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

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.

Mitochondrial Diagnostics: A Multiplexed Assay Platform for Comprehensive Assessment of Mitochondrial Energy Fluxes

“…Rates of ATP synthesis were determined as described previously (Lark et al, 2016) with slight modifications. Buffer for the assay was Buffer D, supplemented with glucose (5mM), hexokinase (1U/ml), glucose-6-phosphate dehydrogenase (G6PDH; 2U/ml), NADP + (2mM) and ADP (0.2mM).…”

Impaired muscle relaxation and mitochondrial fission associated with genetic ablation of cytoplasmic actin isoforms

“…The technique for preparing and permeabilizing muscle fiber bundles has previously been described [86]. 12 month old mice were anesthetized and red portions of the gastrocnemius muscle were dissected and immediately added to ice-cold buffer × ([mM] − 7.23 K 2 EGTA, 2.77 CaK 2 EGTA, 20 imidazole, 20 taurine, 5.7 ATP, 14.3 phosphocreatine, 6.56 MgCl 2 ·6H 2 O, 50 MES, pH 7.1, 295 mosmol/kgH 2 O).…”

“…The technique for preparing and permeabilizing muscle fiber bundles is as described above [86]. 0.2–0.3mg dry weight of mouse muscle was combined with 30ug/ml saponin and incubated 30 minutes at 4°C.…”

“…In these studies, muscle fiber bundles were used to assess mitochondrial respiration as this approach allows for the assessment of mitochondria in their native state [83]. The technique for preparing and permeabilizing muscle fiber bundles has previously been described [84]. Twelve-month-old mice were anesthetized and red portions of the gastrocnemius muscle were dissected and immediately added to ice-cold buffer X [(mM) - oxygen concentration of~350 lM oxygen as previously described [85].…”

Insulin deficiency and intranasal insulin alter brain mitochondrial function: a potential factor for dementia in diabetes

“…Mitochondrial ATP production was measured using an enzymatic system containing hexokinase and glucose-6-phosphate dehydrogenase to convert ATP to NADPH through sequential formation of glucose-6-phaospahate and 6-phosphoglucolactone using glucose and NADP + as previously described (18,19). A Fluorolog 3 (Horiba Scientific, Piscataway, NJ, USA) spectrofluorometer was used to continuously measure the autofluorescence of NADPH.…”