Enhancing Glycolysis Protects against Ischemia-Reperfusion Injury by Reducing ROS Production

Abstract

After myocardial ischemia-reperfusion, fatty acid oxidation shows fast recovery while glucose oxidation rates remain depressed. A metabolic shift aimed at increasing glucose oxidation has shown to be beneficial in models of myocardial ischemia-reperfusion. However, strategies aimed at increasing glucose consumption in the clinic have provided mixed results and have not yet reached routine clinical practice. A better understanding of the mechanisms underlying the protection afforded by increased glucose oxidation may facilitate the transfer to the clinic. The purpose of this study was to evaluate if the modulation of reactive oxygen species (ROS) was involved in the protection afforded by increased glucose oxidation. Firstly, we characterized an H9C2 cellular model in which the use of glucose or galactose as substrates can modulate glycolysis and oxidative phosphorylation pathways. In this model, there were no differences in morphology, cell number, or ATP and PCr levels. However, galactose-grown cells consumed more oxygen and had an increased Krebs cycle turnover, while cells grown in glucose had increased aerobic glycolysis rate as demonstrated by higher lactate and alanine production. Increased aerobic glycolysis was associated with reduced ROS levels and protected the cells against simulated ischemia-reperfusion injury. Furthermore, ROS scavenger N-acetyl cysteine (NAC) was able to reduce the amount of ROS and to prevent cell death. Lastly, cells grown in galactose showed higher activation of mTOR/Akt signaling pathways. In conclusion, our results provide evidence indicating that metabolic shift towards increased glycolysis reduces mitochondrial ROS production and prevents cell death during ischemia-reperfusion injury.

Keywords: heart; metabolic shift; myocardial infarction.

Figure 1

Graphs showing (A) citrate synthase activity in cells grown in galactose media compared to cells grown in glucose media, and (B) mitochondria oxygen consumption per million of cells in glucose and galactose media. (C–E) correspond to the Western blot of NDUFB9 and COXIV for glucose (n = 5) and galactose (n = 4)-treated cells and their quantification. * Statistically different p < 0.05.

Figure 2

Effect of substrate type (glucose vs. galactose) on mitochondrial pool, as quantified from confocal images of H9C2 cells stained with MitoTracker Green. (A) Representative H9C2 cells cultured in glucose-containing media; (B) same in galactose-containing media; (C) Quantification of mitochondrial abundance per cell in both conditions (expressed as arbitrary units of fluorescence per cell surface [in µm²]). Bars correspond to mean ± SEM of n = 78–89 cells per group.

Figure 3

Metabolic profiling from 1H NMR spectra of H9C2 cell extracts. (A) Corresponds to the score plot of the OPLS-DA (orthogonal projection to latent structures discriminant analysis) model able to differentiate between substrates; empty circles correspond to glucose and full circles to galactose-treated cells. Panels (B–E) show bar graphs depicting lactate, alanine, ATP and PCr concentrations respectively for glucose and galactose media. Data in nmols/10⁶ cells. * statistically different p < 0.05.

Figure 4

Representative ¹H-¹³C HSQC spectra of cell extracts after 24 h of culture with 10 mmol/L 1-¹³C-glucose and 3 mmol/L 1-¹³C acetate (A) or 1-¹³C-galactose and 3 mmol/L 1-¹³C acetate (B). (C,D) correspond to the ¹H-¹³C HMBC spectra of the samples shown in (A,B) respectively. The ratio between glutamate C4 and lactate C3 (E) is indicative of the pathway of glucose oxidation while glutamate C5 (F) is indicative of β-oxidation. n = 3 for double label experiments, * statistically different p < 0.05.

Figure 5

(A) ROS levels measured as DCFDA fluorescence and expressed respect to glucose without TBH induction; 8 mmol/L NAC was added when indicated. (B) Corresponds to the quantification of cell death after simulated ischemia-reperfusion, in the presence or absence of 8 mmol/L NAC. * denotes statistical difference p < 0.05 between glucose vs. galactose while $ denotes statistical difference between TBH treated and untreated cells.

Figure 6

Quantification of the phosphorylation state of Akt (A) and mTOR (B). Bars correspond to the average ± SD of the ratios between p-Akt and total Akt, (n = 4, 5), * statistically different p < 0.05.