Abstract
Loss-of-function mutations of the SCN5A gene encoding for the sodium channel α-subunit NaV1.5 result in the autosomal dominant hereditary disease Brugada Syndrome (BrS) with a high risk of sudden cardiac death in the adult. We here engineered human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) carrying the CRISPR/Cas9 introduced BrS-mutation p.A735V-NaV1.5 (g.2204C > T in exon 14 of SCN5A) as a novel model independent of patient´s genetic background. Recent studies raised concern regarding the use of hiPSC-CMs for studying adult-onset hereditary diseases due to cells’ immature phenotype. To tackle this concern, long-term cultivation of hiPSC-CMs on a stiff matrix (27–42 days) was applied to promote maturation. Patch clamp recordings of A735V mutated hiPSC-CMs revealed a substantially reduced upstroke velocity and sodium current density, a prominent rightward shift of the steady state activation curve and decelerated recovery from inactivation as compared to isogenic hiPSC-CMs controls. These observations were substantiated by a comparative study on mutant A735V-NaV1.5 channels heterologously expressed in HEK293T cells. In contrast to mutated hiPSC-CMs, a leftward shift of sodium channel inactivation was not observed in HEK293T, emphasizing the importance of investigating mechanisms of BrS in independent systems. Overall, our approach supports hiPSC-CMs’ relevance for investigating channelopathies in a dish.
Highlights
A pathophysiological reduction of the NaV1.5 current density associated with a prominent transient outward potassium current (Ito) is thought to cause the typical action potentials (APs) notch during early repolarization phase in myocardial cells
Low levels of Kir channel expression were found for hiPSC-CMs resulting in more depolarized resting membrane potentials poorly resembling the properties of native cardiomyocytes[17]
Equivalent cardiac differentiation of WT versus mutant clones was further confirmed by qRT-PCR for MYH6 (α-myosin heavy chain (MyHC) isoform; Fig. 1d, lower left) and SCN5A expression (Fig. 1d, lower right), suggesting that the g.2204C > T mutation did not impact on SCN5A mRNA abundance
Summary
A pathophysiological reduction of the NaV1.5 current density associated with a prominent transient outward potassium current (Ito) is thought to cause the typical AP notch during early repolarization phase (phase 1) in myocardial cells. Extensive electrophysiological investigations of a disease-causing A735V-NaV1.5 mutation introduced into hiPSC-CMs were performed in comparison to both isogenic and non-genetically related hiPSC-CM controls (“wild type” WT) on the single cell level. To bridge the gap to such non-mammalian model, we introduced the A735V-NaV1.5 mutation into another heterologous system that is HEK293T cells. This cell line is well established for investigating channelopathies and provides a relevant comparison to our hiPSC-CM approach. Combining these technologies, we present a novel hiPSC-CM disease model for A735V-NaV1.5 mutation-based BrS, revealing the causative effect of such point mutation irrespective of patients genetic background
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