Expression Of Calsequestrin D307h Mutant Protein Partially Rescues The Abnormal Calcium Handling And Ultra Structural Defects Of Casq2 Null Hearts

Calsequestrin (CASQ2) is the major calcium binding protein located in the cardiac junctional sarcoplasmic reticulum (jSR). Several mutations have been reported in human CASQ2 and have been linked to Catecholamine induced Polymorphic Ventricular Tachycardia (CPVT). To understand how a point mutation CASQ2 D307H that changes the negative aspartate (D) to a positive histidine (H), causes ventricular tachycardia, we generated a mouse model that expresses the D307H mutant protein in a CASQ2 null background (CASQ2 −/− mice). Our results demonstrate that the mutant protein can be stably expressed, properly targeted and localized to the jSR. This refutes a recently reported study that suggested a highly unstable, easily degraded CASQ2 D307H mutant protein (J Clin Invest. 2007, 117(7):1814–23). Expression of the mutant CASQ2 D307H protein did not affect RyR2, DHPR, SERCA2a and calreticulin levels, but significantly restored both triadin and junctin from the very low levels in the CASQ2 −/− mouse. Expression and appropriate targeting of CASQ2 D307H was directly confirmed by electron microscopy: the “tightly” packed arrangement of CASQ2 within the jSR, which was completely missing in CASQ2 −/− mouse myocardium, was restored. This requires both the presence of CASQ2 and its interaction with triadin/junctin. Calcium transients and calcium load in ventricular myocytes isolated from CASQ2 D307H hearts were not significantly different from those of WT cells. Standard beta-adrenergic stimulation protocols did not induce spontaneous calcium releases and triggered beats in isolated CASQ2 D307H myocytes. Studies are in progress using telemetry to examine if the D307H mice in the null background develop VT upon exercise, a hallmark of CPVT linked mutations. Our studies thus far suggest that mutant CASQ2 D307H protein is capable of interacting with either triadin or junctin, or both, allowing it to be localized within the jSR. In conclusion, our data demonstrate that altered protein stability of CASQ2 D307H is not the causative mechanism for the CPVT phenotype. Other mechanisms such as altered interaction between jSR proteins, decreased SR buffering and/or altered regulation of the RyR2 channels by mutant CASQ2 D307H should be explored. This research has received full or partial funding support from the American Heart Association, AHA Great Rivers Affiliate (Delaware, Kentucky, Ohio, Pennsylvania & West Virginia).