Peter Bast

Kir6.2 is required for adaptation to stress

Reaction to stress requires feedback adaptation of cellular functions to secure a response without distress, but the molecular order of this process is only partially understood. Here, we report a previously unrecognized regulatory element in the general adaptation syndrome. Kir6.2, the ion-conducting subunit of the metabolically responsive ATP-sensitive potassium (KATP) channel, was mandatory for optimal adaptation capacity under stress. Genetic deletion of Kir6.2 disrupted KATP channel-dependent adjustment of membrane excitability and calcium handling, compromising the enhancement of cardiac performance driven by sympathetic stimulation, a key mediator of the adaptation response. In the absence of Kir6.2, vigorous sympathetic challenge caused arrhythmia and sudden death, preventable by calcium-channel blockade. Thus, this vital function identifies a physiological role for KATP channels in the heart.

Mapping hypoxia-induced bioenergetic rearrangements and metabolic signaling by 18O-assisted 31P NMR and 1H 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.

Defective metabolic signaling in adenylate kinase AK1 gene knock-out hearts compromises post-ischemic coronary reflow.

Matching blood flow to myocardial energy demand is vital for heart performance and recovery following ischemia. The molecular mechanisms responsible for transduction of myocardial energetic signals into reactive vasodilatation are, however, elusive. Adenylate kinase, associated with AMP signaling, is a sensitive reporter of the cellular energy state, yet the contribution of this phosphotransfer system in coupling myocardial metabolism with coronary flow has not been explored. Here, knock out of the major adenylate kinase isoform, AK1, disrupted the synchrony between inorganic phosphate Pi turnover at ATP-consuming sites and γ-ATP exchange at ATP synthesis sites, as revealed by 18O-assisted 31P NMR. This reduced energetic signal communication in the post-ischemic heart. AK1 gene deletion blunted vascular adenylate kinase phosphotransfer, compromised the contractility-coronary flow relationship, and precipitated inadequate coronary reflow following ischemia-reperfusion. Deficit in adenylate kinase activity abrogated AMP signal generation and reduced the vascular adenylate kinase/creatine kinase activity ratio essential for the response of metabolic sensors. The sarcolemma-associated splice variant AK1β facilitated adenosine production, a function lost in the absence of adenylate kinase activity. Adenosine treatment bypassed AK1 deficiency and restored post-ischemic flow to wild-type levels, achieving phenotype rescue. AK1 phosphotransfer thus transduces stress signals into adequate vascular response, providing linkage between cell bioenergetics and coronary flow.