Project Abstract Heart disease is a major cause of morbidity and mortality, much of it due to ischemic injury. Mitophagy and biogenesis (mitochondrial turnover) are essential for ischemia tolerance and appropriate recovery during reperfusion. Factors that affect mitochondrial turnover include age, sex, time of day, comorbid conditions such as age and metabolic syndrome, nutritional status, exercise, and a long list of drugs and natural products. The overarching hypothesis is that monitoring mitochondrial turnover will allow us to infer a patient’s response to ischemic stress, and that normalizing turnover will improve outcome. In this proposal we develop tools to measure mitochondrial turnover (Aim I), define the regulation of mitochondrial turnover (Aim II), and leverage the knowledge gained to monitor mitochondrial turnover in the human heart during cardiopulmonary bypass (Aim III). Additionally we will measure mitochondrial function in endomyocardial biopsies of heart transplant patients, where it will be possible to relate mitochondrial function to cardiac contractility. In Aim I we will use our novel MitoTimer mice and organelle flow cytometry to develop a proteomic signature of mitophagy and biogenesis. The protein profiles will be used to create a mass spectrometry assay (multiple reaction monitoring, MRM) to infer mitochondrial turnover in tissue extracts including human heart biopsies. This index of mitophagy and biogenesis will be used to assess mitochondrial turnover in rodents in Aim I and in human heart biopsies in Aim III. In Aim II, we will delineate the regulation of mitophagy by PINK1, Parkin, and optineurin; and the regulation of mitochondrial biogenesis by PGC-1alpha, PARIS, and translational machinery. We have established polysome profiling to interrogate translational control of mitochondrial biogenesis. In Aim III we will use paired atrial biopsies (before and after cardiopulmonary bypass) to characterize mitophagy and biogenesis in the human heart’s response to ischemic stress; we will use paired atrial and ventricular biopsies to gain much-needed information about the differences in mitochondrial function and cardiac proteome; and we will correlate mitochondrial respirometry, mtDNA damage, and MRM assays of mitochondrial turnover to correlate with clinical parameters, specifically postoperative atrial fibrillation. These studies will establish the molecular signatures of appropriate mitochondrial turnover during cardiac surgery, providing markers of target engagement that will position us to evaluate therapeutic interventions. Importantly, understanding this pathway will reveal new therapeutic targets that regulate mitochondrial turnover and influence function of the human heart recovering from ischemia.