Patients with very long-chain acyl-CoA dehydrogenase deficiency (VLCADD) can present with life-threatening cardiac arrhythmias. The pathophysiological mechanism is unknown. We reprogrammed fibroblasts from one mildly and one severely affected VLCADD patient, into human induced pluripotent stem cells (hiPSCs) and differentiated these into cardiomyocytes (VLCADD-CMs). VLCADD-CMs displayed shorter action potentials (APs), more delayed afterdepolarizations (DADs) and higher systolic and diastolic intracellular Ca²⁺ concentration ([Ca²⁺]_i) than control CMs. The mitochondrial booster resveratrol mitigated the biochemical, electrophysiological and [Ca²⁺]_i changes in the mild but not in the severe VLCADD-CMs. Accumulation of potentially toxic intermediates of fatty acid oxidation was blocked by substrate reduction with etomoxir. Incubation with etomoxir led to marked prolongation of AP duration and reduced DADs and [Ca²⁺]_i in both VLCADD-CMs. These results provide compelling evidence that reduced accumulation of fatty acid oxidation intermediates, either by enhanced fatty acid oxidation flux through increased mitochondria biogenesis (resveratrol) or by inhibition of fatty acid transport into the mitochondria (etomoxir), rescues pro-arrhythmia defects in VLCADD-CMs and open doors for new treatments.

Adverse effects of drug combinations and their underlying mechanisms are highly relevant for safety evaluation, but often not fully studied. Hydroxychloroquine (HCQ) and azithromycin (AZM) were used as a combination therapy in the treatment of COVID-19 patients at the beginning of the pandemic, leading to higher complication rates in comparison to respective monotherapies. Here, we used human-induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) to systematically investigate the effects of HCQ, AZM, and their combination on the structure and functionality of cardiomyocytes, and to better understand the underlying mechanisms. Our results demonstrate synergistic adverse effects of AZM and HCQ on electrophysiological and contractile function of iPSC-CMs. HCQ-induced prolongation of field potential duration (FPDc) was gradually increased during 7-day treatment period and was strongly enhanced by combination with AZM, although AZM alone slightly shortened FPDc in iPSC-CMs. Combined treatment with AZM and HCQ leads to higher cardiotoxicity, more severe structural disarrangement, more pronounced contractile dysfunctions, and more elevated conduction velocity, compared to respective monotreatments. Mechanistic insights underlying the synergistic effects of AZM and HCQ on iPSC-CM functionality are provided based on increased cellular accumulation of HCQ and AZM as well as increased Cx43- and Nav1.5-protein levels.

Atrial fibrillation (AF) is unexpectedly prevalent in Brugada syndrome (BrS), yet the mechanisms linking SCN5A loss-of-function to atrial instability remain elusive. Here, we combined patient-specific induced pluripotent stem cell-derived atrial cardiomyocytes with label-free high-density microelectrode array (HD-MEA) mapping. We show that SCN5A haploinsufficiency creates an arrhythmogenic substrate driven by the concomitant loss of excitability and heterogeneous Cx40 remodeling. This specific architecture renders mutant atrial syncytia highly susceptible to sustained, high-frequency spontaneous micro-reentry, a reentry-in-a-chip phenotype not recapitulated by pharmacological sodium-channel blockade in controls. Notably, genotype-negative BrS lines lacked spontaneous instability, exhibiting only inducible arrhythmia. Pharmacological profiling demonstrated that rhythm-control agents terminated reentry, whereas rate-control agents solely slowed rotation. This study defines the first human in vitro model of spontaneous atrial reentry, distinguishing primary mutation-driven defects from secondary clinical remodeling and providing a precision platform for anti-arrhythmic drug discovery.