Decreasing O‐GlcNAcylation of 8‐oxoguanosine DNA Glycosylase (OGG1) in Cardiac Myocytes Cultured in High Glucose Leads to Improvement of DNA Repair and Mitochondrial Function

Abstract

DNA damage in cardiac myocytes resulting from increased oxidative stress is an important factor in the pathogenesis of diabetic cardiomyopathy. Although DNA repair machinery has been described in the cardiac myocyte, the regulation of oxidative DNA damage repair has been incompletely investigated. Moreover, in hyperglycemia the O‐GlcNAc post‐translational protein modification has been reported to interfere with the regular intracellular activity of metabolically relevant proteins in either an adaptive or a maladaptive manner. The enzyme 8‐oxoguanosine DNA glycosylase (OGG1) is key in DNA repair. Thus, the objective of this work was to investigate the influence of O‐GlcNAcylation of OGG1 in excessive glucose concentrations, simulating the levels observed in diabetic animals or humans. Neonatal cardiac myocytes (NCM) were isolated and cultured for 72 h in either normal glucose (NG; 5.5 mM glucose, 19.5 mM mannitol) or high glucose (HG; 25 mM glucose). The relationship between OGG1 O‐GlcNAcylation levels, enzymatic functionality and subcellular localization was analyzed. OGG1 was found to be up‐regulated and highly O‐GlcNAcylated in HG cultured NCM and was associated with increased 8‐OHdG (8‐hydroxy‐deoxyguanosine) levels, a common oxidative stress biomarker targeted by OGG1. In addition, decreased DNA quality and mitochondrial function were observed. Interestingly, OGG1 enzymatic activity in cell lysates was lower in HG compared to NG. Furthermore, in vitro experiments with recombinant OGG1 and O‐GlcNac transferase (OGT) demonstrated that O‐GlcNAcylation of OGG1 diminishes enzyme activity. We performed rescue experiments in NCM in HG by either rectifying OGG1 levels or reducing OGG1 O‐GlcNAcylation. NCM were either transduced with adenoviral particles (Adv) carrying OGG1 (Adv‐OGG1) or co‐transduced with Adv‐OGG1 and a dominant negative OGT (Adv‐dnOGT). Co‐transduced NCM showed improved OGG1 enzymatic activity compared to controls cells in spite of HG. These results were associated with lower 8‐OHdG levels. Finally, a different sub‐cellular enzyme distribution and an improvement in mitochondrial function were also detected. In conclusion, these results suggest that O‐GlcNAcylation of OGG1 could be considered a maladaptive phenomenon caused by hyperglycemia, which directly interferes with DNA repair by altering OGG1 activity and translocation, and indirectly leads to mitochondrial dysfunction.Support or Funding InformationThis work was supported by National Institutes of Health grants (5 P01 HL066941‐13), with a Merit Review Award from the Department of Veteran's Affairs (5 I01BX001121‐02) and the P. Robert Majumder Charitable Foundation.