Work

Blebbistatin reduces calcium buffering in cardiomyocytes: Consequences for cellular electrophysiology

Year: 2025

Type: article

Abstract: Blebbistatin is an excitation–contraction uncoupling agent commonly used in cardiac optical mapping; however, it has been reported to influence cardiac myofilament Ca 2+ sensitivity. As primary contributors to Ca 2+ buffering within cardiomyocytes, cardiac myofilaments play a critical role, and even minor disruptions in intracellular Ca 2+ buffering significantly alter the free Ca 2+ concentration. In this study, we investigated the effect of blebbistatin, a myosin II ATPase inhibitor, on intracellular Ca 2+ 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 Ca 2+ handling, in addition to automated high‐throughput patch‐clamp to investigate ion channel function. Comprehensive analysis of Ca 2+ buffering revealed that blebbistatin (10 µmol/l) causes a significant increase in buffer dissociation constant, suggesting decreased affinity of Ca 2+ buffers. Furthermore, systolic and diastolic Ca 2+ levels, sarcoplasmic reticulum Ca 2+ leak and the incidence of spontaneous Ca 2+ release events were significantly higher upon blebbistatin treatment. Although there was lack of impact on I Na and I Ca,L peak density, Ca 2+ ‐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 Ca 2+ with EGTA. Our observations indicate that blebbistatin reduces Ca 2+ buffering, which, in turn, causes changes in cellular electrophysiology in cardiomyocytes. image Key points Intracellular Ca 2+ buffering plays an important role in determining Ca 2+ dynamics in cardiomyocytes. Blebbistatin, an excitation–contraction uncoupling agent widely used in experimental studies, decreases the affinity of intracellular Ca 2+ buffers, as indicated by an increased buffer dissociation constant. Blebbistatin leads to higher systolic and diastolic Ca 2+ levels and increased sarcoplasmic reticulum Ca 2+ leak. Blebbistatin enhances L‐type Ca 2+ current ( I Ca,L ) Ca 2+ ‐dependent inactivation and reduces inward rectifier potassium current ( I K1 ) density, while I Na and I Ca,L remain unchanged. The effects of blebbistatin are mitigated by chelation of intracellular Ca 2+ with EGTA, showing that they are secondary to altered Ca 2+ buffering.

Source: The Journal of Physiology

DOI: 10.1113/jp287545