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Evaluation of Mitochondrial Function Version: 1

Replaced by version: N/A

Edited by: Dale Abel, Ph.D.

Summary

Protocol

Summary: This protocol describes the procedures used by the AMDCC for evaluating mitochondrial function.

Protocol:

Protocols for Mitochondrial Analyses

Saponin-permeabilized fibers. Left ventricular cardiac fibers are prepared using previously described protocols (1,2). Briefly, small pieces (2-5 mg) of cardiac muscle are taken from the left ventricle and permeabilized with 50 μg/ml saponin at 4°C in buffer A containing (in mM) 7.23 K2EGTA, 2.77 K2CaEGTA, 6.56 MgCl2, 20 imidazole, 0.5 dithiothreitol, 53.3 K-methanS, 20 taurine, 5.3 Na2ATP, 15 PCr and 3 KH2PO4, pH 7.1 adjusted at 25°C. The fibers are then washed twice for 10 min in buffer B containing (in mM) 7.23 K2EGTA, 2.77 K2CaEGTA, 1.38 MgCl2, 20 imidazole, 0.5 dithiothreitol, 100

K-methanS, 20 taurine, 3 KH2PO4 and 2 mg/ml BSA, pH 7.1 adjusted at 25°C. Respiration measurements. The respiratory rates of saponin-permeabilized fibers are determined using an oxygen foxy probe in 2 ml solution B at 25°C with continuous stirring. Substrates used include 5 mM glutamate and 2 mM malate, which evaluates the integrity of the TCA cycle and electron transport chain, 10 mM pyruvate and 5 mM malate which evaluates flux through pyruvate dehydrogenase as well as the TCA cycle and electron transport chain and 20 μM palmitoyl-carnitine with 2 mM malate, which evaluates fatty acid beta oxidation. The solubility of oxygen in the solution B is 215 nmol

O2/ml. Oxygen consumption rates are expressed in nmoles O2/min/mg dry weight. The following respiratory parameters are measured:

(1)State 2: Mitochondrial respirations in the presence of substrate but in the absence

of any added ADP.

(2)State 3: Maximal stimulated mitochondrial respirations, which is initiated by

adding 1mM ADP (in the presence of substrate).

(3)State 4: Mitochondrial respiration in the absence of ATP synthesis, which is

achieved by adding the ATP synthase inhibitor oligomycin (1μg/ml). An increase

in state 4 respiration would be suggestive of mitochondrial uncoupling.

(4)Respiratory control ratio (RC) is the ratio of state 3 to state 4 respirations. This

ratio is used as a general index of the viability of the mitochondrial preparation.

Low RC ratios raise the possibility of mitochondrial damage.

ATP measurements. ATP concentrations are determined by bioluminescence with the luciferin/luciferase reaction as described (3), using a commercially available ATP assay kit (Enliten ATP assay system, Promega, Madison WI).

Mitochondrial enzyme activity assays.

Mitochondrial Isolation: Hearts are minced and homogenized on ice in 10% (wt/v) isolation buffer containing (in mM) 20 HEPES (pH 7.4), 140 KCl, 10 EDTA, 5 MgCl2 supplemented with 2-4 mg nagarse. The homogenate is then centrifuged at 500 g for 10 min. The debris is discarded and the supernatant centrifuged at 9,000 g for 35 min. The pellet is then washed with the isolation buffer without nagarse and suspended in 80 μl isolation buffer. Protein concentration is determined using the Micro BCA protein assay kit (Pierce Chemical Co., Rockford, Illinois, USA).

Carnitine palmitoyl transferase. Total CPT (CPT I and CPT II) activity is measured in isolated mitochondria (see above). Mitochondria (~200 μg) are assayed in 1 ml reaction buffer containing (in mM) 20 HEPES, 1 EGTA, 220 sucrose, 40 KCl, 0.1 5,5’-dithio-bis (2-nitrobenzoic acid) (DTNB), 1.3 mg/ml BSA, and 40 μM palmitoyl-CoA, pH 7.4 at 25°C. The reaction is started by adding 1 mM carnitine (omitted in baseline) and monitored at 412 nm for 4 min using an Ultraspec 3000 spectrophotometer. CPT II activity is measured using an identical reaction as total CPT but after adding 10 μM malonyl-CoA to inhibit CPT I activity. CPT I activity is calculated by subtracting the CPT II activity from the total CPT activity.

Citrate synthase. Small frozen pieces of hearts (~10 mg) are homogenized in 20% (wt/v) homogenization buffer containing (in mM) 20 HEPES, 10 EDTA, pH 7.4 on ice. The homogenates are then frozen for 1h to liberate citrate synthase from mitochondrial matrix and diluted 1:10. The reaction is then performed in 1 ml reaction buffer containing (in mM) 20 HEPES, 1 EGTA, 220 sucrose, 40 KCl, 0.1 DTNB, and 0.1 acetyl-CoA, pH 7.4 at 25°C. The reaction is started by adding 0.05 mM oxaloacetate (omitted at baseline) and monitored at 412 nm for 3 min using an Ultraspec 3000 spectrophotometer.

β-Hydroxyacyl-CoA dehydrogenase. Homogenates are prepared as used for the citrate synthase assay and diluted 1:4. The reaction is performed in 1 ml reaction buffer containing (in mM) 20 HEPES, 1 EGTA, 1 KCN, and 0.15 NADH, pH 7.4 at 25°C. The reaction is started by adding 0.1 mM acetoacetyl-CoA (omitted at baseline) and monitored at 340 nm for 4 min using an Ultraspec 3000 spectrophotometer. References

1. Veksler VI, Kuznetsov AV, Sharov VG, Kapelko VI, Saks VA. Mitochondrial

respiratory parameters in cardiac tissue: a novel method of assessment by using saponin-skinned fibers. Biochim Biophys Acta. 1987;892:191-6.

2. Saks VA, Veksler VI, Kuznetsov AV, Kay L, Sikk P, Tiivel T, Tranqui L,

Olivares J, Winkler K, Wiedemann F, Kunz WS. Permeabilized cell and skinned fiber techniques in studies of mitochondrial function in vivo. Mol Cell Biochem.

1998;184:81-100.

3. Ouhabi R, Boue-Grabot M, Mazat JP. Mitochondrial ATP synthesis in

permeabilized cells: assessment of the ATP/O values in situ. Anal Biochem.

1998;263:169-75.