Am J Physiol Cell Physiol 311: C237–C238, 2016;
doi:10.1152/ajpcell.00203.2016.
Editorial Focus
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”
Creed M. Stary
Department of Anesthesiology, Perioperative and Pain Medicine, Stanford University School of Medicine, Stanford, California
ADENOSINE TRIPHOSPHATE,
Address for reprint requests and other correspondence: C. M. Stary, 300
Pasteur Drive, MC 5117, Stanford, CA 94305-5117 (e-mail:
[email protected]).
http://www.ajpcell.org
assessing reduced NADP⫹ (NADPH) bioluminescence and
polarographic O2 consumption (Fig. 1), Gouspillou et al. assessed OXPHOS affinity for ADP directly during physiologic
steady-state conditions, and at a range of substrate concentrations. This approach, in which [ADP] and [ATP] were effectively clamped, served to increase the quantitative resolution of
OXPHOS affinity for ADP by: 1) reducing intersample variability via simultaneous measurements of O2 consumption and
NADPH; and, 2) permitting repeated measures within a single
experiment thereby increasing the fidelity of measurement.
In the current issue of American Journal of Physiology-Cell
Physiology, Lark and colleagues (3) for the first time apply a
similar high-resolution approach to assess OXPHOS efficiency
in intact, living cells. By utilizing the technique in permeabilized skeletal muscle fiber bundles (PmFBs), which retain a
functional mitochondrial reticulum and intracellular energy
transfer systems, the authors assessed OXPHOS efficiency in a
system that more closely approximates the in vivo intracellular
milieu. Using this approach, Lark et al. compared OXPHOS
efficiency in PmFBs and isolated skeletal muscle mitochondria
at lower levels of [ADP] and observed that OXPHOS efficiency was substantially decreased in PmFBs relative to isolated mitochondria (⬃23% vs. 98%, respectively), which more
closely approximated in vivo estimates (⬃50%). Moreover,
Lark et al. observed that OXPHOS efficiency in PmFBs improved as a function of [ADP], to as high as 84%. This finding
is relevant for: 1) the capacity of mitochondria to meet large,
acute changes in ATP demand, such as the transition from rest
to high-intensity exercise; and, 2) previous observations that
treatments which improve OXPHOS efficiency can augment
both exercise efficiency and the capacity for sustained exercise
(1). However, this observation raises several basic physiological questions. For example, what are the cellular mechanisms
that regulate alterations in OXPHOS efficiency with increased
ATP demand, and more broadly, what is the relevance of
mitochondrial uncoupling in the adult, where total body heat
loss is less substantial with a lower body surface area-tovolume ratio? Application of the model developed by Lark et
al. (Fig. 1) may provide answers to these and other fundamental physiological questions, as well as provide a platform for
testing of pharmacological agents with the potential to alter
OXPHOS efficiency in skeletal muscle.
The capability for real-time, high-resolution measurements
of OXPHOS efficiency in living cells also holds relevance in
other organ systems that have high tissue-specific O2/ATP
requirements, such as in the heart and brain. Even transient
disruptions in O2 delivery to these critical organs can reduce
cellular ATP availability, inducing cell death and dysfunction
and resulting in a high potential for death or disability.
0363-6143/16 Copyright © 2016 the American Physiological Society
C237
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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 (O2) 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/O2 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 O2 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 Ca2⫹ 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 O2
consumption and [ATP] require separate, independent measurements, introducing intersample variability, while assessing
O2 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⫹), and simultaneously
Editorial Focus
C238
decay, may enhance the resolution of measurement, further
increasing the ability to quantify and understand the fundamental mechanics of mitochondrial bioenergetics in living systems.
Advancing the development and application of this technique
to other types of living cells may identify universal, cell- and
organ-specific pathways regulating OXPHOS efficiency, further clarifying the role mitochondria play in normal physiological function, and in organ dysfunction and disease.
GRANTS
This was work was supported, in part, by American Heart Association
Grant FTF19970029.
DISCLOSURES
AUTHOR CONTRIBUTIONS
Fig. 1. The technique employed by Lark et al. for high-resolution measurement
of oxidative phosphorylation (OXPHOS) efficiency in permeabilized skeletal
muscle fiber bundles (top), and potential applications in primary cell cultures
from other organ systems (bottom). OXPHOS efficiency is defined as the molar
ratio of ATP produced to O2 consumed. Enzymatic coupling of ATP as
substrate for NADH⫹ reduction to NADPH allows real-time quantification of
ATP via NADPH fluorescence, while O2 consumption is simultaneously
measured polarographically. ATP, adenosine triphosphate; ADP, adenosine
diphosphate; G6PD, glucose-6-phosphate dehydrogenase; H2O2, hydrogen
peroxide; HK, hexokinase; NADP⫹, nicotinamide adenine dinucleotide phosphate; NADPH, reduced nicotinamide adenine dinucleotide phosphate; O2,
molecular oxygen.
However, the physiologic relevance of OXPHOS efficiency in
normal cellular function and in the response to ischemic injury
in these organ systems remains largely undetermined. With
advances in live-cell permeabilization techniques (6), real-time
assessment of OXPHOS efficiency may be possible in primary
cultures from these and other organ systems, providing a
platform for cell-specific drug discovery. Moreover, innovative
imaging techniques, such as two-photon intravital microscopy,
or optical O2 measurement in tissues via phosphorescence
C.M.S. drafted manuscript.
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AJP-Cell Physiol • doi:10.1152/ajpcell.00203.2016 • www.ajpcell.org
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No conflicts of interest, financial or otherwise, are declared by the author.