1Department of Biochemistry and Molecular Biology, Faculty of Medicine, University of Calgary, 2Faculty of Kinesiology, University of Calgary
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Hughey, C. C., Hittel, D. S., Johnsen, V. L., Shearer, J. Respirometric Oxidative Phosphorylation Assessment in Saponin-permeabilized Cardiac Fibers. J. Vis. Exp. (48), e2431, doi:10.3791/2431 (2011).
Investigation of mitochondrial function represents an important parameter of cardiac physiology as mitochondria are involved in energy metabolism, oxidative stress, apoptosis, aging, mitochondrial encephalomyopathies and drug toxicity. Given this, technologies to measure cardiac mitochondrial function are in demand. One technique that employs an integrative approach to measure mitochondrial function is respirometric oxidative phosphorylation (OXPHOS) analysis.
The principle of respirometric OXPHOS assessment is centered around measuring oxygen concentration utilizing a Clark electrode. As the permeabilized fiber bundle consumes oxygen, oxygen concentration in the closed chamber declines. Using selected substrate-inhibitor-uncoupler titration protocols, electrons are provided to specific sites of the electron transport chain, allowing evaluation of mitochondrial function. Prior to respirometric analysis of mitochondrial function, mechanical and chemical preparatory techniques are utilized to permeabilize the sarcolemma of muscle fibers. Chemical permeabilization employs saponin to selectively perforate the cell membrane while maintaining cellular architecture.
This paper thoroughly describes the steps involved in preparing saponin-skinned cardiac fibers for oxygen consumption measurements to evaluate mitochondrial OXPHOS. Additionally, troubleshooting advice as well as specific substrates, inhibitors and uncouplers that may be used to determine mitochondria function at specific sites of the electron transport chain are provided. Importantly, the described protocol may be easily applied to cardiac and skeletal tissue of various animal models and human samples.
1. Reagent Preparation
2. Tissue Preparation
A. Cardiac fibers mechanical preparation
B. Cardiac fiber bundles chemical preparation
C. Cardiac Fiber Bundle Washing
D. Wet weight determination
3. Respirometric OXPHOS Analysis
A. Respirometric Equipment
B. Representative Quality Control/Technique Validation Protocol
4. Representative Results:
Oxygen consumption in properly prepared murine cardiac fibers is evaluated by the quality control protocol as shown in Figure 2. Figures 3-5 provide commonly encountered examples of incorrectly prepared cardiac fibers. The respiratory control ratio (RCR) represents an important index in respirometry. This parameter indicates the coupling between oxygen consumption and oxidative phosphorylation. In permeabilized fiber preparations, the RCR is the rate of respiration in state 3 relative to state 2 or alternatively state 3 over state 4 (induced by oligomycin and/or atractyloside, ATR). Furthermore, RCR can be used as a quality assurance marker and can identify changes in coupling resulting from experimental or pathological interventions5. Well-coupled permeabilized murine cardiac preparations yield an RCR between 3-6 depending on the incubation solution utilized1, 8, 9.
The titration of cytochrome c is used as a validation of proper tissue preparation. Cytochrome c is a protein located in the intermembrane space at the mitochondrial inner membrane10. When the outer membrane of mitochondria is intact, the endogenous cytochrome c remains in the intermembrane space and the titration of exogenous cytochrome c has a negligible effect on respiration (Figure 2). If the outer membrane of mitochondria is damaged, the endogenous cytochrome c can be released from the intermembrane space and will inhibit respiration until exogenous cytochrome c addition (Figure 3). Proper preparations should experience only a slight elevation in oxygen flux following cytochrome c addition in the 5-15% range5. Additionally, this experimental titration allows for the assessment of the pathological or experimental stressor's influence on mitochondrial outer membrane intactness. If a cytochrome c effect is experienced, ensure care during mechanical preparation of the tissue and/or reduce saponin concentration.
The addition of oligomycin and/or ATR is used to assess alterations in leak respiration; oxygen consumption not contributing to ADP phosphorylation11. Additionally, this titration step may be used as a validation of proper sample preparation. Control or wild-type fibers should be sensitive to this addition and experience a significant reduction in oxygen flux. A low RCR and reduced sensitivity to oligomycin and/or ATR resulting in a relatively elevated oxygen flux indicates damage to the inner mitochondrial membrane during preparation (Figure 4). Damage is likely induced during mechanical separation of the cardiac fibers as the inner mitochondrial membrane is less susceptible to insult by saponin relative to the outer mitochondrial membrane. However, both mechanical and chemical preparation may have to be adjusted accordingly to avoid improperly prepared cardiac fibers5, 12.
The saponin-skinned fiber bundles from mice should not remain in the RP solution for more than 6h or the MiR05 solution for greater than 2h. Lack of response to substrate-inhibitor-uncoupler titrations as seen in Figure 5 may be indicative of prolonged incubation periods. Subsequent efforts should minimize periods between animal sacrifice and oxygen consumption measurements.
Figure 1. Mechanical preparation of murine cardiac fiber bundles. A. The entire heart immediately following dissection. B. A 10-25mg sample of the anterior left ventricle. C. Cardiac tissue separated into 1mm diameter and 2-4mm length strips. D. Final cardiac fiber bundles ready for chemical permeabilization with saponin.
Figure 2. Representative results of proper tissue preparation utilizing polarographic assessment. The state 2 oxygen flux is supported by complex I substrates glutamate and malate (M/G) and is significantly stimulated following the addition of ADP (state 3). No stimulatory effect of exogenous cytochrome c addition indicates the outer mitochondrial membrane is intact. Sensitivity of oxygen consumption to oligomycin suggests the inner mitochondrial membrane integrity is intact. Oxygen concentration in a 2 mL closed chamber is identified by the blue line. Oxygen consumption of the cardiac tissue sample in a 2 mL closed chamber is represented by the red line. Malate and glutamate, M/G; Adenosine Diphosphate, ADP; Cytochrome c, Cyto c; Oligomycin, o.
Figure 3. Cytochrome c effect validation test utilizing polarographic assessment. The state 2 oxygen flux is supported by complex I substrates glutamate and malate (M/G) and is significantly stimulated following the addition of ADP (state 3). Stimulatory effect of exogenous cytochrome c addition indicates the outer mitochondrial membrane integrity is compromised. Oxygen concentration in a 2 mL closed chamber is identified by the blue line. Oxygen consumption of the cardiac tissue sample in a 2 mL closed chamber is represented by the red line. Malate and glutamate, M/G; Adenosine Diphosphate, ADP; Cytochrome c, Cyto c.
Figure 4. Inner mitochondrial membrane integrity validation test utilizing polarographic assessment. The state 2 oxygen flux is supported by complex I substrates glutamate and malate (M/G) and a relatively weak stimulation of oxygen consumption following the addition of ADP (state 3). Poor sensitivity of oxygen consumption to oligomycin suggests the inner mitochondrial membrane is damaged. Oxygen concentration in a 2 mL closed chamber is identified by the blue line. Oxygen consumption of the cardiac tissue sample in a 2 mL closed chamber is represented by the red line. Malate and glutamate, M/G; Adenosine Diphosphate, ADP Oligomycin, o.
Figure 5. Polarographic assessment following prolonged incubation in MiR05. Oxygen consumption is insensitive to exogenous addition of glutamate and malate (M/G) and oxygen consumption following the addition of ADP (state 3) is reduced. There is no stimulatory effect of exogenous cytochrome c addition and insensitivity of oxygen consumption to oligomycin is experienced. Lack of response to additions to extramitochondrial incubation solution suggests mitochondrial functional stability is compromised. Oxygen concentration in a 2 mL closed chamber is identified by the blue line. Oxygen consumption of the cardiac tissue sample in a 2 mL closed chamber is represented by the red line. Malate and glutamate, M/G; Adenosine Diphosphate, ADP; Cytochrome c, Cyto c.
1. Notes: Reagent Preparation
Preparation of K2EGTA 100mM Stock Solution
|Name of the reagent||Final Concentration (mM)||g/100 mL H2O|
|Ethylene glycol-bis-(2-aminoethylether)-N,N,N',N'-tetraacetic acid (EGTA)||100||3.805|
|Potassium Hydroxide (KOH)||200||1.15|
Note: Adjust pH to 7.4 at room temperature.
Preparation of Ca2EGTA 100mM Stock Solution
|Name of the reagent||Final Concentration (mM)||g/100 mL H2O||Comments (optional)|
|Ethylene glycol-bis-(2-aminoethylether)-N,N,N',N'-tetraacetic acid (EGTA)||100||3.805||Heat to 80°C and stir mildly.|
|Calcium Carbonate (CaCO3)||100||1.001||The calcium concentration must be precise as calcium regulates the function of various organelles including mitochondria. Ensure complete solubilization of all CaCO3. The final solution must be completely transparent. CaCO3 is initially mixed with EGTA and some water to activate the formation of carbonic acid and CO2 evaporation. This reaction may be accelerated by heating up to 80 °C.|
|Potassium Hydroxide (KOH)||200||1.15||Neutralize with KOH after evaporation of CO2 is completed.|
Note: Adjust pH to 7.4 at room temperature.
Preparation of Relaxation and Preservation Solution (RP Solution)1
|Reagent||Final Concentration (mM)||Per litre||Comments|
|Adenosine 5'-triphosphate disodium salt hydrate (ATP)||5.7||3.14g|
|Magnesium chloride (MgCl2)||6.56||0.624g|
Note: Adjust pH to 7.1 at room temperature
Preparation of Mitochondrial Respirometry Solution (MiR05 Solution)2
|Reagent||Final Concentration (mM)||Per litre||Comments (optional)|
|Ethylene glycol-bis-(2-aminoethylether)-N,N,N',N'-tetraacetic acid (EGTA)||0.5||0.190g||Used as a chelator of calcium|
|Magnesium chloride hexahydrate (MgCl2.6H2O)||3.0||0.610g||The quality of fiber preparation cannot be tested without Mg2+.|
|Taurine||20.0||2.502g||Taurine is a membrane stabilizer and antioxidant. 20mM is the intracellular concentration present in the heart.|
|Potassium phosphate monobasic (KH2PO4)||10.0||1.361g|
|Potassium-lactobionate||60.0||120 mL of 0.5 M
|0.5M K-lactobionate Stock: Add 35.83 g lactobionic acid to 100 mL H2O and pH to 7.0 at RT. Adjust volume to 200 mL with ddH2O. Used to replicate the high intracellular K+ concentration. Previously KCl was used, however, the high Cl- inhibits mitochondrial creatine kinase function.|
|Sucrose||110.0||37.65g||Used as a ROS scavenger.|
|Bovine Serum Albumin (BSA)||1g/L||1g||Used as a membrane stabilizer, antioxidant, and chelator of calcium and free fatty acids.|
Note: Adjust pH to 7.1 at 30°C
3. Notes: Respirometric OXPHOS Analysis
B. Select Substrates, uncouplers and inhibitors
List of Selected Substrates for Mitochondrial Respirometry Analysis
|Substrate||[Stock]||Preparation||Volume per 2 mL||[Final]||Comments|
|Adenosine 5'-diphosphate monopotassium salt dihydrate (ADP)||500mM||246mg/ mL ddH2O. Adjust pH to 7.1 at RT. Store at -80°C in 250 μL aliquots.||20ul||5mM||To maintain constant Mg2++ during respirometry experiments add 0.6 mol MgCl2/mol ADP.|
|Ascorbate||800mM||0.1584g/ mL ddH2O. Store at -20°C in 200 μL aliquots. Light sensitive||5 μL||2mM||Acts as substrate when used in parallel with TMPD. Must correct for oxygen flux for auto-oxidation.|
|Cytochrome C||4mM||50mg/ mL ddH2O. Store at -20°C in 250 μL aliquots.||5 μL||10uM|
|0.1mM||0.254mg/10 mL 100% EtOH. Store in glass vials at -20°C in 500 μL aliquots.||Steps of 1 μL||Acts as an uncoupler. Determines maximal electron transport capacity and any limitation of electron transport by phosphorylation system.|
|Glutamate||2M||0.3742g/ mL ddH2O. Adjust pH to 7.1 at RT. Store at -20°C in 250 μL aliquots.||10ul||10mM||Acts as a substrate for NADH dehydrogenase (complex I).|
|Malate||800mM||0.1073g/ mL ddH2O. Adjust pH to 7.1 at RT. Store at -20°C in 250 μL aliquots.||5ul||2mM||Acts as a substrate for NADH dehydrogenase (complex I). Cannot support respiration alone.|
|Pyruvate||1M||11mg/0.1 mL ddH2O. Prepare fresh.||5 μL||2.5mM||Acts as a substrate for NADH dehydrogenase (complex I).|
|Succinate||1000mM||1.3505g/5 mL ddH2O. Adjust pH to 7.1 at RT. Store at -20°C in 250 μL aliquots.||20ul||10mM||Acts as a substrate for succinate dehydrogenase (complex II).|
|200mM||47.1mg/ mL ddH2O. Add 0.8M ascorbate to final concentration of 10mM to prevent auto-oxidation Store at -20°C in 200 μL aliquots.||5 μL||0.5mM||Autoxidation of stock solution evident by appearance of blue coloring. Acts as substrate when used in parallel with TMPD. Must correct for oxygen flux for auto-oxidation.|
List of Selected Inhibitors for Mitochondrial Respirometry Analysis
|Substrate||[Stock]||Preparation||Volume per 2 mL Chamber||[Final]||Comments|
|Antimycin A||5mM||27.4mg/10 mL 100% EtOH. Store at -20°C in 250 μL aliquots.||1 μL||2.5μM||Inhibitor of coenzyme Q : cytochrome c oxidoreductase (Complex III)|
|Atractyloside||50mM||40mg/ mL ddH2O. Store at -20°C in 250 μL aliquots.||30 μL||0.75mM||Inhibitor of ATP Synthase.|
|Oligomycin||4mg/ mL||4mg/ mL 100% EtOH. Store at -20°C in 200 μL aliquots.||1 μL||Inhibitor of ATP synthase.|
|Potassium cyanide||1M||65.1mg/ mL ddH2O. Prepare fresh. Adjust pH to 7.1 at RT||1 μL||1mM||Inhibitor of cytochrome c oxidase (complex IV). Utilize following TMPD and Ascorbate titration to access autoxidation.|
|Rotenone||0.1mM||0.39mg/10 mL 100% EtOH. Store at -20°C in 250 μL aliquots. Light sensitive.||1 μL||0.05μM||Inhibitor of NADH dehydrogenase (complex I). Higher concentrations may be required, however, to reduce rotenone retention in chamber begin as outlined.|
The saponin-permeabilized cardiac fiber technique offers a unique compromise between in vitro and in vivo assessment of mitochondrial OXPHOS oxygen consumption. Advantages of this technique include increased physiological relevance in comparison to isolated mitochondria as cellular architecture is preserved. While the plasma membrane is degraded, intracellular membrane structures including mitochondria12, 14, sarcoplasmic reticulum14, myofilaments and the cytoskeleton 1, 17 remain intact. Moreover, the interactions between mitochondria and cytoskeleton1, 17 are also unaltered. Mitochondria in permeabilized fibers show increased stability. Rodent fiber preparations can be stored in ice-cold preservations solutions for 6h and human fiber samples show stability for up to 24h18,2. Mitochondria experience rapid equilibration with the incubation solution. This allows direct control over site-specific analysis of the electron transport chain in response to added substrates, inhibitors and uncouplers1, 5. Additionally, this in situ technique requires only a few milligrams of tissue.
Limitations of the saponin-skinned fiber technique cannot be ignored. Cardiac mitochondria are heterogeneous, consisting of two subpopulations; subsarcolemmal and interfibrillar. In situ mitochondrial respirometry assesses the total mitochondrial population without the ability to distinguish between the subpopulations5. Additionally, various cellular metabolites and cytosolic factors regulate mitochondrial function. These cytosolic components of the cardiac fibers are lost during the permeabilization process resulting in the inability to assess mitochondrial at the exact in vivo environment5.
Reporting issues are an important concern. Oxygen consumption measurements may be expressed per wet weight or dry weight, however, mitochondrial density is a major factor in respiratory flux heterogeneity when expressed per tissue mass7. It is important to understand that interpretation of respiratory rates and changes are greatly influenced by the choice of reference state. For direct comparisons of respirometric OXPHOS results in permeabilized fibers, rates must be expressed relative to a common reference marker such as mitochondrial DNA, citrate synthase activity, cytochrome c oxidase activity, and/or cytochrome aa3 content19.
In summary, the permeabilized fiber preparation used in conjunction with respirometry analysis allows for the assessment of integrative function of cardiac mitochondria. Various pathological states and genetic models have utilized this technique. These include the evaluation of drug-induced toxicity, aging, diabetes, congestive heart failure, ischemic injury and oxidative stress on mitochondrial physiology5, 20. Several excellent methodological reviews and manuscripts of the saponin-permeabilized fiber technique and polarographic oxygen consumption assessment have been previously published1, 5, 14, 20 and are highly recommended.
No conflicts of interest declared.
This study was supported by the Canadian Institutes of Health Research and Genome Canada. JS holds salary support awards from the Alberta Heritage Foundation for Medical Research, Heart and Stroke Foundation of Canada and the Canadian Diabetes Association. The laboratory would like to acknowledge the technical assistance of Oroboros Instruments during the acquisition of the saponin-permeabilized fiber technique.
|100% Ethanol||Fisher Scientific||HC600|
|70% Ethanol||Fisher Scientific||HC-1000|
|Adenosine 5′-diphosphate monopotassium salt dihydrate (ADP)||Sigma-Aldrich||A5285|
|Albumin from bovine serum essentially fatty acid–free||Sigma-Aldrich||A-6003|
|Adenosine 5′-triphosphate disodium salt hydrate (ATP)||Sigma-Aldrich||A2383|
|Carbonyl cyanide p-(trifluoromethoxy) phenylhydrazone (FCCP)||Sigma-Aldrich||C2920|
|Ethylene glycol-bis-(2-amin–thylether)-N,N,N′,N′-tetraacetic acid (EGTA)||Sigma-Aldrich||E4378|
|Ketamine||Pfizer Pharma GmbH||Ketaset|
|Magnesium chloride (MgCl2)||Sigma-Aldrich||M9272|
|Magnesium chloride hexahydrate (MgCl2∙6H2O)||Sigma-Aldrich||M9272|
|N,N,N’,N’-Tetramethyl- pphenylenediamine Dihydrochloride (TMPD)||Sigma-Aldrich||T3134|
|Potassium phosphate monobasic||Sigma-Aldrich||P5655|
|Sodium Pentobarbital||Ceva Sante Animale||1715 138||Conc. 54.7 mg/ml|
|Oroboros Oxygraph-2k||Oroboros Instruments|
|15ml polypropylene centrifuge tubes||VWR international||89004-368|
|50ml polypropylene centrifuge tubes||VWR international||89004-364|
|Straight Jewelers Forceps||George Tiemann & Co.||160-50B|
|Curved Jewelers Forceps||George Tiemann & Co.||160-57B|
|Straight Surgery Scissors||George Tiemann & Co.||105-402|
|Sterile Surgical Blade||VWR international||BD371610|
|0.45-μm Syringe filters||VWR international||CA28145-485|
|pH meter||VWR international||CA11388-308|
|Glass Petri dishes||VWR international||89000-300|
|12-well Polystyrene Tissue Culture Plates||VWR international||82050-926|
|Plate Stirrer||VWR international||97042-594|
|Fisherbrand Microbars||Fisher Scientific||14-511-67|
|Weigh Scale||VWR international||CA11278-162|
|10μl Hamilton Micro Syringe||Fisher Scientific||14-815-1|
|25μl Hamilton Micro Syringe||Fisher Scientific||14-824-7|
|50μl Hamilton Micro Syringe||Fisher Scientific||14-824-5|
|Nalgene Squeeze Bottles||Wilkem Scientific||LNA2407-1000|
|Polystyrene Weighing Dishes||VWR international||89106-750|
|Dissecting Microscope||Olympus Corporation|