Function of BRD4 in the pathogenesis of high glucose‑induced cardiac hypertrophy

Abstract

Diabetic cardiomyopathy is one of the major complications of diabetes, and due to the increasing number of patients with diabetes it is a growing concern. Diabetes‑induced cardiomyopathy has a complex pathogenesis and histone deacetylase‑mediated epigenetic processes are of prominent importance. The olfactory bromodomain‑containing protein 4 (BRD4) is a protein that recognizes and binds acetylated lysine. It has been reported that the high expression of BRD4 is involved in the process of cardiac hypertrophy. The aim of the present study was to investigate the function of BRD4 in the process of high glucose (HG)‑induced cardiac hypertrophy, and to clarify whether epigenetic regulation involving BRD4 is an important mechanism. It was revealed that BRD4 expression levels were increased in H9C2 cells following 48 h of HG stimulation. This result was also observed in a diabetic rat model. Furthermore, HG stimulation resulted in the upregulation of the myocardial hypertrophy marker, atrial natriuretic peptide, the cytoskeletal protein α‑actin and fibrosis‑associated genes including transforming growth factor‑β, SMAD family member 3, connective tissue growth factor and collagen, type 1, α1. However, administration of the specific BRD4 inhibitor JQ1 (250 nM) for 48 h reversed this phenomenon. Furthermore, protein kinase B (AKT) phosphorylation was activated by HG stimulation and suppressed by JQ1. In conclusion, BRD4 serves an important role in the pathogenesis of HG‑induced cardiomyocyte hypertrophy through the AKT pathway.

Keywords: bromodomain-containing protein 4; high glucose; cardiac hypertrophy; atrial natriuretic peptide; JQ1.

Figure 1.

Diabetic rats present with cardiac hypertrophy and fibrosis. Wistar rats were used to simulate a diabetic model by intraperitoneal injections of streptozotocin for 8 weeks (n=8). The ideal diabetic model had fasting-blood glucose levels >12 mmol/l. The control group was treated with vehicle (n=8). (A) Body weight and (B) blood glucose were assessed every week. (C) ANP expression levels in the control and DM groups were analyzed by western blot analysis. (D) mRNA expression levels of ANP in the control and DM groups were examined using reverse transcription-quantitative polymerase chain reaction. (E) H&E staining of the control and DM groups. (F) Masson's staining of the control and DM groups. Scale bar, 50 µm. *P<0.05 vs. the DM group; ^##P<0.01 with comparisons shown by lines. DM, diabetes model; ANP, atrial natriuretic peptide; H&E, haemotoxylin and eosin.

Figure 2.

Expression of BRD4 and c-myc is higher in streptozotocin-induced diabetic rats. (A) Protein expression levels of BRD4 and c-myc in the control and DM groups were analyzed using western blot analysis. (B) mRNA expression levels of BRD4 and c-myc in the control and DM groups were examined using reverse transcription-quantitative polymerase chain reaction. (C) IHC analysis of c-myc in the control and DM groups. Scale bar, 50 µm. ^#P<0.05 and ^##P<0.01 with comparisons shown by lines. BRD4, bromodomain-containing protein 4; DM, diabetes model; Con, control; IHC, immunohistochemical.

Figure 3.

High glucose induces H9C2 cell hypertrophy and upregulates BRD4 expression. (A) Western blot analysis of ANP expression in H9C2 cells subsequent to exposure to different glucose concentrations for 48 h. (B) Comparison of H9C2 cell size following exposure to different glucose concentrations for 48 h under a light microscope. (C) Quantified cell size performed using Image J. (D) Western blot analysis of BRD4 and c-myc expression in H9C2 cells subsequent to exposure to different glucose concentrations for 48 h. Scale bar, 50 µm. *P<0.05, ^#P<0.05 and ^##P<0.01 with comparisons shown by lines. BRD4, bromodomain-containing protein 4; ANP, atrial natriuretic peptide.

Figure 4.

JQ1 significantly inhibits HG-induced cardiac hypertrophy, BRD4 and c-myc expression and cardiac fibrosis. H9C2 cells were treated with 250 nM JQ1 for 48 h after pretreatment with or without HG (30 mmol/l) for 48 h. (A) Western blot analysis of ANP, BRD4 and c-myc protein expression in normal glucose, HG (30 mM glucose) and HG (30 mM glucose) + JQ1 (250 nM) groups for 48 h. (B) RT-qPCR analysis of ANP, BRD4 and c-myc mRNA expression. (C) Images of α-actin staining of untreated H9C2 cells and HG-induced H9C2 cells with or without 250 nM JQ1 treatment for 48 h. (D) mRNA expression levels of fibrosis-associated genes including TGF-β, SMAD3, CTGF and COL1A1 were examined using RT-qPCR. Scale bar, 50 µm. *P<0.05, **P<0.01, ^#P<0.05 and ^##P<0.01 with comparisons shown by lines. BRD4, bromodomain-containing protein 4; ANP, atrial natriuretic peptide; HG, high glucose; TGF-β, tumor growth factor β; SMAD3, SMAD family member 3; CTGF, connective tissue growth factor; COL1A1, collagen, type 1, α1; RT-qPCR, reverse transcription-quantitative polymerase chain reaction.

Figure 5.

HG-induced diabetic cardiomyopathy occurs through the AKT signaling pathway. (A) Expression levels of P-AKT in the HG group with or without 250 nM JQ1 treatment for 48 h was examined by western blot analysis. (B) A model of the regulation of cardiac hypertrophy by BRD4 under HG stimulation. **P<0.01 and ^##P<0.01 with comparisons shown by lines. P-, phosphorylated; AKT, protein kinase B; HG, high glucose; P-TEFb, positive transcription elongation factor; CDK9, cyclin-dependent kinase 9; BRD4, bromodomain-containing protein 4; ANP, atrial natriuretic peptide; TGF-β, tumor growth factor β; CTGF, connective tissue growth factor.