Modulation of cytosolic and intra-sarcoplasmic reticulum calcium waves by calsequestrin in rat cardiac myocytes

摘要

Waves of Ca²⁺-induced Ca²⁺ release occur in various cell types and are involved in the pathology of certain forms of cardiac arrhythmia. These arrhythmias include catecholaminergic polymorphic ventricular tachycardia (CPVT), certain cases of which are associated with mutations in the cardiac calsequestrin gene (CASQ2). To explore the mechanisms of Ca²⁺ wave generation and unravel the underlying causes of CPVT, we investigated the effects of adenoviral-mediated changes in CASQ2 protein levels on the properties of cytosolic and sarcoplasmic reticulum (SR) Ca²⁺ waves in permeabilized rat ventricular myocytes. The free [Ca²⁺] inside the sarcoplasmic reticulum ([Ca²⁺]SR) was monitored by fluo-5N entrapped into the SR, and cytosolic Ca²⁺ was imaged using fluo-3. Overexpression of CASQ2 resulted in significant increases in the amplitude of Ca²⁺ waves and interwave intervals, whereas reduced CASQ2 levels caused drastic reductions in the amplitude and period of Ca²⁺ waves. CASQ2 abundance had no impact on resting diastolic [Ca²⁺]SR or on the amplitude of the [Ca²⁺]SR depletion signal during the Ca²⁺ wave. However, the recovery dynamics of [Ca²⁺]SR following Ca²⁺ release were dramatically altered as the rate of [Ca²⁺]SR recovery increased ∼3-fold in CASQ2-overexpressing myocytes and decreased to 30% of control in CASQ2-underexpressing myocytes. There was a direct linear relationship between Ca²⁺ wave period and the half-time of basal [Ca²⁺]SR recovery following Ca²⁺ release. Loading the SR with the low affinity exogenous Ca²⁺ buffer citrate exerted effects quantitatively similar to those observed on overexpressing CASQ2. We conclude that free intra-SR [Ca²⁺] is a critical determinant of cardiac Ca²⁺ wave generation. Our data indicate that reduced intra-SR Ca²⁺ binding activity promotes the generation of Ca²⁺ waves by accelerating the dynamics of attaining a threshold free [Ca²⁺]SR required for Ca²⁺ wave initiation, potentially accounting for arrythmogenesis in CPVT linked to mutations in CASQ2.