Permeabilized muscle fibers

From Bioblast
Jump to: navigation, search
Bioblasts - Richard Altmann and MiPArt by Odra Noel
MitoPedia         Terms and abbreviations         Concepts and methods         Oroboros O2k         MiP and biochemistry         Preprints and history



MitoPedia

Permeabilized muscle fibers

Description

Permeabilized muscle fibers (pfi) are used as a mitochondrial preparation in respirometry to access mitochondrial function comparable to isolated mitochondria (imt). pfi are obtained by selectively permeabilizing the plasma membrane mechanically and chemically (saponin), for the exchange of soluble molecules between the cytosolic phase and external medium, without damaging the mt-membranes.

» MitoPedia topic: Mitochondrial preparations

Abbreviation: pfi

Reference: Pesta 2012 Methods Mol Biol


MitoPedia topics: Sample preparation 


Questions.jpg


Click to expand or collaps


High oxygen in permeabilized fibers versus oxygen limitation under hypoxia

Contributed by Gnaiger E 2012-05-26, edited 2016-08-17.

Respiratory capacity and substrate concentrations

When measuring respiration in permeabilized fibers (pfi), the arguments for the appropriate experimental oxygen regime are quite simple: If you are interested in measuring mitochondrial respiratory capacity (OXPHOS, ET-pathway), 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 or even higher 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. Air-level oxygen pressure, however, is effectively hyperoxic in isolated mitochondria (Gnaiger et al 2000), but may by hypoxic in permeabilized muscle fibers.

Oxygen kinetics of permeabilized fibers

Unfortunately, the oxygen dependence of respiration in permeabilized fibers is about 100-fold higher compared to small living cells and isolated mitochondria (reviewed by Scandurra and Gnaiger 2010) due to artificial diffusion gradients resulting from the spatial constraints (long diffusion distances) in the fiber 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 fiber 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 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 human, rat, mouse and fish heart; human, horse, rat, mouse, and fish skeletal muscle) oxygen limitation has been shown 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. Unfortunately, oxygen limitation of pfi has been ignored by several research groups (Kuznetsov et al 2008; compare Kuznetsov et al 1998; for a discussion see Pesta and Gnaiger 2012).
  • 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:273-6. - Bioblast link
  • Gnaiger E, Méndez G, Hand SC (2000) High phosphorylation efficiency and depression of uncoupled respiration in mitochondria under hypoxia. Proc Natl Acad Sci USA 97:11080-5. - Bioblast link
  • Gnaiger E (2003) Oxygen conformance of cellular respiration. A perspective of mitochondrial physiology. Adv Exp Med Biol 543:39-55. - Bioblast link
  • Scandurra FM, Gnaiger E (2010) Cell respiration under hypoxia: Facts and artefacts in mitochondrial oxygen kinetics. Adv Exp Med Biol 662:7-25. - Bioblast link
  • Pesta D, Gnaiger E (2012) High-resolution respirometry. OXPHOS protocols for human cells and permeabilized fibers from small biopisies of human muscle. Methods Mol Biol 810:25-58. - Bioblast link

Oxygen dependence of ROS production - are permeabilized fibers a valid model?

Gnaiger 2001 Respir Physiol New Fig10.jpg
 Bioblast alert 2012(03)  / Talk:Permeabilized muscle fibers
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 fibers, 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 pO2 levels along the oxygen gradient.
We have tested the oxygen dependence of H2O2 production in a preparation of cardiac mitochondria during the 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 H2O2 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; for the figure legend, click on the figure).
  • Gnaiger E (2001) Bioenergetics at low oxygen: dependence of respiration and phosphorylation on oxygen and adenosine diphosphate supply. Respir Physiol 128:277-97. - Bioblast link


Tissue homogenate versus permeabilized fibers

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 O2k-Set for muscle tissue homogenate preparation, not using any proteases (such as nagarse) nor saponin or digitonin, with merely 15 seconds of mechanical treatment, and completing the entire preparation within 10 min. We optimized the tissue homogenate (thom) preparation to obtain a 100% yield of functionally intact mitochondria. Respiration of muscle thom is less oxygen dependent compared to pfi, but some extent of hyperoxia remains necessary compared to imt to avoid any oxygen limitation.
It is possible to obtain a single thom 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 thom that can be partitioned into several O2k-chambers for technical repeats or application of different protocols, 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 O2k-Tool’.
* PBI-Shredder O2k-Set: Preparation of tissue homogenates for diagnosis of mitochondrial respiratory function. Mitochondr Physiol Network 17.02. - Bioblast link
A summary of test experiments (basic respirometry only) and description of the shredder homogenization procedure is available.
  • Mitochondrial respiration in permeabilized fibers versus homogenate from mouse myocardium. An application study with the PBI-Shredder. MiPNet17.03 (contact us for a pdf file).
A demo experiment on respiration and hydrogen peroxide production of mouse heart homogenate with the O2k-Fluo_LED2-Module is shown in the workshop programme ( download pdf file from IOC66).
Add to the discussion: Talk:Permeabilized muscle fibers


Historical perspectives

The skinned muscle fiber technique was originally introduced by Endo (1967), derived from the mechanically skinned skeletal muscle fiber preparation, first introduced by Natori (1954). It was further adapted and modified by Veksler et al (1987) for cardiac muscle fibers, 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 fibers (Saks et al 1998).
Then as now, it was of great importance “to have the characters closely resembling those of the intact fiber” (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 imt (Saks et al 1988). In addition, oxygen kinetics is shifted by a 100-fold in crease of the p50 in pfi versus imt (Pesta and Gnaiger 2012).
References
  1. Natori H (1954) The property and contraction process of isolated myofibrils. Jikei Med J 1
  2. Saida K, Nonomura Y (1978) Characteristics of Ca2+- and Mg2+-induced tension development in chemically skinned smooth muscle fibers. J Gen Physiol 72.
  3. Endo M (1967) Regulation of contraction relaxation of cycle (in japanese). Gen Assoc Jpn Med Congr Proc. 17:193-7.
  4. 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 892:191-6. - Bioblast link
  5. 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. - Bioblast link
  6. 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. - Bioblast link
  7. Pesta D, Gnaiger E (2012) High-resolution respirometry. OXPHOS protocols for human cells and permeabilized fibers from small biopisies of human muscle. Methods Mol Biol 810:25-58. - Bioblast link


Experimental SOP

Addition of fibers to the O2k chamber

  1. While preparing the permeabilized fibers, perform oxygen calibration and an instrumental O2 background test.
  2. The final state of the O2k chambers before addition of the fibers should be 450 µM O2 in the closed chamber.
  3. Immediately after wet weight determination, quickly open the O2k chamber, instert the fiber and close, obtaining an initial O2 concentration of c. 400 µM.
  4. Restart DatLab to define time zero as the time when fibers have been added.
  5. Immediately add P or G or Oct or Pal or .., then add M. Do not leave fibers without substrate. Do not add M, which may lead to oxaloacetate accumulation, which may prolong establishment of a steady-state after addition of P or G or Oct or Pal. A counter-argument might be (as a basis of our published protocols): In the initial phase without added substrates, endogenous substrates are oxidized preferentially when compared to a state with immediate addition of substrates. The prolonged LEAK state (rather than initial substrate-depleted state) proposed here in the alternative initial phase of the experiment will provide a prolonged evaluation of stabilization of flux (not disturbed by substrate additions), but stress due to substrate limitation may be worse than stress due to high membrane potential.

Reoxygenation using H2O2

Small reoxygenation steps during the experiment can be performed by titrating a few μL of H2O2 (200 mM in H2O, adjust to pH 6 and keep on ice to minimize autoxidation) into MiR06(containing catalase). This is sufficient to raise O2 concentrations from 250 μM again to 400 μM. After injecting H2O2, the time required for the flux to stabilize depends on the step change of O2 concentration; smaller steps require less time for stabilization (Fig. 7 in Doerrier 2018 Methods Mol Biol). Using H2O2 increases total gas pressure with O2 pressure. This can generate bubbles in steps from air saturation to >400 μM. Therefore, O2 gas is applied initially.

Chemicals and media

What is the optimum ADP concentration to stimulate maximum muscle fiber respiration?
Especially in muscle fibers an ADP concentration of 5 mM and higher 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 ET capacity (P versus E or P/E coupling control ratio).
This section needs to be extended. 
Mitochondrial membrane potential of permeabilized fibers


Permeabilized fibers: auxiliary O2k-tools

Auxiliary O2k-tools are offered for fiber preparation (O2k-Dissection Set) and weight determination Microbalance-Set. A Peltier-controlled cooling plate will be added to the OROBOROS O2k-Catalogue.


Tissue storage

Small tissue samples (10 mg wet weight) should be put immediately into a pre-cooled storage medium, e.g. BIOPS, and stored on ice (0 to 4 °C). Isolated mitochondria should be stored at high concentration (50 mg mt-protein/ml) on ice in mitochondrial preservation medium.[1]
Up to 12 hours of cold storage does normally not affect mitochondrial respiratory function of liver (pig: 24 h)[2] and human muscle biopsies (skeletal muscle: 24 h[3]; cardiac muscle: 8-12 h[4]).
Muscle biopsies of horses can be stored for several days without loss of function[5]).
Storage time without loss of function is, therefore, tissue and species dependent and should be evaluated experimentally. When a larger tissue sample is available, separate the sample into small (10 mg) pieces, and apply respirometric SUIT protocols on subsamples in a time course. In particular, evaluate dyscoupling (L/E and P/E coupling control ratios), cytochrome c release,[6] and OXPHOS capacities with various substrate combinations.

References

  1. Gnaiger E, Kuznetsov AV, Schneeberger S, Seiler R, Brandacher G, Steurer W, Margreiter R (2000) Mitochondria in the cold. In: Life in the Cold (Heldmaier G, Klingenspor M, eds) Springer, Heidelberg, Berlin, New York:431-42. - Bioblast link
  2. Kuznetsov AV, Strobl D, Ruttmann E, Königsrainer A, Margreiter R, Gnaiger E (2002) Evaluation of mitochondrial respiratory function in small biopsies of liver. Analyt Biochem 305:186-94. - Bioblast link
  3. Skladal D, Sperl W, Schranzhofer R, Krismer M, Gnaiger E, Margreiter R, Gellerich FN (1994) Preservation of mitochondrial functions in human skeletal muscle during storage in high energy preservation solution (HEPS). In: What is Controlling Life? (Gnaiger E, Gellerich FN, Wyss M, eds) Modern Trends in BioThermoKinetics 3. Innsbruck Univ Press:268-71 - Bioblast link
  4. 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 43:1729–38. - Bioblast link
  5. Votion DM, Gnaiger E, Lemieux H, Mouithys-Mickalad A, Serteyn D (2012) Physical fitness and mitochondrial respiratory capacity in horse skeletal muscle. PLoS One 7: e34890. - Bioblast link
  6. Kuznetsov AV, Schneeberger S, Seiler R, Brandacher G, Mark W, Steurer W, Saks V, Usson Y, Margreiter R, Gnaiger E (2004) Mitochondrial defects and heterogeneous cytochrome c release after cardiac cold ischemia and reperfusion. Am J Physiol Heart Circ Physiol 286:H1633–41. - Bioblast link
The following references describe in detail the basis for working with muscle fibers in the O2k.
  1. Pesta D, Gnaiger E (2012) High-resolution respirometry. OXPHOS protocols for human cells and permeabilized fibers from small biopisies of human muscle. Methods Mol Biol 810:25-58. - Bioblast link
  2. 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 43:1729–38. - Bioblast link
  3. Votion DM, Gnaiger E, Lemieux H, Mouithys-Mickalad A, Serteyn D (2012) Physical fitness and mitochondrial respiratory capacity in horse skeletal muscle. PLoS One 7: e34890. - Bioblast link
  • Preparation of permeabilized muscle fibers for diagnosis of mitochondrial respiratory function. Mitochondr Physiol Network 14.14. - Bioblast link
  • Isolated mitochondria or permeabilized tissues and cells. Mitochondr Physiol Network 11.05. - Bioblast link
  • Mitochondrial respiration medium - MiR06. Mitochondr Physiol Network 14.13. - Bioblast link
  • MitoPedia topic: Permeabilized tissue or cells.