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

• 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.

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