https://www.frontiersin.org/articles/10.3389/fphar.2022.887991/full

Epigenetic Reader Bromodomain Containing Protein 2 Facilitates Pathological Cardiac Hypertrophy via Regulating the Expression of Citrate Cycle Genes

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ORIGINAL RESEARCH article

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Front. Pharmacol., 25 May 2022 | https://doi.org/10.3389/fphar.2022.887991

Epigenetic Reader Bromodomain Containing Protein 2 Facilitates Pathological Cardiac Hypertrophy via Regulating the Expression of Citrate Cycle Genes

The bromodomain and extra-terminal domain proteins (BETs) family serve as epigenetic “readers”, which recognize the acetylated histones and recruit transcriptional regulator complexes to chromatin, eventually regulating gene transcription. Accumulating evidences demonstrate that pan BET inhibitors (BETi) confer protection against pathological cardiac hypertrophy, a precursor progress for developing heart failure. However, the roles of BET family members, except BRD4, remain unknown in pathological cardiac hypertrophy. The present study identified BRD2 as a novel regulator in cardiac hypertrophy, with a distinct mechanism from BRD4. BRD2 expression was elevated in cardiac hypertrophy induced by β-adrenergic agonist isoprenaline (ISO) in vivo and in vitro. Overexpression of BRD2 upregulated the expression of hypertrophic biomarkers and increased cell surface area, whereas BRD2 knockdown restrained ISO-induced cardiomyocyte hypertrophy. In vivo, rats received intramyocardial injection of adeno-associated virus (AAV) encoding siBRD2 significantly reversed ISO-induced pathological cardiac hypertrophy, cardiac fibrosis, and cardiac function dysregulation. The bioinformatic analysis of whole-genome sequence data demonstrated that a majority of metabolic genes, in particular those involved in TCA cycle, were under regulation by BRD2. Real-time PCR results confirmed that the expressions of TCA cycle genes were upregulated by BRD2, but were downregulated by BRD2 silencing in ISO-treated cardiomyocytes. Results of mitochondrial oxygen consumption rate (OCR) and ATP production measurement demonstrated that BRD2 augmented cardiac metabolism during cardiac hypertrophy. In conclusion, the present study revealed that BRD2 could facilitate cardiac hypertrophy through upregulating TCA cycle genes. Strategies targeting inhibition of BRD2 might suggest therapeutic potential for pathological cardiac hypertrophy and heart failure.

Introduction

Epigenetic modifications refer to dynamic and reversible changes in chromatin accessibility and post-translational modifications of histone tails, without altering nucleotide sequence (Prinjha et al., 2012). As the earliest and most in-depth studied epigenetic modification, histone acetylation plays important roles in chromatin remodeling and transcriptional regulation (Scher et al., 2007). Not only governed by the “writer” histone acetyltransferases and the “eraser” histone deacetylases, histone acetylation also relies on “readers”, which are acetyl-binding proteins recognizing the acetylated lysine in histones and recruiting transcriptional regulator complexes to chromatin, eventually regulating gene transcription (Wang et al., 2021). Nearly all known “epigenetic readers” contain bromodomains, which are about 110-amino-acid modules found in many chromatin-associated proteins (Scher et al., 2007). The bromodomain and extra-terminal domain (BET) family, consisting of four proteins (namely BRD2, BRD3, BRD4, and BRDT), is a subfamily of bromodomain-containing proteins (BRDs) designated because of the presence of two bromodomains along with an additional region of homology called ET domain (Gyuris et al., 2009). BET members are implicated in a majority of diseases, including human immunodeficiency virus infection (Boehm et al., 2013; Sharma et al., 2013), inflammatory diseases (Belkina et al., 2013), carcinogenesis (Mertz et al., 2011; Francisco et al., 2013), etc.

Pathological cardiac hypertrophy is characterized by enlargement of the heart and thickening of ventricular walls due to sustain and abnormal hemodynamic stress. Although it is initially an adaptive response to maintain cardiac function, prolonged cardiac hypertrophy exacerbates heart workload and facilitates the onset of heart failure (Tham et al., 2015). Maladaptive cardiac hypertrophy is also an independent predictor of adverse outcomes in patients with heart failure (Tham et al., 2015). Therefore, interventions to prevent pathological cardiac hypertrophy are in urgent need. Accumulating evidences demonstrate that BET inhibitors (BETi) exhibit protective effects against pathological cardiac hypertrophy. A pan-BETi, JQ1, ameliorates cardiomyocyte hypertrophy and left ventricular hypertrophy (LVH) induced by transverse aortic constriction (TAC) in mice (Spiltoir et al., 2013). Another BETi, apabetalone (RVX-208), improves the cardiovascular outcomes of patients with diabetes after acute coronary syndrome, suggesting its therapeutic potential for cardiovascular diseases (Ray et al., 2019). Among the four BET family members, BRD4 has been widely reported to be involved in regulation of cardiac hypertrophy (Van der Feen et al., 2019; Zhu et al., 2020; Li et al., 2021). However, the roles of other BET members in pathological cardiac hypertrophy remain unclear. Considering the broad effects of pan-BET, the possible involvement of other BET members could not be ignored. In our preliminary studies, we measured the mRNA levels of BET members in a cardiomyocyte hypertrophy model induced by β-adrenergic receptor activation. BRD2 was remarkably elevated at the transcriptional level in parallel with BRD4, suggesting that BRD2 is probably associated with the pathological progression of cardiac hypertrophy (Li et al., 2021).

BRD2 and BRD4 share similar structure, with about 80% identity at the amino acid level in human and mouse. The only difference is that BRD4 has a distinct longer C-terminal domain (CTD), which can facilitate transcription reactivation and elongation by recruiting and interacting with the positive-transcriptional elongation factor b (P-TEFb), while BRD2 does not have such structure (Belkina and Denis 2012; Taniguchi 2016). With its CTD function, BRD4 enhances the recruitment of P-TEFb, which phosphorylates Ser2 of RNA Pol II and promotes transcriptional pause release of Pol II (Spiltoir et al., 2013; Stratton and McKinsey 2015). However, it is still uncertain about the exact biological functions of BET family members without CTD, such as BRD2 and BRD3 (Belkina and Denis 2012). Some studies indicate that the main role of BRD2/3 might be to remodel the promoters and enhancers to activate the transcriptional initiation process, whereas BRD4 is primarily involved in transcription elongation (Kanno et al., 2014; Kim et al., 2021).

Considering the existent structural and functional differences between BRD2 and BRD4, it raises our interests to explore the role of BRD2 in cardiovascular diseases. In view of this, the present study attempted to determine the regulatory effect of BRD2 in pathological cardiac hypertrophy, and to explore the possible underlying mechanisms.