Respirometric Oxidative Phosphorylation Assessment in Saponin-permeabilized Cardiac Fibers
Summary
Saponin-permeabilized fiber preparation in conjunction with respirometric oxidative phosphorylation analysis provides integrative assessment of mitochondrial function. Mitochondrial respiration in physiological and pathological states can reflect various regulatory influences including mitochondrial interactions, morphology and biochemistry.
Abstract
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.
Protocol
Discussion
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<…
Divulgaciones
The authors have nothing to disclose.
Acknowledgements
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.
Materials
| Material Name | Tipo | Company | Catalogue Number | Comment |
|---|---|---|---|---|
| 100% Ethanol | Fisher Scientific | HC600 | ||
| 70% Ethanol | Fisher Scientific | HC-1000 | ||
| Adenosine 5′-diphosphate monopotassium salt dihydrate (ADP) | Sigma | A5285 | ||
| Albumin from bovine serum essentially fatty acid–free | Sigma | A-6003 | ||
| Antimycin A | Sigma | A8674 | ||
| Ascobic acid | Sigma | A4403 | ||
| Adenosine 5′-triphosphate disodium salt hydrate (ATP) | Sigma | A2383 | ||
| Atractyloside | Sigma | A6882 | ||
| Calcium carbonate | Sigma | C4830 | ||
| Carbonyl cyanide p-(trifluoromethoxy) phenylhydrazone (FCCP) | Sigma | C2920 | ||
| Cytochrome c | Sigma | C7752 | ||
| Digitonin | Sigma | D141 | ||
| Dithiothreitol | Sigma | D9779 | ||
| Ethylene glycol-bis-(2-aminoethylether)-N,N,N′,N′-tetraacetic acid (EGTA) | Sigma | E4378 | ||
| Glutamic acid | Sigma | 27647 | ||
| HEPES | Sigma | H4034 | ||
| Imidazole | Sigma | I5513 | ||
| Ketamine | Pfizer | Ketaset | ||
| Lactobionic acid | Sigma | 153516 | ||
| Magnesium chloride (MgCl2) | Sigma | M9272 | ||
| Magnesium chloride hexahydrate (MgCl2∙6H2O) | Sigma | M9272 | ||
| Malic acid | Sigma | M1000 | ||
| MES | Sigma | M3671 | ||
| N,N,N’,N’-Tetramethyl- pphenylenediamine Dihydrochloride (TMPD) | Sigma | T3134 | ||
| Oligomycin | Sigma | O4876 | ||
| Phosphocreatine | Sigma | P7936 | ||
| Potassium Chloride | Sigma | P9541 | ||
| Potassium Hydroxide | Sigma | P5958 | ||
| Potassium cyanide | Fluka | 60178 | ||
| Potassium phosphate monobasic | Sigma | P5655 | ||
| Rotenone | Sigma | R8875 | ||
| Saponin | Sigma | 47036 | ||
| Sodium Pentobarbital | Ceva Sante Animale | 1715 138 | Conc. 54.7 mg/ml | |
| Sodium pyruvate | Sigma | P2256 | ||
| Succinic acid | Sigma | S3674 | ||
| Sucrose | Sigma | S7903 | ||
| Taurine | Sigma | T8691 | ||
| Xylazine | Bayer | Rompun | ||
| ddH2O | ||||
| Ice | ||||
| Oroboros Oxygraph-2k | Oroboros Instruments | |||
| Kimwipes | VWR | 21905-026 | ||
| 15ml polypropylene centrifuge tubes | VWR | 89004-368 | ||
| 50ml polypropylene centrifuge tubes | VWR | 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 | BD371610 | ||
| 0.45-μm Syringe filters | VWR | CA28145-485 | ||
| pH meter | VWR | CA11388-308 | ||
| Glass Petri dishes | VWR | 89000-300 | ||
| 12-well Polystyrene Tissue Culture Plates | VWR | 82050-926 | ||
| Plate Stirrer | VWR | 97042-594 | ||
| Fisherbrand Microbars | Fisher Scientific | 14-511-67 | ||
| Weigh Scale | VWR | 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 | 89106-750 | ||
| Dissecting Microscope | Olympus |
Referencias
- Saks, V. A., Veksler, V. I., Kuznetsov, A. V. Permeabilized cell and skinned fiber techniques in studies of mitochondrial function in vivo. Mol Cell Biochem. 184, 81-100 (1998).
- Gnaiger, E., Kuznetsov, A. V., Schneeberger, S., Heldmaier, G., Klingenspor, M. Mitochondria in the cold. Life in the Cold. , 431-442 (2000).
- Rasmussen, H. N., Rasmussen, U. F. Oxygen solubilities of media used in electrochemical respiration measurements. Anal Biochem. 319, 105-113 (2003).
- Visscher, G. D. e., Rooker, S., Jorens, P. Pentobarbital fails to reduce cerebral oxygen consumption early after non-hemorrhagic closed head injury in rats. J Neurotrauma. 22, 793-806 (2005).
- Kuznetsov, A. V., Veksler, V., Gellerich, F. N. Analysis of mitochondrial function in situ in permeabilized muscle fibers, tissues and cells. Nat Protoc. 3, 965-976 (2008).
- Gnaiger, E. Oxygen conformance of cellular respiration. A perspective of mitochondrial physiology. Adv Exp Med Biol. 543, 39-55 (2003).
- Gnaiger, E. Capacity of oxidative phosphorylation in human skeletal muscle: new perspectives of mitochondrial physiology. Int J Biochem Cell Biol. 41, 1837-1845 .
- Sena, S., Hu, P., Zhang, D. Impaired insulin signaling accelerates cardiac mitochondrial dysfunction after myocardial infarction. J Mol Cell Cardiol. 46, 910-918 (2009).
- Boudina, S., Sena, S., O’Neill, B. T. Reduced mitochondrial oxidative capacity and increased mitochondrial uncoupling impair myocardial energetics in obesity. Circulation. 112, 2686-2695 (2005).
- Lenaz, G., Genova, M. L. Structure and organization of mitochondrial respiratory complexes: a new understanding of an old subject. Antioxid Redox Signal. 12, 961-1008 .
- Lemieux, H., Hoppel, C. L. Mitochondria in the human heart. J Bioenerg Biomembr. 41, 99-106 (2009).
- O, . Retarded diffusion of ADP in cardiomyocytes: possible role of mitochondrial outer membrane and creatine kinase in cellular regulation of oxidative phosphorylation. Biochim Biophys Acta. 1144, 134-148 (1993).
- Endo, M., Kitazawa, T., Morad, M. E-C coupling studies in skinned cardiac fibers. Biophysical Aspects of Cardiac Muscle. , 307-327 (1978).
- Veksler, V. I., Kuznetsov, A. V., Sharov, V. G. Mitochondrial respiratory parameters in cardiac tissue: a novel method of assessment by using saponin-skinned fibers. Biochim Biophys Acta. 892, 191-196 (1987).
- Bangham, A. D., Horne, R. W., Glauert, A. M. Action of saponin on biological cell membranes. Nature. , 196-952 (1962).
- Daum, G. Lipids of mitochondria. Biochim Biophys Acta. 822, 1-42 (1985).
- Milner, D. J., Mavroidis, M., Weisleder, N. Desmin cytoskeleton linked to muscle mitochondrial distribution and respiratory function. J Cell Biol. 150, 1283-1298 (2000).
- Skladal, D., Sperl, W., Schranzhofer, R., Skladal, E., Gellerich, F., Wyss, M. Preservation of mitochondrial functions in human skeletal muscle during storage in high energy preservation solution (HEPS). What is Controlling Life?. , 268-271 (1994).
- Kuznetsov, A. V., Wiedemann, F. R., Winkler, K. Use of saponin-permeabilized muscle fibers for the diagnosis of mitochondrial diseases. Biofactors. 7, 221-223 (1998).
- Gnaiger, E., Dykens, J., Will, Y. Polarographic oxygen sensors, the oxygraph and high-resolution respirometry to assess mitochondrial function. Drug-Induced Mitochondrial Dysfunction. , 327-352 (2008).
