RyRCa2+ Leak Limits Cardiac Ca2+ Window Current Overcoming the Tonic Effect of Calmodulin in Mice

Ca2+ mediates the functional coupling between L-type Ca2+ channel (LTCC) and sarcoplasmic reticulum (SR) Ca2+ release channel (ryanodine receptor, RyR), participating in key pathophysiological processes. This crosstalk manifests as the orthograde Ca2+-induced Ca2+-release (CICR) mechanism triggered by Ca2+ influx, but also as the retrograde Ca2+-dependent inactivation (CDI) of LTCC, which depends on both Ca2+ permeating through the LTCC itself and on SR Ca2+ release through the RyR. This latter effect has been suggested to rely on local rather than global Ca2+ signaling, which might parallel the nanodomain control of CDI carried out through calmodulin (CaM). Analyzing the CICR in catecholaminergic polymorphic ventricular tachycardia (CPVT) mice as a model of RyR-generated Ca2+ leak, we evidence here that increased occurrence of the discrete local SR Ca2+ releases through the RyRs (Ca2+ sparks) causea depolarizing shift in activation and a hyperpolarizing shift inisochronic inactivation of cardiac LTCC current resulting in the reduction of window current. Both increasing fast [Ca2+]i buffer capacity or depleting SR Ca2+ store blunted these changes, which could be reproduced in WT cells by RyRCa2+ leak induced with Ryanodol and CaM inhibition.Our results unveiled a new paradigm for CaM-dependent effect on LTCC gating and further the nanodomain Ca2+ control of LTCC, emphasizing the importance of spatio-temporal relationships between Ca2+ signals and CaM function

The role of myosin in vesicle transport during bovine chromaffin cell secretion.

Bovine adrenomedullary cells in culture have been used to study the role of myosin in vesicle transport during exocytosis. Amperometric determination of calcium-dependent catecholamine release from individual digitonin-permeabilized cells treated with 3 microM wortmannin or 20 mM 2,3-butanedione monoxime (BDM) and stimulated by continuous as well as repetitive calcium pulses showed alteration of slow phases of secretion when compared with control untreated cells. The specificity of these drugs for myosin inhibition was further supported by the use of peptide-18, a potent peptide affecting myosin light-chain kinase activity. These results were supported also by studying the impact of these myosin inhibitors on chromaffin granule mobility using direct visualization by dynamic confocal microscopy. Wortmannin and BDM affect drastically vesicle transport throughout the cell cytoplasm, including the region beneath the plasma membrane. Immunocytochemical studies demonstrate the presence of myosin types II and V in the cell periphery. The capability of antibodies to myosin II in abrogating the secretory response from populations of digitonin-permeabilized cells compared with the modest effect caused by anti-myosin V suggests that myosin II plays a fundamental role in the active transport of vesicles occurring in the sub-plasmalemmal area during chromaffin cell secretory activity

Paradoxical Effect of Increased Diastolic Ca 2+ Release and Decreased Sinoatrial Node Activity in a Mouse Model of Catecholaminergic Polymorphic Ventricular Tachycardia: Depressed SAN Automaticity in CPVT

International audienceCatecholaminergic polymorphic ventricular tachycardia is characterized by stress-triggered syncope and sudden death. Patients with catecholaminergic polymorphic ventricular tachycardia manifest sinoatrial node (SAN) dysfunction, the mechanisms of which remain unexplored

Paradoxical Effect of Increased Diastolic Ca 2+

BACKGROUND: Catecholaminergic Polymorphic Ventricular Tachycardia (CPVT) is characterized by stress-triggered syncope and sudden death. CPVT patients manifest sino-atrial node (SAN) dysfunction, the mechanisms of which remain unexplored. METHODS AND RESULTS: We investigated SAN [Ca(2+)](i) handling in mice carrying the CPVT-linked mutation of ryanodine receptor (RyR2(R4496C)) and on their wild-type (WT) littermates. In vivo telemetric recordings showed impaired SAN automaticity in RyR2(R4496C) mice following Isoproterenol (ISO) injection, analogous to what was observed in CPVT patients after exercise. Pacemaker activity was explored by measuring spontaneous [Ca(2+)](i) transients in SAN cells within the intact SAN by confocal microscopy. RyR2(R4496C) SAN presented significantly slower pacemaker activity and impaired chronotropic response under β-adrenergic stimulation, accompanied by the appearance of pauses (in spontaneous [Ca(2+)](i) transients and action potentials) in 75% of the cases. Ca(2+) spark frequency was increased by 2-fold in RyR2(R4496C) SAN. Whole-cell patch-clamp experiments performed on isolated RyR2(R4496C) SAN cells showed that L-type Ca(2+) current (I(Ca,L)) density was reduced by ~50%, an effect blunted with internal Ca(2+) buffering. ISO dramatically increased the frequency of Ca(2+) sparks and waves by ~5 and ~10-fold, respectively. Interestingly, the sarcoplasmic reticulum (SR) Ca(2+) content was significantly reduced in RyR2(R4496C) SAN cells in the presence of ISO, which may contribute to stopping the “Ca(2+)-clock” rhythm generation, originating SAN pauses. CONCLUSIONS: The increased activity of RyR2(R4496C) in SAN leads to an unanticipated decrease on SAN automaticity by Ca(2+)-dependent decrease of I(Ca,L) and SR Ca(2+) depletion during diastole, identifying subcellular pathophysiologic alterations contributing to the SAN dysfunction in CPVT patients