The Role of CASQ2^D307H Mutant protein in Catecholamine Induced Polymorphic Ventricular Tachycardia (CPVT)

Recent studies have documented that mutations in Calsequestrin (CASQ2), a major calcium (Ca²⁺) buffering protein, result in CPVT, a form of ventricular arrhythmia. One of these mutations is a missense mutation, CASQ2^D307H that occurs in the C-terminus region of the protein which has been suggested to bind calcium and also to interact with Triadin1 (TRD1) to regulate Ca²⁺ release through the RyR2. Although the mutation has been associated with CPVT, the exact mechanism by which the mutant CASQ2 induces the disease is unknown. In order to dissect the molecular mechanism that causes the disease we generated a transgenic mouse model that expresses the mutant CASQ2^D307H protein in a CASQ2 Null background by crossing a mouse model that expresses the CASQ2^D307H in the Wildtype background with the CASQ2 Null mouse.

Expression of the mutant CASQ2^D307H protein up to 2 fold does not result in cardiac pathology or any structural modifications. It is targeted to the SR, localizes specifically at the junctional SR (jSR) and partially restores the ultrastructural modifications of the jSR cisternae. Expression of the mutant protein does not induce ER stress and is ubiquitinated at the same rate as the WT CASQ2 indicating that the mutant protein is degraded at the same rate as the WT protein.

Using purified CASQ2^D307H protein, we showed that in the presence of Ca²⁺, CD spectra were identical between the mutant CASQ2^D307H and the WT protein under conditions of high temperature and chemical denaturation. In addition, limited proteolysis using trypsin showed that the mutant protein is more susceptible to proteolysis than the WT CASQ2 in the presence of calcium. These data collectively emphasize the differences in Ca²⁺ dependent conformation between the WT and mutant CASQ2^D307H proteins.

The mutant protein expression did not alter the expression of other SR Ca²⁺ handling proteins including RyR2, SERCA2a, PLB and NCX but increased the expression of TRD1 to ~79 % of WT. Electrophysiological recordings from isolated mutant cardiomyocytes showed no significant alterations in the membrane potential, ICa, SR Ca²⁺ content and Ca²⁺ induced Ca²⁺ transients. When stimulated with isoproterenol, we did not observe extrasystolic Ca²⁺ release events. In mildly anesthetized mice, ECG recordings after adrenergic stimulation showed a significant decrease in the occurrence of ventricular arrhythmia in the mutant mice when compared to CASQ2 Null mice.

The data demonstrate that the mutant protein can be expressed stably, targeted to the jSR, and partially restores the jSR modifications of the CASQ2 Null mice. In addition, the mutant CASQ2^D307H partially rescues TRD1 level in the null hearts and this restoration of TRD1 may be responsible for the reduced occurrence of ventricular arrhythmia.