Functional and structural investigation of catecholaminergic polymorphic ventricular tachycardia associated calmodulin mutations in disease and therapy

Vanderbilt University Medical Center, Nashville TN

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Abstract

PROJECT SUMMARY/ABSTRACT Cardiac arrhythmias are the leading cause of sudden cardiac death. Catecholaminergic Polymorphic Ventricular Tachycardia (CPVT) is a genetic disorder estimated to account for as many as 15% of unexplained deaths. During CPVT, ectopic ventricular arrhythmias are generated by inappropriately-timed calcium release from the sarcoplasmic reticulum (SR) of the cardiomyocyte through the ryanodine receptor (RyR2). Idiopathic ventricular fibrillation (IVF) is an arrhythmia syndrome that is diagnosed in the absence of all other arrhythmia causes and little is known about the pathogenic mechanism. Recently, mutations in the protein calmodulin (CaM) were found in patients with CPVT and IVF. CaM is a calcium binding protein that regulates RyR2; however, the mechanism by which CaM regulates RyR2 is not fully understood. Due to the rarity of CaM mutations in CPVT, even less is understood about the functional and structural consequences of these pathogenic CaM mutations. A recent report identified a CPVT-associated CaM mutation in an EF-hand motif of the C-domain that alters calcium binding in vitro. To determine whether residues in this domain are important for RyR2 regulation, we mutated an adjacent amino acid that does not alter calcium binding. Preliminary data indicate that this mutation also elicits a CPVT-like phenotype in mice. The goal of this proposal is to study two novel pathogenic CaM mutants (F90L and A104V) to gain mechanistic insight into CaM regulation of RyR2. Specific Aim 1 will determine the functional mechanisms by which pathogenic CaM mutations dysregulate RyR2 and contribute to ventricular arrhythmia. To address this aim, mice carrying CaM mutations will be assessed for susceptibility to ventricular arrhythmias. Functional measurements will be made to assess SR calcium release, RyR2 phosphorylation and binding of CaM to RyR2. Specific Aim 2 will determine the structural consequences of pathogenic CaM mutations in regulation of RyR2. To probe the structural consequences of these mutations, calcium binding assays and NMR spectroscopy will be performed in the presence of RyR2 binding peptides over a range of calcium concentrations. Aim 2 will test whether the residues of the EF hand motif are a critical determinant of CaM binding to RyR2 and appropriate RyR2 calcium release. This application focuses on two genetic syndromes that cause familial sudden cardiac death, CPVT and IVF, associated with mutations in CaM. RyR2 dysregulation by CaM is poorly understood; these studies will resolve the importance of the CaM EF-hand motif and mechanisms of RyR2 regulation. This proposal has high potential impact by advancing our understanding of CaM-RyR2 interactions as well as defining mechanisms of pathogenic Ca release in ventricular arrhythmia.