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K201 (JTV-519) alters the spatiotemporal properties of diastolic Ca(2+) release and the associated diastolic contraction during β-adrenergic stimulation in rat ventricular cardiomyocytes.

ABSTRACT: K201 has previously been shown to reduce diastolic contractions in vivo during β-adrenergic stimulation and elevated extracellular calcium concentration ([Ca(2+)](o)). The present study characterised the effect of K201 on electrically stimulated and spontaneous diastolic sarcoplasmic reticulum (SR)-mediated Ca(2+) release and contractile events in isolated rat cardiomyocytes during β-adrenergic stimulation and elevated [Ca(2+)](o). Parallel experiments using confocal microscopy examined spontaneous diastolic Ca(2+) release events at an enhanced spatiotemporal resolution. 1.0 μmol/L K201 in the presence of 150 nmol/L isoproterenol (ISO) and 4.75 mmol/L [Ca(2+)](o) significantly decreased the amplitude of diastolic contractions to ~16% of control levels. The stimulated free Ca(2+) transient amplitude was significantly reduced, but stimulated cell shortening was not significantly altered. When intracellular buffering was taken into account, K201 led to an increase in action potential-induced SR Ca(2+) release. Myofilament sensitivity to Ca(2+) was not changed by K201. Confocal microscopy revealed diastolic events composed of multiple Ca(2+) waves (2-3) originating at various points along the cardiomyocyte length during each diastolic period. 1.0 μmol/L K201 significantly reduced the (a) frequency of diastolic events and (b) initiation points/diastolic interval in the remaining diastolic events to 61% and 71% of control levels respectively. 1.0 μmol/L K201 can reduce the probability of spontaneous diastolic Ca(2+) release and their associated contractions which may limit the propensity for the contractile dysfunction observed in vivo.

SUBMITTER: Elliott EB

PROVIDER: S-EPMC3228956 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Catecholaminergic 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.We investigated SAN [Ca(2+)](i) handling in mice carrying the catecholaminergic polymorphic ventricular tachycardia-linked mutation of ryanodine receptor (RyR2(R4496C)) and their wild-type (WT) littermates. In vivo telemetric recordings showed impaired SAN automaticity in RyR2(R4496C) mice after isoproterenol injection, analogous to what was observed in catecholaminergic polymorphic ventricular tachycardia 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 ?-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 by internal Ca(2+) buffering. Isoproterenol dramatically increased the frequency of Ca(2+) sparks and waves by ?5 and ?10-fold, respectively. Interestingly, the sarcoplasmic reticulum Ca(2+) content was significantly reduced in RyR2(R4496C) SAN cells in the presence of isoproterenol, which may contribute to stopping the "Ca(2+) clock" rhythm generation, originating SAN pauses.The increased activity of RyR2(R4496C) in SAN leads to an unanticipated decrease in SAN automaticity by a Ca(2+)-dependent decrease of I(Ca,L) and sarcoplasmic reticulum Ca(2+) depletion during diastole, identifying subcellular pathophysiological alterations contributing to the SAN dysfunction in catecholaminergic polymorphic ventricular tachycardia patients.

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Project description:AIMS:The EMPA-REG OUTCOME study showed reduced mortality and hospitalization due to heart failure (HF) in diabetic patients treated with empagliflozin. Overexpression and Ca2+ -dependent activation of Ca2+ /calmodulin-dependent kinase II (CaMKII) are hallmarks of HF, leading to contractile dysfunction and arrhythmias. We tested whether empagliflozin reduces CaMKII- activity and improves Ca2+ -handling in human and murine ventricular myocytes. METHODS AND RESULTS:Myocytes from wild-type mice, mice with transverse aortic constriction (TAC) as a model of HF, and human failing ventricular myocytes were exposed to empagliflozin (1 μmol/L) or vehicle. CaMKII activity was assessed by CaMKII-histone deacetylase pulldown assay. Ca2+ spark frequency (CaSpF) as a measure of sarcoplasmic reticulum (SR) Ca2+ leak was investigated by confocal microscopy. [Na+ ]i was measured using Na+ /Ca2+ -exchanger (NCX) currents (whole-cell patch clamp). Compared with vehicle, 24 h empagliflozin exposure of murine myocytes reduced CaMKII activity (1.6 ± 0.7 vs. 4.2 ± 0.9, P < 0.05, n = 10 mice), and also CaMKII-dependent ryanodine receptor phosphorylation (0.8 ± 0.1 vs. 1.0 ± 0.1, P < 0.05, n = 11 mice), with similar results upon TAC. In murine myocytes, empagliflozin reduced CaSpF (TAC: 1.7 ± 0.3 vs. 2.5 ± 0.4 1/100 μm-1 s-1 , P < 0.05, n = 4 mice) but increased SR Ca2+ load and Ca2+ transient amplitude. Importantly, empagliflozin also significantly reduced CaSpF in human failing ventricular myocytes (1 ± 0.2 vs. 3.3 ± 0.9, P < 0.05, n = 4 patients), while Ca2+ transient amplitude was increased (F/F0 : 0.53 ± 0.05 vs. 0.36 ± 0.02, P < 0.05, n = 3 patients). In contrast, 30 min exposure with empagliflozin did not affect CaMKII activity nor Ca2+ -handling but significantly reduced [Na+ ]i . CONCLUSIONS:We show for the first time that empagliflozin reduces CaMKII activity and CaMKII-dependent SR Ca2+ leak. Reduced Ca2+ leak and improved Ca2+ transients may contribute to the beneficial effects of empagliflozin in HF.

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K201 (JTV-519) alters the spatiotemporal properties of diastolic Ca(2+) release and the associated diastolic contraction during β-adrenergic stimulation in rat ventricular cardiomyocytes.

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