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. 2021 Jun;42(6):921-931.
doi: 10.1038/s41401-020-0495-2. Epub 2020 Aug 24.

Melatonin promotes cardiomyocyte proliferation and heart repair in mice with myocardial infarction via miR-143-3p/Yap/Ctnnd1 signaling pathway

Wen-Ya Ma #  1 , Rui-Jie Song #  1 , Bin-Bin Xu #  1 , Yan Xu  1 , Xiu-Xiu Wang  1 , Hong-Yue Sun  1 , Shuai-Nan Li  1 , Shen-Zhen Liu  1 , Mei-Xi Yu  1 , Fan Yang  1 , Dan-Yu Ye  1 , Rui Gong  1 , Zhen-Bo Han  1 , Ying Yu  1 , Djibril Bamba  1 , Ning Wang  1 , Zhen-Wei Pan  1 , Ben-Zhi Cai  2   3   4
Affiliations

Melatonin promotes cardiomyocyte proliferation and heart repair in mice with myocardial infarction via miR-143-3p/Yap/Ctnnd1 signaling pathway

Wen-Ya Ma et al. Acta Pharmacol Sin. 2021 Jun.

Abstract

The neonatal heart possesses the ability to proliferate and the capacity to regenerate after injury; however, the mechanisms underlying these processes are not fully understood. Melatonin has been shown to protect the heart against myocardial injury through mitigating oxidative stress, reducing apoptosis, inhibiting mitochondrial fission, etc. In this study, we investigated whether melatonin regulated cardiomyocyte proliferation and promoted cardiac repair in mice with myocardial infarction (MI), which was induced by ligation of the left anterior descending coronary artery. We showed that melatonin administration significantly improved the cardiac functions accompanied by markedly enhanced cardiomyocyte proliferation in MI mice. In neonatal mouse cardiomyocytes, treatment with melatonin (1 μM) greatly suppressed miR-143-3p levels. Silencing of miR-143-3p stimulated cardiomyocytes to re-enter the cell cycle. On the contrary, overexpression of miR-143-3p inhibited the mitosis of cardiomyocytes and abrogated cardiomyocyte mitosis induced by exposure to melatonin. Moreover, Yap and Ctnnd1 were identified as the target genes of miR-143-3p. In cardiomyocytes, inhibition of miR-143-3p increased the protein expression of Yap and Ctnnd1. Melatonin treatment also enhanced Yap and Ctnnd1 protein levels. Furthermore, Yap siRNA and Ctnnd1 siRNA attenuated melatonin-induced cell cycle re-entry of cardiomyocytes. We showed that the effect of melatonin on cardiomyocyte proliferation and cardiac regeneration was impeded by the melatonin receptor inhibitor luzindole. Silencing miR-143-3p abrogated the inhibition of luzindole on cardiomyocyte proliferation. In addition, both MT1 and MT2 siRNA could cancel the beneficial effects of melatonin on cardiomyocyte proliferation. Collectively, the results suggest that melatonin induces cardiomyocyte proliferation and heart regeneration after MI by regulating the miR-143-3p/Yap/Ctnnd1 signaling pathway, providing a new therapeutic strategy for cardiac regeneration.

Keywords: Ctnnd1; Yap; cardiac repair; cardiomyocyte proliferation; luzindole; melatonin; miR-143-3p; myocardial infarction.

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Conflict of interest statement

The authors declare no competing interests.

Figures

Fig. 1
Fig. 1. Melatonin promotes cell cycle activity in neonatal cardiomyocytes.
a EdU staining was used to detect the proliferative effect of different concentrations of melatonin on cardiomyocytes. n = 5. b Mitosis was visualized with an antibody specific for phospho-histone H3 (pH3). n = 6. c The influence of melatonin on the proliferation of cultured cardiac fibroblasts, as determined by pH3 staining. n = 4. The data represent the mean ± SEM. The data were analyzed by one-way ANOVA followed by Dunnett’s post hoc test. **P < 0.01, ***P < 0.001 versus 0 nM.
Fig. 2
Fig. 2. Administration of melatonin enhances cardiac regenerative capacity in adult mice after MI.
a Cardiac function was measured by echocardiography (n = 9, 5, 6). The data were analyzed by one-way ANOVA followed by the Newman–Keuls multiple comparison test. b Representative heart sections stained with pH3 for evaluating mitosis. The Mann–Whitney two-tailed U test was used for nonparametric analysis. c Representative heart sections stained with Aurora B for evaluating cytokinesis. The Mann–Whitney two-tailed U test was used for nonparametric analysis. d WGA staining for determining the cross-sectional area of cells. The Mann–Whitney two-tailed U test was used for nonparametric analysis. The data represent the mean ± SEM, ***P < 0.001 versus the sham group, ##P < 0.05, ###P < 0.001 versus the MI group.
Fig. 3
Fig. 3. Melatonin reactivates cardiomyocyte proliferation by decreasing miR-143-3p expression.
a MiRNA expression levels were analyzed by qRT-PCR after treatment with melatonin. b The influence of miR-143-3p inhibitor on the proliferation of cultured cardiomyocytes, as determined by EdU and pH3 staining. n = 5. c, d Cardiomyocytes transfected with miR-143-3p mimic were fixed 48 h posttransfection and immunostained for EdU to evaluate DNA synthesis and for pH3 to evaluate mitosis. n = 5. e EdU (n = 3) and pH3 (n = 4) staining were performed to examine the effect of cotreatment with miR-143-3p mimic and melatonin on cardiomyocyte proliferation. The data represent the mean ± SEM. The data were analyzed by two-tailed Student’s t test with or without Welch’s correction. *P < 0.05, ***P < 0.001 versus the control group, ##P < 0.01, ###P < 0.001 versus the mel group.
Fig. 4
Fig. 4. Ctnnd1 and Yap, as targets of miR-143-3p, regulate cardiomyocyte proliferation induced by melatonin.
a The predicted binding site of miR-143-3p for Yap or Ctnnd1. b Yap and Ctnnd1 levels in cardiomyocytes with miR-143-3p upregulation or downregulation were determined by Western blotting. c The luciferase assay was used to analyze the binding between miR-143-3p and Yap1 or Ctnnd1. d Yap and Ctnnd1 levels after melatonin treatment. e Western blotting was used to evaluate the expression of Yap and Ctnnd1. f, g Cardiomyocyte proliferative ability was evaluated by pH3 staining. n = 5. h, i Yap and Ctnnd1 siRNAs abrogated the influence of melatonin on the proliferation of neonatal cardiomyocytes, as determined by EdU and pH3 staining. n = 5. The data represent the mean ± SEM. The data were analyzed by two-tailed Student’s t test with or without Welch’s correction, and the Mann–Whitney U test was used for nonnormally distributed data from two groups. One-way ANOVA with post hoc Newman–Keuls test was used to analyze data from more than two groups. *P < 0.05, **P < 0.01, ***P < 0.001 versus the control group,  #P < 0.05, ###P < 0.001 versus the mel group.
Fig. 5
Fig. 5. Luzindole blocks the effect of melatonin on cardiomyocyte proliferation and cardiac regeneration.
a EdU and pH3 staining for cardiomyocyte proliferation in the presence of melatonin and luzindole. n = 5. One-way ANOVA with the Newman–Keuls multiple comparison test was used for analysis. ***P < 0.001 versus the control group, ###P < 0.001 versus the mel group. b Representative echocardiography and serial echocardiographic measurements of ejection fraction (EF) and fraction shortening (FS) (n = 6, 6, 4, 4). Mann–Whitney two-tailed U test was used for nonparametric analysis. **P < 0.01 versus the MI+mel group. c Representative heart sections stained with pH3 for evaluating mitosis. The Mann–Whitney two-tailed U test was used for nonparametric analysis. ***P < 0.001 versus the MI group, ###P < 0.001 versus the MI+mel group. d Representative heart sections stained with WGA for evaluating the cross-sectional area of the cells. Two-tailed Student’s t test with Welch’s correction. The data are shown as the mean ± SEM, ***P < 0.001 versus the MI group, ##P < 0.01, ###P < 0.001 versus the MI+mel group.
Fig. 6
Fig. 6. Silencing of miR-143-3p inhibits the loss of cardiomyocyte cell cycle re-entry ability induced by luzindole.
a miR-143-3p expression was detected by qRT-PCR. Two-tailed Student’s t test was used for analysis. *P < 0.05 versus the control group, #P < 0.05 versus the mel group. b EdU staining for evaluating DNA synthesis. c pH3 staining for evaluating mitosis. The data are shown as the mean ± SEM. n = 5. One-way ANOVA with the Newman–Keuls multiple comparison test was used for analysis. ***P < 0.001 versus the mel group, ###P < 0.001 versus mel+luzindole group, &&&P < 0.001 versus the luzindole group. d Western blotting for evaluating the expression of Yap and Ctnnd1.
Fig. 7
Fig. 7. Melatonin receptor 2 plays an important role in melatonin-induced cardiac regeneration.
a, b EdU (n = 5) and pH3 (n = 6) staining for evaluating cardiomyocyte proliferation. The data are shown as the mean ± SEM. One-way ANOVA with the Newman–Keuls multiple comparison test was used for analysis. *P < 0.05, **P < 0.01, ***P < 0.001 versus the control group, ###P < 0.001 versus the mel group.
Fig. 8
Fig. 8. Underlying mechanisms of melatonin promoting cardiomyocyte proliferation and heart repair.
Schematic representation showing that melatonin regulates cardiomyocyte proliferation by upregulating Yap and Ctnnd1 through inhibition of miR-143-3p via activation of the melatonin receptor.
See this image and copyright information in PMC

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