A high-resolution method for assessing cellular oxidative phosphorylation efficiency: bringing mitochondrial bioenergetics into focus. Focus on “Direct real-time quantification of mitochondrial oxidative phosphorylation efficiency in permeabilized skeletal muscle myofibers”

ADENOSINE TRIPHOSPHATE, ATP, is a high-energy molecule that plays a central role for a host of fundamental cellular processes.Maintaining adequate ATP availability is paramount for cell survival during both normal physiological states and in response to stress or injury.Mitochondria are fundamental in maintaining the dynamic, yet persistent, demand for ATP by coupling the electrochemical gradient generated by complexes I, II, and III of the electron transport chain (ETC) to the reduction of molecular oxygen (O 2 ) by ATP synthase (complex V).Understanding the role and regulation of this high-energy cellular "currency" has defined the field of mitochondrial bioenergetics since early last century (for review, see ref. 4).More recent advances have highlighted the observed variability in the coupling between ETC flux/O 2 consumption and ATP synthesis, or oxidative phosphorylation (OXPHOS) efficiency.Uncoupling proteins (UCPs) are mitochondrial proton transporters present in the inner membrane that act as a shunt between ETC complexes and ATP synthase (7).Activation of the uncoupling process results in a futile cycle of O 2 consumption without ATP synthesis, with dissipation of oxidation energy as heat.In developing endotherms where heat loss secondary to a higher surface area-to-volume ratio is substantial, UCPs play a critical role in maintaining normothermia.OXPHOS efficiency remains relevant in the adult as flux through the ETC and ATP availability independently coordinate intracellular Ca 2ϩ handling, initiation of apoptosis, and the regulation of oxidant production, processes that determine cellular fate during both normal physiologic functioning and in response to stress (5).Traditional methods to assess OXPHOS efficiency have been technically limited in resolution: direct assessment of O 2 consumption and [ATP] require separate, independent measurements, introducing intersample variability, while assessing O 2 consumption at a known level of [ADP] as substrate for OXPHOS is limited in dynamic range and does not account for other sources of ADP rephosphorylation (e.g., from phosphocreatine/creatine kinase).However, advances in fluorescent imaging techniques have dramatically improved the ability to simultaneously observe intracellular biochemical processes in real time.Recently, Gouspillou and colleagues (2) described an enzymatically coupled approach to measure OXPHOS affinity for ADP in isolated mitochondria.By enzymatically coupling ATP as substrate for the reducing equivalent nicotinamide adenine dinucleotide phosphate (NADP ϩ ), and simultaneously