View source for Permeabilized muscle fibers

{{MitoPedia
|abbr=Pfi
|description='''Permeabilized muscle fibres''' (Pfi) are used as a mitochondrial preparation in respirometry to access mitochondrial function comparable to [[isolated mitochondria]]. Pfi are obtained by selectively permeabilizing the plasma membrane mechanically and [[permeabilization of plasma membrane|chemically]], for the exchange of soluble molecules between the cytosolic phase and external medium, without damaging the [[mitochondrial|mt]]-membranes.

Add MitoPedia topic: [[Mitochondrial preparation]]
|info=[[Pesta 2012 Methods Mol Biol]]
}}
{{MitoPedia methods
|mitopedia method=Respirometry, Fluorometry
}}
{{MitoPedia topics}}
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== 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 ''p''<sub>O2</sub> 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. Air-level oxygen pressure, however, is effectively hyperoxic in isolated mitochondria ([[Gnaiger 2000 Proc Natl Acad Sci USA|Gnaiger et al 2000]]), but may by hypoxic in permeabilized muscle fibres.

=== Oxygen kinetics of permeabilized fibres ===
Unfortunately, the oxygen dependence of respiration in permeabilized fibres is about 100-fold higher compared to small intact cells and isolated mitochondria ([[Scandurra 2010 Adv Exp Med Biol|Scandurra and Gnaiger 2010]]) due to artificial diffusion gradients resulting from the spatial constraints (long diffusion distances) in the fibre preparation ([[Gnaiger 2003 Adv Exp Med Biol|Gnaiger 2003]], [[Pesta 2012 Methods Mol Biol|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 ''J''<sub>O2</sub> in the O2k over prolonged periods of time, when oxygen drops 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. In Pfi from various species and muscle types (including [[Lemieux 2011 Int J Biochem Cell Biol|human]], rat, mouse and fish heart; human, [[Votion 2012 PLoS One|horse]], rat, mouse, and fish skeletal muscle) oxygen limitation has been shown of Pfi respiration below air level ''p''<sub>O2</sub>. 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. Unfortunately, oxygen limitation of Pfi has been ignored by several research groups ([[Kuznetsov 2008 Nat Protoc|Kuznetsov et al 2008]]; compare [[Kuznetsov_1998_BTK|Kuznetsov et al 1998]]; for a discussion see [[Pesta 2012 Methods Mol Biol|Pesta and Gnaiger 2012]]).

* [[Kuznetsov_1998_BTK|Kuznetsov AV, Lassnig B, Margreiter R, Gnaiger E (1998) Diffusion limitation of oxygen versus ADP in permeabilized muscle fibers. In BioThermoKinetics in the Post Genomic Era (Larsson C, Påhlman I-L, Gustafsson L, eds) Chalmers Reproservice, Göteborg: 273-276.]]
* [[Gnaiger 2000 Proc Natl Acad Sci USA|Gnaiger E, Méndez G, Hand SC (2000) High phosphorylation efficiency and depression of uncoupled respiration in mitochondria under hypoxia. Proc Natl Acad Sci USA 97: 11080-11085.]]
* [[Gnaiger 2003 Adv Exp Med Biol|Gnaiger E (2003) Oxygen conformance of cellular respiration. A perspective of mitochondrial physiology. Adv Exp Med Biol 543: 39-55.]]
* [[Scandurra 2010 Adv Exp Med Biol|Scandurra FM, Gnaiger E (2010) Cell respiration under hypoxia: Facts and artefacts in mitochondrial oxygen kinetics. Adv Exp Med Biol 662: 7-25.]]
* [[Pesta 2012 Methods Mol Biol|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.]]

== Oxygen dependence of ROS production - are permeabilized fibres a valid model? ==
[[File:Gnaiger 2001 Respir Physiol New Fig10.jpg|255px|right|thumb|link=]]
 [[Bioblast_alert#Bioblast_alert_2012_.2803.29:_2012-03-19| '''Bioblast alert 2012(03)''']]  / '''[[Talk:Permeabilized muscle fibres]]'''
When studying mitochondrial ROS production, oxygen levels have to be controlled as a critical factor involved in hypoxic or hyperoxic stress. Considering uncontrolled oxygen gradients in permeabilized fibres, can this mitochondrial preparation provide a valid model for the study of mt-ROS production and ROS dynamics?

The topic for uncontrolled oxygen concentration gradients in Pfi, therefore, 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 by application of 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 ''p''<sub>O2</sub> levels along the oxygen gradient. 

We have tested the oxygen dependence of H<sub>2</sub>O<sub>2</sub> production in a preparation of cardiac mitochondria during the [http://www.oroboros.at/?IOC66 O2k-Fluorometry Workshop IOC66]. 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 H<sub>2</sub>O<sub>2</sub> production in the high oxygen range when mitochondrial respiration is fully oxygen-saturated (Boveris 1977). An illustration of the complex interactions of LEAK respiration, membrane potential, hyperoxia and hypoxia is given in the figure (modfied from [[Gnaiger_2001_Respir Physiol|Gnaiger 2001]]; for the figure legend, click on the figure).
* [[Gnaiger_2001_Respir Physiol|Gnaiger E (2001) Bioenergetics at low oxygen: dependence of respiration and phosphorylation on oxygen and adenosine diphosphate supply. Respir Physiol 128: 277-297.]]

=== 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 HRR-Set|PBI-Shredder]] for muscle tissue homogenate preparation, not using any proteases (such as [[nagarse]]) nor [[saponin]] or [[digitonin]], 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 subsamples can 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’. 
* [[MiPNet17.02 PBI-Shredder-Manual|PBI-Shredder HRR-Set: Preparation of tissue homogenates for diagnosis of mitochondrial respiratory function. Mitochondr Physiol Network 17.02.]]

A summary of our first series of test experiments (basic respirometry only) and description of the shredder homogenization procedure is available.
* Mitochondrial respiration in permeabilized fibres versus homogenate from mouse myocardium. An application study with the PBI-Shredder. MiPNet17.03 ([[Bioblast editorial board|contact]] us for a pdf file). 

A demo experiment on respiration and hydrogen peroxide production of mouse heart homogenate with the [[O2k-Fluorescence LED2-Module]] is shown in the workshop programme ([http://www.oroboros.at/?IOC66 download pdf file from IOC66]). 

 '''Add to the discussion: [[Talk:Permeabilized muscle fibres]]'''


== Historical perspectives ==
The skinned muscle fibre technique was originally introduced by Endo (1967), derived from the mechanically skinned skeletal muscle fibre preparation, first introduced by Natori (1954). It was further adapted and modified by Veksler et al (1987) for cardiac muscle fibres, applied by Letellier et al (1992) for the first time for functional diagnosis of mitochondrial myopathies, and is now well established particularly for skeletal muscle fibres (Saks et al 1998). 

Then as now, it was of great importance “to have the characters closely resembling those of the intact fibre” (Natori 1954). The idea was: “from a physiological point of view, it is desirable to study the contractile system in a state as close to the living state as possible” (Saida and Nonomura 1978). 

After removal of the tissue form the species, the procedure allots mechanical separation of muscle tissue in a relaxing solution. Thereafter, muscle tissue is incubated in saponin by gentle agitation. [[Saponin]], a chemical permeabilization agent of plant origin, selectively attacks cholesterol rich (plasma) membranes of cells or muscle ﬁbers, leaving intracellular membrane structures (mitochondria and ER) as well as myoﬁlaments and the cytoskeleton intact. Consequently, the plasma membrane barrier integrity is lost and the cytosol and all solutes are washed out and the composition of the intracellular milieu is quickly equilibrated with the incubation medium. The mitochondria (''in situ'') are then able to use various substrates within a given protocol. 

Plasma membrane permeabilization with [[digitonin]] or [[saponin]] yields effective wash-out of free cytosolic molecules including adenylates, substrates, and cytosolic enzymes, making externally added compounds accessible to the mitochondria. ADP kinetics is fundamentally different in Pfi and isolated mitochondria (Imt) (Saks et al 1988). In addition, oxygen kinetics is shifted by a 100-fold in crease of the ''p''<sub>50</sub> in Pfi versus Imt (Pesta and Gnaiger 2012).

'''References'''
# Natori H (1954) The property and contraction process of isolated myofibrils. Jikei Med J 1
# Saida K, Nonomura Y (1978) Characteristics of Ca2+- and Mg2+-induced tension development in chemically skinned smooth muscle fibers. J Gen Physiol 72.
# Endo M (1967) Regulation of contraction relaxation of cycle (in japanese). Gen Assoc Jpn Med Congr Proc. 17: 193-197.
# [[Veksler 1987 Biochim Biophys Acta|Veksler VI, Kuznetsov AV, Sharov VG, Kapelko VI, Saks VA (1987) Mitochondrial respiratory parameters in cardiac tissue: A novel method of assessment by using saponin-skinned fibers. Biochim Biophys Acta 892: 191-196.]]
# [[Letellier 1992 Pediatr Res|Letellier T, Malgat M, Coquet M,Moretto B, Parrot-Roulaud F, Mazat JP (1992) Mitochondrial myopathy studies on permeabilized muscle fibers. Pediatr Res 32: 17–22.]]
# [[Saks 1998 Mol Cell Biochem|Saks VA, Veksler VI, Kuznetsov AV, Kay L, Sikk P, Tiivel T, Tranqui L, Olivares J, Winkler K, Wiedemann F, Kunz WS (1998) Permeabilized cell and skinned fiber techniques in studies of mitochondrial function in vivo. Mol Cell Biochem 184: 81-100.]]
# [[Pesta 2012 Methods Mol Biol|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.]]


== 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. 

[[Mitochondrial membrane potential of permeabilized fibres]]


== Permeabilized fibres: Auxiliary HRR-Tools ==

'''Auxiliary HRR-Tools''' are offered for fibre preparation ([[HRR-Dissection Set]]) and weight determination [[Microbalance-Set]]. Some additional equipment is not (yet) in our [[O2k-Catalogue OROBOROS]], such as a 'cold plate'.

== References ==

* [[O2k-Publications: Permeabilized Cell or Tissue; Homogenate]]

The following references describe in detail the basis for working with muscle fibers in the O2k.

# [[Pesta 2012 Methods Mol Biol|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.]]
# [[Lemieux 2011 Int J Biochem Cell Biol|Lemieux H, Semsroth S, Antretter H, Hoefer D, Gnaiger E (2011) Mitochondrial respiratory control and early defects of oxidative phosphorylation in the failing human heart. Int J Biochem Cell Biol 43: 1729–1738.]]
# [[Votion 2012 PLoS One|Votion DM, Gnaiger E, Lemieux H, Mouithys-Mickalad A, Serteyn D (2012) Physical fitness and mitochondrial respiratory capacity in horse skeletal muscle. PLoS One, in press.]]
* [[MiPNet14.14 PermeabilizedFibrePreparation|Preparation of permeabilized muscle fibres for diagnosis of mitochondrial respiratory function. Mitochondr Physiol Network 14.14.]]
* [[MiPNet11.05 Mitos-PermeabilizedCells|Isolated mitochondria or permeabilized tissues and cells. Mitochondr Physiol Network 11.05.]]
* [[MiPNet14.13 Medium-MiR06|Mitochondrial respiration medium - MiR06. Mitochondr Physiol Network 14.13.]]
* MitoPedia topic: [[Permeabilized tissue or cells]].

{{Labeling
|preparations=Permeabilized tissue; Homogenate
}}

{{#set:Scientific service=Respirometry | Scientific service=Permeabilized fibres}}

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