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

Summary Depending on metabolic requirements, skeletal muscle mitochondria integrate O2 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-β-oxidation coupled with O2 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).


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: 30 0 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 À20 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 C and B at 30 C to ensure proper mitochondria isolation and coupling.
CRITICAL: Adjust all stock solution pH to 7.4. Note: KCN (final concentration: 10 mM), can also be used to inhibit ETC-Complex IV. However, it should be noted that pyruvate and high oxygen concentrations may revert KCN inhibition (even at concentrations as high as 1 mM).
e. Oligomycin, inhibitor of the H + -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).
Aliquot and store at À20 C. Dilute 1:100 with milli-Q H 2 O just before use. b. 100 mM antimycin A (MW 534,6 g/mol): 5.34 mg in 1 mL EtOH to obtain the 10 mM stock.
Aliquot and store at À20 C. Dilute 1:100 with milli-Q H 2 O just before use. c. 500 mM oligomycin (MW 791 g/mol): 19.8 mg in 1 mL EtOH to obtain the 25 mM stock. Aliquot and store at À20 C. Dilute 1:50 with milli-Q H 2 O just before use. d. 500 mM mM FCCP (MW 254,16 g/mol): 6.35 mg 1 mL EtOH to obtain the 25 mM stock. Aliquot and store at À20 C. Dilute 1:500 with milli-Q H 2 O just before use.
CRITICAL: EtOH stocks may be stored at À20 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.    Storage at À20 C until use. Use at 30 C.

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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
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 13 PBS 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.

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). 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.
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.

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Note: the double centrifugation is required to improve purity of isolated mitochondria.
h. Centrifuge supernatant for 10 min at 10,000 g at 4 C. i. Eliminate supernatant, and suspend mitochondria enriched pellet in 100 mL of buffer B. j. Measure mitochondria protein by Bradford method in a spectrophotometer. A summary for steps a-j is provided in Figure 6.
Note: This and other protocols require coupled mitochondria (Nuevo-Tapioles et al., 2020). When coupling is not required (ETC enzymatic activities, blue native, in-gel activities among others protocols (Nuevo-Tapioles et al., 2020)) h and i steps may be repeated once or twice to enhance mitochondrial purity.
CRITICAL: Buffer B may interfere with protein measurement. Ensure to use buffer B for calibration curve.
CRITICAL: Make sure to maintain mitochondria on ice all the time to avoid uncoupling.

OCR measurement by clark-type electrode
Timing: 1-2 h Measurement of oxygen consumption rate (OCR) in isolated mitochondria from mouse soleus in a Clark-type electrode (Sanchez-Gonzalez et al., 2020). 5. Add 500 mL of respiration buffer to the electrode working chamber.
Note: the electrode chamber volume may vary between Clark-type electrodes and proper volume should be calibrated to each individual instrument.
6. Add 30-100 mg of mitochondrial protein to the chamber. 7. Close chamber (Figure 7) 8. Click GO to start recording the trace. 9. At 30 s inject one substrate and add a label (repeat it in each injection). Note: All injections should be performed with a Hamilton syringe to avoid opening the working chamber. a. 0.5 mM malate + 50 mM palmitoyl-carnitine: 5 mL from a 50 mM malate stock + 5 mL from a 5 mM palmitoyl-carnitine stock. b. 10 mM glutamate/malate: 10 mL from a 500 mM glutamate/malate stock. c. 10 mM succinate: 10 mL from a 500 mM succinate stock CRITICAL: using substrates in c. add 1 mM rotenone (5 mL from a 100 mM rotenone stock) before substrate injection to avoid retrograde electron transfer (RET) (Scialo et al., 2016). Alternatively, following ADP addition, pyruvate (final concentration: 25 mM) may be added to allow a measurement of flux control ratio of fatty acids relative to pyruvate (see (Horscroft et al., 2019)), followed by rotenone to consider RET. Step 2: soleus extraction and weight.
Step 3: soleus wash with cold and sterile PBS.
Step 4: mince soleus in buffer A.
Step 7: pellet nuclear and membrane rests.
Step 10: resuspend mitochondria in buffer B.

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Note: Allow sufficient time to permit the calculation of slopes in between injections. The time indicated here is flexible.
CRITICAL: Consider a 10% of initial oxygen concentration limiting for proper measurement. Ensure to stop the experiment before oxygen becomes limiting.

EXPECTED OUTCOMES
Expected control mitochondria trace using the Oxygraph+ system is presented in Figure 9.

QUANTIFICATION AND STATISTICAL ANALYSIS
Adapted from (Nicholls, 2013) and (Gnaiger, 2020 Note: A control-step includes the inhibition of ETC complexes to correct total O 2 uptake for residual O 2 consumption (Rox) (Gnaiger, 2020).
Note: State III respiration corrected for Leak (P-L) is potentially available for net coupled phosphorylation of ADP to ATP (Gnaiger, 2020  coupling mitochondria. For Skm mitochondria, RCI for glutamate/malate should be > 4 in healthy controls (usually $5-6). 20. Phosphate/Oxygen Ratio (P/O) for malate + palmitoyl-carnitine (trace a in Figure 9) is calculated by dividing the generated ATP moles (= moles of ADP injected at step 10) per moles of consumed oxygen (O) (from the injection of ADP at step 10 until reaching the state IV). This value is representative of coupling ETC O 2 consumption with FFA-b-oxidation.
Note: P/O ratios of $2.5 and $1.5 with NADH-linked substrates or succinate, respectively, are expected. CRITICAL: This value should be compared with P/O ratio for succinate (trace c in Figure 9) to discard late ETC dysfunctions in the sample mitochondria compared to control mitochondria.
Optional: the day after the experiment, enzymatic activities of complexes I, II, III and IV (Santacatterina et al., 2016;Spinazzi et al., 2012) may be performed to discard single ETC complexes dysfunctions in sample mitochondria compared to control mitochondria.
Note: H + -ATP synthase dysfunctions may result in alterations in DJm (Formentini et al., 2012) 22. Maximal Respiration is calculating comparing FCCP-induced respiration with state 1 respiration 23. Statistical analyses are performed using a two-tailed Student's t test. ANOVA and the Tukey's post hoc test is used for multiple comparisons, employing SPSS 17.0 and GraphPad Prism7 software packages. Bonferroni correction may be applied to avoid multiple comparison errors. p<0.05 is considered statistically significant.

LIMITATIONS
At least one Clark-type electrode is required. However, note that Oroboros Oxygraph-2k may also be used to measure oxygen consumption.
Uncoupled mitochondria (or stored mitochondria preparations) do not work in Clark-type electrode, thus same-day mitochondria isolation and oxygen consumption assessment is mandatory.
CRITICAL: mitochondria tend to uncouple over time: use them immediately after isolation.
Coupled mitochondria may be difficult to obtain due to several limitations: a. WAT deposits in soleus muscle (or any tissue) decrease mitochondrial coupling and increase proton leak (see Troubleshooting section). b. Extra force or potter passes in the extraction decrease mitochondrial coupling (see Trouble-shooting section). c. Temperature is a critical feature of the protocol: mitochondria isolation must be performed on ice, pre-cooling all the material at 4 C, including buffers, centrifuges and potters.
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.
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 (Benador et al., 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.

Problem 4
Mitochondria do not respond properly to substrates/inhibitors after few traces (related to '' Step-bystep method details-OCR 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.

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)

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Consider increasing the concentration of mitochondrial protein in the chamber. Mitochondrial activity decreases with aging (Kauppila et al., 2017) or pathologies (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 (lformentini@cbm.csic.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.