Circulating Mitochondrial Transfer Carrying Dihydroorotate Dehydrogenase Reduces Oxidative Cell Death in Prolonged Cold-induced Cardiac Reperfusion Injury in Heart Transplantation

BACKGROUND: Prolonged cold myocardial ischemia/reperfusion injury-driven by mitochondrial dysfunction, oxidative stress, and ferroptosis-limits cardiac transplantation success. Dihydroorotate dehydrogenase (DHODH), a key mitochondrial enzyme for redox homeostasis and ferroptosis suppression, has short ischemic half-life and poor targeting, restricting clinical use. We aimed to engineer cardiomyocyte-targeted mitochondria (DHODH-CT-Mito) for sustained DHODH delivery to mitigate prolonged cold myocardial ischemia/reperfusion. METHODS: Using a Langendorff model, we validated DHODH's transient protective role. DHODH-CT-Mito was designed by fusing translocase of the outer mitochondrial membrane 20 with an ischemic myocardium-targeting peptide. Efficacy/safety were tested in a 2-mo rat allogeneic heart transplant model, assessing homing, membrane potential, immunogenicity, biodistribution, and sustained DHODH activity. RESULTS: DHODH-CT-Mito showed robust myocardial homing, preserved membrane potential, and sustained DHODH activity. Treated grafts had improved long-term function, less fibrosis, and reduced apoptosis. It displayed low immunogenicity (transient inflammation, balanced macrophages) and minimal off-target accumulation. Acute DHODH delivery primed grafts for sustained redox balance. CONCLUSIONS: DHODH-CT-Mito overcomes exogenous DHODH limits, providing durable mitochondrial activity, suppressing ferroptosis, and preserving graft function with low risk-offering a translatable strategy for donor heart preservation.