| Background: Acute myocardial infarction is a leading cause of morbidity and mortality worldwide.Although primary percutaneous coronary intervention is effective to restore coronary blood flow,myocardial reperfusion after an ischemic episode triggers a second wave of lethal injury that currently lacks an effective clinical therapy.It has been well established that exercise training not only reduces risk factors of cardiovascular diseases but also provides direct endogenous cardiovascular protection.However,the mechanisms underlying the cardioprotective effects of exercise are still unclear.Recently,exercise-induced humoral factors(exerkines),such as IL-6 and irisin,have attracted great attention as they are reported to exert paracrine or endocrine effects to orchestrate the multisystemic effects of exercise.The search for exerkines,therefore,has been the focus of interest in recent years,but still many questions remain unanswered,as to the nature and the mechanisms of actions of exerkines.Exosomes that are endogenous small(30-100 nm)membrane vesicles secreted by most cell types have been shown to play important roles in mediating cell-to-cell communications and crosstalks between organs via transmitting a variety of signaling molecules in their payload including proteins,m RNAs,and non-coding RNAs(such as mi RNAs)to target cells.As such,exosomes emerge as novel elements that promote intercellular communication.Recently,it was reported that circulating exosomes increased immediately after one bout of exercise in humans.Importantly,Whitham et al observed an increase of over 300 proteins in plasma exosomes following an acute bout of exercise in healthy humans.These findings indicate that exercise-induced release of exosomes into circulation may play a role in exercise-conferred systemic adaptions.Given the fact that circulating exosomes reflect physiological and pathophysiological condition of the body and are of striking numbers in the order of 1010 ml-1,there is a crucial need to explore the functional effects of exercise-derived circulating exosomes.However,little is known about the role of exosomes in long-term exercise-induced cardioprotection.Aims: 1.To investigate the effects of long-term aerobic exercise on circulating exosomes.2.To determine the role of exercise-derived circulating exosomes in cardioprotection and identify the molecular mechanisms involved.Methods: 1.Circulating exosomes were isolated from the plasma of volunteers with or without exercise training and rats subjected to 4 weeks of swimming exercise or kept as sedentary control 24 h after the last training session using a standard protocol of serial,differential centrifugation,and ultracentrifugation steps.2.Transmission electron microscopy and Western blot were used to identify isolated exosomes.Absolute size distribution of exosomes was examined using nanoparticle tracking analysis(NTA).3.In vivo myocardial ischemia/reperfusion(MI/R,30 min/24 h)and in vitro neonatal rat ventricular cardiomyocytes hypoxia/reoxygenation(H/R,12 h/3 h)models were used to evaluate the cardioprotective actions of circulating exosomes.4.A mi RNA profiling assay between circulating exosomes of exercised and sedentary rats was performed using Illumina Hi Seq 2500 high-throughput sequencing.Quantitative real time-PCR(q RT-PCR)was used to examine expressions of mi RNAs.5.Western blot and luciferase reporter assay were used to identify target genes of selected mi RNA.6.Serotype 9 adeno-associated virus(AAV9)carrying specific sequence targeting selected mi RNA and selected mi RNA agomir were used to inhibit or increase mi R-342-5p in hearts in vivo.7.All of the statistical tests were performed with the Graph Pad Prism software version 5.0.Results: 1.Compared with sedentary controls,exercised rats exhibited reduced myocardial infarct size and serum lactate dehydrogenase(LDH)level(p < 0.01)in response to MI/R,indicating that 4 weeks' exercise training provides cardioprotection against MI/R injury.2.No significant changes in total circulating exosome level were observed in exercised rats or humans compared with the sedentary control 24 h after the last training session.However,preincubation with exosomes isolated from plasma of exercised rats(Exe-exo),but not exosomes isolated from plasma of sedentary rats(Sed-exo),for 48 h attenuated H/R-induced cardiomyocyte injury as indicated by reducedcardiomyocytes apoptosis and LDH release,and increased cardiomyocyte viability compared with vehicle control.3.The infarct size relative to the area at risk was significantly smaller in Exe-exo-treated rats compared with that in vehicle-treated ones 24 h following MI/R(35.6 ± 4.3% vs.58.6 ± 3.5%,p < 0.01;n = 11),concomitantly with decreased serum LDH and improved cardiac function as evidenced by increased ±LVdp/dtmax and decreased left ventricular end-diastolic pressure.No significant differences in myocardial infarct size,serum LDH level or cardiac function were observed between rats pretreated with Sed-exo or vehicle.4.A total of 14 differentially expressed mi RNAs(fold change > 2.0;p < 0.05)were identified between Exe-exo and Sed-exo using Illumina Hi Seq 2500 high-throughput sequencing and 12 of them were further confirmed by q RT-PCR analysis.5.The functional effects of these differentially expressed mi RNAs on cardiomyocytes were evaluated in a model of H/R and mi R-342-5p stood out as a particularly intriguing candidate as it significantly reduced apoptosis,decreased LDH release and increased cell viability in cardiomyocytes subjected to H/R.6.Plasma exosomal mi R-342-5p level increased by 80% in student athletes compared with that in untrained control students.7.Bioinformatic analysis,Western blot and luciferase reporter assay indicated that pro-apoptotic genes Caspase9 and Jnk2 were target genes of mi R-342-5P in cardiomyocytes.Preincubation with mi R-342-5p mimic significantly inhibited H/R-induced cleaved-caspase3,cleaved-caspase9 and phospho(p)-Jnk2(Thr183/Tyr185)in cardiomyocytes.Taken together,these data suggest that mi R-342-5p exerts anti-apoptotic effects via targeting Caspase9 and Jnk2 in cardiomyocytes.8.Phosphorylation of Akt(Ser473)in cardiomyocytes was elevated by mi R-342-5p mimic and decreased by mi R-342-5p inhibitor,while the phosphorylation of Erk1/2(Thr202/Tyr204)was not significantly changed.Bioinformatic analysis,Western blot and luciferase reporter assay indicated that phosphatase gene Ppm1 f was target genes of mi R-342-5P in cardiomyocytes.In addition,down-regulating Ppm1 f protein expression by si RNA silencing not only significantly elevated Akt phosphorylation level but also decreased cardiomyocyte apoptosis induced by H/R.Taken together,these data suggest that mi R-342-5p enhances survival signaling(p-Akt,Ser473)via targeting phosphatase gene Ppm1 f in cardiomyocytes.9.Long-term exercise increased both matured mi R-342-5p and pre-mi R-342-5p in the heart,skeletal muscle,aorta,liver,adipose,kidney and spleen of rats,especially in the aorta,while pre-mi R-342-5p was undetectable in circulating exosomes.The results indicated that the increased circulating exosomal mi R-342-5p following exercise training were from multiple tissues.10.Long-term exercise training increased cardiac mi R-342-5p,concomitantly with decreased cardiac Caspase9,Jnk2 and Ppm1 f expressions,and increased phosphorylation of Akt;these changes were attenuated by AAV-anti-mi R-342-5p treatment one week prior to swim training.In addition,reduced myocardial infarct size and cardiomyocyte apoptosis were observed in exercised MI/R rats,which were significantly blunted by AAV-anti-mi R-342-5p treatment.Moreover,mi R-342-5p agomir treatment 48 h prior to MI/R significantly increased mi R-342-5p level in rat hearts and reduced myocardial infarct size compared with control.Conclusions: 1.Circulating exosomes 24 h after long-term exercise,though whose level has no significant change,confer protective effects against MI/R injury.2.Exercise-derived exosomal mi R-342-5p is a major cardioprotective molecule that inhibits apoptotic signal(Caspase9 and Jnk2)and enhances survival signal(p-Akt)in cardiomyocytes.These findings reveal a novel mechanism underlying long-term exercise-afforded cardioprotection with exosomal mi R-342-5p as a novel cardioprotective exerkine,and highlight the therapeutic potential of mi R-342-5p in the prevention and rehabilitation of cardiac diseases. |
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