Preparation and Respirometric Assessment of Mitochondria Isolated from Skeletal Muscle Tissue Obtained by Percutaneous Needle Biopsy

NOTE: The protocol described was approved by the Institutional Review Board of Wake Forest School of Medicine. Informed consent was obtained in writing. All participants were healthy older adults (65-79 years) of both genders, with BMIs ranging from 23-35.

1. Skeletal Muscle Biopsy

1. As previously described, ⁹ perform all biopsies in the early morning after an O/N fast. Ask the subjects to refrain from taking aspirin, prescription and over-the-counter non-steroidal anti-inflammatory drugs, or other compounds that may affect bleeding, platelets, or bruising for the week prior to the biopsy. Ask the participants to also refrain from any strenuous activity for at least 36 hr prior to the biopsy.
   NOTE: Muscle is obtained from the Vastus lateralis using the percutaneous needle biopsy technique with a percutaneous needle under local anesthesia with 1% lidocaine. No medical complications or other reported adverse events from the procedure have occurred in our clinical research unit.
2. NOTE: The biopsy procedure described is adapted from that of Bergstrom¹⁰.
   1. Briefly, locally administer 1% lidocaine taking care not to infiltrate the muscle. Follow this with a 10 min wait period to permit sufficient numbing.
   2. Take the biopsies from the belly of the muscle (the middle region of muscle between insertion and origin) avoiding subfascial and myotendonous areas.
      1. Use percutaneous needle (a suction assisted reusable device with a side cutting window and inner cutting cylinder) and follow a fascial “pop” or resistance of the fascia as a guide. 
      2. Estimate the depth with the anesthesia needle, then again feel it with the narrow scalpel blade and make a 4-5 mm incision through the fascia.  Advance the needle through the incision until it is inserted into the muscle. 
      3. Collect multiple samples with the window turned in different directions. Apply continuous suction using a 60 cc syringe while advancing and withdrawing muscle samples into the percutaneous needle two to four times in different directions. Discontinue the suction and remove the needle.
         NOTE: Each pass (insertion and removal of needle) should take less than a minute.
      4. Have an assistant apply firm pressure to puncture site for 5 min to establish hemostasis. Disconnect needle from suction tubing and carefully remove muscle samples from the window and barrel.
   3. Make a second pass if more muscle is needed by repeating the above procedure.
   4. Remove any visible blood clots from the muscle sample using forceps, weigh the sample, and immediately place in a tube containing ice-cold DPBS.
      NOTE: The average yield using this methodology is 150 ± 20 mg.

2. Mitochondrial Isolation

1. Freshly prepare the Chappel-Perry (CP) and Mitochondrial Assay Solution (MAS) buffers (Table 1) on the day of the experiment, or aliquot and store them at -20 °C. Prepare the compounds in dimethyl sulfoxide at a concentration of 2.5 mM and aliquot and store at -20 °C. Use the buffer and compound aliquots stored at -20 °C within 2 months from the day of preparation.
2. Remove visible connective tissue from the sample using sharp scissors and tweezers; if needed use a dissecting microscope for this step. Thoroughly wash specimens 3-4 times with ice-cold DPBS buffer to remove blood. Keep the samples on ice-cold DPBS and process as soon as possible, taking care not to exceed more than 45 min from biopsy. Take precaution to carefully remove any tendons or adipose tissue from the muscle sample.
3. Immediately chop the muscle tissue into fine pieces using a sterile pair of scissors and suspend in 500 μl to 1 ml CPI containing proteinase (Nagarse) at a concentration of 0.2 mg/g tissue. Follow this with a 5 min incubation at RT and then transfer to ice.
4. Homogenize the minced tissues treated with Nagarse using an automated homogenizer. Keep the sample on ice throughout this process. Homogenize each tissue sample four times, each time for a pulse of 2 sec, using the automated homogenizer at a speed setting of 10,000 rpm. Wash the probe with 70% ethanol followed by distilled water between tissues.
5. Wash the homogenized tissue with an equal volume of CP I (500 μl to 1 ml) and 2x volume of CP II (1 ml to 2 ml), and collect the content in a centrifuge tube and centrifuge at 600 x g, 4 °C for 10 min. Pass the supernatant through a wetted cheese cloth, collect the filtrate and discard the pellet, thus removing the majority of non-mitochondrial fractions.

3. Washing the Mitochondria

1. Centrifuge the supernatant from the above step at 10,000 x g, 4 °C for 10 min. Suspend the pellet in 4 ml of CP II buffer and further centrifuge at 10,000 x g, 4 °C for 10 min.
   NOTE: On rare occasions, a thin pellet of blood is formed below the mitochondrial pellet. In that case, remove the mitochondrial pellet by gentle aspiration with CPII buffer. This step can be avoided by thoroughly washing away blood at step 2.
2. Suspend the pellet obtained in 2ml of CP I buffer. At this stage use a small aliquot from this suspension for protein estimation. Resuspend the remaining sample in CP I buffer and centrifuge as above. Suspend the final pellet in a minimal amount (200 μl) of Mitochondrial Assay Solution (MAS).
   NOTE: Protein is assayed at this stage because CP I buffer does not have BSA, whereas MAS in which the samples will be later suspended does have BSA in it.

4. Estimate the Mitochondrial Content by Measuring Protein Concentration Using a BCA Protein Assay Kit

NOTE: Use this concentration to calculate the quantity of mitochondria used for loading onto a 24-well microplate for respirometric measurements, or for Western blot experiments. Take into account the dilution factor (10) for calculation of the protein concentration.

5. Perform the XF assays as described by Rogers, GW, et al.¹²

NOTE: Visualize the O₂ consumption rate (OCR) in pMoles O₂/min, or absolute levels of O₂ and pH in the data output.

1. Use 1x MAS to prepare compounds to be injected. Add a 10x concentration of the compounds to the ports A-D to give a final concentration as follows: Port A, ADP [Adenosine 5’ –diphosphate, 2 mM, 50 μl]; port B, oligomycin (2 μM, 55 μl); port C, FCCP [carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone, 6 µM, 60 μl]; and port D, 2 μM antimycin-A (65 μl). Prepare sufficient volume of compounds for the required number of wells.
2. Determine the optimum amount of mitochondria by titration. For example, load 1.0 μg, 2.5 μg, and 5.0 μg of mitochondria per well in a 50 μl volume of ice-cold 1x MAS containing substrate. To minimize variability between wells, first dilute 10x mitochondria in cold 1x MAS + substrate. Next, deliver 50 μl of this suspension to each well (except background correction wells).
3. Centrifuge the plate at 2,000 x g, 20 min at 4 °C. After centrifugation, gently add 450 μl of 1x MAS + succinate (10 mM) and rotenone (2 μM) (initial conditions; see below) to each well. View the mitochondria under the microscope to ensure homogenous adherence to the well prior to transferring the plate to the XF analyzer.
   NOTE: The initial conditions used will depend on whether the respiration is driven by either ETC complex I or complex II. For complex II-driven respiration, use succinate (10 mM) and rotenone (2 μM) as initial conditions. Rotenone blocks complex I and succinate provides fuel for complex II (succinate dehydrogenase). To study respiration driven by complex I, use either pyruvate and malate, each at a final concentration of 5 mM or glutamate and malate each at a final concentration of 10 mM as initial conditions. The latter can help in distinguishing dysfunction among tricarboxylic acid cycle and substrate transport. To study respiration driven by both complex I and complex II, include pyruvate and succinate without rotenone or malate as initial conditions. Use palmitoyl carnitine as a substrate for β-oxidation.
4. Sequentially measure the mitochondrial respiration in real time using the respirometer by programming it as previously described ¹². Use the settings for the respirometer provided in Table 2.
   NOTE: A brief explanation of various mitochondrial respiration states are as follows:
5. State 2 = coupled state with substrate present; State 3 = phosphorylating respiration in the presence of saturating ADP; State 4_o = non-phosphorylating respiration induced by oligomycin; State 3u = maximal uncoupled respiration stimulated by the uncoupler FCCP; Residual non-mitochondrial respiration = respiration after complex III inhibition by antimycin-A. It should also be pointed out that, the length of the OCR measurement in combination with the mitochondrial concentration could influence the data by depleting oxygen during the measurement periods.

6. Western Blot

NOTE: Determine the mitochondrial marker COX IV and whole tissue GAPDH by Western blotting to assure enrichment of mitochondria in the final sample

1. Separate the isolated mitochondria and whole tissue extract by 12% sodium dodecyl sulfate polyacrylamide gels.
2. Transfer the proteins onto a polyvinylidene fluoride (PVDF) membrane.
3. Incubate with COX IV antibody (1:20,000), followed by horseradish peroxidase (HRP)-conjugated secondary antibodies. For GAPDH determination, strip the blot and probe with a GAPDH monoclonal antibody (1:2,000).
   NOTE: If differences in ER contamination are of concern, include ER markers such as ERp72, calnexin, or calreticulin in the analysis.