A novel approach to measure mitochondrial respiration in frozen biological samples
2020; Springer Nature; Volume: 39; Issue: 13 Linguagem: Inglês
10.15252/embj.2019104073
ISSN1460-2075
AutoresRebeca Acín‐Pérez, Ilan Y. Benador, Anton Petcherski, Michaela Veliova, Gloria A. Benavides, Sylviane Lagarrigue, Arianne Caudal, Laurent Vergnes, Anne N. Murphy, Georgios Karamanlidis, Rong Tian, Karen Reue, Jonathan Wanagat, Harold S. Sacks, Francesca Amati, Victor Darley‐Usmar, Marc Liesa, Ajit S. Divakaruni, Linsey Stiles, Orian S. Shirihai,
Tópico(s)Metabolomics and Mass Spectrometry Studies
ResumoResource20 May 2020Open Access Source DataTransparent process A novel approach to measure mitochondrial respiration in frozen biological samples Rebeca Acin-Perez Corresponding Author Rebeca Acin-Perez [email protected] orcid.org/0000-0001-9553-8337 Department of Medicine, Endocrinology, David Geffen School of Medicine, University of California, Los Angeles, CA, USA Metabolism Theme, David Geffen School of Medicine, University of California, Los Angeles, CA, USA Search for more papers by this author Ilan Y Benador Ilan Y Benador Department of Medicine, Endocrinology, David Geffen School of Medicine, University of California, Los Angeles, CA, USA Metabolism Theme, David Geffen School of Medicine, University of California, Los Angeles, CA, USA Nutrition and Metabolism, Graduate Medical Sciences, Boston University School of Medicine, Boston, MA, USA Search for more papers by this author Anton Petcherski Anton Petcherski Department of Medicine, Endocrinology, David Geffen School of Medicine, University of California, Los Angeles, CA, USA Metabolism Theme, David Geffen School of Medicine, University of California, Los Angeles, CA, USA Search for more papers by this author Michaela Veliova Michaela Veliova Department of Medicine, Endocrinology, David Geffen School of Medicine, University of California, Los Angeles, CA, USA Metabolism Theme, David Geffen School of Medicine, University of California, Los Angeles, CA, USA Department of Molecular and Medical Pharmacology, University of California, Los Angeles, CA, USA Search for more papers by this author Gloria A Benavides Gloria A Benavides Department of Pathology and Mitochondrial Medicine Laboratory, University of Alabama at Birmingham, Birmingham, AL, USA Search for more papers by this author Sylviane Lagarrigue Sylviane Lagarrigue Department of Biomedical Sciences, University of Lausanne, Lausanne, Switzerland Search for more papers by this author Arianne Caudal Arianne Caudal Mitochondria and Metabolism Center, University of Washington, Seattle, WA, USA Search for more papers by this author Laurent Vergnes Laurent Vergnes Metabolism Theme, David Geffen School of Medicine, University of California, Los Angeles, CA, USA Department of Human Genetics, David Geffen School of Medicine, University of California, Los Angeles, CA, USA Search for more papers by this author Anne N Murphy Anne N Murphy orcid.org/0000-0002-5222-9902 Department of Pharmacology, University of California, San Diego, CA, USA Search for more papers by this author Georgios Karamanlidis Georgios Karamanlidis Cardiometabolic Disorders, Amgen Research, Thousand Oaks, CA, USA Search for more papers by this author Rong Tian Rong Tian Mitochondria and Metabolism Center, University of Washington, Seattle, WA, USA Search for more papers by this author Karen Reue Karen Reue Metabolism Theme, David Geffen School of Medicine, University of California, Los Angeles, CA, USA Department of Human Genetics, David Geffen School of Medicine, University of California, Los Angeles, CA, USA Search for more papers by this author Jonathan Wanagat Jonathan Wanagat Metabolism Theme, David Geffen School of Medicine, University of California, Los Angeles, CA, USA Department of Medicine, Division of Geriatrics, University of California, Los Angeles, CA, USA Search for more papers by this author Harold Sacks Harold Sacks Department of Medicine, Endocrinology, David Geffen School of Medicine, University of California, Los Angeles, CA, USA Metabolism Theme, David Geffen School of Medicine, University of California, Los Angeles, CA, USA Search for more papers by this author Francesca Amati Francesca Amati Department of Biomedical Sciences, University of Lausanne, Lausanne, Switzerland Search for more papers by this author Victor M Darley-Usmar Victor M Darley-Usmar Department of Pathology and Mitochondrial Medicine Laboratory, University of Alabama at Birmingham, Birmingham, AL, USA Search for more papers by this author Marc Liesa Marc Liesa orcid.org/0000-0002-5909-8570 Department of Medicine, Endocrinology, David Geffen School of Medicine, University of California, Los Angeles, CA, USA Metabolism Theme, David Geffen School of Medicine, University of California, Los Angeles, CA, USA Department of Molecular and Medical Pharmacology, University of California, Los Angeles, CA, USA Molecular Biology Institute, UCLA, Los Angeles, CA, USA Search for more papers by this author Ajit S Divakaruni Ajit S Divakaruni orcid.org/0000-0002-2528-9651 Metabolism Theme, David Geffen School of Medicine, University of California, Los Angeles, CA, USA Department of Molecular and Medical Pharmacology, University of California, Los Angeles, CA, USA Search for more papers by this author Linsey Stiles Corresponding Author Linsey Stiles [email protected] orcid.org/0000-0002-1514-458X Department of Medicine, Endocrinology, David Geffen School of Medicine, University of California, Los Angeles, CA, USA Metabolism Theme, David Geffen School of Medicine, University of California, Los Angeles, CA, USA Search for more papers by this author Orian S Shirihai Corresponding Author Orian S Shirihai [email protected] orcid.org/0000-0001-8466-3431 Department of Medicine, Endocrinology, David Geffen School of Medicine, University of California, Los Angeles, CA, USA Metabolism Theme, David Geffen School of Medicine, University of California, Los Angeles, CA, USA Nutrition and Metabolism, Graduate Medical Sciences, Boston University School of Medicine, Boston, MA, USA Department of Molecular and Medical Pharmacology, University of California, Los Angeles, CA, USA Molecular Biology Institute, UCLA, Los Angeles, CA, USA Search for more papers by this author Rebeca Acin-Perez Corresponding Author Rebeca Acin-Perez [email protected] orcid.org/0000-0001-9553-8337 Department of Medicine, Endocrinology, David Geffen School of Medicine, University of California, Los Angeles, CA, USA Metabolism Theme, David Geffen School of Medicine, University of California, Los Angeles, CA, USA Search for more papers by this author Ilan Y Benador Ilan Y Benador Department of Medicine, Endocrinology, David Geffen School of Medicine, University of California, Los Angeles, CA, USA Metabolism Theme, David Geffen School of Medicine, University of California, Los Angeles, CA, USA Nutrition and Metabolism, Graduate Medical Sciences, Boston University School of Medicine, Boston, MA, USA Search for more papers by this author Anton Petcherski Anton Petcherski Department of Medicine, Endocrinology, David Geffen School of Medicine, University of California, Los Angeles, CA, USA Metabolism Theme, David Geffen School of Medicine, University of California, Los Angeles, CA, USA Search for more papers by this author Michaela Veliova Michaela Veliova Department of Medicine, Endocrinology, David Geffen School of Medicine, University of California, Los Angeles, CA, USA Metabolism Theme, David Geffen School of Medicine, University of California, Los Angeles, CA, USA Department of Molecular and Medical Pharmacology, University of California, Los Angeles, CA, USA Search for more papers by this author Gloria A Benavides Gloria A Benavides Department of Pathology and Mitochondrial Medicine Laboratory, University of Alabama at Birmingham, Birmingham, AL, USA Search for more papers by this author Sylviane Lagarrigue Sylviane Lagarrigue Department of Biomedical Sciences, University of Lausanne, Lausanne, Switzerland Search for more papers by this author Arianne Caudal Arianne Caudal Mitochondria and Metabolism Center, University of Washington, Seattle, WA, USA Search for more papers by this author Laurent Vergnes Laurent Vergnes Metabolism Theme, David Geffen School of Medicine, University of California, Los Angeles, CA, USA Department of Human Genetics, David Geffen School of Medicine, University of California, Los Angeles, CA, USA Search for more papers by this author Anne N Murphy Anne N Murphy orcid.org/0000-0002-5222-9902 Department of Pharmacology, University of California, San Diego, CA, USA Search for more papers by this author Georgios Karamanlidis Georgios Karamanlidis Cardiometabolic Disorders, Amgen Research, Thousand Oaks, CA, USA Search for more papers by this author Rong Tian Rong Tian Mitochondria and Metabolism Center, University of Washington, Seattle, WA, USA Search for more papers by this author Karen Reue Karen Reue Metabolism Theme, David Geffen School of Medicine, University of California, Los Angeles, CA, USA Department of Human Genetics, David Geffen School of Medicine, University of California, Los Angeles, CA, USA Search for more papers by this author Jonathan Wanagat Jonathan Wanagat Metabolism Theme, David Geffen School of Medicine, University of California, Los Angeles, CA, USA Department of Medicine, Division of Geriatrics, University of California, Los Angeles, CA, USA Search for more papers by this author Harold Sacks Harold Sacks Department of Medicine, Endocrinology, David Geffen School of Medicine, University of California, Los Angeles, CA, USA Metabolism Theme, David Geffen School of Medicine, University of California, Los Angeles, CA, USA Search for more papers by this author Francesca Amati Francesca Amati Department of Biomedical Sciences, University of Lausanne, Lausanne, Switzerland Search for more papers by this author Victor M Darley-Usmar Victor M Darley-Usmar Department of Pathology and Mitochondrial Medicine Laboratory, University of Alabama at Birmingham, Birmingham, AL, USA Search for more papers by this author Marc Liesa Marc Liesa orcid.org/0000-0002-5909-8570 Department of Medicine, Endocrinology, David Geffen School of Medicine, University of California, Los Angeles, CA, USA Metabolism Theme, David Geffen School of Medicine, University of California, Los Angeles, CA, USA Department of Molecular and Medical Pharmacology, University of California, Los Angeles, CA, USA Molecular Biology Institute, UCLA, Los Angeles, CA, USA Search for more papers by this author Ajit S Divakaruni Ajit S Divakaruni orcid.org/0000-0002-2528-9651 Metabolism Theme, David Geffen School of Medicine, University of California, Los Angeles, CA, USA Department of Molecular and Medical Pharmacology, University of California, Los Angeles, CA, USA Search for more papers by this author Linsey Stiles Corresponding Author Linsey Stiles [email protected] orcid.org/0000-0002-1514-458X Department of Medicine, Endocrinology, David Geffen School of Medicine, University of California, Los Angeles, CA, USA Metabolism Theme, David Geffen School of Medicine, University of California, Los Angeles, CA, USA Search for more papers by this author Orian S Shirihai Corresponding Author Orian S Shirihai [email protected] orcid.org/0000-0001-8466-3431 Department of Medicine, Endocrinology, David Geffen School of Medicine, University of California, Los Angeles, CA, USA Metabolism Theme, David Geffen School of Medicine, University of California, Los Angeles, CA, USA Nutrition and Metabolism, Graduate Medical Sciences, Boston University School of Medicine, Boston, MA, USA Department of Molecular and Medical Pharmacology, University of California, Los Angeles, CA, USA Molecular Biology Institute, UCLA, Los Angeles, CA, USA Search for more papers by this author Author Information Rebeca Acin-Perez *,1,2,‡, Ilan Y Benador1,2,3, Anton Petcherski1,2, Michaela Veliova1,2,4, Gloria A Benavides5, Sylviane Lagarrigue6, Arianne Caudal7, Laurent Vergnes2,8, Anne N Murphy9, Georgios Karamanlidis10, Rong Tian7, Karen Reue2,8, Jonathan Wanagat2,11, Harold Sacks1,2, Francesca Amati6, Victor M Darley-Usmar5, Marc Liesa1,2,4,12, Ajit S Divakaruni2,4, Linsey Stiles *,1,2,‡ and Orian S Shirihai *,1,2,3,4,12,‡ 1Department of Medicine, Endocrinology, David Geffen School of Medicine, University of California, Los Angeles, CA, USA 2Metabolism Theme, David Geffen School of Medicine, University of California, Los Angeles, CA, USA 3Nutrition and Metabolism, Graduate Medical Sciences, Boston University School of Medicine, Boston, MA, USA 4Department of Molecular and Medical Pharmacology, University of California, Los Angeles, CA, USA 5Department of Pathology and Mitochondrial Medicine Laboratory, University of Alabama at Birmingham, Birmingham, AL, USA 6Department of Biomedical Sciences, University of Lausanne, Lausanne, Switzerland 7Mitochondria and Metabolism Center, University of Washington, Seattle, WA, USA 8Department of Human Genetics, David Geffen School of Medicine, University of California, Los Angeles, CA, USA 9Department of Pharmacology, University of California, San Diego, CA, USA 10Cardiometabolic Disorders, Amgen Research, Thousand Oaks, CA, USA 11Department of Medicine, Division of Geriatrics, University of California, Los Angeles, CA, USA 12Molecular Biology Institute, UCLA, Los Angeles, CA, USA ‡These authors contributed equally to this work *Corresponding author. Tel: +1 310 622 3929; E-mail: [email protected] *Corresponding author. Tel: +1 310 825 8630; E-mail: [email protected] *Corresponding author. Tel: +1 617 230 8570; E-mail: [email protected] The EMBO Journal (2020)39:e104073https://doi.org/10.15252/embj.2019104073 [Correction added on 28 May 2020, after first online publication: the author affiliations have been corrected.] PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract Respirometry is the gold standard measurement of mitochondrial oxidative function, as it reflects the activity of the electron transport chain complexes working together. However, the requirement for freshly isolated mitochondria hinders the feasibility of respirometry in multi-site clinical studies and retrospective studies. Here, we describe a novel respirometry approach suited for frozen samples by restoring electron transfer components lost during freeze/thaw and correcting for variable permeabilization of mitochondrial membranes. This approach preserves 90–95% of the maximal respiratory capacity in frozen samples and can be applied to isolated mitochondria, permeabilized cells, and tissue homogenates with high sensitivity. We find that primary changes in mitochondrial function, detected in fresh tissue, are preserved in frozen samples years after collection. This approach will enable analysis of the integrated function of mitochondrial Complexes I to IV in one measurement, collected at remote sites or retrospectively in samples residing in tissue biobanks. Synopsis Freeze-thawing events cause mitochondrial membrane permeabilization and disrupt mitochondrial functionality in cells and tissues. Reconstitution of maximal mitochondrial respiration allows the analysis of mitochondrial bioenergetics in frozen and thawed crude samples, thus overcoming limitations associated with the current methods. Following reconstitution, mitochondrial maximal respiration can be assessed in frozen isolated mitochondria or total tissue lysate. Respiratory rate measurements from tissue lysates reduce by an order of magnitude the minimal mass of tissue required. Maximal respiration rates in reconstituted frozen samples are comparable to those in fresh ones. Respiratory rates can be normalized per cell, per total protein or per mitochondrial content in frozen specimens. Respiratory rates can be measured in stored samples from clinical and animal studies, and drug toxicity assays. Introduction Mitochondrial oxidative function is an essential parameter to understand metabolism in health and disease. Mitochondria consume 90% of the oxygen that we breathe through Complex IV of the electron transport chain (ETC). The ETC is not only essential to transform the energy of nutrients into a proton gradient used to make ATP, but the ETC is required for all other aspects of mitochondrial-dependent cell metabolism. This is the reason why measuring oxygen consumption, namely respirometry, is the gold standard measurement of mitochondrial function. A major limitation in oxygen consumption measurements is that they require fresh tissue. There are two key reasons for this: (i) the ETC activity is depressed after freeze–thaw through loss of cytochrome c from the inter-membrane space. (ii) Freeze–thaw damages the mitochondrial membranes, which effectively uncouples the ETC activity (oxygen consumption) from ATP synthesis. These problems are a barrier to basic and translational research since samples cannot be stored and assayed together to decrease the cost and variability of the measurements. This current limitation in oxygen consumption methods restricts measurements from samples stored in biobanks, which are essential for translational research. Consequently, establishing reliable high-throughput methods for assessing mitochondrial function independently of the type of sample and specific freezing methods would overcome this limitation. Clinicians have been using spectrophotometric assays to determine the activity of individual ETC complexes or the combination of CI + III or CII + III, in previously frozen samples. These measurements were successfully used in a relatively high-throughput manner to diagnose primary mitochondrial diseases, namely diseases caused by a primary defect in ETC function (Birch-Machin & Turnbull, 2001; Barrientos, 2002; Barrientos et al, 2009). However, this approach cannot provide a single measurement of the coordinated function of the ETC function working at more physiological rates. In this regard, some protocols measure supraphysiological activities by using non-physiological electron donors and acceptors. Consequently, spectrophotometric assays might be less sensitive to detect milder reductions in mitochondrial function, such as the ones associated with age-related cardiovascular metabolic diseases. This explains why there have been several attempts to cryopreserve tissues focusing on the maintenance of the mitochondrial inner membrane integrity, by addition of different reagents at the time of freezing with different outcomes (Kuznetsov et al, 2003; Nukala et al, 2006; Yamaguchi et al, 2007; Larsen et al, 2012; Garcia-Roche et al, 2018). It has been shown that frozen samples do not show coupled respiration as membrane integrity is lost during the freezing process, which results in the uncoupling of electron transport from ATP synthesis. However, despite the loss of coupled respiration, both the enzymatic assays of individual complex activities and the ability of supercomplexes to consume oxygen, after their separation in a blue native gel from frozen samples, support the idea the ETC components are not destroyed by freeze–thawing and the reconstitution of electron transport activity is feasible (Acin-Perez et al, 2008). The question arises: Why is it that isolated mitochondria do not respire using the conventional substrate combination, after freezing and thawing if the ETC components are insensitive to freeze and thaw cycles? We have developed a new approach that reconstitutes maximal mitochondrial respiration in previously frozen samples. Our approach is versatile, as it is amenable to multiple sample types without the need of any special freezing and thawing protocols. In this method, we measure maximal oxygen consumption of the ETC in isolated mitochondria as an integrated unit, using physiological electron donors and acceptors, as in the protocols used for freshly prepared samples. We have also developed a quantitative method to determine mitochondrial content in tissue lysates and intact cells, allowing for evaluation of mitochondrial bioenergetics per cell and also per mitochondria in frozen specimens. Our assay provides a standardized, cost-effective, and widely available test of mitochondrial function, which is one step closer to measure physiological respiration rates in frozen specimens. Results The electron transport system remains intact in mitochondria isolated from previously frozen liver samples To determine whether respiratory capacity can be measured in previously frozen liver mitochondria, we first measured their oxygen consumption rate (OCR) using the conventional mitochondrial assay protocols using the Seahorse XF96 Extracellular Flux Analyzer. Mitochondria (4 μg protein/well) isolated from fresh (mFresh) and frozen (mFrozen) mouse livers were assayed in the presence of pyruvate and malate, as substrates, and sequential injections of ADP, oligomycin, FCCP, and antimycin A/rotenone followed. mFrozen assayed using pyruvate and malate as substrates showed significantly lower OCR in all respiratory states compared to mitochondria isolated from fresh tissue (Fig 1A–C). This suggests that when pyruvate plus malate were used as substrates, the electron transfer from Complex I to Complex III and ultimately to cytochrome c oxidase is impaired in mFrozen (Fig 1A–C). Figure 1. Mitochondria isolated from previously frozen liver maintain intact electron transport system A. Representative traces of oxygen consumption rate (OCR) of mouse liver mitochondria isolated from fresh or frozen tissue sustained by pyruvate + malate. Pyruvate + malate + ADP (PM + ADP), oligomycin (oligo), FCCP, and antimycin A + rotenone (AA + ROT) were sequentially injected to assess mitochondrial respiratory states. B. Pyruvate + malate-dependent state 3 (substrate plus ADP)/state 4 (substrate without ADP) in fresh and frozen liver mitochondria. C. Quantification of maximal respiration rate (MRR) supported by pyruvate + malate in fresh and frozen liver mitochondria. D. Representative traces of OCR of liver mitochondria isolated from fresh or frozen tissue supported by the Complex II substrate succinate + rotenone + ADP (SR + ADP). E. Succinate + rotenone-dependent state 3/state 4 in fresh and frozen liver mitochondria. F. Quantification of the different bioenergetic parameters sustained by succinate + rotenone in fresh and frozen liver mitochondria. G. Representative traces of OCR of liver mitochondria isolated from fresh or frozen tissue sustained by the Complex 1 substrate NADH + ADP. H. NADH-dependent state 3/state 4 in fresh and frozen liver mitochondria. I. MRR driven by NADH in fresh and frozen liver mitochondria. J. Representative traces of OCR of liver mitochondria isolated from fresh or frozen tissue starting in state 1 and sustained by substrates without ADP (state 4) and by substrates with ADP (state 3). Mitochondria were tested for CAT sensitivity. K. Representative traces of OCR of liver mitochondria isolated from fresh or frozen tissue starting in state 1 and sustained by substrates with ADP (state 3). Mitochondria were tested for CAT sensitivity. Data information: Panels (A, D, G, J, and K) are representative seahorse traces including four technical replicates. Biological replicates: (B and C), n = 4; (E and F), n = 6; and (H and I), n = 4. Every biological replicate represents the average of four technical replicates. Data are the mean ± SEM. Download figure Download PowerPoint We reasoned that freeze–thaw impairs TCA cycle function by disrupting the inner and outer mitochondrial membranes (McGann et al, 1988) releasing TCA cycle components from the mitochondrial matrix compartment. While mitochondrial membranes are sensitive to freeze–thaw, published work has shown that the inner membrane mitochondrial supercomplexes maintain assembly and activity after freezing (Acin-Perez et al, 2008). We therefore hypothesized that the electron transport system may remain intact in mFrozen. To test this, we assessed respiration in mFrozen using succinate that feeds directly into the electron transport system through Complex II. To control for non-specific respiration, we confirmed that succinate-dependent mFrozen respiration is inhibited by the Complex II-specific inhibitor 3-nitroproprionic acid in a concentration-dependent manner (Appendix Fig S1A). mFrozen pre-incubated with succinate showed a significantly higher respiratory rate than mFresh. However, as compared to mFresh, the mFrozen sample was insensitive to oligomycin. Lack of an oligomycin response in the mFrozen sample is consistent with increased proton permeability as a result of the broken membrane. This is a consequence of the generation of an uncoupled state by the freezing and thawing, leading to lack of control of respiration by Complex V (Fig 1D–F). This conclusion is supported by the observation that mFresh showed increased respiration upon addition of the uncoupler FCCP that reached the same level as mFrozen before the addition of FCCP. Both mFresh and mFrozen were equally sensitive to the Complex III inhibitor antimycin A (Fig 1E and F). These data suggest that electron transport between Complex II, co-enzyme Q, Complex III, cytochrome c, and Complex IV is functional in mFrozen. Next, to test the capacity of other ETC substrates to fuel respiration in mFrozen, we injected the Complex I substrate, NADH. Since intact mitochondria are not permeable to NADH, it is expected that fresh mitochondria will not respire upon injection of exogenous NADH as the sole substrate. In comparison in mFrozen, the inner membrane is permeable and Complex I has direct access to the injected NADH. Indeed, mFresh responded to NADH injection with no significant increase in oxygen consumption (Fig 1G) while pyruvate plus malate, which stimulate the TCA cycle to produce endogenous NADH (Fig 1A–C), resulted in threefold increase in oxygen consumption. In comparison, mFrozen pre-incubated with NADH respired in a similar pattern to that of succinate. In the presence of NADH, respiration of mFrozen was higher than mFresh, insensitive to ATP synthase inhibition and to FCCP, but was sensitive to inhibition by ETC inhibitors (Fig 1G–I). The progressive decrease in NADH-dependent OCR suggests that NADH is rapidly depleted over time in this assay, as we previously observed (Darley-Usmar et al, 1987). As a final control, we performed respirometry assays in fresh and frozen liver mitochondria starting in state 1 without any substrates or ADP and injecting substrates (state 4), substrates plus ADP (state 3), and the adenine nucleotide transporter (ANT) inhibitor carboxy-atractyloside (CAT; Fig 1J and K). As expected, state 1 was similar in fresh and frozen mitochondria and only fresh mitochondria responded to CAT injection as observed with oligomycin. Taken together, our results demonstrate that mitochondria isolated from freeze–thawed mouse livers maintain an intact electron transport capacity despite the disruption of ATP-coupled respiration and substrate shuttle carriers. Uncoupled electron transport activity in liver mFrozen is equivalent to mFresh Our results show that mFrozen are uncoupled but maintain electron transport capacity. However, mFrozen do not respond to the more commonly used mitochondrial substrates (e.g., pyruvate/malate). We therefore sought to develop a dedicated sequential assay for mFrozen using more compatible substrates. Since mitochondria isolated from frozen tissue are already fully uncoupled, measuring the responses to oligomycin and FCCP is no longer informative (Appendix Fig S1B). Instead, we injected compatible substrates (NADH or succinate) and complex inhibitors (rotenone or antimycin A), respectively. This modification allowed for the inclusion of the substrates needed for Complex IV activity including TMPD/ascorbate and the Complex IV inhibitor, sodium azide. To control for non-mitochondrial oxygen consumption, we confirmed that the TMPD/ascorbate-dependent respiration is inhibited by the Complex IV inhibitor potassium cyanide in a dose-dependent manner (Appendix Fig S1C). Our results show that mFrozen have a robust azide-sensitive respiratory response to TMPD/ascorbate following injections of any of the three substrates (Fig 2A, C, and E and Appendix Fig S1B). Figure 2. RIFS measurement of Complex I, II, and IV activity in fresh and frozen liver mitochondria A. Representative pyruvate + malate seahorse profile using RIFS the respirometry protocol in mouse liver mitochondria isolated from fresh or frozen tissue. Pyruvate + malate (Pyr + Mal), antimycin A + rotenone (AA + ROT), TMPD + ascorbate (TMPD/Asc), and azide were injected sequentially B. Pyruvate + malate- and TMPD/ascorbate (Complex IV, CIV)-dependent respiration in fresh and frozen liver mitochondria. C. Representative succinate + rotenone seahorse profile using the RIFS respirometry protocol in mouse liver mitochondria isolated from fresh or frozen tissue. D. Succinate + rotenone (Succ)- and CIV-dependent respiration in fresh and frozen liver mitochondria. E. Representative NADH seahorse profile using RIFS respirometry protocol in liver mitochondria isolated from fresh or frozen tissue. F. NADH- and CIV-dependent respiration in fresh and frozen liver mitochondria. Data information: Panels (A, C, and E) are representative seahorse traces including four technical replicates. Biological replicates: (B and F), n = 3–4; (D), n = 4–6. Every biological replicate represents the average of four technical replicates. Data are the mean ± SEM. Download figure Download PowerPoint Since comparison of respiratory chain function in mFrozen and mFresh is constrained by the loss of coupling, we compared activities in the presence of FCCP such that respiration is independent from ATP synthase and so does not require ADP. Our results show that in the presence of pyruvate plus malate, mFresh + FCCP had significantly increased respiration compared to mFrozen, confirming that NADH production by the TCA cycle is impaired in mFrozen (Fig 2A and B). There were no significa
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