Abstract 16479: Remote Ischemic Preconditioning: Do Exosomes Play a Requisite Role in Evoking Cardioprotection?
Remote ischemic preconditioning (RIPC) is the phenomenon whereby brief episodes of ischemia-reperfusion applied in distant tissues or organs render the heart resistant to a subsequent sustained ischemic insult. The hallmark of RIPC is inter-organ communication, with recent evidence suggesting that exosomes may serve as the vector for the transport of protective factor(s) from the site of the distant trigger to the cardiomyocytes. To test this hypothesis, rats underwent a standard RIPC stimulus: i.e., 4 5-min episodes of bilateral femoral artery occlusion. Serum (~3 mL per rat) was harvested immediately after RIPC; 1 mL was stored at -80°C, and 2 mL was processed for exosome isolation by prolonged ultracentrifugation (12 hours at 300,000xg). Aliquots of the pellet and supernatant were probed (by immunoblotting) for expression of exosome markers including HSP60 and flotillin. Most importantly, the cardioprotective efficacy of: 1) RIPC serum, 2) the pellet, reconstituted in buffer, 3) supernatant-bottom layer, 4) supernatant-top layer and 5) buffer alone was assessed by transfer of volume-matched aliquots to cultured HL-1 cardiomyocytes. After 1 hour of pretreatment, cells were subjected to 2.5 hours simulated ischemia + 24 hours reoxygenation and % cell death was quantified by Trypan blue staining. As expected, RIPC serum was protective: mean cell death was 34+5%* vs 48+5% in buffer-controls (*p<0.05). As further expected, the exosome-depleted top layer of supernatant failed to confer protection. However, in contrast to the ‘exosome hypothesis’, RIPC with exosome-rich aliquots did not yield consistent cardioprotection: both the pellet and the supernatant-bottom layer had robust expression of HSP60 and flotillin, yet only the supernatant-bottom attenuated cell death (32+3%*; Figure). Thus, while transport of protective factor(s) via exosomes may play a role in RIPC, our results reveal that exosomes per se are not sufficient to evoke remote cardioprotection.
Author Disclosures: J. Wider: Research Grant; Modest; Supported by NIH T32. A.R. Kulek: None. V.V. Undyala: None. K. Przyklenk: Research Grant; Significant; NIH R01.
- © 2015 by American Heart Association, Inc.