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....

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....

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 ± 2.0.......

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

List of materials used in this article

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