Functional hyperactivity in long QT syndrome type 1 pluripotent stem cell-derived sympathetic neurons.

Sympathetic activation is an established trigger of life-threatening cardiac events in long QT syndrome type 1 (LQT1). KCNQ1 loss-of-function variants, which underlie LQT1, have been associated with both cardiac arrhythmia and neuronal hyperactivity pathologies. However, the LQT1 sympathetic neuronal phenotype is unknown. Here, we aimed to study human induced pluripotent stem cell (hiPSC)-derived sympathetic neurons (SNs) to evaluate neuronal functional phenotype in LQT1. We generated hiPSC-SNs from two patients with LQT1 with a history of sympathetically triggered arrhythmia and KCNQ1 loss-of-function genotypes (c.781_782delinsTC and p.S349W/p.R518X). Characterization of hiPSC-SNs was performed using immunohistochemistry, enzyme-linked immunosorbent assay, and whole cell patch clamp electrophysiology, and functional LQT1 hiPSC-SN phenotypes compared with healthy control (WT) hiPSC-SNs. hiPSC-SNs stained positive for tyrosine hydroxylase, peripherin, KCNQ1, and secreted norepinephrine. hiPSC-SNs at 60 ± 2.2 days in vitro had healthy resting membrane potentials (-60 ± 1.3 mV), and fired rapid action potentials with mature kinetics in response to stimulation. Significant hyperactivity in LQT1 hiPSC-SNs was evident via increased norepinephrine release, increased spontaneous action potential frequency, increased total inward current density, and reduced afterhyperpolarization, compared with age-matched WT hiPSC-SNs. A significantly higher action potential frequency upon current injection and larger synaptic current amplitudes in compound heterozygous p.S349W/p.R518X hiPSC-SNs compared with heterozygous c.781_782delinsTC hiPSC-SNs was also observed, suggesting a potential genotype-phenotype correlation. Together, our data reveal increased neurotransmission and excitability in heterozygous and compound heterozygous patient-derived LQT1 sympathetic neurons, suggesting that the cellular arrhythmogenic potential in LQT1 is not restricted to cardiomyocytes.NEW & NOTEWORTHY Here, we present the first study of patient-derived LQT1 sympathetic neurons that are norepinephrine secreting, and electrophysiologically functional, in vitro. Our data reveal a novel LQT1 sympathetic neuronal phenotype of increased neurotransmission and excitability. The identified sympathetic neuronal hyperactivity phenotype is of particular relevance as it could contribute to the mechanisms underlying sympathetically triggered arrhythmia in LQT1.

Functional coculture of sympathetic neurons and cardiomyocytes derived from human-induced pluripotent stem cells.

Sympathetic neurons (SNs) capable of modulating the heart rate of murine cardiomyocytes (CMs) can be differentiated from human stem cells. The electrophysiological properties of human stem cell-derived SNs remain largely uncharacterized, and human neurocardiac cocultures remain to be established. Here, we have adapted previously published differentiation and coculture protocols to develop feeder-free SNs using human-induced pluripotent stem cells (hiPSCs). hiPSC-SNs were characterized in monoculture and coculture with hiPSC-CMs, using antibody labeling, enzyme-linked immunosorbent assay, and whole cell patch-clamp electrophysiology techniques. hiPSC-SNs stained positive for peripherin, tyrosine hydroxylase, and nicotinic acetylcholine receptors, the latter two colocalizing in somas and synaptic varicosities. hiPSC-SNs functionally matured in vitro and exhibited healthy resting membrane potentials (average = -61 ± 0.7 mV), secreted norepinephrine upon activation, and generated synaptic and action currents and inward and outward voltage-dependent currents. All hiPSC-SNs fired action potentials in response to current injection, local application of potassium, or spontaneously, followed by short-medium afterhyperpolarizations. hiPSC-SNs could successfully be maintained in coculture with hiPSC-CMs, and this induced further development of hiPSC-SN action potential kinetics. To test functional coupling between the neurons and cardiomyocytes, the hiPSC-CM beating response to nicotine-induced norepinephrine release was assessed. In neurocardiac cocultures, nicotine exposure significantly increased the hiPSC-CM spontaneous beating rate, but not in hiPSC-CM monocultures, supporting nicotinic neuronal hiPSC-SN stimulation directly influencing hiPSC-CM function. Our data show the development and characterization of electrophysiologically functional hiPSC-SNs capable of modulating the beating rate of hiPSC-CMs in vitro. These human cocultures provide a novel multicellular model to study neurocardiac modulation under physiological and pathological conditions.NEW & NOTEWORTHY We present data on a functional coculture between human-induced pluripotent stem cell-derived sympathetic neurons and cardiomyocytes. Moreover, this study adds significantly to the available data on the electrophysiological function of human-induced pluripotent stem cell-derived sympathetic neurons.