Endogenous RBM4 prevents Ang II-induced cardiomyocyte hypertrophy via downregulating the expression of PTBP1

Abstract

Aberrant gene expression in cardiomyocyte has been revealed to be the fundamental essence of pathological cardiac hypertrophy. However, the detailed mechanisms are not fully understood. The underlying regulators of gene expression involved in cardiac hypertrophy remain to be further identified. Here, we report that the RNA-binding protein RNA-binding motif protein 4 (RBM4) functions as an endogenic protector that is able to fight against cardiomyocyte hypertrophy in vitro. Under pro-hypertrophic stimulation of angiotensin II (Ang II), the protein level of RBM4 in cardiomyocyte and myocardium is elevated. Knockdown of RBM4 can further aggravate cardiomyocyte hypertrophy, while over-expression of RBM4 represses cardiomyocyte hypertrophy. Mechanistically, RBM4 is localized in the nucleus and down-regulates the expression of polypyrimidine tract-binding protein 1 (PTBP1), which has been shown to aggravate cardiomyocyte hypertrophy. In addition, we suggest that the up-regulation of RBM4 in cardiomyocyte hypertrophy is caused by N6-methyladenosine (m6A). Ang II induces m6A methylation of RBM4 mRNA, which further enhances the YTH domain-containing family protein 1 (YTHDF1)-mediated translation of RBM4. Thus, our results reveal a novel pathway consisting of m6A, RBM4 and PTBP1, which is involved in cardiomyocyte hypertrophy.

1. Introduction

Pathological cardiac hypertrophy is the compensatory response of the heart to hemodynamic overload caused by hypertension or valvulopathy, or myocardium loss-induced ventricular volume overload. Sustained pro-hypertrophic stimulation often leads to cardiac dysfunction, irreversible heart failure, and even sudden death. The cardiomyocyte in cardiac hypertrophy is characterized by increased size and aberrantly activated fetal gene (e.g., atrial natriuretic peptide, ANF, and brain natriuretic peptide, BNP) expression, which has been reported to be mediated by several signaling pathways (e.g., calmodulin-dependent kinase II, CaMKII and mTOR signaling), histone modification regulators (e.g., histone acetyltransferase p300 and histone deacetylases, HDACs) as well as transcription factors (e.g., nuclear factor of activated T cells, NFAT, GATA-binding factor 4, GATA4 and myocyte-specific enhancer factor 2, MEF2) [1,2]. In the recent decade, post-transcriptional regulatory mechanisms, such as non-coding RNAs (ncRNAs), alternative splicing and N6-methyladenosine (m6A) are gradually revealed to be closely involved in the cardiomyocyte hypertrophy. Nevertheless, the underlying post-transcriptional mechanisms and related critical regulatory factors still need to be further explored.

RNA-binding motif (RBM) protein is a subfamily of RNA-binding protein (RBP). They usually have at least one RNA-recognition motif (RRM), which is supposed to bind to single-stranded RNA. RBM proteins are generally involved in pre-mRNA splicing, and are also reported to regulate translation and mRNA degradation [3,4]. Notably, some RBM proteins play fundamental roles in the heart. Deficiency or dysregulation of these RBM proteins can cause heart disease. For instance, cardiac-specific RBM20 mediates the splicing of Titin pre-mRNA. Mutation of the RBM20 gene is a critical cause of dilated cardiomyopathy [5,6]. RBM24 is essential for cardiac sarcomere assembly and heart development by mediating alternative splicing and translation initiation in cardiomyocyte [7‒9]. Deletion of RBM24 also leads to dilated cardiomyopathy [10]. Elevated RBM25 aggravates abnormal SCN5A pre-mRNA splicing and heart failure [11]. RBM38 has been reported to rescue cardiac hypertrophy through inhibiting LXR-α expression [12]. RBM4 is a member of RBM protein family that has a regulatory function in alternative splicing, as well as translation [13]. RBM4 has been shown to function in muscle cell differentiation [14] and tumorigenesis [15]. Intriguingly, RBM4 mRNA is highly expressed in heart tissue [16]. Moreover, it has been found to mediate the splicing of various cardiomyocyte hypertrophy-related genes [17]. However, it is still unclear whether RBM4 is involved in cardiomyocyte hypertrophy.