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. 2024 Feb 26:7:0327.
doi: 10.34133/research.0327. eCollection 2024.

Exercise-Induced miR-210 Promotes Cardiomyocyte Proliferation and Survival and Mediates Exercise-Induced Cardiac Protection against Ischemia/Reperfusion Injury

Yihua Bei  1   2   3 , Hongyun Wang  1   2   3 , Yang Liu  4 , Zhuhua Su  1   2   3 , Xinpeng Li  2   3   5 , Yujiao Zhu  1   2   3 , Ziyi Zhang  1   2   3 , Mingming Yin  1   2 , Chen Chen  1   2 , Lin Li  1   2 , Meng Wei  1   2 , Xiangmin Meng  1 , Xuchun Liang  1   2 , Zhenzhen Huang  1   2 , Richard Yang Cao  6 , Lei Wang  7 , Guoping Li  8 , Dragos Cretoiu  9   10 , Junjie Xiao  1   2   3
Affiliations

Exercise-Induced miR-210 Promotes Cardiomyocyte Proliferation and Survival and Mediates Exercise-Induced Cardiac Protection against Ischemia/Reperfusion Injury

Yihua Bei et al. Research (Wash D C). .

Abstract

Exercise can stimulate physiological cardiac growth and provide cardioprotection effect in ischemia/reperfusion (I/R) injury. MiR-210 is regulated in the adaptation process induced by exercise; however, its impact on exercise-induced physiological cardiac growth and its contribution to exercise-driven cardioprotection remain unclear. We investigated the role and mechanism of miR-210 in exercise-induced physiological cardiac growth and explored whether miR-210 contributes to exercise-induced protection in alleviating I/R injury. Here, we first observed that regular swimming exercise can markedly increase miR-210 levels in the heart and blood samples of rats and mice. Circulating miR-210 levels were also elevated after a programmed cardiac rehabilitation in patients that were diagnosed of coronary heart diseases. In 8-week swimming model in wild-type (WT) and miR-210 knockout (KO) rats, we demonstrated that miR-210 was not integral for exercise-induced cardiac hypertrophy but it did influence cardiomyocyte proliferative activity. In neonatal rat cardiomyocytes, miR-210 promoted cell proliferation and suppressed apoptosis while not altering cell size. Additionally, miR-210 promoted cardiomyocyte proliferation and survival in human embryonic stem cell-derived cardiomyocytes (hESC-CMs) and AC16 cell line, indicating its functional roles in human cardiomyocytes. We further identified miR-210 target genes, cyclin-dependent kinase 10 (CDK10) and ephrin-A3 (EFNA3), that regulate cardiomyocyte proliferation and apoptosis. Finally, miR-210 KO and WT rats were subjected to swimming exercise followed by I/R injury. We demonstrated that miR-210 crucially contributed to exercise-driven cardioprotection against I/R injury. In summary, this study elucidates the role of miR-210, an exercise-responsive miRNA, in promoting the proliferative activity of cardiomyocytes during physiological cardiac growth. Furthermore, miR-210 plays an essential role in mediating the protective effects of exercise against cardiac I/R injury. Our findings suggest exercise as a potent nonpharmaceutical intervention for inducing miR-210, which can alleviate I/R injury and promote cardioprotection.

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Conflict of interest statement

Competing interests: The authors declare that they have no competing interests.

Figures

Fig. 1.
Fig. 1.
MiR-210 expression is increased in the heart and blood circulation upon exercise training. (A and B) qRT-PCR of miR-210 expression in the rat (A, n = 4) and mouse (B, n = 6) heart tissues after regular swimming exercise regimen. (C and D) qRT-PCR of miR-210 expression in the serum of rat (C, n = 4) and mouse (D, n = 6) after regular swimming exercise regimen. (E) qRT-PCR of miR-210 expression in the serum of mice at 8 days after finishing the swimming regimen (n = 6). Mice performed a 3-week swimming regimen followed by sham surgery. Serum samples were collected at 8 days after swimming exercise. (F) qRT-PCR of miR-210 expression in the serum of mice with cardiac ischemia/reperfusion (I/R) injury at 8 days after finishing the swimming regimen (n = 6). Mice performed a 3-week swimming regimen followed by cardiac I/R injury for 7 days. (G) qRT-PCR of miR-210 expression in human serum samples from patients with coronary heart diseases before and after 8 weeks of cardiac rehabilitation (n = 20). For statistical analysis, unpaired Student’s t test was performed for (A) to (D) and (F). Mann–Whitney U test was performed for (E). Paired Student’s t test was performed for (G) to compare the difference of human serum miR-210 expression levels before and after the cardiac rehabilitation program. Data are mean ± SD. *P < 0.05; **P < 0.01.
Fig. 2.
Fig. 2.
MiR-210 is involved in exercise-induced cardiomyocyte proliferative activity in vivo. (A) Schematic of rat swimming exercise model illustrating that adult wild-type (WT) versus miR-210 knockout (KO) rats took a swimming regimen or stayed sedentary for 8 weeks. (B) qRT-PCR of miR-210 expression in the heart (n = 4 to 5). (C) The heart weight (HW), body weight (BW), and HW relative to BW or tibia length (TL) were shown (n = 4 to 5). (D) Wheat germ agglutinin (WGA) staining to analyze cross-sectional myocardium area (n = 4 to 5). Scale bar, 50 μm. CM, cardiomyocyte. (E) Immunofluorescent staining for Ki67 and α-actinin for analyzing cardiomyocyte proliferative activity (n = 4 to 5). Scale bar, 100 μm. An enlarged area was shown below. Scale bar, 10 μm. (F) qRT-PCR of ANP and BNP expressions in the heart (n = 4 to 5). For statistical analysis, robust two-way ANOVA followed by post hoc pairwiseMedianTest was performed for (B) and (C) (HW/BW ratio). Two-way ANOVA test followed by Tukey post hoc test was performed for (C) (HW, BW, and HW/TL ratio) to (F). Data are mean ± SD. *P < 0.05; **P < 0.01; ***P < 0.001.
Fig. 3.
Fig. 3.
MiR-210 enhances proliferation but not cell size of cardiomyocytes in vitro. (A) qRT-PCR of miR-210 expression in neonate cardiomyocytes with transfection of miR-210 mimic or inhibitor (n = 4). NRCM, neonatal rat cardiomyocytes; NC, negative controls. (B and C) EdU/α-actinin (B, n = 6) or Ki67/α-actinin (C, n = 6) immunofluorescent stainings in NRCM with miR-210 overexpression or inhibition. Scale bar, 100 μm. For statistical analysis, Mann–Whitney U test was performed for (A) (miR-210 expression in NRCM transfected with miR-210 mimic) and (B) (EdU-positive CM in NRCM transfected with miR-210 inhibitor). Data in other figures were analyzed by unpaired Student’s t test. Data are mean ± SD. *P < 0.05; **P < 0.01; ns, not significant.
Fig. 4.
Fig. 4.
MiR-210 protects cardiomyocytes against OGD/R-induced apoptosis in vitro. (A) qRT-PCR of miR-210 expression in neonate cardiomyocytes stressed with oxygen glucose deprivation/reperfusion (OGD/R) (n = 6). (B) TUNEL labeling in miR-210 mimic or inhibitor-transfected NRCMs under OGD/R stress (n = 4). Scale bar, 100 μm. (C) Western blot for Bax, Bcl-2, and caspase-3 in miR-210 mimic or inhibitor-transfected NRCM upon OGD/R stress (n = 6). For statistical analysis, unpaired Student’s t test was performed for (A) and (B). One-way ANOVA test was performed for (C). Data are mean ± SD. **P < 0.01; ***P < 0.001.
Fig. 5.
Fig. 5.
MiR-210 regulates proliferation of cardiomyocytes by targeting CDK10. (A) Western blot for CDK10 in NRCMs with miR-210 overexpression or inhibition (n = 6). (B) Luciferase reporter assay to evaluate the direct binding of miR-210 with the 3′UTR of CDK10 (n = 6). (C) EdU/α-actinin staining in neonate cardiomyocytes transfected with miR-210 inhibitor and/or CDK10 siRNA (n = 4). Scale bar, 200 μm. (D) TUNEL/α-actinin staining in neonate cardiomyocytes transfected with miR-210 inhibitor and/or CDK10 siRNA under OGD/R stress (n = 4). Scale bar, 100 μm. (E) Western blot for CDK10 in the rat hearts (n = 4 to 5). For statistical analysis, unpaired Student’s t test was performed for (A) and (E). Two-way ANOVA test followed by Tukey post hoc test was performed for (B) to (D). Data are mean ± SD. *P < 0.05; **P < 0.01; ***P < 0.001.
Fig. 6.
Fig. 6.
EFNA3 mediates the effect of miR-210 on cardiomyocyte proliferation and apoptosis. (A) Western blot for EFNA3 in NRCMs with miR-210 overexpression or inhibition (n = 6). (B) Luciferase reporter assay to evaluate the direct binding of miR-210 with the 3′UTR of EFNA3 (n = 6). (C) EdU/α-actinin staining in neonate cardiomyocytes transfected with miR-210 inhibitor and/or EFNA3 siRNA (n = 6). Scale bar, 100 μm. (D) TUNEL/α-actinin staining in neonate cardiomyocytes transfected with miR-210 inhibitor and/or EFNA3 siRNA under OGD/R stress (n = 4). Scale bar, 100 μm. (E) Western blot for EFNA3 in the rat hearts (n = 4 to 5). For statistical analysis, unpaired Student’s t test was performed for (A) and (E). Two-way ANOVA test followed by Tukey post hoc test was performed for (B) and (D). Robust two-way ANOVA followed by post hoc pairwiseMedianTest was performed for (C). Data are mean ± SD. **P < 0.01; ***P < 0.001.
Fig. 7.
Fig. 7.
MiR-210 functions in human embryonic stem cell-derived cardiomyocytes. (A) qRT-PCR of miR-210 expression in human embryonic stem cell-derived cardiomyocytes (hESC-CMs) with miR-210 overexpression or inhibition (n = 3). (B) EdU/α-actinin immunofluorescent staining in hESC-CM with transfection of miR-210 mimic or inhibitor (n = 6). Scale bar, 100 μm. (C) TUNEL staining in miR-210 mimic-transfected hESC-CM under OGD/R stress (n = 6). Scale bar, 100 μm. (D) qRT-PCR of CDK10 and EFNA3 expressions in hESC-CM with transfection of miR-210 mimic or inhibitor (n = 3). For statistical analysis, unpaired Student’s t test was performed for (A), (B), and (D). Two-way ANOVA test followed by Tukey post hoc test was performed for (C). Data are mean ± SD. *P < 0.05; **P < 0.01; ***P < 0.001.
Fig. 8.
Fig. 8.
MiR-210 is essential to mediate exercise-driven cardiac protection against I/R injury. (A) Schematic of rat exercise model followed by ischemia/reperfusion (I/R) injury illustrating that adult WT and miR-210 KO rats received swimming exercise or stayed sedentary for 8 weeks, and then were subjected to I/R surgery. (B) qRT-PCR of cardiac miR-210 expression in the swimming and control (CTL) group after I/R surgery (n = 6). (C) 2,3,5-Triphenyltetrazolium chloride (TTC) staining of rat hearts showing the area at risk (AAR)/left ventricle weight (LV) ratio and the infarct size (INF)/AAR ratio (n = 8 to 10). (D) TUNEL staining in cardiac tissues after I/R injury (n = 6 to 7). Scale bar, 50 μm. (E) Immunofluorescent staining for Ki67/α-actinin for analyzing cardiomyocyte proliferative activity (n = 6 to 7). Scale bar, 50 μm. An enlarged area was shown below. Scale bar, 5 μm. (F) Western blot for CDK10 and EFNA3 in the rat hearts (n = 6). For statistical analysis, two-way ANOVA test followed by Tukey post hoc test was performed for (B) to (F). Data are mean ± SD. *P < 0.05; **P < 0.01; ***P < 0.001.
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