Bupivacaine induces cardiotoxicity in human iPSC-derived cardiomyocytes through mitochondrial dysfunction and oxidative stress-associated apoptosis: attenuation by Intralipid

Abstract Bupivacaine is a widely used local anesthetic, but its cardiotoxicity during local anesthetic systemic toxicity (LAST) remains incompletely characterized in human-relevant cardiac models. In this study, human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs), generated using the GiWi monolayer differentiation protocol, were exposed to bupivacaine for 24 or 48 hours. Cell viability, cytotoxicity, spontaneous beating activity, mitochondrial function, oxidative stress, and apoptotic signaling were evaluated. Bupivacaine reduced cell viability in a dose- and time-dependent manner, with estimated IC₅₀ values of approximately 550 µM at 24 hours and 312 µM at 48 hours, and significantly impaired spontaneous beating rate and rhythm regularity at concentrations ≥ 100 µM. Mechanistically, bupivacaine induced mitochondrial membrane depolarization and mitochondrial fragmentation, accompanied by increased expression of the fission-related proteins DRP1 and FIS1 and decreased expression of the fusion-related proteins MFN2 and OPA1. These changes were associated with increased intracellular reactive oxygen species (ROS), an elevated BAX/BCL2 ratio, increased cytochrome c expression, and enhanced caspase-3 cleavage, indicating activation of the intrinsic mitochondrial apoptotic pathway. Co-treatment with Intralipid significantly restored cell viability, preserved mitochondrial membrane potential, reduced oxidative stress, and attenuated apoptotic signaling in bupivacaine-exposed hiPSC-CMs. These findings suggest that bupivacaine induces cardiotoxicity through a mitochondria–ROS–apoptosis cascade involving dysregulated mitochondrial dynamics, and provide mechanistic cellular evidence supporting Intralipid as a rescue agent for LAST-associated cardiotoxicity.