Benign cardiac troponin I release induced by elevated beat rate is associated with membrane bleb formation

Cardiac troponin I (cTnI), a key regulatory protein of cardiomyocyte contraction, serves as a clinical biomarker for cellular necrosis. Measuring circulating cTnI in preclinical studies is used to assess cardiotoxicity of new chemical entities (NCEs) and gate their advancement into clinical trials. Recent studies showed benign troponin release due to reversible myocyte injury post-exercise, that could be primarily driven by sustained elevations in heart rate. However, mechanisms linking increased heart rate to troponin release without necrotic damage remain undetermined. In this study we investigated the relationship between sustained beat rate increases and cTnI release using human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) and zebrafish models. Beat rate was increased in hiPSC-CMs with forskolin, and a concomitant increase in cTnI release was observed without impacting cell viability. To evaluate the translatability of benign troponin release from in vivo contexts, we established methods for quantifying systemic troponin levels in zebrafish and evaluated the effects of forskolin on heart rate and troponin release. Similarly, forskolin treatment increased heart rate and circulating troponin in zebrafish. To investigate the molecular mechanisms involved, hiPSC-CMs were co-treated with forskolin and (±)-blebbistatin, a Myosin II and membrane blebbing inhibitor. We found (±)-blebbistatin to significantly reduce cTnI release, suggesting membrane bleb formation as a possible mechanism of benign troponin release triggered by elevated beat rates. These findings provide a better understanding of troponin release mechanisms in cardiomyocytes. Further, these results may support refinement of cardiotoxicity assessment of NCEs, preventing misinterpretations that might lead to unnecessary termination of promising therapeutics.