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Modulation of SR Ca Release by Luminal Ca and Calsequestrin in Cardiac Myocytes: Effects of CASQ2 Mutations Linked to Sudden Cardiac Death

Dmitry Terentyev ^*, Zuzana Kubalova , Giorgia Valle , Alessandra Nori , Srikanth Vedamoorthyrao ^*, Radmila Terentyeva ^*, Serge Viatchenko-Karpinski ^*, Donald M. Bers , Simon C. Williams ^¶, Pompeo Volpe  and Sandor Gyorke ^*

^* The Ohio State University Medical Center, Department of Physiology, Dorothy M. Davis Heart and Lung Research Institute, Columbus, Ohio; Institute of Molecular Physiology and Genetics, Slovak Academy of Sciences, Bratislava, Slovak Republic; University of Padova, Padua, Italy; Loyola University Chicago, Maywood, Illinois; and ^¶ Texas Tech University, Lubbock, Texas

Correspondence: Address reprint requests to Sandor Gyorke, PhD, Professor of Physiology and Cell Biology, Associate Director Dorothy M. Davis Heart and Lung Research Institute, The Ohio State University, 473 West 12th Ave., Columbus, OH 43210-1252. E-mail: sandor.gyorke{at}osumc.edu.

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.