Cardioprotection by remote ischemic preconditioning of the rat heart is mediated by extracellular vesicles

Abstract

Remote ischemic preconditioning (RIPC) of the heart is exerted by brief ischemic insults affected on a remote organ or a remote area of the heart before a sustained cardiac ischemia. To date, little is known about the inter-organ transfer mechanisms of cardioprotection by RIPC. Exosomes and microvesicles/microparticles are vesicles of 30-100nm and 100-1000nm in diameter, respectively (collectively termed extracellular vesicles [EVs]). Their content of proteins, mRNAs and microRNAs, render EVs ideal conveyors of inter-organ communication. However, whether EVs are involved in RIPC, is unknown. Therefore, here we investigated whether (1) IPC induces release of EVs from the heart, and (2) EVs are necessary for cardioprotection by RIPC. Hearts of male Wistar rats were isolated and perfused in Langendorff mode. A group of donor hearts was exposed to 3x5-5min global ischemia and reperfusion (IPC) or 30min aerobic perfusion, while coronary perfusates were collected. Coronary perfusates of these hearts were given to another set of recipient isolated hearts. A group of recipient hearts received IPC effluent depleted of EVs by differential ultracentrifugation. Infarct size was determined after 30min global ischemia and 120min reperfusion. The presence or absence of EVs in perfusates was confirmed by dynamic light scattering, the EV marker HSP60 Western blot, and electron microscopy. IPC markedly increased EV release from the heart as assessed by HSP60. Administration of coronary perfusate from IPC donor hearts attenuated infarct size in non-preconditioned recipient hearts (12.9+/-1,6% vs. 25.0+/-2.7%), similarly to cardioprotection afforded by IPC (7.3+/-2.7% vs. 22.1+/-2.9%) on the donor hearts. Perfusates of IPC hearts depleted of EVs failed to exert cardioprotection in recipient hearts (22.0+/-2.3%). This is the first demonstration that EVs released from the heart after IPC are necessary for cardioprotection by RIPC, evidencing the importance of vesicular transfer mechanisms in remote cardioprotection.