Abnormal interactions of calsequestrin with the ryanodine receptor calcium release channel complex linked to exercise-induced sudden cardiac death

Catecholaminergic polymorphic ventricular tachycardia ( CPVT) is a familial arrhythmogenic disorder associated with mutations in the cardiac ryanodine receptor ( RyR2) and cardiac calsequestrin ( CASQ2) genes. Previous in vitro studies suggested that RyR2 and CASQ2 interact as parts of a multimolecular Ca2+- signaling complex; however, direct evidence for such interactions and their potential significance to myocardial function remain to be determined. We identified a novel CASQ2 mutation in a young female with a structurally normal heart and unexplained syncopal episodes. This mutation results in the nonconservative substitution of glutamine for arginine at amino acid 33 of CASQ2 ( R33Q). Adenoviral- mediated expression of CASQ2(R33Q) in adult rat myocytes led to an increase in excitation contraction coupling gain and to more frequent occurrences of spontaneous propagating ( Ca2+ waves) and local Ca2+ signals ( sparks) with respect to control cells expressing wild- type CASQ2 ( CASQ2(WT)). As revealed by a Ca2+ indicator entrapped inside the sarcoplasmic reticulum ( SR) of permeabilized myocytes, the increased occurrence of spontaneous Ca2+ sparks and waves was associated with a dramatic decrease in intra- SR [ Ca2+]. Recombinant CASQ2WT and CASQ2(R33Q) exhibited similar Ca2+- binding capacities in vitro; however, the mutant protein lacked the ability of its WT counterpart to inhibit RyR2 activity at low luminal [ Ca2+] in planar lipid bilayers. We conclude that the R33Q mutation disrupts interactions of CASQ2 with the RyR2 channel complex and impairs regulation of RyR2 by luminal Ca2+. These results show that intracellular Ca2+ cycling in normal heart relies on an intricate interplay of CASQ2 with the proteins of the RyR2 channel complex and that disruption of these interactions can lead to cardiac arrhythmia.