Best, Jabe M.

Key points Mutations in the caveolae scaffolding protein, caveolin‐3 (Cav3), have been linked to the long QT type 9 inherited arrhythmia syndrome (LQT9) and the cause of underlying action potential duration prolongation is incompletely understood. In the present study, we show that LQT9 Cav3 mutations, F97C and S141R, cause mutation‐specific gain of function effects on Cav1.2‐encoded L‐type Ca2+ channels responsible for ICa,L and also cause loss of function effects on heterologously expressed Kv4.2 and Kv4.3 channels responsible for Ito. A computational model of the human ventricular myocyte action potential suggests that the major ionic current change causing action potential duration prolongation in the presence of Cav3‐F97C is the slowly inactivating ICa,L but, for Cav3‐S141R, both increased ICa,L and increased late Na+ current contribute equally to action potential duration prolongation. Overall, the LQT9 Cav3‐F97C and Cav3‐S141R mutations differentially impact multiple ionic currents, highlighting the complexity of Cav3 regulation of cardiac excitability and suggesting mutation‐specific therapeutic approaches. AbstractMutations in the CAV3 gene encoding caveolin‐3 (Cav3), a scaffolding protein integral to caveolae in cardiomyocytes, have been associated with the congenital long‐QT syndrome (LQT9). Initial studies demonstrated that LQT9‐associated Cav3 mutations, F97C and S141R, increase late sodium current as a potential mechanism to prolong action potential duration (APD) and cause LQT9. Whether these Cav3 LQT9 mutations impact other caveolae related ion channels remains unknown. We used the whole‐cell, patch clamp technique to characterize the effect of Cav3‐F97C and Cav3‐S141R mutations on heterologously expressed Cav1.2+Cavβ2cN4 channels, as well as Kv4.2 and Kv4.3 channels, in HEK 293 cells. Expression of Cav3‐S141R increased ICa,L density without changes in gating properties, whereas expression of Cav3‐F97C reduced Ca2+‐dependent inactivation of ICa,L without changing current density. The Cav3‐F97C mutation reduced current density and altered the kinetics of IKv4.2 and IKv4.3 and also slowed recovery from inactivation. Cav3‐S141R decreased current density and also slowed activation kinetics and recovery from inactivation of IKv4.2 but had no effect on IKv4.3. Using the O'Hara–Rudy computational model of the human ventricular myocyte action potential, the Cav3 mutation‐induced changes in Ito are predicted to have negligible effect on APD, whereas blunted Ca2+‐dependent inactivation of ICa,L by Cav3‐F97C is predicted to be primarily responsible for APD prolongation, although increased ICa,L and late INa by Cav3‐S141R contribute equally to APD prolongation. Thus, LQT9 Cav3‐associated mutations, F97C and S141R, produce mutation‐specific changes in multiple ionic currents leading to different primary causes of APD prolongation, which suggests the use of mutation‐specific therapeutic approaches in the future.

Background— Long QT syndrome (LQTS) is the most common cardiac channelopathy with 15 elucidated LQTS-susceptibility genes. Approximately 20% of LQTS cases remain genetically elusive. Methods and Results— We combined whole-exome sequencing and bioinformatic/systems biology to identify the pathogenic substrate responsible for nonsyndromic, genotype-negative, autosomal dominant LQTS in a multigenerational pedigree, and we established the spectrum and prevalence of variants in the elucidated gene among a cohort of 102 unrelated patients with “genotype-negative/phenotype-positive” LQTS. Whole-exome sequencing was used on 3 members within a genotype-negative/phenotype-positive family. Genomic triangulation combined with bioinformatic tools and ranking algorithms led to the identification of a CACNA1C mutation. This mutation, Pro857Arg-CACNA1C, cosegregated with the disease within the pedigree, was ranked by 3 disease-network algorithms as the most probable LQTS-susceptibility gene and involves a conserved residue localizing to the proline, gltamic acid, serine, and threonine (PEST) domain in the II-III linker. Functional studies reveal that Pro857Arg-CACNA1C leads to a gain of function with increased I Ca,L and increased surface membrane expression of the channel compared to wild type. Subsequent mutational analysis identified 3 additional variants within CACNA1C in our cohort of 102 unrelated cases of genotype-negative/phenotype-positive LQTS. Two of these variants also involve conserved residues within Ca v 1.2’s PEST domain. Conclusions— This study provides evidence that coupling whole-exome sequencing and bioinformatic/systems biology is an effective strategy for the identification of potential disease-causing genes/mutations. The identification of a functional CACNA1C mutation cosegregating with disease in a single pedigree suggests that CACNA1C perturbations may underlie autosomal dominant LQTS in the absence of Timothy syndrome.