The Functional and Clinical Phenotypes Caused by Perturbations in CACNA1C-Encoded Cav1.2 L-Type Calcium Channel and the Cav1.2 Interacting Protein, Calmodulin, in the Human Heart

The CACNA1C-encoded alpha-subunit of L-type calcium channel (LTCC; Cav1.2; ICaL) plays a critical role in the human heart. The LTCC, regulated by calmodulins (CaM), is responsible for the influx of Ca2+ ions in response to membrane depolarization and is critical for the plateau phase of the cardiac action potential. The cardiac action potential is mediated by an intricate balance of the opening and closing of specific Na+, K+, and Ca 2+ ion channels. Through many genetic and electrophysiology based studies, hundreds of pathogenic mutations in genes encoding for Na + and K+ ion channels and their ChIPs (channel interacting proteins) have been identified that lead to channel dysfunction capable of causing a potentially life threatening arrhythmia disorder known as long QT syndrome (LQTS). LQTS is an autosomal dominant disorder diagnosed through QT prolongation on an electrocardiogram, is characterized by delayed repolarization of the ventricular myocardium, and manifests clinically as syncope, seizures, or sudden cardiac death (SCD) in the setting of a structurally normal heart. Of the 5 phases of the cardiac action potential, 3 had been implicated in a pure LQTS phenotype, whereas Cav1.2-mediated phase 2 of the cardiac action potential had not yet been linked to isolated LQTS.;Instead, Cav1.2 gain-of-function mutations had been attributed to a multisystem disorder, Timothy syndrome (TS), characterized by cardiac phenotypes of QT prolongation, cardiac hypertrophy, and congenital heart defects, and by extra-cardiac phenotypes of syndactyly, neurological dysfunction, and facial dysmorphisms. This led the field to believe that every gain-of-function mutation in Cav1.2 would lead to TS rather than an isolated phenotype of LQTS. However, we hypothesized that perturbations in Cav1.2 and Cav1.2 ChIPS, such as CaM, could contribute to LQTS pathogenesis and other cardiac-only phenotypes associated with QT prolongation.;Therefore, this doctoral work set out to better understand the molecular, phenotypic, and pleiotropic role(s) of Cav1.2 and CaM in the human heart. Using whole exome sequencing (WES) in different family structures and electrophysiological analyses, we identified a novel p.Pro857Arg - Cav1.2 mutation that leads to increased current density and underlies a pure LQTS phenotype. In addition, we found a novel TS-causing p.Ile1166Thr - Cav1.2 mutation that localized outside of the typical canonical hotspot for TS and led to an increase in Cav1.2 window current when characterized electrophysiologically. We also defined a novel disorder, cardiac-only TS (COTS), in which novel p.Arg518Cys/His - Cav1.2 mutations were attributed to the disease phenotype.;During this thesis, several groups identified mutations in CALM1 and CALM2 leading to LQTS. Therefore, we examined our genetically elusive LQTS cohort and identified several of mutations in CALM1 and CALM2 (4/38; 10.5%). Although three of the four mutations had been functionally characterized previously, our functional analysis of the novel p.E141G-CaM mutation revealed a decrease in Ca 2+ affinity and increase in both late Cav1.2 and Na v1.5 currents. Finally, through WES, we were the first to establish CALM3 as the latest LQTS-susceptibility gene thereby completing the trilogy of the so-called calmodulinopathies.;These studies have greatly expanded the previous knowledge surrounding calcium mediated arrhythmias and LQTS, TS, and COTS genetics. Based on these findings, expanded genetic testing of CACNA1C and CALM1-3 is warranted in cases of LQTS. However, future studies are necessary to further understand the roles of Cav1.2 and CaM in the human heart and to identify genotype-specific treatment strategies for Cav1.2- and CaM-mediated LQTS.