PROJECT SUMMARY/ABSTRACT Novel approaches to reduce infarct size (IS) have stalled in translation. A major obstacle is the fact that the clinical care paradigm for acute myocardial infarction (AMI) has one dominant objective: to open the infarct- related artery as quickly as possible. Only after flow is restored might the treating physician take time to think about adjunctive therapies, but by then the window of opportunity to recruit classical cardioprotection has most likely closed, or at least narrowed significantly. Here we characterize the novel phenomenon of cellular postconditioning: cell therapy delivered 30 min post-reperfusion (or even longer; defining the precise timing is an aim of this R01) can reduce the extent of lethal injury and improve functional recovery. The timing is compatible with standard clinical practice in that the decision to treat can be delayed until after the artery has been opened, if an off-the-shelf product is available. Allogeneic cardiosphere-derived cells (CDCs) are available for immediate use and are currently in phase 2 clinical testing for chronic MI. Here we show preliminary data, from both rats and pigs, that CDCs and their exosomes are cardioprotective when given with a reasonable delay after reflow in AMI. We looked at 48 hr structural and functional endpoints to ensure a focus on acute cardioprotection; otherwise it is impossible to exclude some contribution from longer-term regenerative effects of CDCs, which are evident only weeks after treatment. In pigs subjected to 90 mins of ischemia and 30 mins of reflow, the intracoronary infusion of CDCs decreased IS and also reduced the extent of microvascular obstruction. In rats we see large decreases of IS when CDCs are administered 20 mins after a 45 min ischemic episode, a finding now independently confirmed by a major unaffiliated laboratory. We further show preliminary data that implicate macrophages as key players in the mechanism of cardioprotection. The major focus here is on defining the mechanisms whereby CDCs reduce IS. We test the following overarching hypothesis: CDCs secrete exosomes which modify macrophages so as to enhance efferocytosis. Precisely how macrophages are modulated by CDCs (are RNAs transferred by exosomes involved?), and how macrophages mitigate lethal injury, are major questions addressed here. Although we focus on the cardioprotective effects of CDCs, we expect these to synergize with the regenerative effects, augmenting overall benefit. Two well-established animal models will be subjected to AMI: rats for mechanistic studies, and Yucatan minipigs for translational studies. The role of efferocytosis will be probed both by novel in vitro co-culture assays of macrophages, neutrophils, and stressed cardiomyocytes, as well as by in vivo experiments in transgenic rats to quantify efferocytosis. The work has the potential to elucidate the cardioprotective mechanisms of cell therapy.