Normoxia Cardiac Fibroblasts-derived Exosomes Promote Cardiomyocyte Apoptosis After Anoxia Reoxygenation

Background and ObjectiveIn myocardial ischemia-reperfusion injury,cardiomyocytes,endothelial cells,fibroblasts and neutrophils in the ischemic zone,border zone,and remote area may play different roles.The autophagy of cardiomyocytes under starvation follows the law of the jungle,that is,healthy cells engulf unhealthy cells.Cells can communicate through mediators such as paracrine and exosomes.Therefore,we wonder what effect exosomes derived from non-ischemic fibroblasts have on cardiomyocytes.It is poorly understood that exosomes derived from fibroblasts alleviate or aggravate damage in myocardial ischemia-reperfusion injury.Fibroblasts are activated and highly proliferative after myocardial injury,becoming key components to support the ventricular wall.It has been reported that fibroblasts can protect or exacerbate myocardial damage by paracrine under pathological conditions.We found a lot of microRNAs related to cell survival have a pro-apoptotic effect by analyzing sequencing results of exosomes derived from fibroblasts.Therefore,we hypothesized that exosomes derived from fibroblasts under ischemia-reperfusion setting aggravates myocardial injury by carrying pro-apoptotic microRNAs into cardiomyocytes in the ischemic area.MethodsCardiac fibroblasts isolated from 1-2 days old SD rat were cultured and a sufficient amount of supernatant was collected for differential centrifugation.The marker proteins CD63,CD81 and TSG101 of exosomes were detected by Western blot,and the morphology,particle size and diameter were identified by transmission electron microscopy and nanoparticle tracking analysis system.PKH67 dye was used to specifically label exosomes,and exosomes were respectively added in normoxic and hypoxic cardiomyocytes.Immunofluorescence staining was performed to observe whether exosomes were taken up by cardiomyocytes.The observation and comparison of the number of apoptotic cardiomyocytes were performed by using TUNEL staining.Real-time quantitative PCR(qPCR)and Western blot were used to detect related apoptosis genes and proteins such as Caspase3,Bax and Bcl2.Bioinformatics analysis was carried out for miRNA expression profile in the exosomes derived from rat myocardial fibroblasts.The miRNA data GSE76175 was downloaded from the GEO database.The top 20 high expression miRNAs were selected.The target genes and related pathways were analyzed by using a variety of database at websites,such as Targetscan,Diana tools and Metascape,so as to explore the pathway mechanism of their role.The selected target genes were tested by Western blot and qPCR.ResultsWe extracted and characterized the exosomes from neonatal rat cardiac fibroblasts.Co-culture of neonatal rat cardiomyocytes with exosomes showed thatunder hypoxia,the expression of Cltc protein in cardiomyocytes under hypoxia was up-regulated and exosome uptake was increased.TUNEL staining showed that the percentage of positive apoptotic cardiomyocytes in the anoxia/reoxygenation+exosome group(A/R+EXO)was significantly higher than the control group(14.65%vs 10.86%,P<0.05).Both qPCR and Western blot results showed that the pro-apoptotic gene and protein of Caspase 3 was significantly increased in the A/R+EXO group.By analyzing the sequencing data,it was found that there were 12 microRNAs related to apoptosis signaling pathway,of which 6 were enriched to promote cardiomyocyte apoptosis.Among these 6 miRNAs,5 had a common target gene Rap 1b,and they were rno-miR9a-5p,rno-miR-92b-3p,rno-miR-181a-5p,rno-miR-494-3p and rno-miR-708-5p.ConclusionExosomes derived from normoxia cardiac fibroblasts aggravate cardiomyocyte apoptosis in rats by a group of miRNA mediated-inhibition of Rap 1b gene.