Gene environment-interaction and cardiovascular phenotype in obesity and diabetes

Although a large body of evidence supports the notion that genes determine cardio-metabolic traits and outcomes, the non-genetic regulation of these events has recently gained increasing attention. Plastic chemical modifications of DNA-histone complexes defined epigenetic changes regulate gene expression by modifying chromatin accessibility to transcription factors. In the present thesis, we have investigated the emerging role of epigenetic modifications as fine-tuning regulators of gene expression in diabetic cardiomyopathy, as well as in obesity and diabetes-driven endothelial dysfunction.

Study I: The objective was to investigate whether mitochondrial adaptor p66Shc contributes to obesity-related vascular dysfunction. Oxidative stress and vascular expression of chromatin modifying enzymes were investigated in visceral fat arteries (VFA) from obese and age-matched healthy subjects. VFA from obese patients displayed enhanced mitochondrial reactive oxygen species (ROS) and endothelial dysfunction as well as a significant dysregulation of chromatin modifier enzymes methyltransferase SUV39H1, demethylase JMJD2C and acetyltransferase SRC-1 as compared to control VFA. These changes were associated with reduced methylation and acetylation of histone 3 lysine 9 (H3K9) on p66Shc promoter. Specifically, we demonstrated that obesity-induced downregulation of SUV39H1 orchestrates JMJD2C/SRC-1 recruitment to p66Shc promoter, fostering adverse H3K9 remodeling and p66Shc upregulation.

Study II: We sought to investigate whether epigenetic regulation of pro-oxidant adaptor p66Shc contributes to persistent myocardial dysfunction despite intensive glycemic control (IGC). p66Shc expression was increased in the heart of diabetic mice, and IGC did not revert this phenomenon. Dysregulation of methyltransferase DNMT3b and deacetylase SIRT1 linked to upregulation of miRNAs (miR-218 and miR-34a) drive persistent transcription of the adaptor p66Shc, thereby leading to mitochondrial oxidative stress, myocardial inflammation and left ventricular dysfunction. Our findings showed that adverse epigenetic signatures on p66Shc promoter contribute to left ventricular (LV) dysfunction in the setting of diabetes.

Study III: Here we demonstrate for the first time a protective role of activated protein-1 (AP-1) transcription factor JunD against derangement of ROS homeostasis, inflammation and myocardial impairment in the setting of diabetes-induced hyperglycemia. JunD transcriptional activity was reduced in the heart of wild-type mice with streptozotocin-induced diabetes and was associated with downregulation of free radical scavengers, increased expression of ROS-generating NADPH oxidase and marked increase in myocardial superoxide anion generation. These redox changes were paralleled by activation of NF-κB-dependent inflammatory pathways and left ventricular dysfunction. Interestingly enough, such detrimental changes did not occur in diabetic mice with cardiac-specific overexpression of JunD (α-MHC-JunDtg) and LV function was not impaired, indicating the relevant role of JunD in counteracting hyperglycemia-induced redox changes and cardiac damage in diabetes.

Study IV: Enhancer of zeste homologue 2 (EZH2), a member of the family of SET1 methyltransferase and a catalytic component in the polycomb repressive complex 2, is associated with transcriptional repression through histone H3K27me3 modification. Therefore, we hypothesize that its pharmacological modulation could have an impact on hyperglycemia-driven endothelial dysfunction. We demonstrated that pharmacological inhibition of EZH2 by GSK126 might prevent key hallmarks of diabetic vascular dysfunction, such as oxidative stress and inflammation. Experiments in human aortic endothelial cells showed that GSK126 protects against hyperglycemia-induced oxidative stress and inflammation via restoration of JunD, SOD1 and SOD2 expression and inhibition of Nox4 upregulation. Moreover, GSK126 was able to prevent activation of transcription factor NF-kB and subsequent upregulation of inflammatory adhesion molecules IL-6 and MCP-1.

Altogether, our studies provide novel molecular insights on the regulation of redox and inflammatory pathways implicated in the impairment of obesity and diabetes-induced endothelial and cardiac function. Moreover, by targeting epigenetic changes responsible of derailed pro-oxidant and pro-inflammatory transcriptional programmes, we shed some light on putative pharmacological strategies to reduce the burden of cardiovascular disease in this setting.

List of scientific papers

I. Sarah Costantino, Francesco Paneni, Agostino Virdis, Shafaat Hussain, Shafeeq Ahmed Mohammed, Giuliana Capretti, Alexander Akhmedov, Kevin Dalgaard, Sergio Chiandotto, J Andrew Pospisilik, Thomas Jenuwein, Marco Giorgio, Massimo Volpe, Stefano Taddei, Thomas F Lüscher, Francesco Cosentino. Interplay among H3K9-editing enzymes SUV39H1, JMJD2C and SRC-1 drives p66Shc transcription and vascular oxidative stress in obesity. European Heart Journal. (2019); 40 (4):383-39.
https://doi.org/10.1093/eurheartj/ehx615

II. Sarah Costantino, Francesco Paneni, Katharyn Mitchell, Shafeeq A.Mohammed, Shafaat Hussain, Christos, Gkolfos, Liberato Berrino, MassimoVolpe, Colin Schwarzwald, Thomas Felix Lüscher, Francesco Cosentino. Hyperglycaemia-induced epigenetic changes drive persistent cardiac dysfunction via the adaptor p66Shc. International Journal of Cardiology. (2018); 268: 179-186.
https://doi.org/10.1016/j.ijcard.2018.04.082

III. Shafaat Hussain, Abdul Waheed Khan, Alexander Akhmedov, Sarah Costantino, Francesco Paneni, Kenneth Caidahl, Shafeeq A. Mohammed, Rosa Suades, Camilla Hage, Christos Gkolfos, Hanna Björck, John Pernow, Lars H. Lund, Thomas F. Luscher, Francesco Cosentino. Protective role of AP-1 transcription factor JunD in the diabetic heart. [Manuscript]

IV. Shafaat Hussain, Abdul Waheed Khan, John Pernow, Francesco Cosentino. EZH2 inhibition via GSK126 attenuates high glucose induced oxidative stress and inflammation in human aortic endothelial cells. [Manuscript]