2025 – Blebbistatin reduces calcium buffering in cardiomyocytes: Consequences for cellular electrophysiology

Blebbistatin is an excitation–contraction uncoupling agent commonly used in cardiac optical mapping; however, it has been reported to influence cardiac myofilament Ca²⁺ sensitivity. As primary contributors to Ca²⁺ buffering within cardiomyocytes, cardiac myofilaments play a critical role, and even minor disruptions in intracellular Ca²⁺ buffering significantly alter the free Ca²⁺ concentration. In this study, we investigated the effect of blebbistatin, a myosin II ATPase inhibitor, on intracellular Ca²⁺ buffering and cellular electrophysiology in induced pluripotent stem cell-derived atrial cardiomyocytes. Simultaneous whole-cell ruptured patch-clamp and fluorescence microscopy techniques were used to assess intracellular Ca²⁺ handling, in addition to automated high-throughput patch-clamp to investigate ion channel function. Comprehensive analysis of Ca²⁺ buffering revealed that blebbistatin (10 µmol/l) causes a significant increase in buffer dissociation constant, suggesting decreased affinity of Ca²⁺ buffers. Furthermore, systolic and diastolic Ca²⁺ levels, sarcoplasmic reticulum Ca²⁺ leak and the incidence of spontaneous Ca²⁺ release events were significantly higher upon blebbistatin treatment. Although there was lack of impact on I_Na and I_Ca,L peak density, Ca²⁺-dependent inactivation of I_Ca,L was significantly enhanced, and I_K1 density was significantly smaller after blebbistatin. Importantly, these effects were reversed after chelation of intracellular Ca²⁺ with EGTA. Our observations indicate that blebbistatin reduces Ca²⁺ buffering, which, in turn, causes changes in cellular electrophysiology in cardiomyocytes.