An optimized protocol for coupling oxygen consumption rates with β-oxidation in isolated mitochondria from mouse soleus

Protocol An optimized protocol for coupling oxygen consumption rates with b-oxidation in isolated mitochondria from mouse soleus Depending on metabolic requirements, skeletal muscle mitochondria integrate O 2 consumption and ATP production with lipid, glucose, or amino acid metabolism. Free fatty acids (FFAs) are the main source of energy during rest and mild-intensity exercise. We present a detailed protocol for measuring FFA-b-oxidation coupled with O 2 respiration by a Clark-type electrode in isolated mitochondria from mouse soleus oxidative muscle. We optimized the procedure, including buffer composition, protease treatment, and quantifiable parameters (P/O, OCR, RCR, OSR). Cristina Sa´nchez-Gonza´lez, Laura Formentini cristina.sanchez@cbm. csic.es (C.S.-G.) [email protected] (L.F.) Highlights Nagarse disaggregates muscle fibers facilitating mitochondrial isolation Mitochondrial oxygen consumption is coupled with palmitoyl-carnitine b-oxidation Phosphate/oxygen (P/O) and other ratios (OCR, RCR, OSR) may be easily calculated This protocol uses isolated mitochondria from soleus but can be adapted to any muscle Sa´nchez-Gonza´lez& Formentini, STAR Protocols 2, 100735 September 17, 2021 ª2021 The Author(s). https://doi.org/10.1016/ j.xpro.2021.100735 ll OPEN ACCESS Protocol An optimized protocol for coupling oxygen consumption rates with b-oxidation in isolated mitochondria from mouse soleus Cristina Sa ´nchez-Gonza ´lez 1,4, *and Laura Formentini 1,2,3,5, * 1 Departamento de Biologı ´a Molecular, Centro de Biologı ´a Molecular ‘‘Severo Ochoa’’ (CBMSO), Universidad Auto ´noma de Madrid, c/ Nicolas Cabrera 1, 28049, Madrid, Spain 2 Centro de Investigacio ´n Biome ´dica en red de Enfermedades Raras (CIBERER), ISCIII, Madrid, Spain 3 Instituto de Investigacio ´n Hospital 12 de Octubre, i+12, Universidad Auto ´noma de Madrid, Madrid, Spain 4 Technical contact 5 Lead contact *Correspondence: [email protected]sic.es (C.S.-G.), [email protected].es (L.F.) https://doi.org/10.1016/j.xpro.2021.100735 SUMMARY Depending on metabolic requirements, skeletal muscle mitochondria integrate O 2 consumption and ATP production with lipid, glucose, or amino acid metabolism. Free fatty acids (FFAs) are the main source of energy during rest and mild-intensity exercise. We present a detailed protocol for measuring FFAb-oxidation coupled with O 2 respiration by a Clark-type electrode in isolated mitochondria from mouse soleus oxidative muscle. We optimized the procedure, including buffer composition, protease treatment, and quantifiable parameters (P/O, Phosphate/Oxygen Ratio; OCR, Oxygen Consumption Rate; RCR,Respiration Control Rate; OSR, Oligomycin Sensitive Respiration). For complete details on the use and execution of this protocol, please refer to Sanchez-Gonzalez et al. (2020). BEFORE YOU BEGIN Note: All animal experiments were approved by the Spanish Animal Experiments Committee (PROEX 183/17) in compliance with the European Community Council Directive Guidelines (EU directive 86/609) and ARRIVE Guidelines. All procedures were performed ensuring minimal discomfort and distress to animals. Skeletal muscle mitochondria pre-isolation preparation Timing: 300 1. Prepare a sufficient amount of extraction (A) and respiration (B) buffers, following the recipe (see below). CRITICAL: A and B buffers can be stored at 20C for up to 1 month. 2. Thaw buffer A on ice and B at 30C prior to usage. CRITICAL: Buffer A should be used at 4CandBat30 C to ensure proper mitochondria isolation and coupling. STAR Protocols 2, 100735, September 17, 2021 ª2021 The Author(s). This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/). 1 ll OPEN ACCESS 3. Prepare Digestion buffer (C). Weigh Nagarse (0.2 mg/mL in buffer A (see below) to make buffer C; itcanbestoredat80C for up to 6 months with negligible loss of activity). Note: Nagarse ensures mitochondrial purity, reducing the amounts of endoplasmic vesicles and reticulum (Contreras et al., 2007). Other mild alkaline proteases work similarly. Exact Units of protease/mL should be set up for every muscle. 4. Pre-cool 13PBS, beakers, tubes, and surgical material for soleus extraction. Note: If possible, work inside a laminar flow cabinet to reduce contaminations during soleus dissection. CRITICAL: Use sterilized surgical material by spraying with 70% ethanol (EtOH) 5. Make sure that all of the equipment works well and the centrifuges have been pre-chilled at 4C. CRITICAL: Make sure that no detergent residues are present on potter and glass material. Detergents may uncouple mitochondria. 6. Prepare a sterilized working space, spraying surgical area with 70% EtOH Clark-type electrode preparation Timing: 1–1.5 h 7. Prepare stock solutions for all mitochondria substrates. Skm mitochondria may be energized using substrates: a. glutamate-malate or any NADH-linked substrates of the NADH:ubiquinone oxidoreductase (allow the entrance of electrons from the ETC-Complex I). Pyruvate-malate are the most commonly used alternative. Final concentration: 10 mM b. succinate, natural substrate of the Succinate Dehydrogenase (allow the entrance of electrons from the ETC-Complex II). Final concentration: 10 mM c. malate + palmitoyl-carnitine, coupling oxygen consumption with b-oxidation (Nicholls, 2013). Final concentration: 0.5 mM malate; 0.05 mM palmitoyl-carnitine CRITICAL: Higher concentrations of malate may support NADH-linked electron flow via ETC-complex I. Note: Octanoyl-carnitine or any other FFA-carnitine may be used. Note: TMPD/ascorbate may be used as non-natural substrate of the ETC-Complex IV. Final concentration: 2 mM TMPD; 10 mM ascorbate. Stocks (503) are prepared as follows: a. 1 M malate (MW 134.09 g/mol): 134 mg in 1 mL milli-Q H 2 O. b. 1 M glutamate (MW187.13g/mol):187mgin1mLmilli-QH 2 O. Mixed with malate at 1:1 (v:v) to obtain the 500 mM glutamate/malate stock. c. 500 mM succinate (MW118.09g/mol):59.05mgin1mLmilli-QH 2 O. d. 50 mM malate: dilute 1:20 the 1 M malate stock with milli-Q H 2 O. e. 5 mM palmitoyl-carnitine (MW 436.07 g/mol): 2.18 mg in in 1 mL milli-Q H 2 O. f. 100 mM ADP (MW 427.2): 42.72 mg in 1 mL milli-Q H 2 O. g. 25 mM ADP: dilute 1:4 the 100 mM ADP stock with milli-Q H 2 O. ll OPEN ACCESS 2STAR Protocols 2, 100735, September 17, 2021 Protocol CRITICAL: Adjust all stock solution pH to 7.4. 8. Prepare stock solutions for all mitochondria inhibitors. Skm oxidative phosphorylation (OXPHOS) may be inhibited by using: a. Rotenone or piericidin A, inhibitors of the ETC-Complex I (final concentration: 1 mM) b. Malonate, inhibitor of the ETC-Complex II (final concentration: 100 mM) c. Antimycin A, inhibitor of the ETC-Complex III (final concentration: 1 mM) d. Sodium Azide (NaN 3 ), inhibitor of the ETC-Complex IV (final concentration 100 mM) Note: KCN (final concentration: 10 mM), can also be used to inhibit ETC-Complex IV. However, it should be noted that pyruvate and highoxygenconcentrations may revert KCN inhibition (even at concentrations as high as 1 mM). e. Oligomycin,inhibitoroftheH + -ATP synthase (final concentration: 5 mM) Prepare stock solutions for other compounds: f. FCCP, an ionophore that uncouples ETC electron flow and O 2 consumption from ATP production (final concentration: 0.5 mM) Note: FCCP (or CCCP as an alternative) allows the measurement of maximum ETC electron flow capacity that is not limited by the ATP synthase activity (Gnaiger, 2020). FCCP should be titrated in in 0.25–0.5 mM steps until no further increase in oxygen consumption is observed, as excessive FCCP quickly collapses the proton gradient across the inner mitochondrial membrane, leading to a reduction in measured oxygen flux (Brennan et al., 2006). Stocks (1003) are prepared as follows: a. 100 mMrotenone (MW394,41g/mol):3.94mgin1mLEtOHtoobtainthe10mMstock. Aliquot and store at 20C. Dilute 1:100 with milli-Q H 2 O just before use. b. 100 mMantimycin A (MW 534,6 g/mol): 5.34 mg in 1 mL EtOH to obtain the 10 mM stock. Aliquot and store at 20C. Dilute 1:100 with milli-Q H 2 O just before use. c. 500 mMoligomycin (MW791g/mol):19.8mgin1mLEtOHtoobtainthe25mMstock.Aliquot and store at 20C. Dilute 1:50 with milli-Q H 2 O just before use. d. 500 mMmMFCCP (MW254,16g/mol):6.35mg1mLEtOHtoobtainthe25mMstock.Aliquot and store at 20C. Dilute 1:500 with milli-Q H 2 O just before use. CRITICAL: EtOH stocks may be stored at 20C up to 1 month. Inhibitor final stocks need to be prepared freshly the day of the experiment and stored on ice, protected from light. Figure 1. Scheme of the electrode (platinum cathode and silver anode) ll OPEN ACCESS STAR Protocols 2, 100735, September 17, 2021 3 Protocol CRITICAL: Make sure to adjust pH to 7.4 in all reagent solutions and maintain all reagents on ice. 9. Set the electrode-bath temperature to 30C to ensure constant temperature during measurement Note: Remember that in conditions of O 2 saturation, at 30C, oxygen (O) concentration in liquid media is 445 nmol/mL 10. Set up the Clark type electrode (Figure 1) by placing 1 drop of saturated KCl on the top of the platinum electrode and immediately covering it by Teflon membrane, ensuring no bubble is forming. 11. Immediately place milli-Q water in the electrode working chamber. CRITICAL: Teflon membrane MUST be maintained wet for all the time of the experiment. 12. Turn on the Clark-type electrode (Oxygraph+, Hansatech-instruments, Figure 2). a. Turn on the electrode b. Turn on the computer c. Run ‘‘Oxygraph+’’ (or similar) Software. Figure 3. Oxygraph+ calibration interface Figure 2. Clark-type electrode Scheme (left) and apparatus (right). ll OPEN ACCESS 4STAR Protocols 2, 100735, September 17, 2021 Protocol d. Turn on agitation (Stirrer ON) to ensure homogenous concentration of O 2 in the working chamber. 13. Calibrate electrode with dithionite to set 0% oxygen consumption. a. Click Liquid phase calibration:T30 C; Stirrer 70 rpms; click OK (Figure 3) b. Start recording the trace. c. Add a pinch of dithionite to set 0% of O 2 . Click OK. d. The trace will drop until a plateau. e. Save new calibration. 14. Wash twice the working chamber with 2 mL milli-Q H 2 O, 2 mL EtOH, 2 mL milli-Q H 2 O, to carefully remove all dithionite residues. KEY RESOURCES TABLE REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Anti-Calreticulin (dilution 1:1000) Abcam Cat#ab92516 Anti-NDUFA9 (dilution 1:1000) Abcam Cat#ab14713 Anti-a-Tubulin (dilution 1:1000) Sigma-Aldrich Cat#T5168 Biological samples Healthy oxidative soleus muscle Mus musculus - Chemicals, peptides, and recombinant proteins Sucrose Sigma-Aldrich Cat#84100 Tris-HCl Sigma-Aldrich Cat#10812846001 EDTA Sigma-Aldrich Cat#ED2P KH2PO4 Sigma-Aldrich Cat#P0662 K2HPO4 Sigma-Aldrich Cat#60353 KCl Merck Cat#104936 MgCl2 Sigma-Aldrich Cat#M8266 BSA Nzytech Cat#MB04602 EGTA Sigma-Aldrich Cat# 324626 Nagarse Sigma-Aldrich Cat# P4789 discontinued Bradford (Bio-Rad Protein Assay) Bio-Rad Cat#5000006 Glutamate Sigma-Aldrich Cat#49621 Malate Sigma-Aldrich Cat#2300 Sodium succinate Sigma-Aldrich Cat#S7501 Palmitoyl-carnitine Sigma-Aldrich Cat#P1645 TMPD Sigma-Aldrich Cat# T7394 Sodium ascorbate Sigma-Aldrich Cat#A7631 Rotenone Sigma-Aldrich Cat#R8875 Piericidin A Sigma-Aldrich Cat# 96861 Malonate Sigma-Aldrich Cat# 63409 Antimycin A Sigma-Aldrich Cat#A8474 Sodium azide (NaN3) Sigma-Aldrich Cat#S2002 KCN Sigma-Aldrich Cat# 207810 Oligomycin Sigma-Aldrich Cat#O4876 ADP Sigma-Aldrich Cat#A2754 FCCP Sigma-Aldrich Cat#C2920 Dithionite Merck Cat#1065070500 EtOH Merck Cat#51976 Experimental models: Organisms/strains Mus musculus: C57BL/6 6 month-old mice (males) The Jackson Laboratories Cat#MGI:5656552 (Continued on next page) ll OPEN ACCESS STAR Protocols 2, 100735, September 17, 2021 5 Protocol MATERIALS AND EQUIPMENT Buffers Optional: Nagarse Buffer (C) Continued REAGENT or RESOURCE SOURCE IDENTIFIER Software and algorithms Oxygraph+ Software Hansatech Instruments - GraphPad Prism 7 1992-2016 GraphPad Software, Inc - Other Oxygraph+ (Clark-type electrode) Hansatech Instruments http://www.hansatech-instruments. com/product/oxygraph-system/ Hamilton syringe (25 and 50 mL) Sigma-Aldrich Cat#21492 Cat#24544 KIMBLE Dounce tissue grinder set (Glass to glass homogenizer) Sigma-Aldrich Cat#D9063-1SET Centrifuge Eppendorf Cat#5418 R Moria Iris Forceps FST Cat#11370-31 Moria Iris Forceps FST Cat#11370-32 Halsted-Mosquito Hemostats FST Cat#13008-12 Vannas Spring Scissors FST Cat#15000-00 Extra Fine Bonn Scissors FST Cat#14084-08 Surgical Scissors FST Cat#14001-12 25G needles Sterican 100 Braun Cat#4657853 Extraction Buffer (A) Reagent Final concentration Amount Sucrose 0.32M 11 g Tris-HCl 10 mM 121 mg EDTA 1M 1 mM 100 mL H 2 O milli-Q n/a up to 100 mL Total n/a 100 mL Storage at 20C until use. Use at 4C. Respiration Buffer (B) Reagent Final concentration Amount Sucrose 225 mM 7.7 g KCl 10 mM 74 mg MgCl 2 5 mM 47.6 mg HK 2 PO 4 10 mM 134 mg H 2 KPO 4 10 mM 30 mg EGTA 1 mM 38 mg Tris HCl 10 mM 121 mg BSA 0.05% 50 mg H 2 O milli-Q n/a up to 100 mL Total n/a 100 mL Storage at 20C until use. Use at 30C. ll OPEN ACCESS 6STAR Protocols 2, 100735, September 17, 2021 Protocol Note: A and B buffers were published in (Formentini et al., 2014 ) CRITICAL: Adjust pH to 7.4 in all buffers. STEP-BY-STEP METHOD DETAILS Skeletal muscle mitochondria isolation Timing: [1 h] Isolation of soleus oxidative skeletal muscle mitochondria by centrifugation steps (Sanchez-Gonzalez et al., 2020). 1. Sacrifice mice in CO 2 chamber. 2. Extract soleus muscles from mice hindlimbs using pre-cooled and sterilized surgical material inside a laminar flow cabinet to reduce impurities. Note: To extract soleus, fix the mouse leg in a flexed position to the dissecting table and cover it with cold 13PBS solution. Remove hair, skin and surrounding fascia. Cut Achilles tendon. Separate the soleus muscle from the hindlimb and clean from leftover fascia. (Figure 4, adapted from (Shinin et al., 2009)) CRITICAL: Remove as much fascia as possible, as remaining fascia will complicate the isolation of the entire muscle. Note: To identify soleus, pay attention to muscle color. Being deeply oxidative and enriched in mitochondria, soleus red color is darker than surrounding muscle. Carefully cut the tendon as close as possible to the knee and separate soleus. CRITICAL: Soleus must be extracted with no white adipose tissue (WAT) deposits to ensure coupling of isolated mitochondria. 3. Weigh soleus. Note: Depending on the age of the animals, 2 or more solei are needed to get enough mitochondria for measuring respiration. This also depends on the volume of the chamber and the sensitivity of the Clark-type electrode. This protocol has been optimized for 4 solei (a pool of 2 mice/preparation). Note: Mitochondrial function can be also analyzed in situ in permeabilized mouse soleus fiber bundles (Kuznetsov et al., 2008), allowing 2 respirometer runs per soleus, although the method does not allow the measurement of all the parameters reported in this protocol (see below). Reagent Final concentration Amount Sucrose 0.32M 11 g Tris-HCl 10 mM 121 mg EDTA 1M 1 mM 100 mL Nagarse 0.2 mg/mL 20 mg H 2 O milli-Q n/a up to 100 mL Total n/a 100 mL Storage at 20C until use. ll OPEN ACCESS STAR Protocols 2, 100735, September 17, 2021 7 Protocol 4. For isolating mitochondria (on ice). Adapted from (Fernandez-Vizarra et al., 2010): a. Wash soleus in cold PBS 13in a pre-cooled 50 mL glass beaker. b. Mince muscles in four volumes (depending on muscle weight) of buffer A until pieces are homogeneous with the help of pre-cooled surgical scissors. Optional: Nagarse or other smooth proteases increase mitochondrial purity, reducing the amounts of other organelles. Step b may be performed in buffer C. Incubate 5–10 min on ice to allow Nagarse to act. CRITICAL: excessive time in Nagarse added buffer may disrupt mitochondria. Note: Mitochondria purity may be verified by WB using specific antibodies against mitochondrial complexes (anti-NDUFA9), cytosolic (anti-tubulin) or other organelle (anti-calreticulin) proteins. c. Homogenize in a glass-glass homogenizer (Figure 5): - 10 times with potter A (smoother). - 15 times with potter B (stronger). CRITICAL: It is really important not to pass more times with the potters than necessary. Mitochondria may uncouple if the homogenization step is too strong. Exact conditions of homogenization should be optimized for any potter. d. Immediately transfer the homogenized suspension to previously pre-cooled centrifuge tubes. e. Centrifuge 10 min at 700 g at 4C. f. Discard pellet, contains nucleus and intact cells. g. Repeat eand f steps once. Figure 4. Scheme of soleus extraction 1. Lateral view of the anatomy of mouse hindlimb. 2.Extraction of soleus: remove fascia, insert fine-tip forceps between distal tendons, liberate Achilles tendon from tibial bone, liberate soleus from proximal tendons and extract soleus. 3: Immediately transfer soleus to cold PBS. ll OPEN ACCESS 8STAR Protocols 2, 100735, September 17, 2021 Protocol Clark-type electrode experiments must be performed at constant temperature because O 2 saturation level in media depend on temperature (see above). Electrode needs to be properly calibrated prior to OCR measurements. Substrates and inhibitors might be degraded in freeze/thawing cycles; thus, it is strongly recommended to use compounds freshly prepared. TROUBLESHOOTING Problem 1 How to be sure that electrode is working properly after the manual set up (related to ‘‘Before you begin-Clark-type Electrode preparation’’ section, steps 10–13). Potential solution Verification: Start recording. For Oxygraph+ program: Click GO. Wait for the trace to start. Stop: click Stirrer OFF. Wait for the trace to go down. Click Stirrer ON. Wait for the trace to restore initial level. Note: If no decrease in O 2 (or only a slight decrease) is observed, the electrode does not work properly. Solution: Dismantle the electrode. Clean the electrode with H 2 O milli-Q water. Add a drop of a saturated KCl solution in the platinum electrode. Put a new Teflon membrane. Put correctly the O ring (Figure 3, left panel) adjusting the membrane and avoiding bubbles. Problem 2 How to check if mitochondria are uncoupled (related to ‘‘Step-by-step method details-Skeletal muscle mitochondria isolation’’ section, step 4). Potential solution This can be checked respirometrically by the addition of exogenous cytochrome c (cyt c, 10 mM) to mitochondrial preparations. If mitochondria have become damaged or uncoupled during the preparation step, the addition of cyt c will increase the oxygen flux highlighting the damage of the outer membrane and loss of the endogenous cyt c. See: (Pesta and Gnaiger, 2012) Problem 3 Mitochondria are uncoupled (low RCR) (related to ‘‘Step-by-step method details-Skeletal muscle mitochondria isolation’’ section, step 4). Potential solution A wrong buffer composition/storage may affect mitochondria coupling. Re-do all the buffers following right indications. ll OPEN ACCESS STAR Protocols 2, 100735, September 17, 2021 15 Protocol A too strong process of purification may uncouple mitochondria. Change to milder homogenization. Homogenize in a glass-glass homogenizer: 8 times with potter A (smoother). 8 times with potter B (stronger). Note: In certain cases, it could be recommended to perform only 1 centrifugation for nuclei separation and 1 for mitochondria isolation. Reducing the number of centrifugations reduces the purity of the preparation but increases mitochondrial coupling. Although fundamental interaction between lipid-storages and mitochondria has been recently described (Benadoretal.,2019), the presence of excessive intramuscular adipocyte accumulation may result in excess of lipids during the isolation, thus altering permeability and uncoupling mitochondria (Rial et al., 1983). Carefully eliminate lipid phase with the help of a cotton swab after the first centrifugation (step 4f) to reduce lipid amount into the preparation. Increase the percentage of BSA in the buffer B. Note: BSA will bind lipids reducing their concentration. Obesity and aging decrease RCR, increase mitochondrial proton leaks and alter mitochondrial inner membrane lipid composition (Cadenas, 2018;Formentini et al., 2017a;Sanchez-Gonzalez et al., 2020). Make sure that control animals are lean and young (2–8 months old). Problem 4 Mitochondria do not respond properly to substrates/inhibitors after few traces (related to ‘‘Step-bystep method detailsOCR measurement by Clark-type Electrode’’ section, steps 5–16). Potential solution This could be due to the presence of traces of inhibitors or impurities in the working chamber (bad or difficult cleaning). Clean the electrode working chamber with 2 mL milli-Q H 2 O,2 mL EtOH, 2 mL milli-Q H 2 O. Add 2 mL of PBS + 1% BSA to the working chamber for enhancing the clean effectiveness. Impurities and inhibitors will bind BSA. Repeat steps 1 and 2 twice. Note: increasing the time of the washes also helps in removing inhibitors. Wash the chamber for at least 15 min in 100% EtOH between runs. Problem 5 Electrode is not sensitive (slow slops, instable traces) (related to ‘‘Step-by-step method detailsOCR measurement by Clark-type Electrode’’ section, steps 5–16) Potential solution A well-known cause of failures of oxygen sensors is the appearance of gas bubbles. The unequal rates of the heating of the measuring system’s components are the most probable (but not unique) reason of the diffusive flow of oxygen through the membrane of the sensor: Make sure heating system works properly and constant temperature is maintained Adjust stirring and avoid any vortex, which can change the reading by adding oxygen from the ambient air. Eliminate gas bubbles in the proximity of the Teflon membrane (Figure 2) ll OPEN ACCESS 16 STAR Protocols 2, 100735, September 17, 2021 Protocol Consider increasing the concentration of mitochondrial protein in the chamber. Mitochondrial activity decreases with aging (Kauppila et al., 2017)orpathologies(Balaban et al., 2005;Dillin et al., 2002;Formentini et al., 2012;Formentini et al., 2017b;Nuevo-Tapioles et al., 2020) RESOURCE AVAILABILITY Lead contact Further information and requests for resources and reagents should be directed to and will be fulfilled by the lead contact, Laura Formentini ([email protected]ic.es). Materials availability This study does not need any new reagents. Data and code availability This study does not generate/analyze data sets or code. ACKNOWLEDGMENTS We acknowledge Prof. J.M. Cuezva from CBMSO-UAM for advice. We thank The CBMSO Animal House Facility. This work was supported by grants from Ministerio de Economı ´a, Industria y Competitividad, MINECO, Spain (PID2019-104241RB-I00). S.-G.C. is supported by MINECO (BES-2017079909). 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