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Impaired calcium-calmodulin-dependent inactivation of Cav1.2 contributes to loss of sarcoplasmic reticulum calcium release refractoriness in mice lacking calsequestrin 2.

ABSTRACT: AIMS:In cardiac muscle, Ca(2+) release from sarcoplasmic reticulum (SR) is reduced with successively shorter coupling intervals of premature stimuli, a phenomenon known as SR Ca(2+) release refractoriness. We recently reported that the SR luminal Ca(2+) binding protein calsequestrin 2 (Casq2) contributes to release refractoriness in intact mouse hearts, but the underlying mechanisms remain unclear. Here, we further investigate the mechanisms responsible for physiological release refractoriness. METHODS AND RESULTS:Gene-targeted ablation of Casq2 (Casq2 KO) abolished SR Ca(2+) release refractoriness in isolated mouse ventricular myocytes. Surprisingly, impaired Ca(2+)-dependent inactivation of L-type Ca(2+) current (ICa), which is responsible for triggering SR Ca(2+) release, significantly contributed to loss of Ca(2+) release refractoriness in Casq2 KO myocytes. Recovery from Ca(2+)-dependent inactivation of ICa was significantly accelerated in Casq2 KO compared to wild-type (WT) myocytes. In contrast, voltage-dependent inactivation measured by using Ba(2+) as charge carrier was not significantly different between WT and Casq2 KO myocytes. Ca(2+)-dependent inactivation of ICa was normalized by intracellular dialysis of excess apo-CaM (20 ?M), which also partially restored physiological Ca(2+) release refractoriness in Casq2 KO myocytes. CONCLUSIONS:Our findings reveal that the intra-SR protein Casq2 is largely responsible for the phenomenon of SR Ca(2+) release refractoriness in murine ventricular myocytes. We also report a novel mechanism of impaired Ca(2+)-CaM-dependent inactivation of Cav1.2, which contributes to the loss of SR Ca(2+) release refractoriness in the Casq2 KO mouse model and, therefore, may further increase risk for ventricular arrhythmia in vivo.

SUBMITTER: Kryshtal DO

PROVIDER: S-EPMC4405490 | biostudies-literature | 2015 May

REPOSITORIES: biostudies-literature

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Impaired calcium-calmodulin-dependent inactivation of Cav1.2 contributes to loss of sarcoplasmic reticulum calcium release refractoriness in mice lacking calsequestrin 2.

Kryshtal Dmytro O DO Gryshchenko Oleksiy O Gomez-Hurtado Nieves N Knollmann Bjorn C BC

Journal of molecular and cellular cardiology 20150307

<h4>Aims</h4>In cardiac muscle, Ca(2+) release from sarcoplasmic reticulum (SR) is reduced with successively shorter coupling intervals of premature stimuli, a phenomenon known as SR Ca(2+) release refractoriness. We recently reported that the SR luminal Ca(2+) binding protein calsequestrin 2 (Casq2) contributes to release refractoriness in intact mouse hearts, but the underlying mechanisms remain unclear. Here, we further investigate the mechanisms responsible for physiological release refracto ...[more]

PMID: 25758429

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Project description:BackgroundCatecholaminergic polymorphic ventricular tachycardia (CPVT) is a potentially lethal cardiac arrhythmia syndrome triggered by catecholamines released during exercise, stress, or sudden emotion. Variants in the calsequestrin-2 gene (CASQ2), encoding the major calcium (Ca) binding protein in the sarcoplasmic reticulum (SR), are the second most common cause of CPVT. Recently, several CASQ2 gene variants, such as CASQ2-K180R, have been linked to an autosomal dominant form of Casq2-linked CPVT (CPVT2), but the underlying mechanism is not known.MethodsA K180R mouse model was generated using CRIPSR/Cas9. Heterozygous and homozygous K180R mice were studied using telemetry ECG recordings in vivo. Ventricular cardiomyocytes were isolated and studied using fluorescent Ca indicators and patch clamp. Expression levels and localization of SR Ca-handling proteins were evaluated using Western blotting and immunostaining. Intra-SR Ca kinetics were quantified using low-affinity Ca indicators.ResultsK180R mice exhibit an autosomal dominant CPVT phenotype following exercise or catecholamine stress. Upon catecholamine stress, K180R ventricular cardiomyocytes exhibit increased spontaneous SR Ca release events, triggering delayed afterdepolarizations and spontaneous beats. K180R had no effect on levels of Casq2, Casq2 polymers, or other SR Ca-handling proteins. Intra-SR Ca measurements revealed that K180R impaired dynamic intra-SR Ca buffering, resulting in a more rapid rise of free Ca in the SR during diastole. Steady-state SR Ca buffering and total SR Ca content were not changed. Consistent with the reduced dynamic intra-SR buffering, K180R causes reduced SR Ca release refractoriness.ConclusionsCASQ2-K180R causes CPVT2 via a heretofore unknown mechanism that differs from CASQ2 variants associated with autosomal recessive CPVT2. Unlike autosomal recessive CASQ2 variants, K180R impairs the dynamic buffering of Ca within the SR without affecting total SR Ca content or Casq2 protein levels. Our data provide insight into the molecular mechanism underlying autosomal dominant CPVT2.

Dataset Information

Impaired calcium-calmodulin-dependent inactivation of Cav1.2 contributes to loss of sarcoplasmic reticulum calcium release refractoriness in mice lacking calsequestrin 2.

Publications

Impaired calcium-calmodulin-dependent inactivation of Cav1.2 contributes to loss of sarcoplasmic reticulum calcium release refractoriness in mice lacking calsequestrin 2.

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