Direct real-time quantification of mitochondrial oxidative phosphorylation efficiency in permeabilized skeletal muscle myofibers

Daniel S Lark; Maria J Torres; Chien-Te Lin; Terence E Ryan; Ethan J Anderson; P Darrell Neufer

doi:10.1152/ajpcell.00124.2016

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Direct real-time quantification of mitochondrial oxidative phosphorylation efficiency in permeabilized skeletal muscle myofibers

Journal article

Open access

Peer reviewed

Direct real-time quantification of mitochondrial oxidative phosphorylation efficiency in permeabilized skeletal muscle myofibers

Daniel S Lark, Maria J Torres, Chien-Te Lin, Terence E Ryan, Ethan J Anderson and P Darrell Neufer

American Journal of Physiology: Cell Physiology, Vol.311(2), pp.C239-C245

08/01/2016

DOI: 10.1152/ajpcell.00124.2016

PMCID: PMC5129772

PMID: 27335172

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Abstract

Oxidative phosphorylation (OXPHOS) efficiency, defined as the ATP-to-O ratio, is a critical feature of mitochondrial function that has been implicated in health, aging, and disease. To date, however, the methods to measure ATP/O have primarily relied on indirect approaches or entail parallel rather than simultaneous determination of ATP synthesis and O2 consumption rates. The purpose of this project was to develop and validate an approach to determine the ATP/O ratio in permeabilized fiber bundles (PmFBs) from simultaneous measures of ATP synthesis (JATP) and O2 consumption (JO2 ) rates in real time using a custom-designed apparatus. JO2 was measured via a polarigraphic oxygen sensor and JATP via fluorescence using an enzyme-linked assay system (hexokinase II, glucose-6-phosphate dehydrogenase) linked to NADPH production. Within the dynamic linear range of the assay system, ADP-stimulated increases in steady-state JATP mirrored increases in steady-state JO2 (r(2) = 0.91, P < 0.0001, n = 57 data points). ATP/O ratio was less than one under low rates of respiration (15 μM ADP) but increased to more than two at moderate (200 μM ADP) and maximal (2,000 μM ADP) rates of respiration with an interassay coefficient of variation of 24.03, 16.72, and 11.99%, respectively. Absolute and relative (to mechanistic) ATP/O ratios were lower in PmFBs (2.09 ± 0.251, 84%) compared with isolated mitochondria (2.44 ± 0.124, 98%). ATP/O ratios in PmFBs were not affected by the activity of adenylate kinase or creatine kinase. These findings validate an enzyme-linked respiratory clamp system for measuring OXPHOS efficiency in PmFBs and provide evidence that OXPHOS efficiency increases as energy demand increases.

Mice, Inbred C57BL

Oxidative Phosphorylation

Male

Mitochondria - metabolism

Muscle Fibers, Skeletal - metabolism

Oxygen Consumption - physiology

Animals

Adenosine Triphosphate - metabolism

Adenylate Kinase - metabolism

Glucosephosphate Dehydrogenase - metabolism

Mice

Adenosine Diphosphate - metabolism

NADP - metabolism

Hexokinase - metabolism

Mitochondria - physiology

Muscle Fibers, Skeletal - physiology

Details

Title: Subtitle: Direct real-time quantification of mitochondrial oxidative phosphorylation efficiency in permeabilized skeletal muscle myofibers
Creators: Daniel S Lark - Department of Kinesiology, Brody School of Medicine, East Carolina University, Greenville, North Carolina; East Carolina Diabetes and Obesity Institute, Brody School of Medicine, East Carolina University, Greenville, North Carolina Daniel.lark@vanderbilt.edu
Maria J Torres - Department of Kinesiology, Brody School of Medicine, East Carolina University, Greenville, North Carolina; East Carolina Diabetes and Obesity Institute, Brody School of Medicine, East Carolina University, Greenville, North Carolina
Chien-Te Lin - Department of Physiology, Brody School of Medicine, East Carolina University, Greenville, North Carolina; East Carolina Diabetes and Obesity Institute, Brody School of Medicine, East Carolina University, Greenville, North Carolina
Terence E Ryan - Department of Physiology, Brody School of Medicine, East Carolina University, Greenville, North Carolina; East Carolina Diabetes and Obesity Institute, Brody School of Medicine, East Carolina University, Greenville, North Carolina
Ethan J Anderson - Department of Pharmacology and Toxicology, Brody School of Medicine, East Carolina University, Greenville, North Carolina; and East Carolina Diabetes and Obesity Institute, Brody School of Medicine, East Carolina University, Greenville, North Carolina
P Darrell Neufer - Department of Kinesiology, Brody School of Medicine, East Carolina University, Greenville, North Carolina; Department of Physiology, Brody School of Medicine, East Carolina University, Greenville, North Carolina; East Carolina Diabetes and Obesity Institute, Brody School of Medicine, East Carolina University, Greenville, North Carolina
Resource Type: Journal article
Publication Details: American Journal of Physiology: Cell Physiology, Vol.311(2), pp.C239-C245
Publisher: United States
DOI: 10.1152/ajpcell.00124.2016
PMID: 27335172
PMCID: PMC5129772
ISSN: 0363-6143
eISSN: 1522-1563
Grant note: R01 DK096907 / NIDDK NIH HHS R01 HL122863 / NHLBI NIH HHS
Language: English
Date published: 08/01/2016
Academic Unit: Pharmaceutical Sciences and Experimental Therapeutics; Fraternal Order of Eagles Diabetes Research Center; Health and Human Physiology
Record Identifier: 9984065309602771

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