Dan Ye1, Wei Zhou1, David J Tester1, Michael J Ackerman2. 1. Department of Molecular Pharmacology & Experimental Therapeutics, Windland Smith Rice Sudden Death Genomics Laboratory, Mayo Clinic, Rochester, MN 55905, USA; Department of Cardiovascular Medicine/Division of Heart Rhythm Services, Mayo Clinic, Rochester, MN 55905, USA. 2. Department of Molecular Pharmacology & Experimental Therapeutics, Windland Smith Rice Sudden Death Genomics Laboratory, Mayo Clinic, Rochester, MN 55905, USA; Department of Cardiovascular Medicine/Division of Heart Rhythm Services, Mayo Clinic, Rochester, MN 55905, USA; Department of Pediatric and Adolescent Medicine/Division of Pediatric Cardiology, Mayo Clinic, Rochester, MN 55905, USA. Electronic address: ackerman.michael@mayo.edu.
Abstract
BACKGROUND: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.
BACKGROUND: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. METHODS: 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. RESULTS: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. CONCLUSIONS: 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.