Introduction Increased reactive oxygen species (ROS) production is involved in the pathophysiology of cardiac hypertrophy and failure. Interestingly, a specialised ROS-generating enzyme NADPH oxidase-4 (Nox4) was previously found to have beneficial effects by promoting adaptive remodelling during pressure-overload cardiac hypertrophy. Nox4 modulates intracellular signalling cascades but how it achieves beneficial effects in the chronically overloaded heart remains unclear.
Methods and results To obtain an unbiased global overview of putative Nox4-mediated changes, the proteome of cardiac-specific Nox4 transgenic (TG) and wild-type (WT) mouse hearts was first characterised through a 2D-DIGE approach. TG hearts had a significant over-representation of changes in protein levels of enzymes involved in glucose and fatty acid utilisation. We therefore analysed the metabolome using 1H-NMR and targeted LC-MS approaches. This identified a differential accumulation of glycolytic intermediates in the proximal part of glycolysis both in unstressed and pressure-overloaded TG hearts, as well as an increase in alanine levels (1.4 fold, p = 0.05), confirming significant alterations to metabolism. To specifically quantify glucose uptake, glycolysis, glucose oxidation and fatty acid oxidation rates, ex vivo working heart studies were conducted. TG hearts had a marked increase cf. WT in palmitate oxidation rate in the unstressed as well as pressure-overloaded heart (3.6 fold increase; n = 6/group; p = 0.01). Glucose uptake was unaltered but glycolysis and oxidation rates were decreased, suggesting diversion of glucose away from oxidation. Importantly, an increase in palmitate oxidation was not detrimental either for in vivo cardiac energetics (31P-NMR) or contractile function during pressure-overload hypertrophy. We found that activity of the hexosamine biosynthesis pathway (HBP), an alternative route for glucose metabolism, was increased in TG hearts as assessed by the O-GlcNAc post-translational modification of cardiac proteins by N-acetylglucosamine, the end-product of HBP. O-GlcNAc levels were 2.4 fold higher in TG cf. WT (n = 4/group; p = 0.02). In cultured cardiomyocytes, endogenous Nox4 induced similar changes in HBP and palmitate oxidation (extracellular flux analysis), and it was found that changes in O-GlcNAcylation regulated fatty acid oxidation.
Discussion These results show that Nox4 reprograms substrate utilisation in the heart by directing glucose towards the HBP and inducing a linked increase in fatty acid oxidation. These changes appear to enable the heart to better adapt to chronic pressure overload and may be important in the beneficial effects of Nox4 on cardiac remodelling. These data identify a novel redox mechanism that drives beneficial metabolic reprogramming in the heart and suggest potential new therapeutic approaches to promote adaptation to chronic overload stress.
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