Cellular calcium handling and electrophysiology are modulated by chronic physiological pacing in human induced pluripotent stem cell-derived cardiomyocytes

Electric pacing of human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CM) has been increasingly used to simulate cardiac arrhythmias in vitro and to enhance cardiomyocyte maturity. However, the impact of electric pacing on cellular electrophysiology and Ca²⁺ handling in differentiated hiPSC-CM is less characterized. Here we studied the effects of electric pacing for 24 h or 7 days at a physiological rate of 60 beats/min on cellular electrophysiology and Ca²⁺ cycling in late-stage, differentiated hiPSC-CM (>90% troponin⁺, >60 days postdifferentiation). Electric culture pacing for 7 days did not influence cardiomyocyte cell size, apoptosis, or generation of reactive oxygen species in differentiated hiPSC-CM compared with 24-h pacing. However, epifluorescence measurements revealed that electric pacing for 7 days improved systolic Ca²⁺ transient amplitude and Ca²⁺ transient upstroke, which could be explained by elevated sarcoplasmic reticulum Ca²⁺ load and SERCA activity. Diastolic Ca²⁺ leak was not changed in line-scanning confocal microscopy, suggesting that the improvement in systolic Ca²⁺ release was not associated with a higher open probability of ryanodine receptor (RyR)2 during diastole. Whereas bulk cytosolic Na⁺ concentration and Na⁺/Ca²⁺ exchanger (NCX) activity were not changed, patch-clamp studies revealed that chronic pacing caused a slight abbreviation of the action potential duration (APD) in hiPSC-CM. We found in whole cell voltage-clamp measurements that chronic pacing for 7 days led to a decrease in late Na⁺ current, which might explain the changes in APD. In conclusion, our results show that chronic pacing improves systolic Ca²⁺ handling and modulates the electrophysiology of late-stage, differentiated hiPSC-CM. This study might help to understand the effects of electric pacing and its numerous applications in stem cell research including arrhythmia simulation.<b>NEW & NOTEWORTHY</b> Electric pacing is increasingly used in research with human induced pluripotent stem cell cardiomyocytes (hiPSC-CM), for example to simulate arrhythmias but also to enhance maturity. Therefore, it is mandatory to understand the effects of pacing itself on cellular electrophysiology in late-stage, matured hiPSC-CM. This study provides an electrophysiological characterization of the effects of chronic electric pacing at a physiological rate on differentiated hiPSC-CM.