Rationale:: In cardiomyocytes, Na _V 1.5 and Kir2.1 channels interact dynamically as part of membrane bound macromolecular complexes. Objective:: The objective of this study was to test whether Na _V 1.5 and Kir2.1 preassemble during early forward trafficking and travel together to common membrane microdomains. Methods and Results:: In patch-clamp experiments, coexpression of trafficking-deficient mutants Kir2.1 ^Δ314-315 or Kir2.1 ^R44A/R46A with wild-type (WT) Na _V 1.5 ^WT in heterologous cells reduced inward sodium current compared with Na _V 1.5 ^WT alone or coexpressed with Kir2.1 ^WT . In cell surface biotinylation experiments, expression of Kir2.1 ^Δ314-315 reduced Na _V 1.5 channel surface expression. Glycosylation analysis suggested that Na _V 1.5 ^WT and Kir2.1 ^WT channels associate early in their biosynthetic pathway, and fluorescence recovery after photobleaching experiments demonstrated that coexpression with Kir2.1 increased cytoplasmic mobility of Na _V 1.5 ^WT , and vice versa, whereas coexpression with Kir2.1 ^Δ314-315 reduced mobility of both channels. Viral gene transfer of Kir2.1 ^Δ314-315 in adult rat ventricular myocytes and human induced pluripotent stem cell–derived cardiomyocytes reduced inward rectifier potassium current and inward sodium current, maximum diastolic potential and action potential depolarization rate, and increased action potential duration. On immunostaining, the AP1 (adaptor protein complex 1) colocalized with Na _V 1.5 ^WT and Kir2.1 ^WT within areas corresponding to t-tubules and intercalated discs. Like Kir2.1 ^WT , Na _V 1.5 ^WT coimmunoprecipitated with AP1. Site-directed mutagenesis revealed that Na _V 1.5 ^WT channels interact with AP1 through the Na _V 1.5 ^Y1810 residue, suggesting that, like for Kir2.1 ^WT , AP1 can mark Na _V 1.5 channels for incorporation into clathrin-coated vesicles at the trans -Golgi. Silencing the AP1 ϒ-adaptin subunit in human induced pluripotent stem cell–derived cardiomyocytes reduced inward rectifier potassium current, inward sodium current, and maximum diastolic potential and impaired rate-dependent action potential duration adaptation. Conclusions:: The Na _V 1.5-Kir2.1 macromolecular complex pre-assembles early in the forward trafficking pathway. Therefore, disruption of Kir2.1 trafficking in cardiomyocytes affects trafficking of Na _V 1.5, which may have important implications in the mechanisms of arrhythmias in inheritable cardiac diseases.

Cardiac Nav1.5 and Kir2.1–2.3 channels generate Na (I _Na ) and inward rectifier K (I _K1 ) currents, respectively. The functional I _Na and I _K1 interplay is reinforced by the positive and reciprocal modulation between Nav15 and Kir2.1/2.2 channels to strengthen the control of ventricular excitability. Loss-of-function mutations in the SCN5A gene, which encodes Nav1.5 channels, underlie several inherited arrhythmogenic syndromes, including Brugada syndrome (BrS). We investigated whether the presence of BrS-associated mutations alters I _K1 density concomitantly with I _Na density. Results obtained using mouse models of SCN5A haploinsufficiency, and the overexpression of native and mutated Nav1.5 channels in expression systems — rat ventricular cardiomyocytes and human induced pluripotent stem cell–derived cardiomyocytes (hiPSC-CMs) — demonstrated that endoplasmic reticulum (ER) trafficking–defective Nav1.5 channels significantly decreased I _K1 , since they did not positively modulate Kir2.1/2.2 channels. Moreover, Golgi trafficking–defective Nav1.5 mutants produced a dominant negative effect on Kir2.1/2.2 and thus an additional I _K1 reduction. Moreover, ER trafficking–defective Nav1.5 channels can be partially rescued by Kir2.1/2.2 channels through an unconventional secretory route that involves Golgi reassembly stacking proteins (GRASPs). Therefore, cardiac excitability would be greatly affected in subjects harboring Nav1.5 mutations with Golgi trafficking defects, since these mutants can concomitantly trap Kir2.1/2.2 channels, thus unexpectedly decreasing I _K1 in addition to I _Na .

Human induced pluripotent stem cell–derived cardiomyocytes (hiPSC-CMs) have been used extensively to model inherited heart diseases, but hiPSC-CM models of ischemic heart disease are lacking. Here, our objective was to generate an hiPSC-CM model of ischemic heart disease. To this end, hiPSCs were differentiated into functional hiPSC-CMs and then purified using either a simulated ischemia media or by using magnetic antibody-based purification targeting the nonmyocyte population for depletion from the cell population. Flow cytometry analysis confirmed that each purification approach generated hiPSC-CM cultures that had more than 94% cTnT ⁺ cells. After purification, hiPSC-CMs were replated as confluent syncytial monolayers for electrophysiological phenotype analysis and protein expression by Western blotting. The phenotype of metabolic stress–selected hiPSC-CM monolayers recapitulated many of the functional and structural hallmarks of ischemic CMs, including elevated diastolic calcium, diminished calcium transient amplitude, prolonged action potential duration, depolarized resting membrane potential, hypersensitivity to chemotherapy-induced cardiotoxicity, depolarized mitochondrial membrane potential, depressed SERCA2a expression, reduced maximal oxygen consumption rate, and abnormal response to β ₁ -adrenergic receptor stimulation. These findings indicate that metabolic selection of hiPSC-CMs generated cell populations with phenotype similar to what is well known to occur in the setting of ischemic heart failure and thus provide a opportunity for study of human ischemic heart disease.

Background: Patients with cardiomyopathy of Duchenne Muscular Dystrophy (DMD) are at risk of developing life-threatening arrhythmias, but the mechanisms are unknown. We aimed to determine the role of ion channels controlling cardiac excitability in the mechanisms of arrhythmias in DMD patients.Methods: To test whether dystrophin mutations lead to defective cardiac Na_V1.5–Kir2.1 channelosomes and arrhythmias, we generated iPSC-CMs from two hemizygous DMD males, a heterozygous female, and two unrelated control males. We conducted studies including confocal microscopy, protein expression analysis, patch-clamping, non-viral piggy-bac gene expression, optical mapping and contractility assays.Results: Two patients had abnormal ECGs with frequent runs of ventricular tachycardia. iPSC-CMs from all DMD patients showed abnormal action potential profiles, slowed conduction velocities, and reduced sodium (I_Na) and inward rectifier potassium (I_K1) currents. Membrane Na_V1.5 and Kir2.1 protein levels were reduced in hemizygous DMD iPSC-CMs but not in heterozygous iPSC-CMs. Remarkably, transfecting just one component of the dystrophin protein complex (α1-syntrophin) in hemizygous iPSC-CMs from one patient restored channelosome function, I_Na and I_K1 densities, and action potential profile in single cells. In addition, α1-syntrophin expression restored impulse conduction and contractility and prevented reentrant arrhythmias in hiPSC-CM monolayers.Conclusions: We provide the first demonstration that iPSC-CMs reprogrammed from skin fibroblasts of DMD patients with cardiomyopathy have a dysfunction of the Na_V1.5–Kir2.1 channelosome, with consequent reduction of cardiac excitability and conduction. Altogether, iPSC-CMs from patients with DMD cardiomyopathy have a Na_V1.5–Kir2.1 channelosome dysfunction, which can be rescued by the scaffolding protein α1-syntrophin to restore excitability and prevent arrhythmias.Funding: Supported by National Institutes of Health R01 HL122352 grant; ‘la Caixa’ Banking Foundation (HR18-00304); Fundación La Marató TV3: Ayudas a la investigación en enfermedades raras 2020 (LA MARATO-2020); Instituto de Salud Carlos III/FEDER/FSE; Horizon 2020 - Research and Innovation Framework Programme GA-965286 to JJ; the CNIC is supported by the Instituto de Salud Carlos III (ISCIII), the Ministerio de Ciencia e Innovación (MCIN) and the Pro CNIC Foundation), and is a Severo Ochoa Center of Excellence (grant CEX2020-001041-S funded by MICIN/AEI/10.13039/501100011033). American Heart Association postdoctoral fellowship 19POST34380706s to JVEN. Israel Science Foundation to OB and MA [824/19]. Rappaport grant [01012020RI]; and Niedersachsen Foundation [ZN3452] to OB; US-Israel Binational Science Foundation (BSF) to OB and TH [2019039]; Dr. Bernard Lublin Donation to OB; and The Duchenne Parent Project Netherlands (DPPNL 2029771) to OB. National Institutes of Health R01 AR068428 to DM and US-Israel Binational Science Foundation Grant [2013032] to DM and OB.

The voltage-gated channel, hERG1, conducts the rapid delayed rectifier potassium current (I_Kr) and is critical for human cardiac repolarization. Reduced I_Kr causes long QT syndrome and increases the risk for cardiac arrhythmia and sudden death. At least two subunits form functional hERG1 channels, hERG1a and hERG1b. Changes in hERG1a/1b abundance modulate I_Kr kinetics, magnitude, and drug sensitivity. Studies from native cardiac tissue suggest that hERG1 subunit abundance is dynamically regulated, but the impact of altered subunit abundance on I_Kr and its response to external stressors is not well understood. Here, we used a substrate-driven human-induced pluripotent stem cell–derived cardiomyocyte (hiPSC-CM) maturation model to investigate how changes in relative hERG1a/1b subunit abundance impact the response of native I_Kr to extracellular acidosis, a known component of ischemic heart disease and sudden infant death syndrome. I_Kr recorded from immatured hiPSC-CMs displays a 2-fold greater inhibition by extracellular acidosis (pH 6.3) compared with matured hiPSC-CMs. Quantitative RT-PCR and immunocytochemistry demonstrated that hERG1a subunit mRNA and protein were upregulated and hERG1b subunit mRNA and protein were downregulated in matured hiPSC-CMs compared with immatured hiPSC-CMs. The shift in subunit abundance in matured hiPSC-CMs was accompanied by increased I_Kr. Silencing hERG1b’s impact on native I_Kr kinetics by overexpressing a polypeptide identical to the hERG1a N-terminal Per-Arnt-Sim domain reduced the magnitude of I_Kr proton inhibition in immatured hiPSC-CMs to levels comparable to those observed in matured hiPSC-CMs. These data demonstrate that hERG1 subunit abundance is dynamically regulated and determines I_Kr proton sensitivity in hiPSC-CMs.

The hERG1 potassium channel conducts the cardiac repolarizing current, I _Kr . hERG1 has emerged as a therapeutic target for cardiac diseases marked by prolonged action potential duration (APD). Unfortunately, many hERG1 activators display off-target and proarrhythmic effects that limit their therapeutic potential. A Per-Arnt-Sim (PAS) domain in the hERG1 N-terminus reduces I _Kr by slowing channel activation and promoting inactivation. Disrupting PAS activity increases I _Kr and shortens APD in human induced pluripotent stem cell–derived cardiomyocytes (hiPSC-CMs). We thus hypothesized that the hERG1 PAS domain could represent a therapeutic target to reduce arrhythmogenic potential in a long QT syndrome (LQTS) background. To test this, we measured the antiarrhythmic capacity of a PAS-disabling single-chain variable fragment antibody, scFv2.10, in a hiPSC-CM line derived from a patient with Jervell and Lange Nielsen (JLN) syndrome. JLN is a severe form of LQTS caused by autosomal recessive mutations in KCNQ1 . The patient in this study carried compound heterozygous mutations in KCNQ1 . Corresponding JLN hiPSC-CMs displayed prolonged APD and early afterdepolarizations (EADs). Disrupting PAS with scFv2.10 increased I _Kr , shortened APD, and reduced the incidence of EADs. These data demonstrate that the hERG1 PAS domain could serve as a therapeutic target to treat disorders of cardiac electrical dysfunction.