Epigenetic regulation of cardiac muscle-specific genes in H9c2 cells by Interleukin-18 and histone deacetylase inhibitor m-carboxycinnamic acid bis-hydroxamide

Abstract

Interleukin-18 (IL-18) elicited a robust hypertrophy response in H9c2 cardiomyocytes as judged by their accelerated rates of protein synthesis and increased cell size. Evidently, IL-18 treatment also induced a cardiac hypertrophy-specific program of gene expression in H9c2 cardiomyocytes since they elicited enhanced expression of atrial naturetic factor (ANF), desmin, and skeletal alpha-actin genes accompanied by a canonical switch in the transcription of alpha- and beta-myosin heavy chain (MyHC) genes. Co-treatment of H9c2 cells with m-carboxycinnamic acid bis-hydroxamide (CBHA), an inhibitor of histone deacetylases, significantly blocked both morphological and molecular manifestations of IL-18-induced cardiac hypertrophy in vitro. IL-18 treatment led to activation of phosphoinositide-3-kinase and phosphorylated Akt/protein kinase B, while CBHA blunted this pathway via inducing the expression of its upstream regulator, PTEN (phosphatase and tensin homolog). The architecture of bulk chromatin of H9c2 cells exposed to IL-18 and/or CBHA was significantly altered as judged by the extent of covalent modifications of its constituent histones. The chromatin immuno-precipitation (ChIP) assays revealed that IL-18-induced specific epigenetic changes in the chromatin of ANF, desmin, skeletal alpha-actin, and MyHC genes that were largely neutralized by CBHA. We demonstrate for the first time that 'histone code' of the entire approximately 50 kb genomic DNA encoding the alpha- and beta-MyHC genes and the intergenic DNA that generates anti-beta-MyHC RNA was uniquely modulated by pro- and anti-hypertrophy signals of IL-18 and CBHA, respectively.

Panhistone deacetylase inhibitors inhibit proinflammatory signaling pathways to ameliorate interleukin-18-induced cardiac hypertrophy

“…Thus subcellular redistribution in response to external signals mechanistically links HDACs to maladaptive cardiac hypertrophy. Consistent with such a mechanism some pan-HDAC inhibitors have been shown to preserve cardiac function in the face of pathological stressors in rodents (21,24,33,40).…”

“…We have reported previously that IL-18-treated H9c2 cardiac myocytes became highly enlarged while they concomitantly elicited induction of a number of cardiac hypertrophyspecific genes, and both physical and molecular manifestations of IL-18-induced hypertrophy were greatly attenuated by CBHA (40). Based on our published data in H9c2 cells, combined with the current observations in BALB/c mice, we posit that IL-18 treatment induces cardiac hypertrophy-specific gene expression in vivo and in vitro that was attenuated by two distinct pan-HDACIs, CBHA and TSA.…”

“…H9c2 cells were purchased from American Type Culture Collection (Bethesda, MD) and were grown in Dulbecco's minimum essential medium containing 10% fetal bovine serum (Hyclone, Logan, UT), 2 mM glutamine, and 1% penicillin-streptomycin. Cells were allowed to reach ϳ80% confluence in complete culture medium and incubated for additional 24 h in serum-free medium prior to experimental treatments, as outlined previously (40). Replicate cultures of H9c2 cells were incubated in media supplemented with IL-18 (0.1 g/ml), CBHA (1 M), or TSA (100 nM).…”

“…These data were normalized against 18S ribosomal RNA and were presented as fold changes in treated versus control. Nonspecific PCR amplifications were eliminated by assessing melting curve patterns, as detailed previously (40).…”

“…The blots were sequentially reacted with primary antibodies (used after 1:1,000 dilution) followed by horseradish peroxidase-conjugated anti-rabbit IgG antibodies (diluted 1:10,000; Santa Cruz Biotechnology, Santa Cruz, CA). Chemi-luminescence signals developed using ECL Plus kit (Amersham-Pharmacia Biotech, Piscataway, NJ) were quantified as detailed previously (39,40). The blots probed with PTEN antibodies were stripped and reprobed with an anti-actin antibody (1:10,000) to determine equivalency of protein loading; the blots developed with anti-phospho-Akt were stripped and reprobed with anti-Akt antibody (1:10,000, Cell Signaling) to determine total Akt.…”