Heart transplantation (HTx) is the ultimate treatment for end-stage heart failure, but its use is limited by the availability of donor hearts. As a result, a substantial number of HTx candidates die awaiting transplant. Donation after circulatory death (DCD) offers a promising additional source for HTx. However, its adoption is hindered due to obligatory warm ischemic injury during death process and subsequent reperfusion injury, as well as unsuitable preservation modality during transport. The EVHP provides normothermic perfusion platform for satisfactory preservation of DCD hearts and offers the possibility to deliver therapeutic agents that improve resuscitation of DCD hearts prior to transplantation. Given stem cell-derived paracrine protection following myocardial ischemia, our goal is to determine potential efficacy of post-ischemic administration of stem cell secretions (conditioned medium [CM], extracellular vesicles [EVs], or engineered EVs) using the EVHP on recovery of DCD hearts prior to transplantation, thereby increasing the number of available donor hearts. Our recent studies have reported that adding MSC-CM or MSC-EVs to storage solution protected heart graft performance after 6h-cold storage using an in vivo mouse HTx model and MSC-CM restored cold ischemia- impaired cardiac transcriptome profile. These results suggest therapeutic potential of MSC secretions on donor heart preservation. Our published work and preliminary data have also shown: 1) using human MSCs or their CM improved heart functional recovery in the EVHP following acute ischemia/reperfusion (I/R); 2) MSC-CM prevented mitochondrial damage in donor hearts from cold ischemia and restored clock gene period 2 (Per2, with a role in mitochondrial metabolic regulation); 3) warm ischemia resulted in abnormal mitochondrial structure in mouse and human DCD hearts, with decreased Per2, and 4) Per2 knockdown worsened mitochondrial injury and abolished MSC CM-mediated mitochondrial preservation in myocytes during stress. Collectively, we hypothesize that post-ischemic administration of MSC secretions using the EVHP ameliorates warm ischemia-induced functional deterioration of DCD hearts via Per2-elicited mitochondrial improvement. We expect that a novel approach using the engineered EVs to deliver Per2 will promote mitochondrial recovery and myocardial resuscitation in DCD hearts. We propose two aims: 1. test the hypothesis that ex vivo application of MSC secretions using the EVHP ameliorates warm ischemia-induced myocardial damage via mitochondrial preservation in DCD hearts; and 2. determine the role of Per2 in improving mitochondrial performance in DCD hearts and demonstrate beneficial effects of Per2-loaded MSC-EVs on resuscitation of DCD hearts. The translational potential of MSC-derived therapy and the engineered EVs will also be assessed in human-like porcine DCD hearts. The results from this study can provide foundation for potential clinical trials in that MSC secretio...