Mapping hypoxia-induced bioenergetic rearrangement and metabolic signalling by ¹⁸O-assisted ³¹P NMR and ¹ NMR spectroscopy

Brief hypoxia or ischemia perturbs energy metabolism inducing paradoxically a stress-tolerant state, yet metabolic signals that trigger cytoprotection remain poorly understood. To evaluate bioenergetic rearrangements, control and hypoxic hearts were analyzed with ¹⁸O-assisted ³¹P NMR and ¹H NMR spectroscopy. The ¹⁸O-induced isotope shift in the ³¹P NMR spectrum of CrP, βADP and βATP was used to quantify phosphotransfer fluxes through creatine kinase and adenylate kinase. This analysis was supplemented with determination of energetically relevant metabolites in the phosphomonoester (PME) region of ³¹P NMR spectra, and in both aromatic and aliphatic regions of ¹H NMR spectra. In control conditions, creatine kinase was the major phosphotransfer pathway processing high-energy phosphoryls between sites of ATP consumption and ATP production. In hypoxia, creatine kinase flux was dramatically reduced with a compensatory increase in adenylate kinase flux, which supported heart energetics by regenerating and transferring β- and γ-phosphoryls of ATP. Activation of adenylate kinase led to a build-up of AMP, IMP and adenosine, molecules involved in cardioprotective signaling. ³¹P and ¹H NMR spectral analysis further revealed NADH and H⁺ scavenging by α-glycerophosphate dehydrogenase (αGPDH) and lactate dehydrogenase contributing to maintained glycolysis under hypoxia. Hypoxia-induced accumulation of α-glycerophosphate and nucleoside 5′-monophosphates, through αGPDH and adenylate kinase reactions, respectively, was mapped within the increased PME signal in the ³¹P NMR spectrum. Thus, ¹⁸O-assisted ³¹P NMR combined with ¹H NMR provide a powerful approach in capturing rearrangements in cardiac bioenergetics, and associated metabolic signaling that underlie the cardiac adaptive response to stress.