Neufer 2016 Abstract MitoFit Science Camp 2016
|Direct real-time quantification of mitochondrial oxidative phosphorylation efficiency in permeabilized skeletal muscle myofibers.|
Oxidative phosphorylation (OXPHOS) efficiency, defined as the ATP: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 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 (r2 = 0.91, p < 0.0001, n=57 data points). ATP:O ratio was <1 under low rates of respiration (15 μM ADP) but increased to >2 at moderate (200 μM ADP) and maximal (2000 μM ADP) rates of respiration with an inter-assay 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.
Labels: MiParea: Respiration
Tissue;cell: Skeletal muscle, Blood cells Preparation: Permeabilized tissue, Isolated mitochondria
Regulation: ADP Coupling state: OXPHOS
Event: C1 MitoFit Science Camp 2016
1-Vanderbilt Univ, Nashville, TN; 2-East Carolina Univ, Greenville, NC, US. - [email protected]