Modulation of SR Ca release by luminal Ca and calsequestrin in cardiac myocytes: effects of CASQ2 mutations linked to sudden cardiac death

Cardiac calsequestrin (CASQ2) is an intrasarcoplasmic reticulum (SR) low-affinity Ca-binding protein, with mutations that are associated with catecholamine-induced polymorphic ventricular tachycardia (CPVT). To better understand how CASQ2 mutants cause CPVT, we expressed two CPVT-linked CASQ2 mutants, a truncated protein (at G112+5X, CASQ2^DEL) or CASQ2 containing a point mutation (CASQ2^R33Q), in canine ventricular myocytes and assessed their effects on Ca handling. We also measured CASQ2-CASQ2 variant interactions using fluorescence resonance transfer in a heterologous expression system, and evaluated CASQ2 interaction with triadin. We found that expression of CASQ2^DEL or CASQ2^R33Q altered myocyte Ca signaling through two different mechanisms. Overexpressing CASQ2^DEL disrupted the CASQ2 polymerization required for high capacity Ca binding, whereas CASQ2^R33Q compromised the ability of CASQ2 to control ryanodine receptor (RyR2) channel activity. Despite profound differences in SR Ca buffering strengths, local Ca release terminated at the same free luminal [Ca] in control cells, cells overexpressing wild-type CASQ2 and CASQ2^DEL-expressing myocytes, suggesting that a decline in [Ca]_SR is a signal for RyR2 closure. Importantly, disrupting interactions between the RyR2 channel and CASQ2 by expressing CASQ2^R33Q markedly lowered the [Ca]_SR threshold for Ca release termination. We conclude that CASQ2 in the SR determines the magnitude and duration of Ca release from each SR terminal by providing both a local source of releasable Ca and by effects on luminal Ca-dependent RyR2 gating. Furthermore, two CPVT-inducing CASQ2 mutations, which cause mechanistically different defects in CASQ2 and RyR2 function, lead to increased diastolic SR Ca release events and exhibit a similar CPVT disease phenotype.