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Exercise training, in addition to reducing cardiovascular risk factors, also confers direct cardioprotection against myocardial ischemia/reperfusion (MI/R) injury in animal models and has been associated with improved survival following a heart attack in humans. However, the underlying mechanisms are still unclear. Exosomes are nanometer-sized vesicles secreted by multiple cell types into the blood, where they can transmit signals throughout the body. This study sought to evaluate the functional effects of exercise-derived circulating exosomes in the context of MI/R injury and identify the molecular mechanisms involved.
Circulating exosomes were isolated from the serum of volunteers with or without exercise training and rats subjected to 4 weeks of swimming exercise or sedentary littermates. In vivo MI/R and in vitro neonatal rat ventricular cardiomyocytes hypoxia/reoxygenation (H/R) models were used to evaluate the cardioprotective actions of circulating exosomes. A miRNA profiling assay between circulating exosomes of exercised and sedentary rats was performed using Illumina HiSeq 2500 high-throughput sequencing. Western blot and luciferase reporter assay were used to identify target genes of miR-342-5p. Rats were intramyocardially injected with serotype 9 adeno-associated virus (AAV9) carrying specific sequence targeting miR-342-5p one week prior to 4 weeks of swimming exercise to inhibit miR-342-5p in hearts in vivo.
Nanoparticle tracking analysis and Western blot showed no significant changes in total level of circulating exosomes in long-term exercise rats compared with sedentary ones. However, exercise-derived circulating exosomes afforded profound cardioprotective effects in MI/R rats as evidenced by reduced infarct size (from 57.8 ± 2.7% to 31.6 ± 4.0% in relation to the area at risk) and improved cardiac function. miRNA array and qRT-PCR analysis identified 12 differentially expressed miRNAs in exercise-derived circulating exosomes from exercised rats and rowing athletes, and miR-342-5p stood out as the most potent cardioprotective molecule in the context of H/R injury. Moreover, inhibition of miR-342-5p almost abolished the cardioprotective effects of exercise-derived circulating exosomes. More importantly, in vivo inhibition of miR-342-5p with AAV9 attenuated exercise-afforded cardioprotective effects in MI/R rats. Mechanistically, miR-342-5p significantly inhibited the apoptotic signaling via targeting Caspase9 and Jnk2, and enhanced the survival signaling (p-Akt) via targeting phosphatase Ppm1f in cardiomyocytes.
Our findings reveal a novel endogenous cardioprotective mechanism that exercise-derived circulating exosomes protect the heart against MI/R injury via miR-342-5p.