bilateral cavernous sinus and mediastinal involvement in a child

We report a 12-year-old boy who presented with incomplete right ophthalmoplegia, exophthalmos and headache. Initial CT and MRI revealed a mass in the right cavernous sinus. During tumour work-up, CT identified additional tumour within the mediastinum. Biopsy of the mediastinal lesion identified Burkitt's lymphoma. We report on this case because radiologists and clinicians should be alerted to identify sites of primary Burkitt's lymphoma outside of the central nervous system if clinical symptoms indicate, or imaging shows, CNS lesions. Primary CNS involvement in Burkitt's lymphoma is rar