Optimization of cell permeabilization in electron flow based mitochondrial function assays

The biochemical bases for the high mass-specific metabolic rates of flying insects remain poorly understood. To gain insights into mitochondrial function during flight, metabolic rates of individual flying honeybees were measured using respirometry, and their thoracic muscles were fixed for electron microscopy. Mitochondrial volume densities and cristae surface densities, combined with biochemical data concerning cytochrome content per unit mass, were used to estimate respiratory chain enzyme densities per unit cristae surface area. Despite the high content of respiratory enzymes per unit muscle mass, these are accommodated by abundant mitochondria and high cristae surface densities such that enzyme densities per unit cristae surface area are similar to those found in mammalian muscle and liver. These results support the idea that a unit area of mitochondrial inner membrane constitutes an invariant structural unit. Rates of O(2) consumption per unit cristae surface area are much higher than those estimated in mammals as a consequence of higher enzyme turnover rates (electron transfer rates per enzyme molecule) during flight. Cytochrome c oxidase, in particular, operates close to its maximum catalytic capacity (k(cat)). Thus, high flux rates are achieved via (i) high respiratory enzyme content per unit muscle mass and (ii) the operation of these enzymes at high fractional velocities.

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Mitochondrial Function in Oxidative and Glycolytic Bovine Skeletal Muscle Postmortem

Meat and Muscle Biology ◽

10.22175/mmb.10698 ◽

2019 ◽

Vol 3 (2) ◽

Author(s):

P. Ramos ◽

L. Bell ◽

S. Wohlgemuth ◽

T. Scheffler

Keyword(s):

Cytochrome C ◽

Oxidative Phosphorylation ◽

Outer Membrane ◽

Functional Properties ◽

Mitochondrial Function ◽

Complex I ◽

Fixed Effects ◽

Citrate Synthase ◽

Electron Flow ◽

Coupling Efficiency

ObjectivesMitochondrial function in postmortem muscle is affected by decreasing oxygenation. Functional properties relating to energy production and integrity of mitochondria may influence development of meat quality characteristics. Therefore, the objective was to evaluate changes in mitochondrial function in oxidative and glycolytic muscles during the first 24h postmortem.Materials and MethodsSteers (n = 6) of primarily Angus (80 to 100%) genetics were harvested at approximately 18.5 mo and 630 kg live weight. Samples from the longissimus lumborum (LL) and diaphragm (Dia) were collected at 1, 3, and 24h postmortem. Fresh-preserved muscle samples were permeabilized using saponin, and muscle bundles (2–4 mg) were transferred to a high-resolution oxygraph for respiration measurements (oxygen consumption rate, OCR, pmol/sec/mg of tissue). Samples were assessed in duplicate under hyperoxia. First, pyruvate and malate were added to support the TCA cycle and assess leak respiration. Then, ADP was added to support electron flow through complex I. The influence of glutamate on NADH production (complex I) was tested, followed by complex II activation by succinate. Integrity of the mitochondria outer membrane was tested with cytochrome c. Next, an uncoupler (FCCP) was added to force the electron transport system (ETS) to maximum capacity. Citrate synthase (CS) activity (nmol/min/mg tissue) was determined in frozen samples and used as a marker of mitochondria content. Subsequently, respiration data were normalized to CS activity (pmol/sec/U CS) to account for differences in mitochondria content. Coupling efficiency of oxidative phosphorylation was calculated as 1– (Leak/ADP-stimulated oxidative phosphorylation capacity). Raw and normalized OCR were analyzed in a randomized block design, with slaughter date as block and fixed effects of muscle, time, and the interaction. Time was considered a repeated measure.ResultsMuscle type affected (P = 0.0002) leak OCR, with Dia showing a higher rate than LL. After ADP was added, mitochondria from Dia exhibited higher OCR at all times tested and at all steps, with OCR being 4 times higher after FCCP addition. Mitochondrial content, evidenced by greater (P < 0.0001) CS activity in Dia, largely explained differences in OCR between muscles. After OCR was normalized to CS activity, the 1 and 3h postmortem OCR from Dia and LL were similar (P > 0.05). However, at 24h postmortem, OCR after ADP, glutamate, and FCCP additions were greater (P < 0.05) in Dia mitochondria. Time, but not muscle, affected cytochrome c response. At 1h postmortem, cytochrome c increased OCR by 6.6%, supporting that mitochondria outer membrane integrity is not compromised. However, cytochrome c response at 3h postmortem increased 52.4%, indicating outer membrane damage. Coupling efficiency is different between muscles (P = 0.005) with Dia exhibiting greater efficiency.ConclusionDespite inherent metabolic differences between the LL and Dia, mitochondria from both muscles are intact and coupled at 1h postmortem. However, by 24h postmortem, functional properties of LL mitochondria are reduced compared to Dia. Declining mitochondrial function may be associated with calcium overload, mitochondrial fragmentation, and protease activation.

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Redox regulation of mitochondrial functional activity by quinones

Physiology International ◽

10.1556/2060.103.2016.4.4 ◽

2016 ◽

Vol 103 (4) ◽

pp. 439-458 ◽

Cited By ~ 4

Author(s):

NG Krylova ◽

TA Kulahava ◽

VT Cheschevik ◽

IK Dremza ◽

GN Semenkova ◽

...

Keyword(s):

Membrane Potential ◽

Electron Transport ◽

Mitochondrial Membrane Potential ◽

Mitochondrial Function ◽

Mitochondrial Membrane ◽

Reduction Potential ◽

Electron Flow ◽

Rat Liver Mitochondria ◽

Ros Generation ◽

Intact Cells

Quinones are among the rare compounds successfully used as therapeutic agents to correct mitochondrial diseases and as specific regulators of mitochondrial function within cells. The aim of the present study was to elucidate the redox-dependent effects of quinones on mitochondrial function. The functional parameters [respiratory activity, membrane potential, and reactive oxygen species (ROS) generation] of isolated rat liver mitochondria and mitochondria in intact cells were measured in the presence of eight exogenously applied quinones that differ in lipophilicity and one-electron reduction potential. The quinones affected the respiratory parameters of mitochondria, and dissipated the mitochondrial membrane potential as well as influenced (either decreased or enhanced) ROS generation, and restored the electron flow during electron transport chain inhibition. The stimulation of ROS production by juglone and 2,5-di-tert-butyl-1,4-benzoquinone was accompanied by a decrease in the acceptor control and respiration control ratios, dissipation of the mitochondrial membrane potential and induction of the reverse electron flow under succinate oxidation in isolated mitochondria. Menadione and 2,3,5-trimethyl-1,4-benzoquinone, which decreased the mitochondrial ROS generation, did not affect the mitochondrial potential and, vice versa, were capable of restoring electron transport during Complex I inhibition. In intact C6 cells, all the quinones, except for coenzyme Q10, decreased the mitochondrial membrane potential. Juglone, 1,4-benzoquinone, and menadione showed the most pronounced effects. These findings indicate that quinones with the reduction potential values E1/2 in the range from −99 to −260 mV were effective redox regulators of mitochondrial electron transport.

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