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Picard 2011 PLoS One

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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) updated 2023-01-06
  • Myofiber respiration was measured under hyperoxygenated conditions by pre-bubbling the measurement buffer with pure O2 to minimize diffusion limitations at low PO2 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 are likely to be partially hypoxic, whereas imt were effectively hyperoxic under these experimental conditions, which may explain the lower H2O2 production observed in pfi.
  • 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. 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. The low P-L control efficiency in permeabilized fibers is an unresolved puzzle, and confirmation of this puzzle does not foster an argument as to its physiological relevance.
  • .. 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 measurements in the pfi assay (oxygen limitation?).
  • 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. 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. See 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 must be avoided in 'standard' methods of isolating mitochondria.


Communicated by Gnaiger E (2023-01-04)


Critical comments (with reference to lack of further evidence) or validation of isolated mitochondria by comparison with alternative models of mitochondrial preparations or in vivo studies:
1. Lai N, M Kummitha C, Rosca MG, Fujioka H, Tandler B, Hoppel CL (2019) Isolation of mitochondrial subpopulations from skeletal muscle: Optimizing recovery and preserving integrity. Acta Physiol (Oxf) 225:e13182. doi: 10.1111/apha.13182. PMID: 30168663
Ref 31: "Nevertheless, the lower integrity observed in the previous rat studies could be related to the use of nagarse which has been shown to produce a loss of mitochondrial matrix constituent.31 Homogenization conditions, i.e., rpm and loose pestle, of the tissue samples and the gentle resuspension of the pellet after each centrifugation are important factors affecting the integrity of mitochondria. … The degree of mitochondrial integrity obtained with current protocols has been questioned,31 with less disruptive approaches involving permeabilized fibers being favored.53 Other studies reported bioenergetics differences between permeabilized fiber and isolated mitochondria suggesting that the disruption of mitochondrial structure during the isolation process might alter mitochondria function probably because of a loss of matrix constituents.31 Nevertheless, inner and outer mitochondrial membrane integrity was not evaluated in those studies. In a heart study by our group, it was shown that whereas the isolated mitochondria become spheroid their cristae were 77% lamelliform, as were those in the SSM in in situ fibers.54 In a heart muscle study, the respiratory capacity in isolated mitochondria was the same as that observed in fibers.55"
2. Newsom SA, Stierwalt HD, Ehrlicher SE, Robinson MM (2021) Substrate-specific respiration of isolated skeletal muscle mitochondria after 1 h of moderate cycling in sedentary adults. Med Sci Sports Exerc 53:1375-84. doi: 10.1249/MSS.0000000000002615. PMID: 34127633 - «Bioblast link»
Ref 46: "Differences between fiber preparations and mitochondrial isolation are critical considerations as exercise alters fatty acid uptake at the cell membrane, which is removed during cell fractionation (46). The isolation procedure represents a larger fraction of mitochondria because it starts from 90–100 milligrams of muscle tissue, versus 2–3 milligrams for fiber preparations. Direct comparisons between respiration rates reveal similar qualitative findings such as with aging and caloric restriction (47). Also, intermyofibrillar mitochondria have higher respiratory rates compared to subsarcolemmal mitochondria (48). Our isolation includes protease treatment to liberate both populations. Extrapolating our lipid respiration rates to whole body results in approximately 0.64 grams fat oxidation per kg lean mass per minute (assuming mitochondria at 5% cell volume (49) and wet-to-dry protein conversion of 4.35 (50)) which align with whole-body indirect calorimetry measures. We measured fat oxidation of 2.04 μmol fatty acids per kg lean mass per min in these participants (data not provided), indicating good integration of the measures of isolated mitochondria to whole body physiology. Our approach provides insight into mitochondrial physiology while attempting to minimize technical and biologic variability (26)."
3. Aoki T, Okuma Y, Becker LB, Hayashida K, Shinozaki K (2021) Methodological Issue of Mitochondrial Isolation in Acute-Injury Rat Model: Asphyxia Cardiac Arrest and Resuscitation. Front Med (Lausanne) 12;8:666735. doi: 10.3389/fmed.2021.666735. PMID: 33912580
"Picard et al. (5) investigated the potential complications arising from isolation methods that involve structural and functional disruption of the mitochondria. They attributed the functional alterations induced by mitochondrial isolation to disruption of the mitochondrial morphology caused by mechanical homogenization as well as loss of soluble proteins and other molecules from the mitochondrial matrix. This is significant under experimental conditions given that animal models with acute injuries likely have mitochondrial disruption in nature. However, although Picard et al. discussed a potential risk of sampling bias as a result of the loss of disrupted mitochondria during the isolation process, no studies have shown such a bias in acute-injury animal models."
4. Krajčová A, Urban T, Megvinet D, Waldauf P, Balík M, Hlavička J, Budera P, Janoušek L, Pokorná E, Duška F (2020) High resolution respirometry to assess function of mitochondria in native homogenates of human heart muscle. PLoS One 15:e0226142. doi: 10.1371/journal.pone.0226142. PMID: 31940313 - «Bioblast link»
Ref 41: "Because isolation of mitochondria disrupts native environment of mitochondrial network and intracellular communication with other organelles[41], the generalizability of the results to in vivo environment have been questioned [29,42]. In 1987, Veksler et al.[43] described the technique of permeabilisation of cardiac muscle fibers by saponin, which interacts with cholesterol and creates pores in plasma membrane which becomes permeable for small molecules (such as for ADP, or other substrates and inhibitors needed in SUIT respirometry protocol), so that cytosol is washed out from otherwise intact cells[17,44]. This in situ technique is therefore more representative of in vivo mitochondrial arrangement than isolated mitochondria and allows determination of mitochondrial functional parameters in small endomyocardial biopsy samples[43,45,46] stored for up to 24 hours[17]. Major disadvantage of permeabilisation is that the process is manually demanding and takes at least ~2 hours[17]. Due to the challenges of assessing citrate synthase activity from permeabilised fibers recovered from the respirometry chamber [17,18], mitochondrial functional indices were normalized to wet weight of manually isolated fibers blotted on a filter paper to remove excess of fluid[17,18]. A recent study showed a relatively large variability (CV of 15.2%) between the measurements of two bundles of skeletal muscle fibers from the same subject[18], roughly the same variability as we have seen in isolated mitochondria in our study, and twice the variability we have seen by using homogenates in this study."
5. Tyrrell DJ, Bharadwaj MS, Jorgensen MJ, Register TC, Molina AJ (2016) Blood cell respirometry is associated with skeletal and cardiac muscle bioenergetics: Implications for a minimally invasive biomarker of mitochondrial health. Redox Biol 10:65-77. doi: 10.1016/j.redox.2016.09.009. PMID: 27693859 - «Bioblast link»
Vastus lateralis muscle fibers were permeabilized and analyzed by high resolution respirometry [34] to examine bioenergetic capacity in a manner that maintains potential differences in mitochondrial content and architecture [35]. In addition, we examined respiratory control in isolated vastus lateralis mitochondria [36] to determine whether blood based measures might be related to differences in intrinsic electron transport chain function. Similar methods using isolated organelles were carried out for analysis of cardiac muscle mitochondrial function. … The comparison of blood based bioenergetic profiles with respiratory analyses of both permeabilized muscle fibers and isolated mitochondria from freshly isolated tissues in a well phenotyped non-human primate model with high variability in age and metabolic health are major strengths of the present study."
6. Porter C, Hurren NM, Cotter MV, Bhattarai N, Reidy PT, Dillon EL, Durham WJ, Tuvdendorj D, Sheffield-Moore M, Volpi E, Sidossis LS, Rasmussen BB, Børsheim E (2015) Mitochondrial respiratory capacity and coupling control decline with age in human skeletal muscle. Am J Physiol Endocrinol Metab 309:E224-32. doi: 10.1152/ajpendo.00125.2015. Erratum in: Am J Physiol Endocrinol Metab 309:E884. PMID: 26037248 - «Bioblast link»
Ref 25: "More recent data suggest that isolation of mitochondria from skeletal muscle has deleterious effects on mitochondrial morphology and function (25). .. However, in the current study we show in permeabilized muscle fibers, where the entire mitochondrial pool is studied in situ, that aging is associated with a decline in mitochondrial respiratory capacity, in line with respiration (3, 38) and ATP production rate measurements made in isolated mitochondria (17, 33, 35)."
7. Glancy B, Willis WT, Chess DJ, Balaban RS (2013) Effect of calcium on the oxidative phosphorylation cascade in skeletal muscle mitochondria. Biochemistry 52:2793-809. doi: 10.1021/bi3015983. PMID: 23547908
Ref 83: "The lower dynamic range of our measures could be due to a lower maximum rate of oxidative phosphorylation or due to a higher ‘resting’ rate. Combining the measured cytochrome a,a3 content of 11.8 ± 1.6 nmol cyt a/g wet weight for the porcine vastus intermedius with the measured in vitro Jo at ∼66 μM ADP (259.2 nmol O2/min/nmol cyt a) approaches the in vivo value reported by Wüst et al. (∼65 ml O2/kg muscle/min) while at ∼17 μM ADP – Ca2+, the rates were 15.2 ml O2/kg muscle/min (in vitro) and 3.8 ml O2/kg muscle/min (in vivo) suggesting that the maximum rate is similar under both conditions and that the discrepancy primarily resided in the resting rates (note that ‘resting’ here is defined as the 17 μM ADP, no Ca2+ condition). The reasons for this may be several-fold. First, in order to make simultaneous, quantitative measurements of Jo, NADH, and ΔΨ, we used isolated mitochondria removed from their native, reticular environment. Though these mitochondria were of high purity (21) and functional integrity, it is likely that the isolation process alters mitochondrial function to some degree (83)."
8. Perry CG, Kane DA, Lanza IR, Neufer PD (2013) Methods for assessing mitochondrial function in diabetes. Diabetes 62:1041-53. - «Bioblast link»
Ref 69: "However, comparisons of isolated mitochondria to permeabilized muscle fiber bundles can be a powerful approach for isolating the effects of cytosolic structures in regulating mitochondrial function as highlighted previously (54,66–69)."
  • 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).

Isolated mitochondria or homogenate

Isolated mitochondria or tissue homogenates used anyway without comparison with alternative models of mitochondrial preparations:
1. Haji G, Wiegman CH, Michaeloudes C, Patel MS, Curtis K, Bhavsar P, Polkey MI, Adcock IM, Chung KF; COPDMAP consortium (2020) Mitochondrial dysfunction in airways and quadriceps muscle of patients with chronic obstructive pulmonary disease. Respir Res 21:262. doi: 10.1186/s12931-020-01527-5. PMID: 33046036
Ref 24: "Isolation of mitochondria from biopsies was performed using the douncer method which potentially can cause mechanical damage to the isolated mitochondria. Disruption of tissue and cellular material might lead to loss or alteration of ETC complex proteins or induce ROS production [24]."
2. Kras KA, Hoffman N, Roust LR, Benjamin TR, DE Filippis EA, Katsanos CS (2019) Adenosine Triphosphate Production of Muscle Mitochondria after Acute Exercise in Lean and Obese Humans. Med Sci Sports Exerc 51:445-53. doi: 10.1249/MSS.0000000000001812. PMID: 30363008
Ref 40: "A limitation of the present study is that measurements were performed in isolated mitochondria and, according to available evidence, isolation of muscle mitochondria appears to alter the structure and function of mitochondria (40). However, isolation of mitochondria from muscle is necessary in order to study mitochondrial subpopulations that are known to exhibit biochemical, functional, and proteomic differences (6, 7). Importantly, data in our study were contrasted only in mitochondria that were isolated using exactly the same procedures and, therefore, our findings reflect differences in mitochondria function associated with the experimental manipulation rather than the mitochondrial isolation procedure. We have previously found that the enzyme nagarse, which is important to isolate functional IMF mitochondria, modifies (i.e., enhances) the function of isolated mitochondria. For that reason, we did not contrast data isolated with and without nagarse (i.e., SS vs IMF mitochondria). Consequently, our comparisons in mitochondrial function are limited to the responses within a given mitochondrial subpopulation."
3. Djafarzadeh S, Jakob SM (2017) Isolation of Intact Mitochondria from Skeletal Muscle by Differential Centrifugation for High-resolution Respirometry Measurements. J Vis Exp 8:55251. doi: 10.3791/55251. PMID: 28362420 - «Bioblast link»
Ref 18: "Some of the limitations of isolated mitochondria, based on differential centrifugation are i) the possibly damaged mitochondria during the homogenization and isolation procedure 19, ii) the large amounts of the tissue specimens required for the mitochondrial isolation, iii) the possible loss and alterations in certain mitochondrial electron transport chain complexes subunits during homogenization and centrifugation steps 18, and iv) the increased production of the reactive oxygen species during homogenization and centrifugation steps, which may interfere with mitochondrial function 18. The advantages of the dounce homogenization and the differential centrifugation method to isolate crude mitochondria for respiratory experiments with respect to existing methods such as density gradient centrifugation 20 are that the method is quick and mitochondria are isolated within a short period of time."
4. Holzem KM, Vinnakota KC, Ravikumar VK, Madden EJ, Ewald GA, Dikranian K, Beard DA, Efimov IR (2016) Mitochondrial structure and function are not different between nonfailing donor and end-stage failing human hearts. FASEB J 30:2698-707. doi: 10.1096/fj.201500118R. PMID: 27075244 O2k
Ref 36: "Mitochondrial isolation yields range from 20–40% of initial mitochondria, which potentially leads to selection bias, and respiration measurements can differ between isolated mitochondria and permeabilized fiber preparations (35, 36). These findings suggest that mitochondrial isolation leads to apparent respiratory dysfunction in diseased tissues; however, our results show no respiratory dysfunction failing isolated mitochondria, which is inconsistent with selection for dysfunctional mitochondria."
5. Margulies SS, Kilbaugh T, Sullivan S, Smith C, Propert K, Byro M, Saliga K, Costine BA, Duhaime AC (2015) Establishing a clinically relevant large animal model platform for TBI therapy development: using Cyclosporin A as a case study. Brain Pathol 25:289-303. O2k
Ref 63: "We made three improvements in our measurements of mitochondrial respiration. First, we transitioned from tissue mitochondrial isolation techniques to new protocols developed to utilize tissue homogenates for mitochondrial respiration. Homogenates potentially have several advantages over isolated mitochondria preparations: reduced disruption of intracellular and intercellular signaling pathways 94, preservation of mitochondrial structure and function 63, 64, and decreased mitochondria population loss, which may be >60% with isolation techniques 62. Thus, homogenates may provide a more relevant and reliable assessment of in vivo function. As a second technological improvement in year 2, we upgraded our traditional Clark‐type oxygen electrode (Hansatech Instruments, Norfolk, UK) that measures oxygen consumption in nmols to a more sensitive instrument (Oxygraph‐2k, OROBOROS Instruments, Innsbruck, Austria) that measures oxygen consumption in pmol, providing a more consistent and nuanced readout of mitochondrial respiration. Third, we upgraded our respiration protocol by adding the substrate succinate to allow us to measure oxidative phosphorylation via integrated convergent complex I and II electron flow 33, whereas our year 1 standard respiration protocol only utilized NADH‐linked substrates (pyruvate + malate)."
6. Bharadwaj MS, Tyrrell DJ, Lyles MF, Demons JL, Rogers GW, Molina AJ (2015) Preparation and respirometric assessment of mitochondria isolated from skeletal muscle tissue obtained by percutaneous needle biopsy. J Vis Exp 96:52350. doi: 10.3791/52350. PMID: 25741892
Ref 13: "Potential limitations of the technique described here arise from the use of isolated organelles and the use of a plate-based format. For example, Picard et al. have demonstrated that isolated mitochondria possess functional characteristics that differ fundamentally from those of intact mitochondria in permeabilized myofibers. They proposed that mitochondrial isolation techniques result in altered bioenergetic function, such as significantly increased RCR compared to permeabilized myofibers accompanied by greater reactive oxygen species production 13. Compared to permeabilized muscle fibers, isolation of mitochondria does require longer preparation time. Also, loss of cellular content diminishes physiological relevance, something that is retained in whole cells and even permeabilized fibers. The use of plate-based respirometry with the described technique permits replicate runs per sample. However, mitochondria must adhere to the bottom of each well. This configuration is different from their normal environment and may affect functional characteristics."

Belief in a paradigm

Without further testing, avoiding isolated mitochondria is emphasized by reference to Picard et al (2011), propagating uncritically an experimental paradigm:
1. Su Y, Claflin DR, Huang M, Davis CS, Macpherson PCD, Richardson A, Van Remmen H, Brooks SV (2021) Deletion of neuronal CuZnSOD accelerates age-associated muscle mitochondria and calcium handling dysfunction that is independent of denervation and precedes sarcopenia. Int J Mol Sci 22:10735. doi: 10.3390/ijms221910735. PMID: 34639076
Ref 30: "One consideration with the common means of measuring mitochondrial function is the potential for damaging mitochondria during isolation procedures that may induce alterations in the function [30,31]. Differential effects of the isolation procedures on muscle fibers with differences in the structure of their mitochondrial networks are easy to imagine."
2. Dia M, Gomez L, Thibault H, Tessier N, Leon C, Chouabe C, Ducreux S, Gallo-Bona N, Tubbs E, Bendridi N, Chanon S, Leray A, Belmudes L, Couté Y, Kurdi M, Ovize M, Rieusset J, Paillard M (2020) Reduced reticulum-mitochondria Ca2+ transfer is an early and reversible trigger of mitochondrial dysfunctions in diabetic cardiomyopathy. Basic Res Cardiol 115:74. doi: 10.1007/s00395-020-00835-7. PMID: 33258101
Ref 47: "Our OCR data in intact versus permeabilized cardiomyocytes, a relevant protocol to assess mitochondrial bioernergetics [13, 47], support that the decreased mitochondrial bioenergetics in the HFHSD cardiomyocytes do not rely on alteration of the electron transport chain but rather on the deficient reticulum-mitochondria Ca2+ transfer."
3. Underwood E, Redell JB, Zhao J, Moore AN, Dash PK (2020) A method for assessing tissue respiration in anatomically defined brain regions. Sci Rep 10:13179. doi: 10.1038/s41598-020-69867-2. PMID: 32764697
Ref 26: "Traditionally, mitochondrial respiration is assessed following isolation from bulk tissue. This technique has a number of disadvantages, including mechanical disruption of the mitochondrial network, and assaying activity in the absence of the mitochondria’s native intracellular environment26–28."
4. Rieger B, Krajčová A, Duwe P, Busch KB (2019) ALCAT1 Overexpression Affects Supercomplex Formation and Increases ROS in Respiring Mitochondria. Oxid Med Cell Longev 2019:9186469. doi: 10.1155/2019/9186469. PMID: 31885824
Ref 31: "In isolated mitochondria, the reactive oxygen species (ROS) H2O2 increased in ALCAT1 overexpressing cells [29]. However, isolated mitochondria tend to lose their ROS detoxifying system which might result in higher ROS levels [31]."
5. Hsu CC, Tsai HH, Fu TC, Wang JS. Exercise Training Enhances Platelet Mitochondrial Bioenergetics in Stroke Patients: A Randomized Controlled Trial (2019) J Clin Med 8:2186. doi: 10.3390/jcm8122186. PMID: 31835774
Ref 17: "However, mitochondria do not work as independent units [17]. Electron transport complexes are interconnected on the mitochondrial inner membrane and turn into respiratory supercomplexes or respirasomes [18]. A polarographic oxygen sensor can measure the mitochondrial respiration of intact cells [19]. Furthermore, individual complexes of the ETC can also be surveyed by addition of exogenous substrates and inhibitor into permeabilized cells [20]. In order to determine the capacities of platelet mitochondria oxidative phosphorylation (OXPHOS) and ETC in stroke patients, the development of novel protocols to evaluate ET effects on mitochondrial OXPHOS and ETC activities are needed."
6. Thome T, Salyers ZR, Kumar RA, Hahn D, Berru FN, Ferreira LF, Scali ST, Ryan TE (2019) Uremic metabolites impair skeletal muscle mitochondrial energetics through disruption of the electron transport system and matrix dehydrogenase activity. Am J Physiol Cell Physiol 317:C701-13. doi: 10.1152/ajpcell.00098.2019. PMID: 31291144 O2k
Ref 28: "Because isolation procedures could alter the functional/physiological properties of the mitochondria (28), additional experiments were undertaken to examine mitochondrial energetics in cultured, intact, mature myotubes."
7. Tomas C, Brown AE, Newton JL, Elson JL (2019) Mitochondrial complex activity in permeabilised cells of chronic fatigue syndrome patients using two cell types. PeerJ 7:e6500. doi: 10.7717/peerj.6500. PMID: 30847260
"When mitochondria are isolated from cells, the architecture and morphology of the mitochondria is altered (Bach et al., 2003; Mitra et al., 2009; Sarin et al., 2013; Hagenbuchner et al., 2013), but permeabilisation of the cell membrane allows the architecture and morphology of mitochondria to remain normal, an advantage over the use of isolated mitochondria as mitochondrial function has previously been shown to have a strong relationship with structure (Saks et al., 1998; Picard et al., 2011)."
8. Puurand M, Tepp K, Klepinin A, Klepinina L, Shevchuk I, Kaambre T (2018) Intracellular Energy-Transfer Networks and High-Resolution Respirometry: A Convenient Approach for Studying Their Function. Int J Mol Sci 19:2933. doi: 10.3390/ijms19102933. PMID: 30261663 O2k
Ref 12: "However, isolation of mitochondria disrupts their normal morphology and interactions with other cellular structures. Furthermore, there is evidence that isolated mitochondria possess several functional characteristics that differ considerably from those of intact mitochondria in permeabilized myofibers and cells [12]. Clearly, the isolated mitochondria do not provide information regarding their function in the intracellular environment under physiological settings, because functional connections between mitochondria and other cellular structures (e.g., ATPases, cytoskeleton), essential for normal function in vivo, are destroyed by the isolation procedure. Therefore, better understanding of bioenergetic processes can be derived from experimental models where the mitochondrial function is directly assessable, but also as undisrupted as possible. In these terms, selective permeabilization of the cellular outer membrane offers several advantages."
9. Li R, Steyn FJ, Stout MB, Lee K, Cully TR, Calderón JC, Ngo ST (2016) Development of a high-throughput method for real-time assessment of cellular metabolism in intact long skeletal muscle fibre bundles. J Physiol 594:7197-213. doi: 10.1113/JP272988. PMID: 27619319
"The use of intact muscle fibres for assessing mitochondrial function circumvents some limitations associated with the disruption of mitochondrial structure and function that can occur during the preparation of isolated mitochondria (Picard et al. 2010; Picard et al. 2011)."
10. Lark DS, Torres MJ, Lin CT, Ryan TE, Anderson EJ, Neufer PD (2016) Direct real-time quantification of mitochondrial oxidative phosphorylation efficiency in permeabilized skeletal muscle myofibers. Am J Physiol Cell Physiol 311:C239-45. doi: 10.1152/ajpcell.00124.2016. PMID: 27335172 O2k
Ref 34: "isolated mitochondria lack the functional reticulum and intracellular energy transfer systems normally present in vivo that influence the properties of the respiratory system (i.e., Km for ADP, etc.) (32). Permeabilized skeletal muscle myofiber bundles (PmFBs), on the other hand, retain the native mitochondrial reticulum (34) and energy transfer systems and thus better reflect in vivo energetics (32)."
11. Layec G, Gifford JR, Trinity JD, Hart CR, Garten RS, Park SY, Le Fur Y, Jeong EK, Richardson RS (2016) Accuracy and precision of quantitative 31P-MRS measurements of human skeletal muscle mitochondrial function. Am J Physiol Endocrinol Metab 311:E358-66. doi: 10.1152/ajpendo.00028.2016. PMID: 27302751
Ref 50: "Similarly, respiration measurements in isolated mitochondria, although providing a more direct measurement, may be affected by the biased selection of a particular mitochondrial population and the disruption of mitochondrial interactions during the isolation procedure, which in turn can alter mitochondrial function (26, 50). However, this issue appears to have been overcome by the development of the in vitro permeabilized fiber technique, which seems to preserve most of the functional properties of the skeletal muscle mitochondria (26, 50)."
12. Cheng AJ, Yamada T, Rassier DE, Andersson DC, Westerblad H, Lanner JT (2016) Reactive oxygen/nitrogen species and contractile function in skeletal muscle during fatigue and recovery. J Physiol 594:5149-60. doi: 10.1113/JP270650. PMID: 26857536
"Many studies of mitochondrial ROS production are performed with in vitro measurements on isolated mitochondria. This approach is problematic because the techniques used to isolate mitochondria from muscle alter mitochondrial structure and function by, for instance, causing fragmentation, loss of soluble matrix enzymes and altered respiratory rates and O2 •−/H2O2 production (Schwerzmann et al. 1989; Picard et al. 2011 a,b)."
13. Layec G, Bringard A, Le Fur Y, Micallef JP, Vilmen C, Perrey S, Cozzone PJ, Bendahan D (2016) Mitochondrial Coupling and Contractile Efficiency in Humans with High and Low V˙O2peaks. Med Sci Sports Exerc 48:811-21. doi: 10.1249/MSS.0000000000000858. PMID: 26694849
Ref 37: "However, mitochondrial efficiency measured in isolated mitochondria during maximal ADP-stimulated respiration was unrelated to the training status (34, 43) or unaltered after 6 weeks of high-intensity training (44). Of note, a caveat with this approach is the alteration of the mitochondrial function during the isolation procedure and the supra-physiological level of O2 surrounding the mitochondria in vitro, which is known to modulate mitochondrial efficiency (37)."
14. Christiansen LB, Dela F, Koch J, Yokota T (2015) Tissue-specific and substrate-specific mitochondrial bioenergetics in feline cardiac and skeletal muscles. J Vet Med Sci 77:669-75. O2k
Ref 27: "Most previous studies have assessed mitochondrial function across different muscle types by using isolated mitochondria in animals and humans. However, disadvantages of using isolated mitochondria are the necessity of larger amounts of tissue due to lower mitochondrial yield and absence of interaction between the mitochondria and surrounding structures within the cytosol [27, 31]."
15. Chu MJ, Hickey AJ, Tagaloa S, Zhang L, Dare AJ, MacDonald JR, Yeong ML, Bartlett AS, Phillips AR (2014) Ob/ob mouse livers show decreased oxidative phosphorylation efficiencies and anaerobic capacities after cold ischemia. PLoS One 9:e100609. O2k
Ref 34: "Mitochondrial respiration is temperature-dependant, in particular LEAK state respiration, which decreases relative to phosphorylating respiration flux (and therefore raises the RCR) [33]. The process of mitochondrial isolation disrupts mitochondrial structure and introduces bias by preferential selection of healthy mitochondria [34]. Our study adjusts for these confounding factors by analyzing mitochondrial function at 37°C and in permeabilised tissues."
16. Sanderson TH, Reynolds CA, Kumar R, Przyklenk K, Hüttemann M (2013) Molecular mechanisms of ischemia-reperfusion injury in brain: pivotal role of the mitochondrial membrane potential in reactive oxygen species generation. Mol Neurobiol 47:9-23. doi: 10.1007/s12035-012-8344-z. PMID: 23011809
"A recent study analyzing mitochondrial morphology and function showed that the structure of isolated mitochondria is clearly different compared to the morphology found in vivo (Picard et al. 2011)."
17. Clerc P, Polster BM (2012) Investigation of mitochondrial dysfunction by sequential microplate-based respiration measurements from intact and permeabilized neurons. PLoS One 7:e34465. doi: 10.1371/journal.pone.0034465. PMID: 22496810
Ref 29: "However, the use of tissue homogenization/density gradient centrifugation to isolate mitochondria is a disadvantage, as it may influence mitochondrial structure/function and likely selects for a subpopulation of mitochondria [28], [29]."
18. Schuh RA, Jackson KC, Khairallah RJ, Ward CW, Spangenburg EE (2012) Measuring mitochondrial respiration in intact single muscle fibers. Am J Physiol Regul Integr Comp Physiol 302:R712-9. doi: 10.1152/ajpregu.00229.2011. PMID: 22160545
Ref 27: "In an elegant study, Picard et al. (27) found that there are alterations in the stoichiometry of proteins within the electron transport chain in response to the isolation process. In addition, the isolation process further results in enhanced reactive oxygen species production in response to substrate delivery (27). The data confirm the importance of studying mitochondrial function in preparations that result in minimal disruption to the cell."
19. Pharaoh G, Brown J, Ranjit R, Ungvari Z, Van Remmen H (2021) Reduced adenosine diphosphate sensitivity in skeletal muscle mitochondria increases reactive oxygen species production in mouse models of aging and oxidative stress but not denervation. JCSM Rapid Commun 4:75-89. doi: 10.1002/rco2.29. PMID: 36159599
Ref 13: "This method uses permeabilized muscle fibres, which more closely model the function of mitochondria in vivo than isolated mitochondria by maintaining the complex mitochondrial networks found in skeletal muscle.13"
20. Kluza J, Peugnet V, Daunou B, Laine W, Kervoaze G, Rémy G, Loyens A, Maboudou P, Fovez Q, Grangette C, Wolowczuk I, Gosset P, Garçon G, Marchetti P, Pinet F, Pichavant M, Dubois-Deruy E (2021) A New Strategy to Preserve and Assess Oxygen Consumption in Murine Tissues. Int J Mol Sci 23:109. doi: 10.3390/ijms23010109. PMID: 35008535
Ref 10: "Moreover, isolation procedures often altered cell and organelle structures and functions, notably through disrupting the mitochondrial network, due to the loss of intracellular interactions [10,11]."
21. Daussin FN, Cuillerier A, Touron J, Bensaid S, Melo B, Al Rewashdy A, Vasam G, Menzies KJ, Harper ME, Heyman E, Burelle Y (2021) Dietary Cocoa Flavanols Enhance Mitochondrial Function in Skeletal Muscle and Modify Whole-Body Metabolism in Healthy Mice. Nutrients 13:3466. doi: 10.3390/nu13103466. PMID: 34684467
Ref 32: "Unlike the previous studies on isolated mitochondria, the assessment of mitochondrial function in permeabilised fibre preserves mitochondrial morphology, functional interactions with other intracellular components [1] and avoids mitochondrial structure disruption during the mitochondrial isolation process [32]. Therefore, it is important to study mitochondrial function in tissue preparations where mitochondrial structure is preserved, and all of the mitochondrial pool is represented to better evaluate the response to a treatment."

Using a paradigm

Without further testing, apparent discrepancies are ‘explained’ by reference to Picard et al (2011):
1. Lewis MT, Blain GM, Hart CR, Layec G, Rossman MJ, Park SY, Trinity JD, Gifford JR, Sidhu SK, Weavil JC, Hureau TJ, Jessop JE, Bledsoe AD, Amann M, Richardson RS (2021) Acute high-intensity exercise and skeletal muscle mitochondrial respiratory function: role of metabolic perturbation. Am J Physiol Regul Integr Comp Physiol 321:R687-98. doi: 10.1152/ajpregu.00158.2021. PMID: 34549627
Ref 60: "Specifically, membrane integrity and maximal flux are essential for mitochondrial survival and subsequent regaining of function, respectively, while phosphorylative flux can be acutely impaired simply by ultrastructural changes as a result of high-intensity exercise (32, 53–55). Importantly, this may explain why prior studies which, in contrast with our previously established findings (1) and that with FENT in the current study, failed to reveal a negative impact of exercise on mitochondrial respiration (55, 56). Indeed, of note, these previous studies, showing no impact of exercise, were performed in isolated mitochondria, which do not retain the mitochondrial reticulum and, thus, the complete ultrastructure, reflective of that found in vivo, as do the permeabilized fibers employed in the current study (57–60)."
2. Zuhra K, Szabo C. Reply to Giamogante et al (2021) The effect of low cyanide on O2 consumption is best observed in physiological, rather than reductionist, systems. Proc Natl Acad Sci U S A 118:e2113369118. doi: 10.1073/pnas.2113369118. PMID: 34544879
Ref 6: "In their letter, Giamogante et al. (4) present OCR measurements in isolated mouse liver mitochondria and detect no significant activating effect of 1 nM cyanide (although a hint for a ∼5% activating effect may be present, this is not statistically significant with the large standard generated from n = 2 to 4 replicates used). In our own studies we avoided using isolated mitochondria because such preparations often respond differently than intact cells (5, 6). When in our experiments we tested the effect of 0.1 nM cyanide in isolated bovine CcOX we also only observed a slight (∼10%) activation (1). Thus, the stimulating effect of low cyanide on OCR is best observable in intact cell-based systems, as opposed to reductionist models."
3. Berg OK, Kwon OS, Hureau TJ, Clifton HL, Thurston TS, Le Fur Y, Jeong EK, Trinity JD, Richardson RS, Wang E, Layec G (2020) Skeletal Muscle Mitochondrial Adaptations to Maximal Strength Training in Older Adults. J Gerontol A Biol Sci Med Sci 75:2269-2277. doi: 10.1093/gerona/glaa082. PMID: 32253421
Ref 18: "This discrepancy among these previous investigations is intriguing, and might stem from the investigational techniques employed. For example, mitochondrial isolation disrupts the organelle’s structure, potentially altering mitochondrial functional characteristics (18), prompting the development of the permeabilized muscle fiber technique (19) that better preserves the properties of the mitochondria. … in young healthy subjects, strength training has been documented to improve muscle state 3CI+CII respiration utilizing permeabilized muscle fibers (8,13). However, in older subjects, results are more equivocal, with some studies reporting increased enzymatic activities or oxidation rate (15,30), but not all (6,17). In this regard, it is interesting to note that the latter studies used an isolated mitochondria preparation (6,17) that disrupts the mitochondria and the respiratory complex interactions (18,19), further suggesting that qualitative adaptations within the mitochondria may play a role in the change in muscle respiratory capacity with strength training."
4. Hart CR, Layec G, Trinity JD, Kwon OS, Zhao J, Reese VR, Gifford JR, Richardson RS (2018) Increased skeletal muscle mitochondrial free radical production in peripheral arterial disease despite preserved mitochondrial respiratory capacity. Exp Physiol 103:838-50. doi: 10.1113/EP086905. PMID: 29604234
"For example, Hou and colleagues (Hou et al., 2002) reported preserved peak mitochondrial ATP production rates in isolated mitochondria from patients with PAD with similar average ABIs (~0.67) as the patients assessed in the current study (Table 1). However, evidence suggests that the mitochondrial isolation procedure itself may selectively damage the organelles of interest, potentially confounding the assessment of mitochondrial function (Picard et al., 2011)."
5. Yao CH, Liu GY, Wang R, Moon SH, Gross RW, Patti GJ (2018) Identifying off-target effects of etomoxir reveals that carnitine palmitoyltransferase I is essential for cancer cell proliferation independent of β-oxidation. PLoS Biol 16:e2003782. doi: 10.1371/journal.pbio.2003782. PMID: 29596410
Ref 63: "The 37 % difference between basal respiration and 200 μM etomoxir treatment is smaller than the 65% difference observed in Fig 2B, likely due to the absence of fatty acid oxidation and the reduced basal respiration of isolated mitochondria [63, 64]."
6. Distefano G, Standley RA, Zhang X, Carnero EA, Yi F, Cornnell HH, Coen PM (2018) Physical activity unveils the relationship between mitochondrial energetics, muscle quality, and physical function in older adults. J Cachexia Sarcopenia Muscle 9:279-94. doi: 10.1002/jcsm.12272. PMID: 29368427
Ref 55: "Many have also used isolated mitochondria,15, 18, 20 an experimental approach that may confound assays of mitochondrial function in ageing.55"
7. Rontoyanni VG, Nunez Lopez O, Fankhauser GT, Cheema ZF, Rasmussen BB, Porter C (2017) Mitochondrial Bioenergetics in the Metabolic Myopathy Accompanying Peripheral Artery Disease. Front Physiol 8:141. doi: 10.3389/fphys.2017.00141. PMID: 28348531 O2k
"Further, more recent evidence has highlighted that the reticular structure and network formed by mitochondria in situ is significantly disrupted by isolation from skeletal muscle (Picard et al., 2011a,b). Importantly, this may lead to erroneous data concerning mitochondrial functionality (Picard et al., 2010). This may also explain the contradictory findings between mitochondrial respiratory capacity in isolated mitochondria and in muscle fiber bundles from PAD patients discussed in the following section. … The discrepant findings may reflect certain limitations with studying mitochondrial respiration in isolated mitochondria than in permeabilized muscle fibers, such as low yields and disruption of the mitochondrial network (Picard et al., 2010, 2011a,b)."
8. Cartee GD, Hepple RT, Bamman MM, Zierath JR (2016) Exercise Promotes Healthy Aging of Skeletal Muscle. Cell Metab 23:1034-47. doi: 10.1016/j.cmet.2016.05.007. PMID: 27304505
"In contrast to this view however, a high reliance on isolated organelles, a procedure that potentiates muscle mitochondrial ROS (Picard et al., 2011c) and which exaggerates the impact of aging (Picard et al., 2010), has skewed perception of the changes in mitochondrial ROS in aging skeletal muscle."
9. Hughes MC, Ramos SV, Turnbull PC, Nejatbakhsh A, Baechler BL, Tahmasebi H, Laham R, Gurd BJ, Quadrilatero J, Kane DA, Perry CG (2015) Mitochondrial bioenergetics and fiber type assessments in microbiopsy vs. Bergstrom percutaneous sampling of human skeletal muscle. Front Physiol 6:360. O2k
"Loss of PmFB integrity could impair respiratory assessments given it has previously been established that preservation of mitochondrial structure and morphology are critical for optimizing respiratory assessments in PmFB (Veksler et al., 1987; Kuznetsov et al., 2008; Picard et al., 2011)."
10. Gifford JR, Garten RS, Nelson AD, Trinity JD, Layec G, Witman MA, Weavil JC, Mangum T, Hart C, Etheredge C, Jessop J, Bledsoe A, Morgan DE, Wray DW, Rossman MJ, Richardson RS (2016) Symmorphosis and skeletal muscle V̇O2 max : in vivo and in vitro measures reveal differing constraints in the exercise-trained and untrained human. J Physiol 594:1741-51. doi: 10.1113/JP271229. PMID: 26614395
"As acknowledged in this prior work, this observation, which disagrees with the findings of the present study, is physiologically improbable and is potentially the result of altered mitochondrial function, inherent to the isolated mitochondria technique (Picard et al. 2011)."
11. Wessels B, Ciapaite J, van den Broek NMA, Nicolay K, Prompers JJ (2014) Metformin impairs mitochondrial function in skeletal muscle of both lean and diabetic rats in a dose-dependent manner. PLoS One 9:e100525. O2k
Ref 37: "However, when comparing our results with the ex vivo animal studies of Kane et al. [34] and Kristensen et al. [21], in which metformin did not affect mitochondrial respiration in either oxidative or glycolytic muscle from rats or mice after 2–4 weeks of metformin treatment at ∼300 mg/kg/day, it seems that all except methodological differences can be excluded. In the current study mitochondria were isolated from a whole TA muscle to allow comparison with the in vivo data, while Kane et al. and Kristensen et al. used permeabilized muscle fibers. It has recently been reported that the respiratory response in permeabilized fibers can be different from that of isolated mitochondria [37]."
12. Sanderson TH. Phosphorylation of mammalian cytochrome c and cytochrome c oxidase in the regulation of cell destiny: respiration, apoptosis, and human disease. Adv Exp Med Biol (2012) 748:237-64. doi: 10.1007/978-1-4614-3573-0_10. PMID: 22729861
"It has been shown that not only mitochondrial morphology changes dramatically after isolation but also that, in contrast to the other ETC complexes, specifically COX activity is significantly increased (Picard et al. 2011), which may account for a loss of ETC flux control by COX in isolated mitochondria."


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

HRR: Oxygraph-2k 

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