Exosomes/microvesicles microRNA-423-3p derived from cardiac fibroblasts mediates the cardioprotective effects of ischemic postconditioning.

Aims: A recent study reported the cardioprotective effects mediated by cardiac fibroblasts (CFs) during acute phase of ischemia-reperfusion injury. Little is known about whether exosomes/microvesicles mediate this beneficial effect and whether ischemia postconditioning (Postcon) can regulate this process. Here, we aimed to investigate the cardioprotective effect of CFs-exosomes/microvesicles and whether Psotcon can regulates this effect.

Methods and Results: By using a transwells co-culture system, we found that hypoxia-reoxygenation (H/R) significantly increased the exosomes/microvesicles secretion of CFs and CFs protected H9C2 cells against H/R injury and Postcon could amplify these effects. Inhibition of CFs exosomes/microvesicles secretion led to a significant abrogation on the amplified protective effect of H/R-Postcon. We further demonstrated that Postcon enhanced the cardioprotective effect of CFs-exosomes/microvesicles both in vitro and in vivo. To detect the underlying mechanism, exosomes/microvesicles microRNAs were analyzed by RNA sequencing and quantitative polymerase chain reaction, our results revealed that miR-423-3p expression was selectively enhanced by Postcon in CFs exosomes/microvesicles. By co-culture H9C2 cells with CFs-exosomes/microvesicles enriching with miR-423-3p, we demonstrated that H/R-Postcon exerted cardioprotective effects by up-regulation of miR-423-3p in CFs-exosomes/microvesicles. RNA-fluorescence in situ hybridization and qPCR demonstrated that the decreasing of miR-423-3p is closely related to IRI, by inhibited miR-423-3p expression with its antagomir in vivo, we demonstrated that miR-423-3p plays an essential mediate role in I/R-Postcon-induced cardioprotection against I/R in vivo, Postcon may exert cardioprotective effect by up-regulation of miR-423-3p in CFs exosomes/microvesicles. Gain- and loss-of-function approaches suggested that rescuing the down-regulated miR-423-3p might be a potential strategy to protect the cardiomyocytes against H/R. Using computational predictions tools and luciferase reporter assay, we demonstrated that miR-423-3p regulates the expression of RAP2C in H9C2 cells, and knockdown of RAP2C by siRNA obviously increased cell viability and reduced apoptosis in H9C2 cells under H/R.

Conclusions: In conclusion, we demonstrated, for the first time, that CFs participate in cardioprotective effects via an exosomes/microvesicles pathway during the acute phase of ischemia-reperfusion injury and Postcon can enhance this effect by upregulating the expression of CFs exosomes/microvesicles miR-423-3p, which targets the downstream effector RAP2C.