EphrinA1, a receptor tyrosine kinase ligand expressed in the plasma membrane of healthy cardiomyocytes, is reduced following acute myocardial ischemia (I)/reperfusion (R) injury in mice. Intramyocardial injection of recombinant EphrinA1-Fc at the time of LAD ligation attenuated infarct injury by 46% and completely preserved cardiac function in I/R (30min I/24hrs R). This was accompanied by reduced inflammation, attenuation of apoptosis, enhanced autophagy, and modulation of metabolic pathways that promote more efficient energy utilization. To examine the mechanism by which this occurs, mitochondrial structure and function was examined in 10 wk-old B6129SF2/J male mice given intramyocardial EphrinA1-Fc or IgG-Fc at LAD occlusion and subsequently reperfused for 24hrs. Permeabilized cardiac muscle fibers were isolated from a portion of the infarct margin of the left ventricle to assess mitochondrial function and energetics. EphrinA1-Fc prevented the 30% loss (p<0.005) in mitochondrial respiratory capacity in the absence of changes in H 2 O 2 emitting potential. Moreover, using a titrated creatine kinase energetic clamp, we found that EphrinA1-Fc improved the mitochondrial thermodynamic force-flow relationship by increasing OXPHOS conductance (+55% vmax, p<0.005), without changes in ADP affinity. Another small portion of the infarct margin was fixed and processed for transmission electron microscopy imaging which revealed that EphrinA1-Fc treatment prevented the MI-induced loss of sarcomere alignment and mitochondrial organization along the Z disks, as well as disorganization of the cristae. There was also significantly less lipid droplet accumulation and preservation of intermitochondrial junctions. Morphometric analyses of mitochondria are in progress as are Western blot assays to identify the proteins involved. Myocardial strain analyses are also underway to explore and pinpoint regional changes in contractile function. Collectively, these data demonstrate that intramyocardial injection of EphrinA1-Fc attenuates I/R injury by preserving the mitochondrial network structure and bioenergetic capacity. The robust cardioprotection afforded by ephrinA1-Fc offers promise as a novel therapeutic for the treatment of acute MI.