Study 1: Myocardial ischemia–reperfusion (I/R) injury is one of the leading causes of death and disability world- wide. Mitochondrial fission has been shown to be involved in cardiomyocyte death. However, molecular machinery involved in mitochondrial fission during I/R injury has not yet been completely understood. In this study, we aimed to investigate molecular mechanisms of controlling activation of dynamin-related protein 1 (Drp1, a key protein in mitochondrial fission) during ischemia-reperfusion (I/R) injury of HL1 cardiomyocytes. I/R injury induced cardiomyocyte death accompanied by the increases of mitochondrial fission, reactive oxygen species (ROS) production and activated Drp1 (pSer616 Drp1), and decrease of inactivated Drp1 (pSer637 Drp1) while mitochondrial fusion protein levels were not significantly changed. Blocking Drp1 activity with mitochondrial division inhibitor mdivi1 attenuated cell death, mitochondrial fission, and Drp1 activation after A/R. Trolox, a ROS scavenger, decreased pSer616 Drp1 level and mitochondrial fission after I/R. Immunoprecipitation assay further indicates that cyclin dependent kinase 1 (Cdk1) and protein kinase C isoform delta (PKCd) bind Drp1, thus increasing mitochondrial fission. Inhibiting Cdk1 and PKCd attenuated the increases in pSer616 Drp1, mitochondrial fission, and cardiomyocyte death. FK506, a calcineurin inhibitor, blocked the decrease in expression of inactivated pSer637 Drp1 and mitochondrial fission. Our findings reveal the following novel molecular mechanisms controlling mitochondrial fission during I/R injury of cardiomyocytes. Study 2: Hyperglycemia can blunt the cardioprotective effects of isoflurane in the setting of ischemia–reperfusion injury. Previous studies suggest that reactive oxygen species (ROS) and increased mitochondrial fission play a role in cardiomyocyte death during ischemia–reperfusion injury. To investigate the role of high glucose concentration in ROS production and mitochondrial fission during ischemia–reperfusion (with and without anesthetic protection), we used the novel platform of human-induced pluripotent stem-cell (iPSC)– derived cardiomyocytes (CMs). Cardiomyocyte differentiation from iPSC was characterized by the expression of CM-specific markers using immunohistochemistry and by measuring contractility. iPSC-CMs were exposed to varying glucose conditions (5, 11, and 25 mM) for 24 hours. Mitochondrial permeability transition pore opening, cell viability, and ROS generation endpoints were used to assess the effects of various treatment conditions. Mitochondrial fission was monitored by the visualization of fragmented mitochondria using confocal microscopy. Expression of activated dynamin-related protein 1, a key protein responsible for mitochondrial fission, was assessed by Western blot. Cardiomyocytes were successfully differentiated from iPSC. Elevated glucose conditions (11 and 25 mM) significantly increased ROS generation, whereas only the 25-mM high glucose condition induced mitochondrial fission and increased the expression of activated dynamin-related protein 1 in iPSC-CMs. Isoflurane delayed mitochondrial permeability transition pore opening and protected iPSC-CMs from oxidative stress in 5- and 11-mM glucose conditions to a similar level as previously observed in various isolated animal cardiomyocytes. Scavenging ROS with Trolox or inhibiting mitochondrial fission with mdivi-1 restored the anesthetic cardioprotective effects in iPSC-CMs in 25-mM glucose conditions.