Identification of molecular mechanisms underlying couplon protein degradation in a mouse model of CPVT2.

Funding Acknowledgements Type of funding sources: Public grant(s) – National budget only. Main funding source(s): - Grant from the Spanish Ministerio de Economía y Competitividad - FPI Severo Ochoa Predoctoral grant Introduction Catecholaminergic polymorphic ventricular tachycardia-2 (CPVT2) is an arrhythmogenic inherited autosomal recessive disorder caused by mutations in the gene encoding cardiac calsequestrin (CASQ2). The homozygous mouse model CASQ2R33Q/R33Q closely mimics the clinical phenotype of patients that carried this mutation, which causes a substantial imbalance in the homeostasis of intracellular calcium, resulting in polymorphic ventricular tachycardia through triggered activity. CASQ2-R33Q and triadin (TRDN) levels are markedly reduced with no changes in the mRNA expression levels. So far, the mechanisms underlying the decrease of these proteins are not completely understood. Deciphering these mechanisms will help us to develop new or personalized therapeutic options for CPVT patients. Purpose The purpose of this study was to identify the molecular mechanisms regulating the decrease of the couplon proteins in the CASQ2R33Q/R33Q mice to find new therapeutic options. Methods Mass spectrometry proteomics and Western blot analysis were performed to explore the role of the main cell proteolytic systems – the proteasome, autophagy and calpain. Experiments were performed in adult mice and in isolated neonatal cardiomyocytes treated with inhibitors of these systems. To explore the role of calcium homeostasis in the degradation of these proteins, mice were treated with a RYR2 inhibitor. Results CASQ2-R33Q and TRDN proteins showed a shorter half-life in CASQ2R33Q/R33Q mice. Mass spectrometry proteomics revealed an increase in proteins that participate in Endoplasmic reticulum stress (ER stress) and changes in calcium release and uptake regulators. Western blot analysis of myocardial tissue from adult mice demonstrated an alteration in the activity of the proteasome, the autophagy flux and the calpain enzyme. Experiments in isolated neonatal cardiomyocytes showed that CASQ2-R33Q, but not TRDN, degradation can be blocked with proteasome, ERAD (ER associated degradation) or autophagy inhibitors. In contrast, both CASQ2-R33Q and TRDN degradation were prevented when the calpain enzyme is inhibited. Inhibition of RYR2 in vivo induced an increase in CASQ2-R33Q and TRDN protein levels. Conclusions We propose that changes in calcium homeostasis lead to the activation of some of the main cell proteolytic systems – proteasome and calpain. CASQ2-R33Q is degraded by the proteasome through the ERAD pathway that is activated as a consequence of ER stress. Calpain inhibition is the only mechanism that recovers both CASQ2-R33Q and TRDN protein levels. In the near future, in vivo ECGs and in vitro calcium transient experiments will determine their therapeutic potential for the treatment of recessive CPVT. [ABSTRACT FROM AUTHOR]