Dataset Information

JTV519 (K201) reduces sarcoplasmic reticulum Ca²? leak and improves diastolic function in vitro in murine and human non-failing myocardium.

ABSTRACT:

Background and purpose

Ca²? leak from the sarcoplasmic reticulum (SR) via ryanodine receptors (RyR2s) contributes to cardiomyocyte dysfunction. RyR2 Ca²? leak has been related to RyR2 phosphorylation. In these conditions, JTV519 (K201), a 1,4-benzothiazepine derivative and multi-channel blocker, stabilizes RyR2s and decrease SR Ca²? leak. We investigated whether JTV519 stabilizes RyR2s without increasing RyR2 phosphorylation in mice and in non-failing human myocardium and explored underlying mechanisms.

Experimental approach

SR Ca²? leak was induced by ouabain in murine cardiomyocytes. [Ca²?]-transients, SR Ca²? load and RyR2-mediated Ca²? leak (sparks/waves) were quantified, with or without JTV519 (1?µmol·L?¹). Contribution of Ca²? -/calmodulin-dependent kinase II (CaMKII) was assessed by KN-93 and Western blot (RyR2-Ser(2814) phosphorylation). Effects of JTV519 on contractile force were investigated in non-failing human ventricular trabeculae.

Key results

Ouabain increased systolic and diastolic cytosolic [Ca²?](i) , SR [Ca²?], and SR Ca²? leak (Ca²? spark (SparkF) and Ca²? wave frequency), independently of CaMKII and RyR-Ser(2814) phosphorylation. JTV519 decreased SparkF but also SR Ca²? load. At matched SR [Ca²?], Ca²? leak was significantly reduced by JTV519, but it had no effect on fractional Ca²? release or Ca²? wave propagation velocity. In human muscle, JTV519 was negatively inotropic at baseline but significantly enhanced ouabain-induced force and reduced its deleterious effects on diastolic function.

Conclusions and implications

JTV519 was effective in reducing SR Ca²? leak by specifically regulating RyR2 opening at diastolic [Ca²?](i) in the absence of increased RyR2 phosphorylation at Ser(2814) , extending the potential use of JTV519 to conditions of acute cellular Ca²? overload.

SUBMITTER: Sacherer M

PROVIDER: S-EPMC3449255 | biostudies-literature | 2012 Oct

REPOSITORIES: biostudies-literature

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JTV519 (K201) reduces sarcoplasmic reticulum Ca²⁺ leak and improves diastolic function in vitro in murine and human non-failing myocardium.

Sacherer M M Sedej S S Wakuła P P Wallner M M Vos M A MA Kockskämper J J Stiegler P P Sereinigg M M von Lewinski D D Antoons G G Pieske B M BM Heinzel F R FR

British journal of pharmacology 20121001 3

<h4>Background and purpose</h4>Ca²⁺ leak from the sarcoplasmic reticulum (SR) via ryanodine receptors (RyR2s) contributes to cardiomyocyte dysfunction. RyR2 Ca²⁺ leak has been related to RyR2 phosphorylation. In these conditions, JTV519 (K201), a 1,4-benzothiazepine derivative and multi-channel blocker, stabilizes RyR2s and decrease SR Ca²⁺ leak. We investigated whether JTV519 stabilizes RyR2s without increasing RyR2 phosphorylation in mice and in non-failing human myocardium and explored underl ...[more]

PMID: 22509897

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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.