Talk:Permeabilized muscle fibers

From Bioblast

MiPNet discussion forum: recovery of permeabilized fibers (2016-05-31)

Stephanie Wohlgemuth

I would like to post a question to the community concerning "recovery of permeabilized muscle fibers" from the O2k chamber after a SUIT experiment for subsequent analyses, such as measurement of Citrate Synthase activity, or processing for Western Blot analysis. Specifically, can the fibers (~ 2.6 mg wwt) be recovered directly into a cell lysis buffer? How effective is this with regard to cell lysis, dilution of cellular protein, and the actual immunoblot?

Dominique Votion

We have stopped to try to recover tissue in the chambers for several reasons:
- Sometimes, the tissue sticks on the stopper and is difficult to get back;
- We obtained unreliable results with CS activity (but we may not exclude another methodological problem);
- It is really time consuming especially when you try to perform several runs in a day;
- It appears more efficient to keep a small sample of the original tissue in order to perform complementary analysis
- And last but not least, can we be sure that there is no interference with the tests we want to perform on these recovered tissues after the SUIT protocols?

Jan Nehlin

I work daily with immunoblotting and have had experience with human muscle satellite stem cells, helping to check protein expression in mitochondria (pls see [1] and [2]).
If the protein is abundant, and the antibody against CS and any other protein of interest is sensitive enough, it should not be a problem. However, there is an improvement in mitochondria protein presence if one could isolate mitochondria from the cell extracts, and thereafter proceed with analysis. Mitochondrial proteins are rather diluted in a whole cell extract.
Seahorse (now Agilent) had a detergent that they claimed disrupted cell membranes, without disrupting mitochondrial membranes. This would allow for an improved isolation of mitochondria using ultracentrifugation or Miltenyi kits. The problem is that me, and others, do not know for sure if individual mitochondria that have defects or are very large (as a result of fusion) in the cells, may have more sensitive membranes and be prone to disrupt easier than the ones that have no defects, and are smaller. This is extremely important when studying samples from different age groups. But if the assays pursued are not age-dependent, perhaps there is no issue to worry about.
For lysis we have used RIPA buffer with protease inhibitors, overnight freezing at -80ยบC and 5x mechanical disruption through 1 ml 21xg syringes. Any more details about the procedures, pls. let me know, kind regards, Jan

Marco Spinazzi

I agree with Dominique Dominique that respiration rates of permeabilized fibers should not be normalized by CS activity obtained from the recovered fibers for the reasons already indicated. I would add also some caution to normalize the data on CS or any other parameter is not obtained from another piece of the sample, unless the researcher can univocally document that the heterogeneity of the muscle fibers does not lead to differences within distinct portions of the sample. In general, any normalization in fact should be done in exactly the same sample where the parameter to be normalized has been measured.
Many papers do not even mention how the normalization was done, which could well explain, at least in part, many of the inconsistencies which exist in the literature using this technique. Best regards, Marco.

Are permeabilized fibers a valid model for measurement of ROS production?

Careful planning

We are struggling with the issue of what oxygen concentration to use with our permeabilized muscle fibers. I know from Leslie who attended the O2k-Workshop last December that you are recommending high oxygen concentrations. However, much of the published work (eg Neufer's work) was done at normal oxygen partial pressure. Also, the work of Wilson and others suggests that intramitochondrial oxygen concentration is usually very low and that OXPHOS is very sensitive to oxygen concentration. I'm concerned about the ability to compare to the current literature and the physiological relevance of using these higher oxygen concentrations. We're also looking at other measures (H2O2 production) using a setup that currently does not allow us to do these measures at higher oxygen concentrations and would not be able to relate these results to mito function if we use high O2 for the respirometry. Any thoughts?? (2012-03-20)
I can certainly see your argument about O2 saturation, but this may not be physiological. With the O2k-Fluorescence Module I can at least do the ROS assay under the exact conditions of the respirometry. Iโ€™ll need to ponder the question of preparation and O2 concentration further. One consideration is that I am comparing subjects before and after an intervention so changes could be meaningful as long I am consistent about conditions. I will definitely need to discuss the caveat that it may not reflect physiological conditions no matter how I do it. Iโ€™ll take a look at the PBI-Shredder information. Again, I really appreciate your input and will look at the references and workshop info more carefully before I decide which way to go.
--Irene Schauer - MiPNet Lab US (2012-03-23)

Unquestionable: pf are a well tested and highly useful model

I am not surprised with your conclusion at this stage that pf may not provide an optimum model for studies of ROS production especially because we have to work with such high 02 levels. But without doubt, pf experiments have a great value to predict athletic abilities, to detect subclinical myopathies and recently, I had convincing results in horses confirmed for a genetic defect causing exertional myopathies. A most interesting aspect was that (1) we get these results before the conformation of the genetic disorder, and (2) for one of these horses (with a history of exercise intolerance), the โ€œstandardโ€ exercise test did not reveal the existing myopathy. With the numerous trials performed with HRR on pf, I became convinced of the usefulness of pf and HRR but we have to be careful with respect to the purpose of the experiments we do.
--Dominique Votion - MiPNet Lab BE (2012-03-23)

Add to the discussion: pf versus imt - or homogenate?

I have gone to the Bioblast page and read with interest the posting about the applicability of permeabilized myofibers for ROS measurements. One thing I would like to know is if there is room for a response on the Bioblast page? In particular, we have found that mitochondrial isolation markedly potentiates mitochondrial ROS generation relative to permeabilized myofibers (Picard et al., PLoS One 2011). As such, I would be interested to know how the ROS production in the mitochondria obtained using the PBI-Shredder compares to that observed in permeabilized myofibers before concluding either way whether this newer method is superior to permeabilized myofibers.
--Russel T. Hepple - MiPNet Lab CA (2012-03-23)
I am a PhD student from Victoria University in Melbourne, one of my supervisors is Tony Hickey from Auckland. I have just read the latest bioblast alert about whether permeabilised fibers are a valid way to measure H2O2 production from mitochondria, I had been wondering about what the hyperoxic environment would have on this. I am more interested in skeletal muscle mitochondrial function, and I was wondering what your thoughts were on the findings of Martin Picard and his group (PLoS ONE) that H2O2 production from isolated mitochondria was markedly higher than permeabilised fibers? I guess there are limitations to either method, and we make the best of what we can. From reading Picard's paper I had thought permeabilised fibers were a better model for representing maximal physiological function (in skeletal muscle at least) but perhaps not?
if your pf preparation method is reasonably well refined and constant, I wonder if the heterogeneity of local oxygen pressure would also become fairly consistent? And thus a simple way around this problem would be to index H2O2 measurements by the chamber oxygen concentration at the time of measurement?
--Chris Hedges - MiPNet Lab AU (2012-03-24)
I speculate (as a testable hypothesis), that the higher ROS generation in isolated mitochondria may be partially due to the effectively higher oxygen pressure experienced by the total population of the isolated mitochondria. I do not argue that elevated pO2 is 'physiological' - quite to the contrary, emphasizing that 'normoxic' air level pO2 (20 kPa) is hyperoxic for incubations of isolated mitochondria and for most cultured cells (Gnaiger et al 2000). But while a concentration (cO2) of 100 ยตM is actually hyperoxic for isolated mitochondria (at a defined pO2 of 10 kPa), oxygen-limited respiration of permeabilized fibers indicates that the same pO2 measured in the external incubation medium is effectively hypoxic for a mt-subpopulation (at undefined low pO2 at various positions in the O2-gradient from 10 kPa towards 0 kPa). To avoid such a hypoxic/anoxic core in pf, we have to increase experimental oxygen concentrations artificially, and maintain them in the range of 450 to 200 ยตM. The exact optimum range for O2-saturated respiration and the critical pO2, below which respiration declines, can be easily evaluated, as described by Pesta and Gnaiger (2012). But pf cannot be used to evaluate the oxygen dependence of mitochondrial respiration due to the inherent heterogeneity of local oxygen pressure. If H2O2 production is strongly oxygen dependent, then pf can neither be used to evaluate the oxygen dependence of mitochondrial H2O2 production.
Although oxygen kinetics of fibers does not quantify oxygen kinetics of mitos, differences in oxygen kinetics between groups may reflect differences in imt-oxygen kinetics. Since the shift of oxygen kinetics from imt to pf involves two orders of magnitude (probably due to spatial diffusion constraints), it may be difficult to exclude possible differences of spatial diffusion constraints in different groups as a major factor causing a difference in oxygen kinetics of fiber oxygen consumption or fiber hydrogen peroxide production. Importantly, even during a respirometric run, some mitochondria may change their position in the pf (e.g. a small subpopulation of mitochondria may become effectively isolated), which is quantitatively reflected in a biphasic oxygen kinetics (as shown in Figure 9 in Pesta and Gnaiger (2012).
--Erich Gnaiger - MiPNet Lab AT (2012-03-24)
  • Gnaiger E, Mรฉndez G, Hand SC (2000) High phosphorylation efficiency and depression of uncoupled respiration in mitochondria under hypoxia. Proc Natl Acad Sci U S A 97: 11080-11085. - ยปBioblast linkยซ
I think we discussed something like that at the end of the session with the heart homogenate experiments. It could be neat to test this out whether there is anything meaningful to be gained from ((H2O2/O2)/tissue mass), well ..... more think through. Also as the O2 drops we have sometimes had an increase in H2O2 signal. It has never been consistent though.
We tried homogenate the other day, it was terrible, and this was due to our method (our homogeniser is pretty brutal). Erich is using a new homogeniser, it shreds the tissue and passes it through a grill so it is isn't sheared a thousand times by spinning blades or a potter. However we got safranin to work with this rather damaged prep.
Another approach is to try and get rid of the oxygen gradient within the fibers. We have found that fish white muscle isn't so oxygen dependent, probably as there is so few mitochondria the localised drain on oxygen withtin the fibers is not inhibited by the impaired diffusion. In hearts another story, and any mammalian muscle is packed with mitochondria compared to fish.
We were thinking myoglobin may work to lower/lessen the diffusion issues. Problem is it is coloured and changes absorbance with O2. So that is a no go. The other thing is blebbistatin (Chris Perry told me about this), appears to relax the fibers and relieve some of the diffusion issues. Another option is a protease specific to collagen, actin, desmin etc. Low conc trypsin? I'm sure the mitochondria would be digested at 37 ยฐC. Collagenase needs Ca2+, and this will rupture mitochondria.
Erich? Do you know if anyone has tried a Biops like media "it is supposed to relax fibers". It would be very expensive in the long run, but perhaps a neat experiement and should mimick the cytosol in terms of ATP and PCr? If super contraction is the issue for O2 diffusion this could solve it and be an MPU!
--Anthony Hickey - MiPNet Lab NZ
Tissue mass of pf added to the O2k-chamber does not appear to determine the oxygen dependence of pf oxygen consumption (human and rat skeletal and cardiac muscle). But this needs testing in different tissues and different species. In our hands, blebbistatin did not change the oxygen kinetics of pf oxygen consumption sufficiently to resolve the issue of a requirement of very high oxygen concentration to obtain maximum oxygen flux (Lemieux et al 2011, human heart, data now shown).
We published the option of using trypsin to obtain an isolated mt-preparation in the O2k-chamber, starting with pf. Since the oxygen kinetics of respiration shifted 100-fold to the high oxygen affinity of isolated mitochondria (imt) with monophasic hyperbolic kinetics, we concluded that all mitochondria became effectively isolated by trypsin added to the chamber. But, as Tony explains, Ca2+ is required, and this damaged the 'isolated' mitochondria to the extent of depressing maximum oxygen flux to about half of the pf. The traces are shown in the original publication (Kuznetsov et al 1998), which is available as a pdf if you follow the link.
We need to join forces to improve pf incubation conditions (media which also improve the sensitivity of fluorescence signals, pre-treatment of pf, temperature transitions, ..). This is a mega-task, and usually we are time limited when starting a big experimental programme. During an experimental programme, however, it is too late, since we cannot 'improve' a project by the sudden switch to an optimized protocol. This explains, why improvements are implemented slowly in established labs, wheres new labs have a choice to pick the best protocol, and then may be quickly ahead of others in terms of quality of results.
--Erich Gnaiger - MiPNet Lab AT (2012-03-25)

Fragmentation and physiological pO2: pf versus imt

It is our view that one of the reasons contributing to the elevated ROS in isolated mitochondria versus pf is the fragmented mitochondrial morphology resulting from mitochondrial isolation. As you alluded in your response to me, we have some testable hypotheses here (higher ROS production by isolated mitochondria is due to their fragmented morphology rather than a function of pO2). In regard to your contention that the more homogenous pO2 for isolated mitochondria than pf, I would counter that mitochondria within the most central parts of myocytes will normally encounter lower pO2 than the mitochondria that are immediately beneath the sarcolemma because of their closer proximity to the oxygen source. Consistent with this view, there is a marked decrease in pO2 in going from capillary blood to the myocyte interior during muscle contractions (see work from R. Richardson in the 1990s), with most studies finding mean capillary pO2 to be approximately 40-43 mmHg whereas intramyocyte pO2 is 3-5 mmHg. In this situation, the higher and more homogenous pO2 with isolated mitochondria may in fact be less physiological than in pf because in this latter preparation the mitochondria remain in their in vivo location and subject to the normally occurring gradients in pO2 from sarcolemma to the deepest regions within a myocyte.
--Russel T. Hepple - MiPNet Lab CA (2012-03-25)
Several factors exert an effect on mt-ROS production under experimental and physiological conditions, and our discussion may help to better understand their relative importance. Homogenate prepared by the 'gentle' shredder technique may contain more fragmented mitochondria compared to pf, but significantly less compared to conventional imt preparations (preliminary observations, Andrea Eigentler showed some confocal images of 'shredder versus fiber' preps during our O2k-Fluorometry Workshop). As Chris Hedges pointed out above, there are different limitations to different methods, and the gentle homogenate preparation may provide one more (not ideal) model.
In the context of homogenous versus heterogenous pO2, we have to clarify the aim and limitations of using experimental systems to analyze in vivo systems. (1) A homogenous experimental pO2 is a necessary condition to exclude artefacts in the study of kinetic relationships between fluxes (oxygen consumption, hydrogen peroxide production) and the substrate activity (pO2). If the oxygen gradients in a heterogenous system and the distribution of mitochondria along these gradients are not quantified, then such a heterogenous system is inadequate for kinetic studies (Scandurra and Gnaiger 2010), independent of the extent of heterogeneities in vivo. (2) Even if mitochondria are oxygen limited at maximum aerobic exercise to some extent 'in vivo' (Gnaiger et al 1998), substrate (pO2) needs to be increased experimentally to saturating levels (or extrapolated on the basis of a defined kinetic function) for the measurement of OXPHOS capacity. Measurement of partially oxygen limited respiration in pf would not allow us to address the question, if OXPHOS capacity or oxygen supply are limiting in exercising muscle in vivo, hence we need to extablish kinetic oxygen saturation (hyperoxia) in such respiratory studies with pf (Boushel et al 2011). (3) Since oxygen gradients are not well defined in pf due to the disrupted microcirculation in the fiber bundles, we cannot use them as a good model of in vivo oxygen gradients. If respiration is oxygen limited in pf at 200 to 100 ยตM O2 by 10-20%, this may be due to anoxia for 5-10% of the mitochondria and various degrees of hypoxia for another mt-fraction whereas most mitochondria are exposed to hyperoxia compared to intramyocyte pO2. Although most studies on imt do not care about physiological intramyocyte pO2, the homogenous experimental model allows us to maintain a defined pO2 in the respiration chamber, at selected levels all the way from hyperoxia to deep hypoxia (Gnaiger et al 2000). Hyperoxia is non-physiological in pf and imt, but only the homogenous system allows us to set experimental conditions to conform to a physiological state. Oxygen kinetics of a 'gentle homogenate preparation' of cardiac tissue shows us that the artificial gradients of pf are avoided (preliminary observations).
--Erich Gnaiger - MiPNet Lab AT (2012-03-26)

Respiration of permeabilized muscle fibers from mice

Beatrice Chabi

We have been working on Tibialis muscle fibers from mice and we are struggling to get a good stimulation with succinate following PM and ADP addition or CCCP following oligomycine addition. We would like some input from colleagues working on mice skeletal muscle, to get some insights on what we can improve in our experimental design.

Vivien Chavanelle

We got a decent increase in O2 consumption (+10%) after succinate addition following PM and ADP addition using the following conditions :
- Muscle : 1 to 3 mg white gastrocnemius from diabetic mice
- Medium : MirO5 + 20 mM creatine monohydrate
- 250ยตM < O2 concentration < 450 ยตM
- Temperature : 37ยฐC
- Substrate concentrations : P [5mM] ; M [2 mM] ; ADP [2,5 mM] along with MgCl2 [1,5 mM] and S [10 mM]

Chris Hedges

I work mainly with rat permabilised muscle, which I imagine will be similar enough to mouse! It's difficult to comment on the lack of succinate stimulation following PM and ADP - lots of things could cause this, though complex II is typically pretty bullet proof in my experience! I have seen situations where there is an initial increase in respiration which doesn't stabilise in some preparations from less experienced users, we attribute this to incompletely permeabilised tissue, but again this typically affects all states, not just addition of succinate. If you're using the inhibitor malonate, then you may not be completely washing this out - we will 'wash' chambers with liver homogenate occasionally to remove some inhibitors.
Regarding the issue of little uncoupling after oligomycin use, I believe that is something that is more commonly seen, and has been discussed before on the oligomycin page We and others have seen the same thing, and at least we have yet to resolve it - instead we opt to use atractyloside, though I believe others have successfully used smaller doses of oligomycin. Even with atractyloside we occasionally see this. I have now taken to designing my experiments based on whether I am interested more in uncoupled or leak states as I'm not confident they can both be reliably determined.
My suggestions would be to try a Rotenone+Succinate protocol to see if you can achieve respiration with complex II without PM, and if you use malonate to revise your washing protocol. For the oligomycin issue, try uncoupling your fibers without adding oligomycin or atractyloside - they may not have much greater capacity for uncoupling in the first place. If they can uncouple in the absence of oligomycin, but not when it is present, then you may have to revise your design (or hopefully someone else has some useful information for both of us!)

Pedro Neves

In my opinion this can be that the membrane has not been permeabilized, there is PM response but not succinate.
"CCCP following oligomycine addition" I think Luiz reported some lower ETS respiration following the addtion of oligomycine. Maybe check the concentration of oligomycine being used.

Deniz Senyilmaz

I am not working with mouse muscle fibers, but I work with permeabilized Drosophila larval body wall, which is mostly made up of muscle tissue. For some reason, after oligomycin, I couldn't get a nice induction with CCCP either. I swapped the uncoupling reagent from FCCP to DNP (dinitrophenol). Even though they have the same function as ionophores, DNP successfully induced uncoupled respiration in permeabilized Drosophila larval body wall.
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