Isolation of Mitochondria from Minimal Quantities of Mouse Skeletal Muscle for High Throughput Microplate Respiratory Measurements

Summary

Here, we present a modification of a previously reported method that allows for the isolation of high quality and purified mitochondria from smaller quantities of mouse skeletal muscle. This procedure results in highly coupled mitochondria that respire with high function during microplate based respirometirc assays.

Abstract

Dysfunctional skeletal muscle mitochondria play a role in altered metabolism observed with aging, obesity and Type II diabetes. Mitochondrial respirometric assays from isolated mitochondrial preparations allow for the assessment of mitochondrial function, as well as determination of the mechanism(s) of action of drugs and proteins that modulate metabolism. Current isolation procedures often require large quantities of tissue to yield high quality mitochondria necessary for respirometric assays. The methods presented herein describe how high quality purified mitochondria (~ 450 µg) can be isolated from minimal quantities (~75-100 mg) of mouse skeletal muscle for use in high throughput respiratory measurements. We determined that our isolation method yields 92.5± 2.0% intact mitochondria by measuring citrate synthase activity spectrophotometrically. In addition, Western blot analysis in isolated mitochondria resulted in the faint expression of the cytosolic protein, GAPDH, and the robust expression of the mitochondrial protein, COXIV. The absence of a prominent GAPDH band in the isolated mitochondria is indicative of little contamination from non-mitochondrial sources during the isolation procedure. Most importantly, the measurement of O₂ consumption rate with micro-plate based technology and determining the respiratory control ratio (RCR) for coupled respirometric assays shows highly coupled (RCR; >6 for all assays) and functional mitochondria. In conclusion, the addition of a separate mincing step and significantly reducing motor driven homogenization speed of a previously reported method has allowed the isolation of high quality and purified mitochondria from smaller quantities of mouse skeletal muscle that results in highly coupled mitochondria that respire with high function during microplate based respirometirc assays.

Introduction

The primary function of mitochondria is to produce ATP from oxidative phosphorylation. However, mitochondria have many other important cellular functions including but not limited to: the production and detoxification of reactive oxygen species, the regulation of cytoplasmic and mitochondrial calcium, organelle trafficking, ionic homeostasis, and involvement in apoptosis^1,2. Therefore, it is not surprising that dysfunctional mitochondria play a role in many disease pathologies, such as aging, neurodegenerative diseases, cardiovascular disease, cancer, obesity, and diabetes^3,4. Importantly, skeletal muscle mitochondria specifically are involved in many of these pathologies^3-5.

Mitochondrial respiration assays using isolated mitochondria allow for the assessment of electron transport chain and oxidative phosphorylation function, and the determination of mechanism(s) of action of drugs and proteins that modulate metabolism. Mitochondrial isolation procedures exist for multiple tissue and cell types for a variety of species^6,7. However, these procedures often require large quantities of tissue/cells for a high quality mitochondria yield necessary for classic respirometric assays.

Microplate based respirometirc assays allow for high throughput measurements using minimal quantities of isolated mitochondria, often just several µg per well⁸. Therefore, we present a modification of previously published methods⁷ to allow for high quality mitochondria to be isolated from smaller quantities of mouse skeletal muscle for use in microplate based respirometirc assays. In addition, methods are provided to establish the quality of the mitochondrial isolation preparation and the integrity of the mitochondrial membranes. Given that skeletal muscle mitochondria are involved in many pathological conditions, the measurement of O₂ consumption in mechanistically driven studies is becoming more prevalent in biomedical research^9,10.

Protocol

Animal studies were performed under an approved protocol by the Institutional Animal Care and use Committee at Virginia Polytechnic Institute and State University.

1. Setup (Time: ~45 min)

1. Thaw frozen stores of 0.25% Trypsin, Isolation Buffer for Mitochondria (IBM) 1 and IBM2 in a 37 °C water bath.
2. Rinse glassware and dissecting instruments in 70% ethanol followed by high purity water.
3. Prepare 0.05% trypsin solution from 0.25% trypsin stock by diluting 1 part trypsin in 4 parts IBM1.
4. Mix protease and phosphatase inhibitor cocktail with cell lysis buffer (1:100 ratio) in a 1.5 ml microcentrifuge tube with screw cap.
5. Aliquot 5 ml (5 ml/mouse) of 0.05% trypsin into a 15 ml plastic tube.
6. Set motor driven tissue homogenizer to 80 RPM.

2. Isolation of Skeletal Muscle Mitochondria (Time: ~90 min)

1. Euthanize a mouse by CO₂ inhalation, followed by cervical dislocation.
2. Remove red muscle from the quadriceps and gastrocnemius muscle, which includes the soleus (~75-100 mg total) as described in the following steps:
   1. Peel the skin towards the mouse.
   2. Remove the fat pad over the quadriceps origin point. Cut the quadriceps tendon that is attached to the patella with fine tipped scissors.
      Note: The quadriceps is identified by its anatomical position on the anterior portion of the proximal femur.
   3. Slowly snip the aponeurosis between the bone and the quadriceps, while avoiding the femoral artery, to liberate the quadriceps from the bone. Cut the tendon with fine tipped scissors at the origin point on the femur to liberate the quadriceps muscle and place the quadriceps muscle in chilled PBS.
   4. Remove visible adipose tissue over the quadriceps with scissors. Flip the quadriceps over so that the portion of the muscle that was overlying the femur is facing up. Open the quadriceps muscle with forceps in a fanning motion. Remove the two visible red muscle portions from each lobe with fine tipped scissors and place in a beaker containing 5 ml of chilled IBM1.
      Note: Quadriceps muscle appear as two lobes with a strip of red muscle located near the lateral edge of each lobe.
   5. Cut the skin overlying the Achilles tendon with fine tipped scissors and peel the skin toward the mouse. Cut the exposed Achilles tendon and peel the muscle towards the body of the mouse.
   6. Cut the tendon at the lateral and medial condyles of the femur with fine tipped scissors to liberate the gastrocnemius and place the gastrocnemius attached to its tendons in chilled PBS.
   7. Flip the gastrocnemius over and peel off the soleus muscle and place into the beaker containing 5 ml of chilled IBM1. Fan open the gastrocnemius. Remove the three visual red muscle portions (two lateral and one medial superficial red strips) with fine tipped scissors and transfer them to the beaker containing 5 ml of chilled IBM1.
3. Remove another ~75-100 mg of red muscle from the other leg of the mouse (as described in step 2.2) and place into a 1.5 ml microcentrifuge tube with protease and phosphatase inhibitor cocktail and cell lysis buffer (50 mM Tris-HCL, 1 mM EDTA, 150 mM NaCl, 1% Sodium dodecyl sulfate, 0.5% Sodium deoxycholate, 1% Polyoxyethylene (9) nonylphenylether, branched. Immediately flash-freeze this second sample in liquid nitrogen for later use in Western blotting (see step 6.1).
4. Place a pre-chilled, flat plastic surface on ice in a large bucket. Pour a drop of IBM1 onto the plastic surface and use tweezers to place all of the sectioned red muscle (step 2.2.4 and 2.2.7) in IBM1 droplet. Mince the muscle tissue for 2 min using 3 single edge razor blades by changing razors every 40 sec.
5. Transfer the minced tissue to a new beaker with 5 ml of fresh IBM1 by holding the plastic surface over the beaker and scraping the muscle into the beaker with a razor blade. Take this solution and drain it through a 100 µm cell strainer placed onto a 50 ml conical tube.
6. Blot the tissue with a delicate task wiper and then transfer it into 5 ml of the 0.05% trypsin solution. Use tweezers to remove the tissue from the task wiper.
7. Incubate the muscle tissue on ice for 30 min in 0.05% trypsin solution.
8. Spin the 15 ml conical tube containing trypsin/muscle mixture at 200 x g for 3 min at 4 °C.
9. Pour the trypsin supernatant into a waste container and resuspend the pellet with 3 ml of ice cold IBM1. Transfer the tissue to a 45 ml glass homogenizer tube. Rinse the 15 ml conical tube with another 1.5 ml IBM1 to gather any remaining sample and add this to the glass homogenizer tube.
10. Place the glass homogenizer tube into a beaker or plastic container filled halfway with ice so the glass homogenizer moves minimally within the beaker.
11. Homogenize the tissue/IMB1 mixture with the PTFE pestle attached to a motor driven tissue homogenizer with 10 passes at 80 rpm. Hold the bottom of each pass for ~2 sec.
12. Transfer the tissue homogenate to a new 15 ml conical tube and rinse the glass homogenizer tube with 6.5 ml of IBM1. Pour the IBM1 into the 15 ml conical tube containing tissue homogenate.
13. Spin the 15 ml conical tube at 700 g for 10 min at 4 °C. Gently pour the supernatant into a glass high strength centrifuge tube and discard the pellet.
14. Spin the supernatant from the above step at 8,000 x g for 10 min at 4 °C.
15. Remove the IBM1 supernatant and re-suspend the pellet by slowly adding 500 µl of IBM2. Cut off the end of a pipette tip and gently homogenize the pellet in IBM2 with mixing and stirring motions. Add another 4.5 ml of IBM2 to the mixture after the pellet is fully suspended.
    Note: Avoid excessive trituration while breaking larger pellet pieces as this may damage the mitochondrial membranes.
16. Spin the IBM2/tissue homogenate at 8,000 x g for 10 min at 4 °C.
    Note: This step may be repeated in a clean high strength centrifuge tube for more accurate protein quantification of isolated mitochondria in Step 4 since residual BSA may contribute to total protein content.
17. Remove the supernatant and gently, but completely re-suspend the pellet by adding two 25 µl increments of IBM2 with a pipette tip with its point cut off. Gently stir and mix the pellet after each addition of 25 µl of IBM2. Place this mitochondrial stock on ice.

3. Homogenization of Whole Tissue Lysate (Time: ~45 min; Can Be Done During Step 7)

1. Remove the muscle from that was placed in the liquid nitrogen from step 2.3 and incubate at room temperature until fully thawed.
2. Mince the tissue for ~30 sec on ice with fine tipped scissors.
3. Add two 100 µl scoops of Zirconium Oxide Beads to the 1.5 ml microcentrifuge tube with screw cap from step 3.2 that contains the minced tissue. Homogenize the tissue in a bead mill tissue homogenizer using two to three 5 min cycles at a speed setting of 4-6 (medium setting).
4. Spin the mixture from step 3.3 at 12,000 x g for 10 min at 4 °C. Remove the supernatant using a 1,000 µl pipette tip after the spin and transfer into a 1.5 ml microcentrifuge tube. Discard the pellet and place the supernatant on ice.

4. Protein Determination (Time: ~30 min)

1. Determine the protein concentration of the whole tissue lysate (step 3.4) and mitochondrial stock (step 2.17) using the BCA Protein assay kit according to the manufacturer’s specifications.

5. Mitochondria Membrane Integrity; Citrate Synthase (CS) Activity (Time: ~15 min)

1. Make two separate 1: 20 dilutions of the mitochondrial stock in high purity water. Add 0.1% of Triton 100-X to one sample and sonicate it at a low setting (765 Watts, amplitude of 2). Leave the other dilution on ice. Return the sample to ice after sonication.
2. Perform a citrate synthase assay with both the non-sonicated and sonicated mitochondrial dilutions using a spectrophotometric assay as previously described^11,12.
3. Determine the percentage of intact mitochondria membranes by using the following equations:
   (Non-sonicated CS activity ÷ Sonicated CS activity)* 100

         100- N (percent attained from above)

6. Mitochondria Isolation Quality; Western Blotting (Time: According to Lab Protocol)

1. Perform Western blotting on the whole tissue lysate and isolated mitochondria as described previously¹².
   Note: Use primary antibodies for GAPDH (1: 1,000 dilution in 5% BSA/TBS-T) and COXIV (1:1,000 dilution in 5% non-fat milk/TBS-T) followed by anti-goat, and rabbit secondary antibodies, respectively (1:10,000).

7. Respirometric Assay Run (Time: ~90 min)

1. Perform desired respirometric assays using microplate based O₂ consumption measuring technology as previously described (see companion article and Rogers et al⁸).
2. Determine the respiratory control ratio (RCR) by dividing State 3 by State 4o (RCR) or dividing State 3u by State 4o. Use either the middle (average) point, or the highest (State 3) and lowest rates (State 4o) from the point-to-point traces when determining RCR.

Results

Citrate synthase activity serves as a measure for membrane integrity since citrate synthase is located in the inner mitochondrial membrane, and thus should not be present in suspensions of mitochondria with intact membranes. Figure 1 represents citrate synthase activity in non-sonicated mitochondrial samples compared with sonicated samples from the same isolation. Sonicating the mitochondria results in a statistically significant increase in citrate synthase activity (P <0.01). Importantly, 92.5 ...

Discussion

The methods presented herein provide a detailed description of a mitochondrial isolation procedure from minimal quantities (~75-100 mg) of mouse skeletal muscle. This isolation procedure is able to yield high functioning, pure mitochondria (~450 µg) as evidenced by O₂ consumption rates, RCR values, maximal citrate synthase activity and protein expression from immunoblotting. Importantly, the mitochondria isolated from this procedure can be used for multiple respirometirc assays with microplate based O

Materials

Name	Company	Catalog Number	Comments
Essentially Fatty	Sigma Aldrich	A6003	N/A
Acid Free- BSA
Tris/HCl	Promega	H5123	N/A
KCL	Sigma Aldrich	P9541	N/A
Tris Base	Promega	H5135	N/A
EDTA	Sigma Aldrich	E6511	N/A
EGTA	Sigma Aldrich	E4378	N/A
Sucrose	Sigma Aldrich	S7903	N/A
D-Mannitol	Sigma Aldrich	63559	N/A
Trypsin-EDTA (0.25%), phenol red	Thermo Scientific	25200-056	N/A
Sodium Chloride White Crystals or Crystalline Powder ≥99.0 %	Fisher Scientific	BP3581	N/A
Sodium dodecyl sulfate	Sigma Aldrich	L3771	N/A
Sodium deoxycholate	Sigma Aldrich	D6750	N/A
Polyoxyethylene (12) nonylphenyl ether, branched	Sigma Aldrich	238651	N/A
Single Edge Razor Blades	Fisher Scientific	12-640	N/A
Falcon- 100 uM Nylon Cell Strainers	Fisher Scientific	352360	N/A
Halt Protease & Phosphatse Inhibitor Cocktail	Thermo Scientific	1861284	N/A
1.5 ml microcentrifuge tubes with screw cap	Thermo Scientific	3474	N/A
Zirconium Oxide beads	Fisher Scientific	C9012112	N/A
GAPDH antibody (1D4)	Santa Cruz Biotechnology	sc-59540	N/A
Anti- COXIV antibody	Cell Signaling	4844s	Any mitochondrial inner membrane protein will suffice
Peroxidase conjugated affinipure Donkey, Anti Rabbit IgG (H+L)	Jackson ImmunoResearh	711-035-152	N/A
Peroxidase conjugated affinipure Goat, Anti Mouse IgG (H+L)	Jackson ImmunoResearh	115-001-003	N/A
Triton-X100	Sigma Aldrich	X100	N/A
Pierce BCA Protein Assay Kit	Thermo Scientific	23225	N/A
Pyruvic Acid, 98%	Sigma Aldrich	107360	Store at 4 °C,pH to 7.4 with KOH prior to use in respirometric assay
Succinic Acid	Sigma Aldrich	S9512	Store at room temperature, pH to 7.4 with KOH prior to use in respirometric assay
L(-) Malic Acid, BioXtra, ≥95%	Sigma Aldrich	M6413	Store at room temperature, to 7.4 with KOH prior to use in respirometric assay
L-Glutamic acid	Sigma Aldrich	G1251	Store at room temperature, to 7.4 with KOH prior to use in respirometric assay, to 7.4 with KOH prior to use in respirometric assay
Palmitoyl L-carnitine chloride	Sigma Aldrich	P1645	Store at -20 °C
Oligomycin A, ≥ 95% (HPLC)	Sigma Aldrich	75351	Store at -20 °C
Carbonyl cyanide 4-(trifluoromethoxy)	Sigma Aldrich	C2920	Store at 2-8 °C
phenylhydrazone
≥98% (TLC), powder [FCCP]
Antimycin A from streptomyces sp.	Sigma Aldrich	A8674	Store at -20 °C
Adenosine 5′-diphosphate monopotassium salt dehydrate [ADP]	Sigma Aldrich	A5285	Store at -20 °C, to 7.4 with KOH prior to use in respirometric assay
Rotenone	Sigma Aldrich	R8875	Store at room temperature