Loss of microRNA-128 promotes cardiomyocyte proliferation and heart regeneration.
The goal of replenishing the cardiomyocyte (CM) population using regenerative therapies following myocardial infarction (MI) is hampered by the limited regeneration capacity of adult CMs, partially due to their withdrawal from the cell cycle. Here, we show that microRNA-128 (miR-128) is upregulated in CMs during the postnatal switch from proliferation to terminal differentiation. In neonatal mice, cardiac-specific overexpression of miR-128 impairs CM proliferation and cardiac function, while miR-128 deletion extends proliferation of postnatal CMs by enhancing expression of the chromatin modifier SUZ12, which suppresses p27 (cyclin-dependent kinase inhibitor) expression and activates the positive cell cycle regulators Cyclin E and CDK2. Furthermore, deletion of miR-128 promotes cell cycle re-entry of adult CMs, thereby reducing the levels of fibrosis, and attenuating cardiac dysfunction in response to MI. These results suggest that miR-128 serves as a critical regulator of endogenous CM proliferation, and might be a novel therapeutic target for heart repair.RNA sequencing (RNA-seq) in mouse cardiac ventricles was performed on postnatal days 1, 7, and 28 (P1, P7, and P28) to identify potential miRNAs involved in the regulation of postnatal heart growth. MiR-128 was robustly upregulated in P7 hearts as compared to P1, which was further confirmed by quantitative PCR (qPCR) array (Supplementary Fig. 1A). As previously reported15, miR-128 was predominantly expressed in brain tissue but was also expressed in the heart (Supplementary Fig. 1B). Its expression in adult myocardium was further confirmed by in situ hybridization (ISH) (Supplementary Fig. 1C). To investigate the role of miR-128 in cell cycle withdrawal during heart growth, mouse hearts were harvested and sectioned at P1, P7, and P28 (Fig. 1a). As neonates (P1) progress to adulthood (P28), CMs underwent a maturation process characterized by suppression of cell proliferation as evidenced by decreased numbers of Ki67+ CMs (Fig. 1a, b). In addition, cardiac mass increased from P1 to P28 primarily due to an increase in CM size rather than in number (Fig. 1c, d). Interestingly, we found that miR-128 expression was significantly increased during heart development (Fig. 1e). Furthermore, the level of miR-128 was found to be significantly elevated in P7 and P28 hearts compared with P1 hearts. In order to examine whether the postnatal upregulation of miR-128 occurs specifically in the CMs, we isolated CMs from P1 and P28 hearts, respectively, and found significantly higher expression of miR-128 in P28 CMs when compared with P1 CMs (Fig. 1f). Moreover, the expression of miR-128 in CMs was significantly higher than in non-CMs (e.g., cardiac fibroblasts, CFs) (Fig. 1g). These data indicate a potential role for miR-128 in regulating CM proliferation.All research protocols conformed to the Guidelines for the Care and Use of Laboratory Animals published by the National Institutes of Health (National Academies Press, eighth edition, 2011). All animal use protocols and methods of euthanasia (pentobarbital overdose followed by thoracotomy) used in this study were approved by the University of Cincinnati Animal Care and Use Committee. An independent review and approval of cell and chemical drug used in this study was conducted by the Institutional Biosafety Committee (IBC). Mice were maintained on a C57BL/6 background and their genotype was determined by PCR from tail DNA. Both male and female mice were randomized in different experiment groups.This work was supported by the National Natural Science Foundation of China (Nos. 81330007, U1601227 to Dr. Xi-Yong Yu.), the National Institutes of Health grants HL107957, HL110740, HL136025, and HL130042 (to Dr. Yigang Wang), the Science and Technology Programs of Guangdong Province (No. 2014A050503047 to Dr. Xiyong Yu and Dr. Yigang Wang, 2015B020225006 to Dr. Xi-Yong Yu), Guangzhou Science and Technology Program (No. 201604010087 to Dr. Xi-Yong Yu.), and the American Heart Association Catalyst (17CCRG33671128) to Dr. Sakthivel Sadayappan and Dr. Yigang Wang. We thank Anne Schaefer (Icahn School of Medicine at Mount Sinai, USA) for providing the miR-128flox/flox mice and Gary E. Shull for manuscript editing.The authors declare no competing financial interests.Wei Huang, Yuliang Feng and Jialiang Liang contributed equally to this work.Electronic supplementary materialSupplementary Information accompanies this paper at 10.1038/s41467-018-03019-z.Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.