Mitochondrial Isolation from Skeletal Muscle

Summary

This protocol describes a procedure to study the respiration of mitochondria isolated from skeletal muscles. This method was adapted from Scorrano et al. (2007). The mitochondrial isolation procedure requires about 2 hours. The mitochondrial respiration can be completed in about 1 hour.

Abstract

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 cascades^2,3. Past and current researchers have isolated mitochondria from rat and mice tissues such as liver, brain and heart^4,5. 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).

Protocol

1. Preparation of Buffers

1. Turn on centrifuge 5804R and set to 4°C. Turn on Isotemp 3006D water bath and set to 37°C.
2. Prepare the following solutions before muscle isolation:
   • PBS: Dissolve phosphate buffered saline (PBS) tablets in distilled water (5 tablets/liter). Mix well.
   • PBS plus 10 mM EDTA: To prepare a 100 ml solution, add 2 ml of 500 mM EDTA to 98 ml of PBS.
   • 8X Mitochondria buffer: 10.28 g of sucrose for a final concentration of 0.6 M, 400 mg of free-fatty acid bovine serum albumin (BSA) for a final concentration of 0.8%, 2.08 g of HEPES for a final concentration of 160 mM, pH to 7.4 and QS to 50 ml with ....

Discussion

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 subst.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
95% CO2 / 5% O2 mix	Local Gas Supplier
Adenosine 5′-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
Centrifuge 5804R	Eppendorf
Compressed nitrogen	Local Gas Supplier
D-mannitol	Sigma-Aldrich	M9647
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
Glutamic acid	Sigma-Aldrich	G5889
HEPES sodium salt	Sigma-Aldrich	H7006
Isotemp 3006D	Fisher Scientific
Magnesium chloride	Sigma-Aldrich	M8266
Male Sprague Dawley Rats	Harlan Laboratories	300-500g
Malic acid	Sigma-Aldrich	M9138
Minifuge	ISC Bioexpress	C1301P
Oligomycin	Sigma-Aldrich	O4876
Oxygen electrode disc	Hansatech	S1
Oxygraph	Hansatech
Oxygraph Plus V1.01 Software	Hansatech
pH-meter	Mettler Toledo	1225506149
Phosphate-buffered saline (PBS)	Sigma-Aldrich	P4417
Potassium chloride	Sigma-Aldrich	P3911
Potassium phosphate	Sigma-Aldrich	P8416
Potter-Elvehjem homogenizers	Fisher Scientific	08-414-14A
PTFE (0.0125mm × 25mm) membrane	Hansatech	S4
SKIL 3320 drill press	Hardware store
Sucrose	Sigma-Aldrich	S5016