Optogenetic mitochondrial preconditioning enhances cardiomyocyte survival under stress

Mitochondria play a central role in preconditioning-mediated cytoprotection, yet the specific role of mitochondrial membrane potential (??m) in this process remains incompletely understood. In this study, we employed a next-generation, mitochondrial-targeted optogenetic system (mOpto) to induce precisely controlled (partial and transient) ??m depolarization and investigate its role in enhancing cardiomyocyte resilience to stress. Human AC16 cardiomyocytes expressing mOpto were subjected to low-intensity LED illumination for preconditioning, followed by exposure to stressors including FCCP, H2O2, or simulated ischemia-reperfusion. mOpto-preconditioned cells exhibited significantly improved viability, attenuated ??m depolarization, and reduced reactive oxygen species (ROS) production compared to non-preconditioned controls. Notably, this cytoprotective effect occurred independently of canonical ROS signaling and mitochondrial ATP-sensitive potassium channel (mitoKATP) activation. Transcriptional analysis revealed coordinated mitochondrial and metabolic reprogramming, including upregulation of genes involved in lipid biosynthesis, mitochondrial quality control, energy homeostasis, and a shift toward mitochondrial fusion. Importantly, mOpto preconditioning conferred similar cytoprotective effects in human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs), underscoring the translational potential of this approach. These findings demonstrate that mOpto-mediated transient ??m depolarization induces a preconditioning effect that enhances cardiomyocyte resilience through the establishment of a mitochondrial "memory" and dynamic remodeling of mitochondrial function. Overall design: RNA-Seq profiling of AC16 cells, including Control and expressing mOpto subjected to FCCP treatment with and without preconditioning