Identification of subcellular energy fluxes by P NMR spectroscopy in the perfused heart: contractility induced modifications of energy transfer pathways

The identification of subcellular fluxes of exchange of ATP, phosphocreatine (PCr) and Pi between mitochondria, cytosol and ATPases and pathways of energy transfer in a whole organ is a challenge specially in the myocardium where 50% of creatine kinases (CK) are found in close vicinity of ATP producing (mito-CK) and utilizing ( MM-bound CK) reactions. To dissect their contribution in cardiac energy transfer we recently developed a new experimental31P NMR spectroscopy approach. This led to identify three kinetically different subcellular CKs and to evidence experimentally the CK shuttle in a rat heart perfused in isovolumy. Here we show that a decreased energy demand alters energetic pathways : two CKs (cytosolic and MM-bound) functioning at equilibrium and a non mitochondrial ATP↔Pi exchange was sufficient to describe NMR data. Mito-CK fluxes was not detected anymore. This confirms the dependence of energy pathways upon cardiac activity. Indeed the subcellular localization and activity of CKs may have important bioenergetic consequences for the in vivo control of respiration at high work: free ADP estimated from global CK equilibrium might not always adequately reflect its concentration at the ANT.