Talk:Permeabilized muscle fibers

Are permeabilized fibres 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)

• This question added to the initiation of the Bioblast alert 2012(3) discussion.

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. I’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. I’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: Pfi are a well tested and highly useful model

I am not surprised with your conclusion at this stage that Pfi 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, Pfi 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 “standard” exercise test did not reveal the existing myopathy. With the numerous trials performed with HRR on Pfi, I became convinced of the usefulness of Pfi 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: Pfi 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)

• Picard M, Taivassalo T, Ritchie D, Wright KJ, Thomas MM, Romestaing C, Hepple RT (2011) Mitochondrial structure and function are disrupted by standard isolation methods. PLoS One 6: e18317.

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 fibres 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 fibres? 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 fibres were a better model for representing maximal physiological function (in skeletal muscle at least) but perhaps not?

--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 p_O2 is 'physiological' - quite to the contrary, emphasizing that 'normoxic' air level p_O2 (20 kPa) is hyperoxic for incubations of isolated mitochondria and for most cultured cells (Gnaiger et al 2000). But while a concentration (c_O2) of 100 µM is actually hyperoxic for isolated mitochondria (at a defined p_O2 of 10 kPa), oxygen-limited respiration of permeabilized fibres indicates that the same p_O2 measured in the external incubation medium is effectively hypoxic for a mt-subpopulation (at undefined low p_O2 at various positions in the O₂-gradient from 10 kPa towards 0 kPa). To avoid such a hypoxic/anoxic core in Pfi, we have to increase experimental oxygen concentrations artificially, and maintain them in the range of 450 to 200 µM. The exact optimum range for O₂-saturated respiration and the critical p_O2, below which respiration declines, can be easily evaluated, as described by Pesta and Gnaiger (2012). But Pfi 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 Pfi can neither be used to evaluate the oxygen dependence of mitochondrial H₂O₂ production.

--Gnaiger Erich 20:17, 24 March 2012 (CET)

• 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.