Hui Luo1,2, Xiaohui Li3, Tangzhiming Li1, Lin Zhao1, Jingni He1, Lihuang Zha1, Qiangqiang Qi1, Zaixin Yu1. 1. Department of Cardiology, Xiangya Hospital, Central South University, 87 Xiangya Road, Changsha, Hunan, China. 2. Department of Cardiology, The First Hospital of Changsha, Hunan Changsha, China. 3. Department of Pharmacology, School of Pharmaceutical Sciences, Central South University, Changsha, Hunan, China.
Abstract
AIMS: A recent study reported the cardioprotective effects mediated by cardiac fibroblasts (CFs) during acute phase of ischaemia-reperfusion injury (IRI). Little is known about whether exosomes/microvesicles mediate this beneficial effect and whether ischaemia post-conditioning (Postcon) can regulate this process. Here, we aimed to investigate the cardioprotective effect of CFs-exosomes/microvesicles and whether Postcon can regulate this effect. METHODS AND RESULTS: By using 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 analysed 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 upregulation 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 upregulation 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 Ras-related protein Rap-2c (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 IRI and Postcon can enhance this effect by upregulating the expression of CFs exosomes/microvesicles miR-423-3p, which targets the downstream effector RAP2C. Published on behalf of the European Society of Cardiology. All rights reserved.
AIMS: A recent study reported the cardioprotective effects mediated by cardiac fibroblasts (CFs) during acute phase of ischaemia-reperfusion injury (IRI). Little is known about whether exosomes/microvesicles mediate this beneficial effect and whether ischaemia post-conditioning (Postcon) can regulate this process. Here, we aimed to investigate the cardioprotective effect of CFs-exosomes/microvesicles and whether Postcon can regulate this effect. METHODS AND RESULTS: By using 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 analysed 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 upregulation 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 upregulation 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 Ras-related protein Rap-2c (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 IRI and Postcon can enhance this effect by upregulating the expression of CFs exosomes/microvesicles miR-423-3p, which targets the downstream effector RAP2C. Published on behalf of the European Society of Cardiology. All rights reserved.