The histone 3 lysine 9 methyltransferase inhibitor chaetocin improves prognosis in a rat model of high salt diet-induced heart failure

Abstract

Histone acetylation has been linked to cardiac hypertrophy and heart failure. However, the pathological implications of changes in histone methylation and the effects of interventions with histone methyltransferase inhibitors for heart failure have not been fully clarified. Here, we focused on H3K9me3 status in the heart and investigated the effects of the histone H3K9 methyltransferase inhibitor chaetocin on prognoses in Dahl salt-sensitive rats, an animal model of chronic heart failure. Chaetocin prolonged survival and restored mitochondrial dysfunction. ChIP-seq analysis demonstrated that chronic stress to the heart induced H3K9me3 elevation in thousands of repetitive elements, including intronic regions of mitochondria-related genes, such as the gene encoding peroxisome proliferator-activated receptor-gamma coactivator 1 alpha. Furthermore, chaetocin reversed this effect on these repetitive loci. These data suggested that excessive heterochromatinization of repetitive elements of mitochondrial genes in the failing heart may lead to the silencing of genes and impair heart function. Thus, chaetocin may be a potential therapeutic agent for chronic heart failure.

Figure 1. Chaetocin improved the prognosis of rats in a model of heart failure.

(A) Systolic and diastolic blood pressures (SBP and DBP, respectively). (B) Heart weight/body weight (HW/BW) ratios are shown for 13-week-old rats in the control group [NS (−), n = 11], normal salt diet with chaetocin group [NS (ch+), n = 7], HF group [HS (−), n = 17], and treatment group [HS (ch+), n = 17]. (C) Quantitative real-time PCR analysis of natriuretic peptide precursor A (Nppa, ANP) mRNA expression in left ventricular tissues for 13-week-old rats in the control group (n = 5), normal salt diet with chaetocin group (n = 4), HF group (n = 10), and treatment group (n = 14). (D) Echocardiographic measurements of fractional shortening (FS) and posterior wall thickness (PWT). (E) Hematoxylin-eosin-stained (upper panel, scale bars: 2 mm) or Elastica van Gieson (EvG) with picrosirius red-stained sections of cardiac tissue (middle panel, scale bars: 200 μm) and quantification of the fibrotic area (lower bar). (F) Kaplan-Meier survival analysis of DS rats in the control group (n = 9), normal salt diet with chaetocin group (n = 12), HF group (n = 17), and treatment group (n = 13). Data are presented as the mean ± SD. *P < 0.05 and **P < 0.01 versus the control group; ^#P < 0.05 and ^##P < 0.01 versus the HF group; ^†P < 0.05 and ^††P < 0.01 versus the normal salt diet with chaetocin group; n.s.: not significant.

Figure 2. Gene ontology analysis of the set of 50 genes that were downregulated in failing hearts and restored with chaetocin; restoration of mitochondrial function with chaetocin.

(A) Singular enrichment analysis of the “cellular component” category. Hyp* means p-value calculated using the corrected hypergeometric distribution. (B) Mitochondrial respiration (each group, n = 5). Substrates: pyruvate/malate. (C) Mitochondrial DNA content (control group [n = 5], HF group [n = 5], and treatment group [n = 7]). (D) Quantitative real-time PCR analysis of peroxisome proliferator-activated receptor gamma, coactivator 1 alpha (Pgc1α) mRNA expression in left ventricular tissues (each group, n = 4) at 13 weeks of age. Data are presented as the mean ± SD. **P < 0.01 versus the control group; ^#P < 0.05 versus the HF group.

Figure 3. Heart failure increased H3K9me3 levels on repetitive elements.

(A) Number of repetitive elements in which heart failure caused an increase (HF-up) or decrease (HF-down) in H3K9me3 compared with control samples (left). The restoration percentage of H3K9 trimethylation state following treatment with chaetocin (right). (B) H3K9me3 levels in the intronic repetitive regions of Pgc1α. Red squares indicate the region that was identified as being enriched in H3K9me3 repetitive elements in rats with heart failure. The black boxes indicate the repetitive loci. The bars indicate the H3K9me3 read alignments of the repetitive elements. RPM, reads per million mapped.

Figure 4

Representative data showing enrichment of H3K9me3 repetitive loci in regions in close proximity to Acadm (A) and Ndufs4 (B) in the HF group. Red squares indicate the region that was identified as being enriched in H3K9me3 repetitive elements in rats with heart failure. The black boxes indicate the repetitive loci. The blue bars indicate H3K9me3 read alignments. The mRNA level of each gene was determined with the real-time quantitative PCR and is shown as the fold change versus the control group. **P < 0.01 versus the control group; ^#P < 0.05 and ^##P < 0.01 versus the HF group.