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, betaADP and betaATP 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 beta- and gamma-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
alpha-glycerophosphate dehydrogenase (
alphaGPDH) and
lactate dehydrogenase contributing to maintained glycolysis under
hypoxia.
Hypoxia-induced accumulation of
alpha-glycerophosphate and
nucleoside 5'-monophosphates, through
alphaGPDH 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.