Pathophysiology of R222Q mutant SCN5a channels

Abstract

Consistent with the role of sodium channels in initiating action potentials throughout myocardial tissue, mutations in SCN5a are associated with arrhythmias and cardiomyopathy. The present studies aim to advance understanding of the mechanism underlying the arrhythmias and dilated cardiomyopathy (DCM) observed in patents with an SCN5a mutation (c.665G>A/R222Q) located in one of the voltage sensor regions. Induced pluripotent stem cells (iPSCs) were generated from a patient heterozygous for the R222Q mutation. Isogenic controls were generated by corrected the mutation. Directed differentiation techniques were used to produce ventricular CMs characterized by the expression of cTnT (Control: 85.10±2.90%, Mutant: 83.14±4.46%) and ventricular-specific MLC2v (Control: 45.62±7.02%, Mutant: 43.92±3.86%). The SCN5a gene undergoes extensive alternative splicing, which is developmentally regulated, resulting in the R222Q loci being expressed exclusively in adult SCN5a mRNA. Consistent with an immature phenotype in our iPSC-derived CMs, about half of the SCN5a mRNA is the adult isoform (Control: 54.40±9.29%, Mutant: 48.45±6.21%, n=4), indicating that ~25% of the SCN5a channels expressed in mutant CMs contain the R222Q mutation. Optical mapping of our 2-dimentional tissue model (monolayers) reveal slower conduction velocities in mutant compared to control monolayers (13.14±0.74cm/s, n=10, vs. 25.09±1.99cm/s, n=7, p<0.05). Treatment with flecainide (10 μM), a sodium channel blocker associated with arrhythmia reduction and DCM reversal in R222Q patients results in conduction slowing (Control: 25.09±1.99cm/s to 18.47±3.20cm/s, n=7, p<0.05, Mutant: 13.14±0.74cm/s to 8.92±0.64cm/s, n=10, p<0.05) and a reduction in maximum capture rates (Control: 3.22±0.14Hz to 1.42±0.13Hz, n=5, p<0.05, Mutant: 3.43±0.18 to 2.38±0.22Hz, n=10, p<0.05). We are currently performing electrophysiological studies to further assess the mechanisms underlying R222Q-associated cardiomyopathy and arrhythmias and advancement of therapeutic options for R222Q patients.