Picard 2011 PLoS One

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.

» PMID: 21512578 Open Access

Picard M, Taivassalo T, Ritchie D, Wright KJ, Thomas MM, Romestaing C, Hepple RT (2011) PLoS One

Abstract: Mitochondria regulate critical components of cellular function via ATP production, reactive oxygen species production, Ca2+ handling and apoptotic signaling. Two classical methods exist to study mitochondrial function of skeletal muscles: isolated mitochondria and permeabilized myofibers. Whereas mitochondrial isolation removes a portion of the mitochondria from their cellular environment, myofiber permeabilization preserves mitochondrial morphology and functional interactions with other intracellular components. Despite this, isolated mitochondria remain the most commonly used method to infer in vivo mitochondrial function. In this study, we directly compared measures of several key aspects of mitochondrial function in both isolated mitochondria and permeabilized myofibers of rat gastrocnemius muscle. Here we show that mitochondrial isolation i) induced fragmented organelle morphology; ii) dramatically sensitized the permeability transition pore sensitivity to a Ca2+ challenge; iii) differentially altered mitochondrial respiration depending upon the respiratory conditions; and iv) dramatically increased H₂O₂ production. These alterations are qualitatively similar to the changes in mitochondrial structure and function observed in vivo after cellular stress-induced mitochondrial fragmentation, but are generally of much greater magnitude. Furthermore, mitochondrial isolation markedly altered electron transport chain protein stoichiometry. Collectively, our results demonstrate that isolated mitochondria possess functional characteristics that differ fundamentally from those of intact mitochondria in permeabilized myofibers. Our work and that of others underscores the importance of studying mitochondrial function in tissue preparations where mitochondrial structure is preserved and all mitochondria are represented. • Keywords: Isolated mitochondria, Permeabilized myofibers, ROS

• O2k-Network Lab: CA Montreal Hepple RT, FR Villeurbanne Romestaing C, US FL Gainesville Hepple RT

Selected quotes and comments

Communicated by Gnaiger E (2022-12-18)

• Myofiber respiration was measured under hyperoxygenated conditions by pre-bubbling the measurement buffer with pure O₂ to minimize diffusion limitations at low P_O2 in permeabilized myofibers [48].

» [48] Gnaiger 2003 Adv Exp Med Biol

Comment: The importance of avoiding O₂ limitation of respiration was well recognized by Picard et al (2011), but was ignored in parallel measurements of H₂O₂ production (see below).

• The substrate addition protocol assessing O₂ flux was added sequentially as follows, with each step interspersed with a period of stabilization between injections: 10 mM glutamate + 2 mM malate (GM), 2 mM adenosine diphosphate (ADP), 10 µM succinate (SUCC), 10 µM cytochrome c, 10 µM antimycin A (AA), 5 mM ascorbate + 0.5 mM N,N,N’,N’-tetramethyl-p-phenylenediamine (TMPD).

Comment: 10 µM succinate is either a typo (and should read 10 mM succinate) or is ineffective in stimulating the S-pathway.

» MitoPedia: Succinate

• Mitochondrial H₂O₂ emission was measured as a surrogate for reactive oxygen species (ROS) production. .. measurements performed as described previously [45] and adapted from [49].

» [45] Picard 2008 Am J Physiol Regul Integr Comp Physiol

» [49] Anderson 2006 Am J Physiol Cell Physiol

Comment: These references indicate that oxygen levels were not monitored or controlled in H₂O₂ emission experiments. Thus pfi but not imt are likely to be partially hypoxic under these experimental conditions, which may explain the lower H₂O₂ production observed in pfi. See Komlódi T, Sobotka O, Gnaiger E (2021) Facts and artefacts on the oxygen dependence of hydrogen peroxide production using Amplex UltraRed. https://doi.org/10.26124/bec:2021-0004.

• First, upon Ca²⁺ stress, most or all isolated mitochondria undergo mPTP opening almost simultaneously (within 5–10 seconds), whereas in permeabilized myofibers, mitochondria exhibiting a broad range of sensitivities undergo mPTP opening at different times (several minutes apart), causing a gradual and progressive inversion of the Ca²⁺ uptake signal. Second, we also found that time to mPTP opening was 98 % shorter in isolated mitochondria compared to permeabilized myofibers (21 vs 977 seconds, respectively) (Figure 2D) and the amount of Ca²⁺ necessary to trigger opening of the mPTP was 42 % lower in the isolated preparations (Figure 2E), demonstrating a marked sensitization of the mPTP to a Ca²⁺ challenge in isolated mitochondria.

Comment: It is surprising that in the previous publication Picard 2008 Am J Physiol Regul Integr Comp Physiol such striking differences have not been noted, but threshold values were not expressed in the same units: "Threshold values for PTP opening were approximately threefold higher in fibers from WG compared with those from Sol (124±47 vs. 30.4±6.8 pmol Ca²⁺/mU citrate synthase). A similar phenomenon was also observed in isolated mitochondria (threshold: 121±60 vs. 40±10 nmol Ca²⁺/mg protein in WG and Sol)".

• Compared to permeabilized myofibers, respiration of isolated mitochondria was lower under basal (77% lower) and state II (GM; 53% lower) conditions (Figure 3B). However, comparison of state 3 respiration, after activation of both complex I (by glutamate and malate) and complex II (by succinate), revealed similar maximal respiration rates between the two methods.

Comment: A higher rate of LEAK respiration ('state II, GM') per OXPHOS capacity ('state 3') is classically interpreted as dyscoupling (BEC 2020.1). The (P-L control efficiency) = (P-L)/P is non-linearly related to the classical respiratory acceptor control ratio RCR. The RCR is a primary quality control criterium for evaluation of the quality of mitochondrial isolation procedures. As such, the high RCR and P-L control efficiency in isolated mitochondria provide a potential argument for the higher quality compared to pfi, which should be discussed. The low RCR<5 in pfi is in striking contrast to the large scope of activity of muscle in vivo, whereas the RCR>9 in imt comes closer to in vivo values.

• .. direct stimulation of Complex IV yielded an 82 % higher respiration rate in isolated mitochondria (Figure 3B). {Unfortunately, Figure 3B does not show the results on pfi.}

Comment: If this is not an artefact of the correction for autoxidation (ascorbate, TMPD), then the higher CIV activity in isolated mitochondria (imt) casts doubts on the validity of such measurements in the pfi assay.

• To determine the relative activity of each complex, we calculated the stoichiometry of respiration rates between complexes I, II and IV.

Comment: It is a common misunderstanding, that N-linked and S-linked respiration reflects the activity of Complexes CI and CII. Only the CIV step in a respiratory protocol reflects - after uncoupler titration - the single step and thus CIV activity (Gnaiger_2020_BEC_MitoPathways).

• We found a reduced activity of complex I relative to that of complex II and complex IV in isolated mitochondria compared to permeabilized myofibers (Figure 3D).

Comment: A reduced activity of complex I would indicate a possible injury in imt. But the reduced activity relative to CIV is due to the increased CIV activity in imt, indicating a possible problem in pfi (see above).

• On this basis, we suggest that isolated mitochondria may better represent stressed organelles than mitochondria functioning under normal circumstances in vivo.

Comment: RCR results do not support this conclusion. However, the low H₂O₂ production measured in pfi may be due to O₂ diffusion limitation in pfi incubated under uncontrolled O₂ levels (probably near air saturation), whereas H₂O₂ in imt is elevated due to effectively hyperoxic stress when imt are incubated under such conditions. O₂-controlled conditions mimicking the intracellular conditions in muscle in vivo can easily be established in experiments with imt The ABC of hypoxia.

• .. the protease Nagarse used in the isolation medium to maximize the recovery of intermyofibrillar mitochondria enters

the mitochondria where it exerts insidious and non-specific proteolytic activity.

Comment: Nagarse should be avoided in proper methods of isolating mitochondria.

Cited by

• Komlódi T, Schmitt S, Zdrazilova L, Donnelly C, Zischka H, Gnaiger E. Oxygen dependence of hydrogen peroxide production in isolated mitochondria and permeabilized cells. MitoFit Preprints (in prep).

Labels:

Stress:Oxidative stress;RONS  Organism: Rat  Tissue;cell: Skeletal muscle  Preparation: Permeabilized tissue, Isolated mitochondria 

HRR: Oxygraph-2k 

MitoFit 2021 AmR 

• See discussion: Talk:Permeabilized muscle fibers