Human pluripotent stem cells (PSC) have been used for disease modelling, after differentiation into the desired cell type. Electrophysiologic properties of cardiomyocytes derived from pluripotent stem cells are extensively used to model cardiac arrhythmias, in cardiomyopathies and channelopathies. This requires strict control of the multiple variables that can influence the electrical properties of these cells. In this article, we report the action potential variability of 780 cardiomyocytes derived from pluripotent stem cells obtained from six healthy donors. We analyze the overall distribution of action potential (AP) data, the distribution of action potential data per cell line, per differentiation protocol and batch. This analysis indicates that even using the same cell line and differentiation protocol, the differentiation batch still affects the results. This variability has important implications in modeling arrhythmias and imputing pathogenicity to variants encountered in patients with arrhythmic diseases. We conclude that even when using isogenic cell lines to ascertain pathogenicity to variants associated to arrythmias one should use cardiomyocytes derived from pluripotent stem cells using the same differentiation protocol and batch and pace the cells or use only cells that have very similar spontaneous beat rates. Otherwise, one may find phenotypic variability that is not attributable to pathogenic variants.

The antidiabetic agent class of sodium-glucose cotransporter 2 (SGLT2) inhibitors confer unprecedented cardiovascular benefits beyond glycemic control, including reducing the risk of fatal ventricular arrhythmias. However, the impact of SGLT2 inhibitors on the electrophysiological properties of cardiomyocytes exposed to stimuli other than hyperglycemia remains elusive. This investigation tested the hypothesis that the SGLT2 inhibitor empagliflozin (EMPA) affects cardiomyocyte electrical activity under hypoxic conditions. Rat neonatal and human induced pluripotent stem cell (iPSC)-derived cardiomyocytes incubated or not with the hypoxia-mimetic agent CoCl2 were treated with EMPA (1 μM) or vehicle for 24 h. Action potential records obtained using intracellular microelectrodes demonstrated that EMPA reduced the action potential duration at 30%, 50%, and 90% repolarization and arrhythmogenic events in rat and human cardiomyocytes under normoxia and hypoxia. Analysis of Ca2+ transients using Fura-2-AM and contractility kinetics showed that EMPA increased Ca2+ transient amplitude and decreased the half-time to recover Ca2+ transients and relaxation time in rat neonatal cardiomyocytes. We also observed that the combination of EMPA with the Na+/H+ exchanger isoform 1 (NHE1) inhibitor cariporide (10 µM) exerted a more pronounced effect on Ca2+ transients and contractility than either EMPA or cariporide alone. Besides, EMPA, but not cariporide, increased phospholamban phosphorylation at serine 16. Collectively, our data reveal that EMPA reduces arrhythmogenic events, decreases the action potential duration in rat neonatal and human cardiomyocytes under normoxic or hypoxic conditions, and improves cytosolic calcium handling at least partially independent of NHE1. Moreover, we provided further evidence that SGLT2 inhibitor-mediated cardioprotection may be partly attributed to its cardiomyocyte electrophysiological effects.

Sodium glucose cotransporter 2 inhibitors (SGLT2i) emerged as promising antidiabetic drugs that decreasing glucose reabsorption as well as effectively reduce heart failure hospitalization and cardiovascular death in type 2 diabetes patients. Recently, it has been shown that the SGLT2i also exert beneficial cardiovascular effects in non‐diabetic heart failure patients. However, the cardioprotective mechanisms of these drugs remain unknow. The present study aimed to test the hypothesis that the cardioprotective effects of SGLT2i empagliflozin (EMPA) may be associated with changes of cardiac electrical activity and Ca2+ homeostasis in hypoxia‐induced injury ventricular myocytes. Cardiomyocytes isolated from neonatal rats submitted to chemical hypoxia (200 μM CoCl2) or normoxia were incubated with EMPA (1 μM) or vehicle during 24 hours. Electrophysiological recordings were obtained using multielectrode array (MEA) system and intracellular microelectrode. Cytoplasmic calcium transients were investigated via fura‐2 fluorescence. Compared with vehicle, MEA analysis showed a significant decrease in field potential duration in EMPA‐group in both conditions [hypoxia: 83 ± 4 (N=19) vs. 37 ± 3 (N=44); normoxia: 213 ± 5 (N=53) vs. 62 ± 4 (N=36) ms, p<0.001]. EMPA reduced action potential duration at 30%, 50% and 90% repolarization (APD30, APD50 and APD90, respectively) in hypoxia [APD30: 109 ± 2 (N=41) vs. 98 ± 1 (N=30); APD50: 150 ± 3 (N=41) vs. 134 ± 1 (N=30); APD90: 292 ± 4 (N=41) vs. 271 ± 5 (N=30) ms, p<0.05] and normoxia [APD30: 94 ± 2 (N=30) vs. 81 ± 1 (N=39); APD50: 129 ± 2 (N=30) vs. 111 ± 1 (N=39); APD90: 259 ± 4 (N=30) vs. 244 ± 4 (N=39) ms, p<0.05]. No differences were observed in amplitude and resting membrane potential. Action potential recordings also revealed that the percentage of arrhythmic cells was lower in EMPA‐group in both conditions [hypoxia: 19 (N=41) vs. 10 (N=30) %; normoxia: 7 (N=30) vs. 3 (N=39) %]. In addition, EMPA significantly increased in Ca2+ transient amplitude in hypoxia [F/F0: 0.16 ± 0.01 (N=21) vs. 0.21 ± 0.01 (N=19), p<0.01] and normoxia [F/F0: 0.17 ± 0.01 (N=19) vs. 0.23 ± 0.01 (N=20) μM, p<0.01]. These results suggest that EMPA modulates electrical activity, reduces arrhythmic events and improves Ca2+ transient, which may contribute to cardioprotective effects of SGLT2i. Support or Funding Information FAPESP and CNPq