No abstract

Cardiac hypertrophy and fibrosis are significant risk factors for chronic heart failure (HF). Since pharmacotherapy agents targeting these processes have not been established, we investigated the effect of alphamagostin (α-man) on cardiomyocyte hypertrophy and fibrosis in vitro. Primary cultured cardiomyocytes and cardiac fibroblasts were prepared from neonatal rats. After α-man treatment, phenylephrine (PE) and transforming growth factor-beta (TGF-β) were added to the cardiomyocytes and cardiac fibroblasts to induce hypertrophic and fibrotic responses, respectively. Hypertrophic responses were assessed by measuring the cardiomyocyte surface area and hypertrophic gene expression levels. PE-induced phosphorylation of Akt, extracellular signal-regulated kinase (ERK)1/2, and p38 was examined by Western blotting. Fibrotic responses were assessed by measuring collagen synthesis, fibrotic gene expression levels, and myofibroblast differentiation. In addition, TGF-β-induced reactive oxygen species (ROS) production was investigated. In cultured cardiomyocytes, α-man significantly suppressed PE-induced increases in the cardiomyocyte surface area, and the mRNA levels (atrial natriuretic factor (ANF) and brain natriuretic peptide (BNP)). Treatment with α-man significantly suppressed PE-induced Akt phosphorylation, but not ERK and p38 phosphorylation. In cultured cardiac fibroblasts, α-man significantly suppressed TGF-β-induced increases in L-proline incorporation, mRNA levels (POSTN and alpha-smooth muscle actin (α-SMA)), and myofibroblast differentiation. Additionally, it significantly inhibited TGF-β-induced reduced nicotinamide adenine dinucleotide phosphate oxidase4 (NOX4) expression and ROS production in cardiac fibroblasts. Treatment with α-man significantly ameliorates hypertrophy by inhibiting Akt phosphorylation in cardiomyocytes and fibrosis by inhibiting NOX4-generating ROS in fibroblasts. These findings suggest that α-man is a possible natural product for the prevention of cardiac hypertrophy and fibrosis.

Protein arginine methyltransferase 5 (PRMT5) is a well-known epigenetic regulatory enzyme. However, the role of PRMT5-mediated arginine methylation in gene transcription related to cardiac fibrosis is unknown. Here we show that fibroblast-specific deletion of PRMT5 significantly reduced pressure overload-induced cardiac fibrosis and improved cardiac dysfunction. Both the PRMT5-selective inhibitor EPZ015666 and knockdown of PRMT5 suppressed the expression of α-smooth muscle actin (α-SMA) induced by transforming growth factor-β (TGF-β) in cultured cardiac fibroblasts. TGF-β stimulation promoted the recruitment of the PRMT5/Smad3 complex to the promoter site of α-SMA. It also increased PRMT5-mediated H3R2 symmetric dimethylation, and this increase was inhibited by knockdown of Smad3. TGF-β stimulation also increased H3K4 tri-methylation mediated by the WDR5/MLL1 lysine methylase complex, which recognizes H3R2 symmetric dimethylation. Finally, treatment with EPZ015666 significantly improved pressure overload-induced cardiac fibrosis and dysfunction. These findings reveal that PRMT5 regulates TGF-β/Smad3-dependent fibrotic gene transcription through histone methylation crosstalk and plays a critical role in cardiac fibrosis and dysfunction.

Background An intrinsic histone acetyltransferase (HAT), p300, is required for acetylation and the transcriptional activity of GATA4, as well as pathological left ventricular hypertrophy (LVH) and the development of heart failure (HF) in vivo. Recently, studies of histone modification have been performed within the flexible tails, such as H3K9 and H3K14. Although most previously studied histone modifications are within the flexible tails of histones, H3K122 is reportedly a novel site of the histone globular domain acetylated by p300, and its acetylation activates gene transcriptions by destabilizing histone-DNA binding and increasing the accessibility of transactional factors to DNA. However, little is known about the extent histone modifications directly affect LVH and HF. Hypothesis We hypothesized that p300 could induce epigenetic changes by acetylation of the globular domain as well as tail domain of histone during the development of LVH and HF. Methods First, to investigate whether the acetylation of H3K122 in the globular domain of histones as well as those of H3K9 and H3K14 in the tail domain of histones increased in cardiomyocytes hypertrophy, western blotting and chromatin-immunoprecipitation (ChIP) assays were performed using neonatal rat cultured cardiomyocytes with phenylephrine (PE) stimulus. Second, neonatal rat cultured cardiomyocytes were treated with p300 knockdown by siRNA or curcumin, a p300-specific HAT inhibitor. Third, to investigate the role of p300 HAT activity in histone acetylation in vivo, we utilized mice overexpressing p300 in the heart, which induced LVH. Final, to investigate whether these acetylation changes during the development of LVH and HF, in vivo ChIP assay was performed using hypertensive heart disease model of Dahl salt-sensitive rats. Results Western blotting indicated that treatment with PE increased the acetylation of H3K122 as well as those of H3K9 and H3K14 in cardiomyocytes hypertrophy. ChIP assay demonstrated that PE increased the recruitment of acetylated H3K122 and H3K9 onto ANF and BNP promoters containing the GATA element and peaks of acetylation of these domains were 4 hours after PE stimulation. Next, these acetylations were significantly inhibited by p300 knockdown by siRNA or treatment with curcumin. Conversely, in vivo ChIP assays in mice overexpressing p300 indicated that p300 overexpression increased recruitment of acetylated H3K122 and H3K9 onto ANF and BNP promoters containing the GATA element. Next, in hypertensive heart disease model of Dahl salt-sensitive rats, in vivo ChIP assays reviled that acetylation of H3K9 was increased around ANF and BNP promoters containing the GATA element at the LVH stage but that of H3K122 was increased at the HF stage. Conclusion Our data indicate that acetylation of H3K122 in globular domain of histones by p300 is the key event of the transition from LVH to HF.