Isolation of Intact Mitochondria from Skeletal Muscle by Differential Centrifugation for High-resolution Respirometry Measurements

Summary

Here, a quadriceps muscle specimen is taken from an anaesthetized pig and mitochondria are isolated by differential centrifugation. Then, the respiratory rates of mitochondrial respiratory chain complexes I, II and IV are determined using high-resolution respirometry.

Abstract

Mitochondria are involved in cellular energy metabolism and use oxygen to produce energy in the form of adenosine triphosphate (ATP). Differential centrifugation at low- and high-speed is commonly used to isolate mitochondria from tissues and cultured cells. Crude mitochondrial fractions obtained by differential centrifugation are used for respirometry measurements. The differential centrifugation technique is based on the separation of organelles according to their size and sedimentation velocity. The isolation of mitochondria is performed immediately after tissue harvesting. The tissue is immersed in an ice-cold homogenization medium, minced using scissors and homogenized in a glass homogenizer with a loose-fitting pestle. The differential centrifugation technique is efficient, fast and inexpensive and the mitochondria obtained by differential centrifugation are pure enough for respirometry assays. Some of the limitations and disadvantages of isolated mitochondria, based on differential centrifugation, are that the mitochondria can be damaged during the homogenization and isolation procedure and that large amounts of the tissue biopsy or cultured cells are required for the mitochondrial isolation.

Introduction

Mitochondrial bioenergetics and respiratory capacities can be studied not only in permeabilized cells or fibers but also in isolated mitochondria. In the present study, we describe a protocol to isolate intact skeletal muscle mitochondria using differential centrifugation for high-resolution respirometry measurements.

To isolate intact mitochondria for respirometry, the tissue is homogenized and mitochondria are isolated by a conventional differential centrifugation method. The differential centrifugation method is based on sequential centrifugations (in a series of increasing speed) of tissue homogenates was first introduced by Pallade and co-workers almost 70 years ago ¹. The tissue is first minced using scissors and homogenized mechanically in a glass homogenizer with a loose-fitting pestle. Afterwards the homogenate is centrifuged at low speed and the resulting pellet that contains unbroken tissue, cellular debris and nuclei is discarded. Then, the supernatant is centrifuged several times at high speed and the mitochondrial enriched fraction is collected. The advantages of the differential centrifugation method to isolate mitochondria are that: i) the method is fast and mitochondria can be isolated within 1-1.5 h (respiratory experiments should be performed as quick as possible); ii) it is inexpensive; and iii) it is very efficient and the mitochondria obtained by differential centrifugation are pure enough for respirometry assays. The disadvantages of differential centrifugation method to isolate mitochondria are that i) mitochondria might get damaged and uncoupled during homogenization; ii) the contamination of mitochondria with other cellular components (could be solved by further washing the mitochondrial pellet with additional centrifugation steps); iii) the possibility of selecting different mitochondrial subpopulations, e.g., during differential centrifugations steps, mitochondria with lower dense can be excluded ⁷; and iv) the mitochondrial cellular surrounding is missing and only the theoretical maximal respiration can be measured. Another method to isolate mitochondria for respirometry assays is the density gradient centrifugation ². In this technique, the tissue extract is layered over a solution of sucrose or a Percoll gradient (with higher density at the bottom of the centrifugation tube) and centrifuged at a certain speed, causing the mitochondria to be isolated from other cellular components according to their densities. This method is often used to isolate brain mitochondria with very low contamination from synaptosomes. However, the rat liver mitochondria isolated by density gradient centrifugation are highly contaminated with other cellular organelles ³. One of the limitations of this method is that the sucrose gradient present in the centrifugation tube might rupture some mitochondria (osmotic shock).

Depending on the type of tissue; there are some important factors to consider for the isolation of intact mitochondria by differential centrifugation. The first necessity is to homogenize tissues in a gentle manner. Soft tissues such as kidney, brain and liver require gentle mechanical forces applied during homogenization. This contrasts with hard tissues such as cardiac and skeletal muscle that require much stronger mechanical forces. The minced tissue is usually treated with proteinase prior to the homogenization to soften the tissue. All buffers used during homogenization and centrifugation should be ice cold and have a physiological relevant pH with an ionic and osmotic strength compatible with cytosol ^4,5.

One of the advantages of studying isolated mitochondrial bioenergetics is that cellular plasma membranes do not need to be permeabilized with detergents such as digitonin or saponin ^4,6, which might compromise the mitochondrial outer membrane integrity. Another advantage of the isolated mitochondria is the absence of other cytosolic factors, which may interfere with the analysis of the mitochondrial functions such as oxygen consumption. The disadvantages of using the isolated mitochondria are the possible selection of certain mitochondrial populations during the centrifugation steps, damage to the mitochondria during homogenization, and the requirement for high quantities of biological samples in order to obtain a good yield of isolated mitochondria ^7,8.

After the isolation procedure, the respiratory rates of mitochondrial complexes I-, II- and IV-dependent (states 2, 3 and 4) are determined using high-resolution respirometry. For complex I-driven respiration, glutamate and malate are added followed by adenosine diphosphate (ADP). For complex II-driven respiration, succinate is added followed by ADP. For complex IV-driven respiration, ascorbate and tetramethylphenylendiamine (TMPD) are added followed by ADP ^9,10,11,12. State 2 refers to oxygen consumption in the presence of substrates alone. State 3 refers to oxygen consumption in the presence of substrates and ADP. State 4 refers to oxygen consumption after ADP depletion. The respiratory control ratio (RCR) is an index of coupling of oxygen consumption ATP production and is calculated as the ratio between state 3 and state 4 ^13,15.

In summary, we describe a protocol to isolate functional and intact skeletal muscle mitochondria by differential centrifugation and use these isolated mitochondria for functional and bioenergetic studies such as high-resolution respirometry.

Protocol

The quadriceps muscle biopsy is taken from an anaesthetized pig, from which mitochondria are isolated by differential centrifugation. The pig is used afterwards for another experiment. The study is performed in accordance with the National Institutes of Health guidelines for the care and use of experimental animals and with the approval of the Animal Care Committee of the Canton Bern, Switzerland.

1. Skeletal Muscle Homogenization and Mitochondrial Isolation

1. Excise 5-10 g quadriceps muscle specimen from an anaesthetized pig. In the present assay, use porcine skeletal muscle. This protocol can also be used to isolate mitochondria from other species (e.g., rat, mice, human, etc.).
   NOTE: This step is performed by a medical specialist with expertise in surgery.
   1. Sedate pigs with intramuscular ketamine (20 mg/kg) and xylazine (2 mg/kg), before anesthesia is induced with intravenous midazolam (0.5 mg/kg, plus atropine 0.02 mg/kg). Maintain anesthesia with continuous intravenous infusions of propofol (4-8 mg/kg per h) and fentanyl (30 µg/kg per h) during surgery.
2. Immediately immerse the tissue sample in a beaker containing 20 mL of ice-cold mitochondrial isolation buffer (Table 1).
   NOTE: Buffers used during homogenization and centrifugations should be ice cold and have a physiological relevant pH with an ionic and osmotic strength compatible with cytosol.
3. Weigh the tissue sample in the beaker on an analytical tared balance. Tare the balance with the beaker containing 20 mL of isolation buffer.
4. Place the beaker on an ice bucket.
   NOTE: Perform all of the next steps for the homogenization procedure at the ice bucket temperature and keep all buffers on the ice bucket.
5. Mince the skeletal muscle in the beaker (keep on ice) into 1-2 mm small pieces for 3-4 min using fine scissors.
6. Rinse the minced tissue in the beaker twice with ice cold isolation buffer (20 mL each washing step).
7. Suspend the tissue in 10 volumes of the isolation buffer per tissue weight (g) containing 5 mg protease/g tissue.
8. Place the beaker in an ice bath on the magnetic stirrer and stir for 10 min.
9. Dilute the suspension in the beaker with additional 10 volumes of the isolation buffer per tissue weight (g) supplemented with 0.2% (weight/volume) defatted bovine serum albumin (BSA).
10. Decant all of the isolation buffer supplemented with 0.2% BSA and rinse the tissue in the beaker twice with ice cold isolation buffer supplemented with 0.2% BSA (20 mL each washing step).
11. Resuspend the tissue in 10 volumes of isolation buffer per tissue weight (g) supplemented with 0.2% with defatted BSA.
12. Homogenize the tissue with a semi-automatic glass homogenizer (kept on ice) with a loose-fitting pestle (10 strokes).
    NOTE: Homogenize the tissue always in the same manner (the same number of strokes, e.g., 10 strokes). A higher number of strokes may damage and uncouple the mitochondria. Preferably, homogenize the tissue using an automated homogenizer. Manually (and subjectively) homogenized tissue samples in glass homogenizers may produce different qualities of isolated mitochondria.
13. Centrifuge the homogenized tissue at 4 °C for 10 min at 10,000 x g.
    NOTE: All centrifugation steps are performed in centrifuges with fixed angle rotors.
14. Discard the supernatant using a serological pipet and resuspend the pellet in BSA-supplemented ice-cold isolation medium (10 mL/g tissue).
15. Centrifuge the suspension at 4 °C for 10 min at 350 x g.
16. Reserve the supernatant using a serological pipet and discard the pellet (cellular debris).
17. Filter the supernatant into a beaker (kept on ice) through two layers of gauze (17 threads/cm²) to remove cell debris.
18. Centrifuge the filtered suspension at 4 °C for 10 min at 7,000 x g.
19. Discard the supernatant and resuspend the crude mitochondrial pellet in the isolation buffer (5 mL/g tissue) supplemented with 0.2% (weight/volume) BSA and centrifuge the suspension at 4 °C for 10 min at 7,000 x g.
20. Discard the supernatant and resuspend the crude mitochondrial pellet in wash buffer (Table 1). Centrifuge the suspension again at 4 °C for 10 min at 7,000 x g.
21. Discard the supernatant and resuspend the crude mitochondrial pellet in wash buffer and centrifuge the suspension again at 4 °C for 10 min at 7,000 x g.
22. Discard the supernatant and resuspend the crude mitochondrial pellet in 1.0 mL of wash buffer.
23. Determine the protein concentration of the mitochondrial suspension and keep the concentrated mitochondrial suspension on ice.
    NOTE: Protein concentration can be measured using any standard method.
24. Just prior to respirometry assays, resuspend the mitochondrial suspension in the respiration buffer to a final concentration of 0.4 mg/mL mitochondrial protein.

2. High-resolution Respirometry

1. Pipette 2.1 mL of respiration buffer (Table 1) ¹⁶ into a high-resolution oxygraph chamber and stir the buffer continuously using a magnetic stirring bar present in the chamber (700 rpm) at 37 °C for 1 h until a stable oxygen flux signal of the polarographic oxygen sensor is obtained.
   NOTE: Polarographic oxygen electrodes within each oxygraph chamber measure the oxygen concentration and calculate oxygen consumption (flux) within each chamber. The oxygen concentration and oxygen consumption rates (flux) are displayed real-time online in a computer using software for data acquisition and analysis. In addition, in order to ensure accurate and reliable results, instrumental oxygen background correction should be performed according to the manufacturer's instructions.
   NOTE: Accurately 2.1 mL of respiration buffer is added into the chamber for calibration of a final chamber volume of 2.0 mL after closing the stoppers.
2. Perform an air calibration of the polarographic oxygen sensor according to the manufacturer's protocols ¹⁷.
   NOTE: During air calibration, with a gas phase present, the media oxygen concentration will equilibrate on the order of 15-20 min. Depending on the temperature, barometric pressure, and solubility of media -oxygen concentration can be calculated.
3. After air calibration, aspirate the respiration medium from the oxygraph chamber and add 2.1 mL of isolated mitochondrial suspension (0.4 mg/mL mitochondrial protein) from step 1.24 to a chamber of the oxygraph.
   NOTE: The volume of respiration buffer depends on the instrument set up and manufacturer. For high resolution respirometry, the 2.1 mL of respiration buffer is added into the chamber for calibration of a final chamber volume of 2.0 mL after closing the stoppers.
4. Close the oxygraph chamber by insertion of the stopper.
5. Stir the mitochondrial suspension continuously using a magnetic stirring bar present in the chamber (700 rpm) at 37 °C and record cellular respiration at baseline for 3-5 min until a stable oxygen flux signal is achieved.
   NOTE: The oxygen concentration and oxygen flux signal are displayed real-time online in a computer using software for data acquisition and analysis ¹⁷.
6. Afterwards, inject substrates and inhibitors for mitochondrial respiration through the titanium injection ports of the stoppers using the following standard protocols.

3. Complex I-dependent Respiration

1. Prepare an oxygraph chamber containing the isolated mitochondrial suspension (0.4 mg/mL) following the procedure described in steps 2.1-2.5 and wait for a stable signal.
2. Inject 12.5 µL of 0.8 M malate (5 mM final concentration) and 10 µL of 2 M glutamate (10 mM final concentration) into the oxygraph chamber containing isolated mitochondrial suspension (0.4 mg/mL) through the titanium injection port of the chamber stopper and record cellular respiration for 3-5 min until a stable oxygen flux signal is achieved.
   NOTE: All the injections in the following steps are performed through the titanium injection ports of stoppers using syringes.
3. Inject 10 µL of 0.05 M ADP (0.25 mM) into the oxygraph chamber through the titanium injection port of the chamber stopper and record mitochondrial respiration until the oxygen flux signal increases, then decreases and stabilizes (3-5 min).
   NOTE: The plateau of respiration after ADP addition indicates that ADP concentration was high and saturating. Depending on the amount of mitochondria used during respiration, ADP concentration should be titrated for a stable state 3 oxygen consumption (maximal respiration). Addition of ADP to the mitochondrial suspension will induce an increase in oxygen consumption and the oxygen flux signal will increase (state 3 respiration). After the ADP is consumed, the oxygen flux signal will decrease (state 4 respiration).

4. Complex II-dependent Respiration

1. Prepare an oxygraph chamber containing isolated mitochondrial suspension (0.4 mg/mL) following the procedure described in steps 2.1-2.5 and wait for a stable signal.
2. Inject 2 µL of 0.2 mM rotenone (0.2 µM) 'CAUTION' into the oxygraph chamber through the titanium injection port of the chamber stopper using a syringe and record the cellular respiration for 3-5 min until a stable oxygen flux signal is achieved.
   NOTE: Rotenone is a dangerous poison and may have a significant impact on human health.
3. Inject 20 µL of 1 M succinate (10 mM) into the oxygraph chamber and record mitochondrial respiration for 3-5 min until a stable oxygen flux signal is achieved.
4. Inject 10 µL of 0.05 M ADP (0.25 mM) into the oxygraph chamber through the titanium injection port of the chamber stopper and record cellular respiration until the oxygen flux signal increases, stabilizes, then decreases and stabilizes (3-5 min).
   NOTE: Addition of ADP to the mitochondrial suspension will induce an increase in oxygen consumption and the oxygen flux signal will increase (state 3 respiration). After the ADP is consumed, the oxygen flux signal will decrease (state 4 respiration).

5. Complex IV-dependent Respiration

1. Prepare an oxygraph chamber containing isolated mitochondrial suspension (0.4 mg/mL) following the procedure described in steps 2.1 to 2.5 of the protocol and wait for a stable signal.
2. Then inject 2.5 µL of 0.8 mM ascorbate (1 mM). Immediately thereafter inject 2.5 µL of 0.2 M TMPD (0.25 mM) and 10 µL of 0.05 M ADP (0.25 mM) into the oxygraph chamber and record cellular respiration until the oxygen flux signal increases and stabilizes (3-5 min).
3. Finally inject 10 µL of 1 M sodium azide (5 mM) 'CAUTION' into the oxygraph chamber and record cellular respiration until the oxygen flux signal decreases and stabilizes.
   NOTE: Sodium azide is a dangerous poison and may have a significant impact on human health.

6. Cytochrome C Test

1. Prepare an oxygraph chamber containing isolated mitochondrial suspension (0.4 mg/mL) following the procedure described in steps 2.1 to 2.5 of the protocol and wait for a stable signal.
2. Inject 12.5 µL of 0.8 M malate (5 mM final concentration) and 10 µL of 2 M glutamate (10 mM final concentration) into the oxygraph chamber containing isolated mitochondrial suspension (0.4 mg/mL) through the titanium injection port of the chamber stopper and record cellular respiration for 3-5 min until a stable oxygen flux signal is achieved.
3. Inject 10 µL of 0.5 M ADP (2.5 mM) into the oxygraph chamber through the titanium injection port of the chamber stopper and record mitochondrial respiration until the oxygen flux signal increases, then decreases and stabilizes (3-5 min).
4. Finally, inject 5 µL of 4 mM cytochrome c (10 µM) into the oxygraph chamber and record cellular respiration for 5 min until a stable oxygen flux signal is achieved.

Results

Complex I-dependent respiration

Isolated mitochondrial complex I-dependent respiratory rates (states 2, 3 and 4) are determined using high-resolution respirometry (Figure 1, a representative diagram). Mitochondrial complex I substrates, glutamate and malate, are added followed by the addition of ADP. State 2 refers to oxygen consumption in the presence of the substrates alone. State 3 refers to ...

Discussion

In the present study we describe a protocol to isolate high-quality, intact and tightly coupled skeletal muscle mitochondria by differential centrifugation which can be used for functional studies such as high-resolution respirometry.

In order to isolate intact and tightly coupled mitochondria, there are some critical points to be considered within the present protocol. After harvesting the skeletal tissue, it should be immediately immersed in ice-cold mitochondrial isolation buffer. All centr...

Materials

Name	Company	Catalog Number	Comments
ADP	Sigma	A 4386	Chemical
Antimycin A	Sigma	A 8674	Chemical, dissolve in ethanol
Ascorbate	Merck	1.00127	Chemical
ATP	Sigma	A 7699	Chemical
BSA	Sigma	A 6003	Chemical
EGTA	fluka	3779	Chemical
Glutamate	Sigma,	G 1626	Chemical
Hepes	Sigma	H 7523	Chemical
KCl	Merck	1.04936	Chemical
KH2PO4	Merck	1.04873	Chemical
K-lactobionate	Sigma	L 2398	Chemical
MgCl2	Sigma	M 9272	Chemical
Morpholinopropane sulphonic acid (MOPS)	Merck	1.06129	Chemical
O2k-Core: Oxygraph-2k	Oroboros Instruments	10000-02	High-resolution respirometry instrument
Proteinase, bacterial	Sigma	P 8038	Chemical
Sodium azide	Sigma	S2002	Chemical
Rotenone	Sigma	R 8875	Chemical, dissolve in ethanol
Succinate	Sigma	S 2378	Chemical
Schuett homogen-plus semiautomatic homogeniser	schuett-biotec GmbH	3.201 011	Tissue homogenizer
Taurine	Sigma	T 8691	Chemical
TMPD	Sigma	T 3134	Chemical