Foell, Jason D.

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— Congenital long-QT syndrome (LQTS) is a primary arrhythmogenic syndrome stemming from perturbed cardiac repolarization. LQTS, which affects ≈1 in 3000 persons, is 1 of the most common causes of autopsy-negative sudden death in the young. Since the sentinel discovery of cardiac channel gene mutations in LQTS in 1995, hundreds of mutations in 8 LQTS susceptibility genes have been identified. All 8 LQTS genotypes represent primary cardiac channel defects (ie, ion channelopathy) except LQT4, which is a functional channelopathy because of mutations in ankyrin-B. Approximately 25% of LQTS remains unexplained pathogenetically. We have pursued a “final common pathway” hypothesis to elicit novel LQTS-susceptibility genes. With the recent observation that the LQT3-associated, SCN5A -encoded cardiac sodium channel localizes in caveolae, which are known membrane microdomains whose major component in the striated muscle is caveolin-3, we hypothesized that mutations in caveolin-3 may represent a novel pathogenetic mechanism for LQTS. Methods and Results— Using polymerase chain reaction, denaturing high-performance liquid chromatography, and direct DNA sequencing, we performed open reading frame/splice site mutational analysis on CAV3 in 905 unrelated patients referred for LQTS genetic testing. CAV3 mutations were engineered by site-directed mutagenesis and the molecular phenotype determined by transient heterologous expression into cell lines that stably express the cardiac sodium channel hNa v 1.5. We identified 4 novel mutations in CAV3 -encoded caveolin-3 that were absent in >1000 control alleles. Electrophysiological analysis of sodium current in HEK293 cells stably expressing hNa v 1.5 and transiently transfected with wild-type and mutant caveolin-3 demonstrated that mutant caveolin-3 results in a 2- to 3-fold increase in late sodium current compared with wild-type caveolin-3. Our observations are similar to the increased late sodium current associated with LQT3-associated SCN5A mutations. Conclusions— The present study reports the first CAV3 mutations in subjects with LQTS, and we provide functional data demonstrating a gain-of-function increase in late sodium current.