Representative oxygraph trace measuring endogenous OXPHOS and ADP channelling from myofibrils to the mitochondria within permeabilised fibres.

The upper trace shows the O2 concentration (μM; blue) and mass specific O2 flux (pmols s-1 mg-1; red). The lower trace shows concurrent fluorescent measurement of total ROS (O-2. + H2O2) with enzyme coupled detection system Amplex UltraRed, super oxide dismutase and horse radish peroxidase. Step wise addition of substrates allows measurement of different respiratory states. Glutamate and malate (GM) produced NADH-stimulated Leak (CI Leak); ATP stimulates oxidative phosphorylation (OXPHOS) respiration through endogenous production of ADP by cellular ATPases (CI ADP-limited OXPHOS); succinate is a complex II substrate (CI + CII ADP-limited OXHOS); phosphoenolpyruvate (PEP) is a substrate for endogenous pyruvate kinase (PK) and a reaction that consumes ADP (ADP trap); additional titration of exogenous PK further tests ADP channelling towards mitochondria. Finally, addition of creatine stimulates mitochondrial creatine kinase (mCK), promoting local turnover of ADP within the intermembrane space and relieving inhibition of the ADP trapping enzyme system. Artefacts in the traces (red and green) during the addition of substrates or O2 have been omitted for clarity.