An epigenetic circuit linking oxidative stress and DNA hydroxymethylation in heart failure

Abstract

Abstract Background Heart failure is a major cause of morbidity and mortality worldwide, but the underlying molecular mechanisms remain not well defined. Reactive oxygen species (ROS) in heart failure (HF) alter multitudes of mitochondrial enzymes and metabolites, such as α-ketoglutarate and its oxidised form L-2-hydroxyglutarate (L-2HG). These metabolites are cofactors for ten eleven translocation (TET) enzymes that convert DNA's 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC). Aim We hypothesize that oxidative stress during heart failure alters mitochondrial function through epigenetic remodeling, and that reduction of oxidative stress will improve cardiac function. Methods and results Targeted LC-MS/MS analysis of dilated cardiomyopathy (DCM) hearts obtained from MLP−/− and WT littermate controls showed a significant increase in the oxidized metabolite L-2HG (∼30%, p=0.004), with significant reduction of multiple TCA cycle intermediates. RNA sequencing revealed a significant reduction in mRNA levels of IDH2 in human TTN related DCM and MLP−/− HF mice. The altered activity of IDH2 contributes to ROS production in these hearts and to the production of L-2HG. No alteration in the mitochondria structures was observed. HF biopsies show decreased TET activity most likely due to increased L-2HG levels. Whole genome single base pair 5mC and 5hmC deep sequencing of gDNA from explanted human DCM heart biopsies and MLP−/− mouse model revealed significantly altered global distribution of both 5mC and 5hmC in comparison to control samples. Genes involved in hypertrophy, such as Myh7 and Fhl2 were among the top genes with differential 5mC levels. Global loss of 5hmC level was observed, especially in the intronic regions of genes involved in redox hemostasis. Reducing oxidative stress in vivo in MLP−/− using a small molecule (AZ14117925) improves heart function (EF) by 13% [EF=(Treated:47,44%, Placebo: 34,54%), n=5 (males per group), p=0.0373, unpaired t-test]. Additional bioinformatic analysis revealed that reduction of ROS most likely leads to activation of TET and activation of pro-survival pathways, anti-oxidative stress response, and significantly less activation of apoptotic pathways. Conclusion Alterations in TCA cycle metabolites may underlie changes in DNA methylation and gene expression in end-stage human DCM and mouse models and indicate a role for epigenetic regulation of mitochondrial function in HF. NRF2 activation may pose a novel therapeutic approach to treat this devastating disease. Funding Acknowledgement Type of funding source: Private company. Main funding source(s): AstraZeneca