Long QT syndrome caveolin-3 mutations differentially modulate K(v) 4 and Ca(v) 1.2 channels to contribute to action potential prolongation.

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 Cav 1.2-encoded L-type Ca2+ channels responsible for ICa,L and also cause loss of function effects on heterologously expressed Kv 4.2 and Kv 4.3 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. Mutations 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 Cav 1.2+Cav β2cN4 channels, as well as Kv 4.2 and Kv 4.3 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.

Tyan L ，Foell JD ，Vincent KP ，Woon MT ，Mesquitta WT ，Lang D ，Best JM ，Ackerman MJ ，McCulloch AD ，Glukhov AV ，Balijepalli RC ，Kamp TJ ... -  《-》

Caveolin-3 suppresses late sodium current by inhibiting nNOS-dependent S-nitrosylation of SCN5A.

Mutations in CAV3-encoding caveolin-3 (Cav3) have been implicated in type 9 long QT syndrome (LQT9) and sudden infant death syndrome (SIDS). When co-expressed with SCN5A-encoded cardiac sodium channels these mutations increased late sodium current (INa) but the mechanism was unclear. The present study was designed to address the mechanism by which the LQT9-causing mutant Cav3-F97C affects the function of caveolar SCN5A. HEK-293 cells expressing SCN5A and LQT9 mutation Cav3-F97C resulted in a 2-fold increase in late INa compared to Cav3-WT. This increase was reversed by the neural nitric oxide synthase (nNOS) inhibitor L-NMMA. Based on these findings, we hypothesized that an nNOS complex mediated the effect of Cav3 on SCN5A. A SCN5A macromolecular complex was established in HEK-293 cells by transiently expressing SCN5A, α1-syntrophin (SNTA1), nNOS, and Cav3. Compared with Cav3-WT, Cav3-F97C produced significantly larger peak INa amplitudes, and showed 3.3-fold increase in the late INa associated with increased S-nitrosylation of SCN5A. L-NMMA reversed both the Cav3-F97C induced increase in late and peak INa and decreased S-nitrosylation of SCN5A. Overexpression of Cav3-F97C in adult rat cardiomyocytes caused a significant increase in late INa compared to Cav3-WT, and prolonged the action potential duration (APD90) in a nNOS-dependent manner. Cav3 is identified as an important negative regulator for cardiac late INa via nNOS dependent direct S-nitrosylation of SCN5A. This provides a molecular mechanism for how Cav3 mutations increase late INa to cause LQT9. This article is part of a Special Issue entitled "Na(+) Regulation in Cardiac Myocytes".

Cheng J ，Valdivia CR ，Vaidyanathan R ，Balijepalli RC ，Ackerman MJ ，Makielski JC ... -  《-》

The interaction of caveolin 3 protein with the potassium inward rectifier channel Kir2.1: physiology and pathology related to long qt syndrome 9 (LQT9).

Vaidyanathan R ，Vega AL ，Song C ，Zhou Q ，Tan BH ，Berger S ，Makielski JC ，Eckhardt LL ... -  《-》

Discovery and characterization of a monogenetic insult, caveolin-3-V37L, that precipitated oligo-proteomic perturbations governing repolarization reserve.

Caveolin-3 (Cav-3) is an essential scaffolding protein for caveolae formation in cardiomyocytes and targets multiple long QT syndrome (LQTS)-associated ion channels. Mutations in CAV3 have caused an LQT3-like accentuation in late sodium current, INa (Nav1.5). Here, we characterize a novel CAV3-V37L variant and determine whether it is the substrate for the patient's LQTS. The proband was a 39-year-old female with drug-induced, sudden cardiac arrest (SCA) with profound QT prolongation (QTc > 600 ms). Genetic testing revealed a rare CAV3-V37L variant of uncertain significance (VUS). Whole-cell patch clamp technique was used to measure IKs, IKr, INa, and ICa, L currents co-expressed with either CAV3-WT or CAV3-V37L in TSA201 cells and to measure the action potential duration (APD) in control human induced pluripotent stem cells-derived cardiomyocytes (hiPSC-CMs) overexpressed with CAV3-WT or CAV3-V37L. CAV3-V37L did not affect Nav1.5 late current. Instead, CAV3-V37L resulted in 1) ICa, L with slower inactivation, a 1.5 fold increase in peak ICa, L current density and a 1.1 fold increase in ICa, L persistent current, 2) dramatically reduced IKs peak current density by 74.9%, 3) significantly reduced IKr peak current density by 31.1%, and 4) significantly prolonged the APD in hiPSC-CMs. These functional validation assays enabled the promotion of CAV3-V37L from VUS status to a likely pathogenic variant. Although Nav1.5 was spared, this monogenetic insult precipitated an oligo-proteomic impact with a concomitant gain-of-function of ICa, L and loss-of-function of both IKs and IKr culminating in a marked prolongation of the cardiomyocyte's action potential duration.

Ye D ，Zhou W ，Tester DJ ，Ackerman MJ ... -  《-》

Long-QT syndrome-associated caveolin-3 mutations differentially regulate the hyperpolarization-activated cyclic nucleotide gated channel 4.

Motloch LJ ，Larbig R ，Darabi T ，Reda S ，Motloch KA ，Wernly B ，Lichtenauer M ，Gebing T ，Schwaiger A ，Zagidullin N ，Wolny M ，Hoppe UC ... -  《Physiology International》