Epigenetic abnormalities in cardiac hypertrophy and heart failure

Abstract

Epigenetics refers to the heritable regulation of gene expression through modification of chromosomal components without an alteration in the nucleotide sequence of the genome. Such modifications include methylation of genomic DNA as well as acetylation, methylation, phosphorylation, ubiquitination, and SUMOylation of core histone proteins. Recent genetic and biochemical analyses indicate that epigenetic changes play an important role in the development of cardiac hypertrophy and heart failure, with dysregulation in histone acetylation status, in particular, shown to be directly linked to an impaired contraction ability of cardiac myocytes. Although such epigenetic changes should eventually lead to alterations in the expression of genes associated with the affected histones, little information is yet available on the genes responsible for the development of heart failure. Current efforts of our and other groups have focused on deciphering the network of genes which are under abnormal epigenetic regulation in failed hearts. To this end, coupling chromatin immunoprecipitation to high-throughput profiling systems is being applied to cardiac myocytes in normal as well as affected hearts. The results of these studies should not only improve our understanding of the molecular basis for cardiac hypertrophy/heart failure but also provide essential information that will facilitate the development of new epigenetics-based therapies.

Fig. 1

Epigenetic changes at different levels of chromatin structure. CpG sites within genomic DNA undergo methylation, and core histones in nucleosomes undergo acetylation (Ac), methylation (Met), ubiquitination (Ub), sumoylation (S), or phosphorylation (P). Higher order chromatin structure is also dynamically modified by chromatin-remodeling complexes

Fig. 2

Epigenetic changes and transcriptional activity. Suppression of gene expression (OFF) is correlated with the methylation (Met) of genomic DNA, deacetylation of histones, and methylation of H3-K9. In contrast, activation of gene expression (ON) is associated with unmethylated genomic DNA, acetylated (Ac) histones, and methylated H3-K4

Fig. 3

The differential chromatin scanning (DCS) method. DNA fragments bound to acetylated (Ac) histones are purified by immunoprecipitation (IP) and subjected to TAG adaptor ligation (green bars) and PCR amplification. The tester DNA is then digested with XmaI, ligated to the first subtraction adaptor (red bars), and annealed with an excess amount of the driver DNA. Given that only the tester-specific fragments self-anneal, PCR with the first subtraction primer selectively amplifies these fragments. The products are subjected to a second round of subtraction PCR with the second subtraction adaptor and primer to ensure the fidelity of the subtraction. Reproduced from Kaneda et al. [17]

Fig. 4

Identification of Itpr3 as a target of histone deacetylase (HDAC) in cardiomyocytes. a One of the DCS clones (H9C2T-2_D09; red rectangle) was mapped to chromosome 20p12, spanning intron 21 and exon 22 of Itpr3. Exons are denoted by black boxes, the arrow indicates the direction of transcription, and blue triangles depict distance markers separated by 50 kbp. b Chromatin immunoprecipitates were prepared from H9C2 cells treated (+) or not (−) with 300 nM inhibitor trichostatin A (TSA) for 24 h. The amount of DNA corresponding to the H9C2T-2_D09 sequence in each chromatin immunoprecipitation (ChIP) product relative to that in the corresponding original sample before immunoprecipitation (PreIP) was then determined by real-time PCR. c The amount of Itpr3 mRNA relative to that of Gapdh mRNA in H9C2 cells treated or not with TSA was determined by quantitative reverse transcription (RT)-PCR. All data are means ± SD of triplicates from representative experiments that were performed at least twice. P values for the indicated comparisons were determined by Student’s t test. Reproduced from Kaneda et al. [18]

Fig. 5

Chromosomal distribution of HDAC targets. The genome fragments (red dots) isolated by the DCS method were mapped to rat chromosomes. Reproduced from Kaneda et al. [18]