High Palmitate Alters Glutathione Redox During Severe Oxidant Stress in the Heart; An Evaluation of Altered Glutathione Redox Environment on Cardiac Function and Mitochondrial Energetics.

Abstract

When the heart is under stress, e.g. ischemia-reperfusion (IR), it produces an enormous amount of reactive oxygen species (ROS). Glutathione, which is an endogenous antioxidant derived from the ‘nicotinamide adenine dinucleotide hydrogen’ (NADH) of the ‘tricarboxylic acid’ cycle, can fight against this oxidative stress to balance the redox environment, and convert the produced ROS to H2O. In this study, we hypothesized that elevated levels of fatty acids, such as palmitate, can contribute to maintain the ratio of reduced (GSH) and oxidized (GSSG) glutathione during cardiac stress. Therefore, the aim was to evaluate the potential effects of high levels of palmitate in hearts exposed to stress induced by IR or diamide pro-oxidant on cardiac function and mitochondrial energetics. Cardiac function was assessed in isolated working hearts perfused with low and high concentrations of palmitate. Following that, mitochondrial respiration and hydrogen peroxide (H2O2) emission were determined using a high-resolution respirometry and fluorometry (Oxygraph-2k, OROBOROS Instruments, Innsbruck, Austria). Liquid chromatography tandem mass spectrometry was used to measure GSH and GSSG levels, and GSH/GSSG. Changes in the expression of various genes related to mitochondrial dynamics and GSH were evaluated using real time-reverse transcription polymerase chain reaction. Lastly, protein S-glutathionylation was determined using western blotting. We observed that although IR and diamide treatments could significantly alter the cardiac function, including the cardiac output, cardiac power, and aortic flow, only the diamide treatment altered the levels of GSH, GSSG, and GSH/GSSG. In addition, diamide treatment altered mitochondrial respiration and H2O2 emission. The presence of high palmitate during diamide treatment resulted in lower levels of GSSG as compared to hearts perfused with low palmitate. Also, mRNA expression of ‘glutathione reductase’ (grs) and ‘glutathione synthetase’ (gss), two genes related to GSH synthesis, were different in hearts perfused with low and high palmitate levels, respectively. However, in-depth studies should be performed to evaluate the role of palmitate in GSH synthesis and oxidation.