- high-resolution terminology - matching measurements at high-resolution
Permeabilized muscle fibers
Description
Permeabilized muscle fibres
Add MitoPedia topic: Mitochondrial preparation
Abbreviation: Pfi
Reference: Pesta 2012 Methods Mol Biol
MitoPedia methods:
Respirometry,
Fluorometry
High oxygen in permeabilized fibres versus oxygen limitation under hypoxia
Respiratory capacity and substrate concentrations
When measuring respiration in permeabilized fibres (Pfi), the arguments for the appropriate experimental oxygen regime are quite simple: If you are interested in measuring mitochondrial respiratory capacity (OXPHOS, ETS), substrate concentrations must be evaluated such that a limitation of respiratory flux by non-saturating substrate concentration can be excluded. Kinetic control studies, therefore, provide the basis of critically examining the appropriate substrate concentration for carbon substrates, ADP, inorganic phosphate and oxygen. Even if ADP concentrations in muscle tissues never reach 5 mM in vivo, we have to increase the ADP concentration to such high values in Pfi, if the kinetics tells us that more physiological 100 µM do not support maximum respiratory flux.
The same argument applies for oxygen supply. 200 µM (20 kPa) is far higher than physiological intracellular pO2 in muscle tissue. Nevertheless, isolated mitochondria are most frequently studied in the range of 10 to 20 kPa, rather than at physiological 0.5 to 3 kPa. This is fine as long as a concentration-independent saturating concentration range is obtained, where high substrate concentrations are neither inhibiting nor damaging.
Oxygen kinetics of permeabilized fibres
Unfortunately, oxygen kinetics of permeabilized fibres is shifted far to the right due to artificial diffusion gradients resulting from the spatial constraints (long diffusion gradients) in the fibre preparation (Gnaiger 2003, Pesta and Gnaiger 2012). When incubating Pfi below air-level oxygen pressures at a physiological temperature of 37 °C, therefore, it must be shown that the preparation is not oxygen limited due to the spatial configuration of fibre bundles and development of a hypoxic core. Control experiments should be shown (rather than neglecting the problem of oxygen limitation) with traces of JO2 in the O2k over prolonged periods of time, when oxygen levels drop from air level to the minimum experimental values, which would provide proof that oxygen flux is stable and not progressively limited by the declining oxygen concentration. We have many such tests in Pfi from various species and muscle types (including human, rat, mouse and fish heart; human, horse, rat, mouse, and fish skeletal muscle) providing direct evidence for the general problem of oxygen limitation of Pfi respiration below air level pO2. Undefined hypoxic limitation of respiration of a core population of mitochondria is not a satisfactory condition, hence hyperoxic incubation presents the best compromise for respiratory studies with Pfi.
Oxygen dependence of ROS production - are permeabilized fibres a valid model?
The topic for oxygen concentration is becoming an even more disturbing issue when studying not only oxygen consumption but also ROS production. Should then oxygen levels be reduced to the more physiological range when working with Pfi? In Pfi, ROS production may be artificially increased due to the high oxygen pressures prevailing around a peripheral mt-subpopulation. On the other hand, ROS may be reduced in a central hypoxic subpopulation, and again increased if reductive stress increases ROS production at some intermediary pO2 levels along the oxygen gradient.
We have tested the oxygen dependence of H2O2 production in a preparation of cardiac mitochondria during a recent [O2k-Fluorometry workshop]. For illustration and discussion, we used a classical simple substrate (succinate+rotenone or the non-physiological condition of succinate only), and showed quantitatively the pronounced oxygen dependence of H2O2 production in the high oxygen range when mitochondrial respiration is fully oxygen-saturated.
Tissue homogenate versus permeabilized fibres
The conclusion at this stage is that Pfi may not provide an optimum model for studies of ROS production. On the other hand, isolated mitochondria (Imt) require too much tissue for most of our studies on biopsies and on mice. To address these problems, we initiated a project on an alternative approach, using a high-quality mt-preparation with a high mt-yield. To achieve this goal, we tested the PBI-Shredder for muscle tissue homogenate preparation, not using any proteases (such as nagarse) nor saponin or digitonine, with merely 15 seconds of mechanical treatment, completing the entire preparation within 10 min, and obtaining a yield of 50% (we hope to optimize this for a 100% yield. It is possible to obtain a single preparation with 2-4 mg of tissue, but also increase the amount of tissue to 50 mg in a single preparation, which then yields a homogenate that can be partitioned into several O2k-chambers and be stored away for other tests. These tests convinced us, such that we are now offering the PBI-Shredder world-wide as an ‘Auxiliary HRR-Tool’. A summary of our first series of test experiments (basic respirometry only) and description of the shredder homogenization procedure is available ([MiPNet17.03. A simple demo experiment with the O2k-Fluorescence LED2-Module is shown in the workshop programme (download pdf file from IOC66).
Chemicals and Media
What is the optimum ADP concentration to stimulate maximum muscle fibre respiration?
Especially in muscle fibers an ADP concentration up to 5 mM needs to be applied to be in the saturating range, an ADP titration might be required to evaluate, whether the applied concentration is saturating.
Pesta 2012 Methods Mol Biol: At a high apparent Km for ADP of 0.5 mM, flux at 2.5 and 5 mM ADP is ADP-limited by 13% and 7% (assuming L/P=0.2). 2.5 mM ADP is saturating in many cases, yet a further increase of ADP concentration provides a test for saturating [ADP]. This is particularly important for evaluation of OXPHOS versus ETS capacity (P versus E or P/E coupling control ratio).
This section needs to be extended.
References
The following references describe in detail the basis for working with muscle fibers in the O2k.
- Pesta D, Gnaiger E (2012) High-resolution respirometry. OXPHOS protocols for human cells and permeabilized fibres from small biopisies of human muscle. Methods Mol Biol 810: 25-58.
- MiPNet14.14 PermeabilizedFibrePreparation|Preparation of permeabilized muscle fibres for diagnosis of mitochondrial respiratory function. Mitochondr Physiol Network 14.14.]]
- Isolated mitochondria or permeabilized tissues and cells. Mitochondr. Physiol. Network 11.05.
- Mitochondrial respiration medium - MiR06. Mitochondr Physiol Network 14.13.
