Crosstalk between redox regulatory pathways and epigenetic processes in tissue and cell culture models of cardiovascular complications
Date issued
Authors
Editors
Journal Title
Journal ISSN
Volume Title
Publisher
License
Abstract
Oxidative stress is caused by an imbalance between the production of reactive oxygen species (ROS) and the neutralization of these species by antioxidant systems, leading to a disruption of redox signaling, oxidative damage and potential pathological consequences, including cardiovascular complications. There is a growing body of evidence that ROS influence epigenetic pathways by affecting the function or levels of epigenetic modulators, such as histone modifying enzymes. Since epigenetic modifications are increasingly recognized as major players in cardiovascular disease development and progression, investigation of the interplay between redox signaling and epigenetic regulation is of particular interest in this context.
A readout assay for the detection of protein S-nitros(yl)ation, a redox modification, was established using an approach consisting of light-induced homolysis of nitros(yl)ated proteins and subsequent immuno-spin trapping of generated protein radicals via 5,5 dimethyl-1 pyrroline N-oxide (DMPO) and a respective antibody.
In order to investigate ROS-induced epigenetic changes, a suitable model system with elevated ROS formation had to be established. Culturing endothelial cells under hyperglycemic conditions is known to generate increased oxidative stress. However, no substantial effects on ROS formation and histone methylation and acetylation patterns could be observed in the endothelial cell line EA.hy926 upon hyperglycemia, possibly due to a systematical technical error that, however, could not be identified despite multiple methodological variations.
In mice deficient in the antioxidant protein glutathione peroxidase-1 (GPx-1) endothelial dysfunction and enhanced ROS levels have been reported previously, an effect that was further potentiated by aging. Epigenetic analysis of this model led to the hypothesis of potential dityrosine cross-linking between histone 3 and histone 4 accompanied by enhanced histone 3 lysine 9 dimethylation upon increased oxidative stress. However, upon further investigation by mass spectrometry and exclusion of antibody cross-reactivity to IgGs in the animal samples this assumption was revealed to be false.
In a published study by our group it was demonstrated that empagliflozin, a selective sodium-glucose co transporter 2 inhibitor (SGLT2i), reduced glucotoxicity and thereby prevented the development of endothelial dysfunction, reduced oxidative stress and exhibited anti-inflammatory effects in ZDF rats, an animal model for type 2 diabetes mellitus (T2DM). Investigation of involved epigenetic mechanisms by chromatin immunoprecipitation (ChIP) analysis revealed an effect of empagliflozin treatment on expression of glucotoxicity- and inflammation-markers in diabetic animals via altered histone methylation patterns.
Finally, the interplay of increased ROS formation and epigenetic alterations was studied in H9c2 cardiomyocytes with a doxorubicin-induced cardiotoxic phenotype. It was discovered that doxorubicin treatment affected the expression of certain epigenetic modulators in correlation with increased oxidative stress markers. Given that epigenetic changes are reversible, they represent potential intervention targets as well as biomarkers that can be addressed for drug discovery. Thus, establishing a comprehensive understanding of the interplay of ROS and epigenetic mechanisms in cardiovascular related diseases may lead to the development of novel and precisely targeted treatment options.