Human induced pluripotent stem cell-derived atrial cardiomyocytes carrying an SCN5A mutation identify nitric oxide signaling as a mediator of atrial fibrillation

Abstract

Mutations in SCN5A, encoding the cardiac sodium channel, are linked with familial atrial fibrillation (AF) but the underlying pathophysiologic mechanisms and implications for therapy remain unclear. To characterize the pathogenesis of AF-linked SCN5A mutations, we generated patient-specific induced pluripotent stem cell-derived atrial cardiomyocytes (iPSC-aCMs) from two kindreds carrying SCN5A mutations (E428K and N470K) and isogenic controls using CRISPR-Cas9 gene editing. We showed that mutant AF iPSC-aCMs exhibited spontaneous arrhythmogenic activity with beat-to-beat irregularity, prolonged action potential duration, and triggered-like beats. Single-cell recording revealed enhanced late sodium currents (I Na,L ) in AF iPSC-aCMs that were absent in a heterologous expression model. Gene expression profiling of AF iPSC-aCMs showed differential expression of the nitric oxide (NO)mediated signaling pathway underlying enhanced I Na,L . We showed that patient-specific AF iPSC-aCMs exhibited striking in vitro electrophysiological phenotype of AF-linked SCN5A mutations, and transcriptomic analyses supported that the NO signaling pathway modulated the I Na,L and triggered AF.

RNA-Seq Analyses Reveal Roles of the HVCN1 Proton Channel in Cardiac pH Homeostasis

“…The RNA-seq analysis was performed as previously described ( Hong et al, 2021 ). We first extracted RNA with Maxwell ® RSC simplyRNA Cells Kit (Promega AS1390) based on manufacturer’s instructions, using Maxwell ® RSC Instrument (Promega AS4500), and then RNA was quantified using Qubit 4.0 Fluorometer (Invitrogen) with the Qubit RNA HS Assay Kit (REF Q32855) analyzed for integrity using the Agilent 4200 TapeStation RNA ScreenTape assay (Agilent 5067–5576, 5067–5577).…”

Calmodulin Interactions with Voltage-Gated Sodium Channels

“…The cardiac sodium channel Na v 1.5, encoded by the SCN5A gene, plays a critical role in the fast depolarization of the cardiac action potential [47]. Cardiac sodium channel dysfunction caused by Na v 1.5 mutations was reported to remodel abnormal action potential underlying arrhythmias [48].…”

Inherited and Acquired Rhythm Disturbances in Sick Sinus Syndrome, Brugada Syndrome, and Atrial Fibrillation: Lessons from Preclinical Modeling

“…In families with affected subjects, the main channels to be mutated in SSS are Nav1.5 and HCN, whose genes SCN5A and HCN are responsible for the generation of I Na and I f currents, as observed by genetic screening [144][145][146][147][148][149][150]. It is noteworthy to mention that I Na alterations are not only manifest in SSS, but also in other cardiac arrhythmias and might often generate a mixed clinical picture (as an example, [151,152]). This could be explained by the relatively complex architecture of the channels encoded by SCN5A genes, i.e., Nav1.5, (four homodomains, characterized each by six transmembrane regions with voltage sensitivity (S1-S4) and pore modulation (S5 and S6) [153]), thus mutations occurring in several genetic points could induce protein loss-of-function.…”

“…The administration of the NO blocker N-ethylmaleimide was, in fact, able to reduce the late I Na current. More interestingly from a translational point of view, the drug ranolazine (20 µM) rescued the aberrant arrhythmogenic behavior observed in these AF-iPS-cardiomyocytes [152], suggesting a possible, personalized pharmacological treatment for these AF patients.…”

“…In addition, ranolazine rescue the arrhythmic behavior of AF iPS-cardiomyocytes. [152] In vitro modeling with patient iPS-cardiomyocytes with unknown genetic etiology…”