miR-199a-3p suppresses Vldlr expression to promote cardiomyocyte proliferation

Abstract

The proliferative capacity of cardiomyocytes is limited in adult mammals, and replacing lost tissue following acute ischemic injury is challenging. Previous studies have demonstrated that miR-199a-3p can promote cardiomyocyte proliferation, but the exact mechanism by which this occurs remains unclear, although multiple targets of miR-199a-3p have been identified. We recently showed that very-low-density-lipoprotein receptor (Vldlr) inhibits cardiomyocyte proliferation, and in this study we aim to test whether Vldlr is a functional target gene of miR-199a-3p. 3′UTR reporter assays demonstrate that miR-199a-3p directly binds to the 3′UTR of Vldlr and inhibits its translation. Overexpressing Vldlr blunts the pro-proliferative effect of miR-199a-3p on cardiomyocytes, suggesting that Vldlr is indeed a functional target of miR-199a-3p. Mechanistically, Vldlr reduces S807/811 phosphorylation of RB1, and inhibiting CDK4/6 to prevent RB1 phosphorylation can block the pro-proliferative effect of both Vldlr knockdown and miR-199a-3p, suggesting that RB1 phosphorylation is required for the cardiomyocyte proliferation induced by miR-199a-3p and Vldlr knockdown. The findings of this study reveal Vldlr as a novel functional target of miR-199a-3p in cardiomyocytes and identify RB1 as a downstream effector of cardiomyocyte proliferation. The identification of the role of the miR-199a-3p-Vldlr-RB1 axis in cardiomyocyte proliferation may provide potential therapeutic targets for cardiac regenerative medicine.

1. Introduction

The adult mammalian heart lacks regenerative capacity, mainly because cardiomyocytes (CMs) exit the cell cycle during maturation [1]. The loss of cardiomyocytes in cardiac diseases such as myocardial infarction cannot be compensated with new cardiomyocytes, thus compromising the contractility of the remaining myocardium, ultimately leading to heart failure and death when the extent of injury is severe [2]. Numerous studies have identified various regulators of cardiomyocyte proliferation in cultured cardiomyocytes and in small animal models [2]; however, only a few have shown effectiveness in promoting adult cardiomyocyte proliferation in large animal models [3,4]. Elucidating the molecular mechanism by which these factors promote adult cardiomyocyte proliferation in large animals may provide a better understanding of human cardiomyocyte regeneration and cardiac regeneration, as well as potential pharmaceutical targets suitable for human patients.

MicroRNAs (miRNAs), small noncoding RNAs that have diverse functions, including the promotion of mRNA degradation and the inhibition of mRNA translation, have been demonstrated to play multifaceted roles in cardiac development, disease control and regeneration [5]. A functional screening revealed that miR-199a-3p can induce robust proliferation of neonatal, as well as adult, rodent cardiomyocytes [6]. Importantly, in a swine model, miR-199a-3p effectively promoted adult cardiomyocyte proliferation after myocardial infarction while causing lethal arrhythmia, possibly due to uncontrolled dedifferentiation of cardiomyocytes [3]. Given the robust ability of miR-199a-3p to induce cardiomyocyte proliferation, there has been a persistent quest to identify its functional targets. Critical transcription factors, including Homer1 and Hopx [6–8]; transcription suppressors, such as NACC2 [9]; regulators of Hippo pathways, including TAOK1 and β-TrCP [10]; and cell membrane receptors, such as Cd151 [11], have been found to be functional targets of miR-199a-3p in cardiomyocytes. However, most microRNAs function by targeting multiple mRNAs, and there may be unidentified targets of miR-199a-3p that are critical in regulating the CM cell cycle. In addition, the deleterious effects of overexpressing miR-199a-3p in adult swine hearts, including tachyarrhythmia, are attributed to possible arrhythmogenic targets and/or coexpressed miR-199a-5p [3]. Therefore, identifying novel functional targets of miR-199a-3p will not only elucidate the mechanisms by which miR-199a-3p regulates cardiomyocyte proliferation but also shed light on the core cell cycle regulation of cardiomyocyte. This may lead to the discovery of novel and specific therapeutic targets for cardiac regenerative medicine, thereby avoiding the off-target effects of miR-199a-3p.

Our previous research on signaling from noncardiomyocytes regulating cardiomyocyte proliferation identified very-low-density-lipoprotein receptor (Vldlr) as a plasma membrane receptor on cardiomyocytes that inhibits proliferation when the suppressive ligand TSP-1 is abundant in the adult heart [12]. Interestingly, both miR-199a-3p and Vldlr have been shown to regulate YAP activity in cardiomyocytes [10,12], and miR-199a-3p, as well as other pro-proliferative microRNAs, may target multiple components of the signaling pathways involved in cardiomyocyte proliferation.