Authors
Sobitov I, Ritzenhoff K, Gaulrapp M, Becker L, Liutkute A, Seibertz F, Fakuade FE, Mason FE, Voigt N
Journal
The Journal of Physiology
Citation
J Physiol. 2025 Aug 25
Abstract
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 INa and ICa,L peak density, Ca2+-dependent inactivation of ICa,L was significantly enhanced, and IK1 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. KEY POINTS: Intracellular Ca2+ buffering plays an important role in determining Ca2+ dynamics in cardiomyocytes. Blebbistatin, an excitation-contraction uncoupling agent widely used in experimental studies, decreases the affinity of intracellular Ca2+ buffers, as indicated by an increased buffer dissociation constant. Blebbistatin leads to higher systolic and diastolic Ca2+ levels and increased sarcoplasmic reticulum Ca2+ leak. Blebbistatin enhances L-type Ca2+ current (ICa,L) Ca2+-dependent inactivation and reduces inward rectifier potassium current (IK1) density, while INa and ICa,L remain unchanged. The effects of blebbistatin are mitigated by chelation of intracellular Ca2+ with EGTA, showing that they are secondary to altered Ca2+ buffering.
DOI
