RNA-seq studies on the porcine model of heart donation after circulatory death
Heart transplantation is a life-saving procedure but demand for donor hearts is not met by availability of suitable organs. New dynamic preservation strategies by means of Normothermic Machine Perfusion (NMP) open new avenues to preserve donor graft integrity even after circulatory death, but it is unknown how NMP cardiac cell biology during perfusion. Using our porcine model in the setting of a cardiac donation after circulatory death (DCD, whereby organ retrieval occurs after a cardiac arrest; https://pubmed.ncbi.nlm.nih.gov/30790198/), the objective of our project was to compare, in a randomized and blinded design, the time-dependent recovery of cardiac function and viability during 6 hours of ex-vivo heart perfusion in DCD hearts receiving standard perfusate with DCD hearts receiving EVHP perfusate supplemented with cardioprotective agents. To this purpose, left ventricular tissue was collected for RNA-seq studies. Because preclinical and clinical evidence suggests an association between circulating extracellular vesicles (EVs) -encapsulated miRNAs and cardiac injury (https://pubmed.ncbi.nlm.nih.gov/27383837/; https://pubmed.ncbi.nlm.nih.gov/34104945/ ) and because EVs may beneficially regulate cellular function in a paracrine fashion through the transfer of their cargo of small RNAs and proteins (https://pubmed.ncbi.nlm.nih.gov/29949381/; https://pubmed.ncbi.nlm.nih.gov/33599250/ ), another specific aim of this project was to profile the miRNA cargo present in EVs released from the porcine myocardium upon ischemia-reperfusion injury. EVs were isolated from the perfusate 1 hour after the onset of reperfusion using size exclusion chromatography. The presence of EVs was confirmed by western blotting using protein EV markers CD9, CD81, and TSG101.