Department of Physiology, University of Kentucky
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Garcia-Cazarin, M. L., Snider, N. N., Andrade, F. H. Mitochondrial Isolation from Skeletal Muscle. J. Vis. Exp. (49), e2452, doi:10.3791/2452 (2011).
Mitochondria are organelles controlling the life and death of the cell. They participate in key metabolic reactions, synthesize most of the ATP, and regulate a number of signaling cascades2,3. Past and current researchers have isolated mitochondria from rat and mice tissues such as liver, brain and heart4,5. In recent years, many researchers have focused on studying mitochondrial function from skeletal muscles.
Here, we describe a method that we have used successfully for the isolation of mitochondria from skeletal muscles 6. Our procedure requires that all buffers and reagents are made fresh and need about 250-500 mg of skeletal muscle. We studied mitochondria isolated from rat and mouse gastrocnemius and diaphragm, and rat extraocular muscles. Mitochondrial protein concentration is measured with the Bradford assay. It is important that mitochondrial samples be kept ice-cold during preparation and that functional studies be performed within a relatively short time (~1 hr). Mitochondrial respiration is measured using polarography with a Clark-type electrode (Oxygraph system) at 37°C7. Calibration of the oxygen electrode is a key step in this protocol and it must be performed daily. Isolated mitochondria (150 μg) are added to 0.5 ml of experimental buffer (EB). State 2 respiration starts with addition of glutamate (5mM) and malate (2.5 mM). Then, adenosine diphosphate (ADP) (150 μM) is added to start state 3. Oligomycin (1 μM), an ATPase synthase blocker, is used to estimate state 4. Lastly, carbonyl cyanide p-[trifluoromethoxy]-phenyl-hydrazone (FCCP, 0.2 μM) is added to measurestate 5, or uncoupled respiration 6. The respiratory control ratio (RCR), the ratio of state 3 to state 4, is calculated after each experiment. An RCR ≥4 is considered as evidence of a viable mitochondria preparation.
In summary, we present a method for the isolation of viable mitochondria from skeletal muscles that can be used in biochemical (e.g., enzyme activity, immunodetection, proteomics) and functional studies (mitochondrial respiration).
1. Preparation of Buffers
2. Muscle Isolation
3. Homogenization/Mitochondrial Isolation
4. Electrode Calibration
NOTE: Complete electrode calibration during mitochondrial isolation.
5. Mitochondrial Respiration
6. Representative Results:
Figure 1. shows a representative tracing of oxygen consumption by skeletal muscle mitochondria. After the electrode signal is stabilized, the mitochondria sample is added to the Oxygraph chamber. State 2 respiration starts with addition of glutamate and malate. Addition of ADP increases oxygen consumption, defining state 3 respiration. ATP synthase is blocked by addition of oligomycin to obtain state 4 respiration. Finally, FCCP is added to uncouple mitochondrial respiration (state 5). Table 1 shows representative oxygen consumption rates for states 2, 3, 4 and 5. The respiratory control ratio (RCR) is calculated for each experiment. RCR ≥4 is considered as evidence of a viable mitochondria preparation.
Table 1. Mitochondrial respiration rates. Representativeoxygen consumption rates from skeletal muscle mitochondria. Values are normalized to the amount of mitochondria added to the chamber. The respiratory control ratio (RCR) is determined by dividing state 3 by state 4. An RCR≥4 represents a viable mitochondria preparation.
We present a protocol to isolate viable mitochondria from skeletal muscles. If yield is a problem, the protocol can be modified by incubating the isolated muscle in 5 ml of PBS/10mM EDTA/0.01% trypsin for 30 minutes in ice. To assure complete muscle digestion with trypsin, the muscle needs to be fully minced. After the 30-minute incubation, the PBS/10mM EDTA/0.01% trypsin solution must be completely replaced with 3 ml of isolation buffer 1 (IB1). In addition, the use of trypsin may interfere with some mitochondrial substrates during the respiration protocol. All the substrates used in this protocol result in good mitochondrial preparations when using trypsin.
For mouse skeletal muscles, it is best to combine the gastrocnemius muscles from both legs, or use the whole diaphragm. For rat skeletal muscles, a small section of gastrocnemius or diaphragm is sufficient. Excessive muscle mass can complicate the isolation procedure and result in lower yields. For isolation of very small muscles such as the extraocular muscles, it is necessary to pool muscles from more than 1 animal to obtain the necessary muscle mass.
This protocol refers to two different supernatants: SN1 and SN2. These supernatants are the result of differential centrifugation and contain non-mitochondrial proteins. Proteins from these supernatants and from the final mitochondrial pellet can be used in western blots to verify the purity of the mitochondrial preparation. Western blots using mitochondrial and cytoplasmic antibodies confirm the purity of your mitochondrial preparation in the final pellet.
Cleaning the mitochondrial respiration equipment is critical for a successful experiment. After each experiment, the chambers must be cleaned thoroughly as follows: rinse chambers five times with distilled water, rinse once with 70% ethanol to remove ethanol-soluble substrates, rinse with a saturated solution of PBS/BSA for about 5 minutes, finally, rinse chambers 5 times with distilled water. Clark-type electrodes must be properly cleaned after every use by rinsing several times with distilled water and softly scrubbed with a toothbrush. Then, they must be completely dried with lent-free wipes and stored in a desiccator. Once a week, the electrodes should be polished using the Oxygraph polishing kit.
Mitochondrial substrates are prepared as stock solutions, aliquoted and stored at -20o C. We do not recommend the re-use of substrates. In our experience, most of the substrates are stable at -20o C for several months, except for oligomycin that must be replaced every 2 months or so. Always refer to the manufacturer's specifications for more details.
Finally, this protocol can also be used to isolate mitochondria from heart. As for other muscles, the size of the animal (rat vs. mouse) will determine what fraction of the organ is necessary to obtain sufficient mitochondria.
No conflicts of interest declared.
This work was supported by a grant from the National Eye Institute (R01 EY12998) to F.H. Andrade.
|95% CO2 / 5% O2 mix||Local Gas Supplier|
|Adenosine 5€²-diphosphate sodium salt||Sigma-Aldrich||A2754|
|Blue Rizla Paper||Hansatech||890101|
|Bradford protein assay||Bio-Rad||500-0006|
|Carbonylcyanide p-trifluoromethoxyphenylhydrazone (FCCP)||Sigma-Aldrich||C2920|
|Compressed nitrogen||Local Gas Supplier|
|Ethlyene-glycol-bis-tetraacetic acid (EGTA)||Sigma-Aldrich||E3889|
|Ethylenediaminetetraacetic acid (EDTA)||Bio-Rad||161-0728|
|Free fatty acid bovine serum albumin||Sigma-Aldrich||A8806|
|HEPES sodium salt||Sigma-Aldrich||H7006|
|Isotemp 3006D||Fisher Scientific|
|Male Sprague Dawley Rats||Harlan Laboratories||300-500g|
|Oxygen electrode disc||Hansatech||S1|
|Oxygraph Plus V1.01 Software||Hansatech|
|Phosphate-buffered saline (PBS)||Sigma-Aldrich||P4417|
|Potter-Elvehjem homogenizers||Fisher Scientific||08-414-14A|
|PTFE (0.0125mm Ãƒ€” 25mm) membrane||Hansatech||S4|
|SKIL 3320 drill press||Hardware store|