High-Resolution Fluoro-Respirometry of Equine Skeletal Muscle
Summary
Horses have an exceptional aerobic exercise capacity, making equine skeletal muscle an important tissue for both the study of equine exercise physiology as well as mammalian mitochondrial physiology. This article describes techniques for the comprehensive assessment of mitochondrial function in equine skeletal muscle.
Abstract
Mitochondrial function-oxidative phosphorylation and the generation of reactive oxygen species-is critical in both health and disease. Thus, measuring mitochondrial function is fundamental in biomedical research. Skeletal muscle is a robust source of mitochondria, particularly in animals with a very high aerobic capacity, such as horses, making them ideal subjects for studying mitochondrial physiology. This article demonstrates the use of high-resolution respirometry with concurrent fluorometry, with freshly harvested skeletal muscle mitochondria, to quantify the capacity to oxidize substrates under different mitochondrial states and determine the relative capacities of distinct elements of mitochondrial respiration. Tetramethylrhodamine methylester is used to demonstrate the production of mitochondrial membrane potential resulting from substrate oxidation, including calculation of the relative efficiency of the mitochondria by calculating the relative membrane potential generated per unit of concurrent oxygen flux. The conversion of ADP to ATP results in a change in the concentration of magnesium in the reaction chamber, due to differing affinities of the adenylates for magnesium. Therefore, magnesium green can be used to measure the rate of ATP synthesis, allowing the further calculation of the oxidative phosphorylation efficiency (ratio of phosphorylation to oxidation [P/O]). Finally, the use of Amplex UltraRed, which produces a fluorescent product (resorufin) when combined with hydrogen peroxide, allows the quantification of reactive oxygen species production during mitochondrial respiration, as well as the relationship between ROS production and concurrent respiration. These techniques allow the robust quantification of mitochondrial physiology under a variety of different simulated conditions, thus shedding light on the contribution of this critical cellular component to both health and disease.
Introduction
The mitochondria of eukaryotic cells produce the majority of the ATP used by the cells for work and maintenance1. A key step in the mitochondrial production of ATP is the conversion of oxygen to water, and thus the metabolic capacity of mitochondria and the associated cells is frequently quantified through the measurement of oxygen consumption2. However, mitochondrial physiology is more complex than the simple process of oxygen consumption, and reliance on this endpoint exclusively provides an incomplete assessment of the impact of mitochondrial function and dysfunction on cellular health. Full characterization of mitochondrial function requires the assessment of not only oxygen consumption, but also the production of ATP as well as reactive oxygen species (ROS).
Additional measures of key mitochondrial functions can be accomplished concurrently with the measurement of respiration through the use of specific fluorophores. Tetramethylrhodamine methylester (TMRM) is a cationic fluorophore that accumulates in the mitochondrial matrix in proportion to the mitochondrial transmembrane voltage potential, resulting in a decrease in fluorescent intensity due to this accumulation3. TMRM can be used as an indicator of relative changes in mitochondrial membrane potential, or can be used to quantify precise changes in transmembrane voltage with additional experiments to determine constants that allow conversion of the fluorescent signal to mV. Magnesium green (MgG) is a fluorophore that fluoresces when bound with Mg2+, and is used for measurements of ATP synthesis based on the differential affinity of ADP and ATP for magnesium divalent cation4. Investigators must determine the specific affinity/dissociation constants (Kd) for both ADP and ATP under specific analytical conditions to convert the changes in MgG fluorescence to a change in ATP concentration. Amplex UltraRed (AmR) is the fluorophore used to measure the production of hydrogen peroxide and other ROS during mitochondrial respiration5. The reaction between H2O2 and AmR (which is catalyzed by horseradish peroxidase) produces resorufin, which is detectable through fluorescence at 530 nM. Each of these assays can be added individually to assays of real-time mitochondrial respiration, to provide concurrent measurements of the respective aspects of mitochondrial physiology, thus providing a direct link between respiration and mitochondrial output.
Horses are capable of very high rates of mass-specific oxygen consumption, due in part to the very high mitochondrial content of equine skeletal muscle, making this tissue highly relevant for studying mitochondrial physiology. With the development of high-resolution respirometry, studies using this novel technology have helped define the contributions of equine skeletal muscle mitochondria to both the remarkable exercise capacity of horses and the pathophysiology of skeletal muscle diseases6,7,8,9,10,11,12,13,14. Studies of equine skeletal muscle mitochondrial function are particularly advantageous, as obtaining large amounts of this tissue is non-terminal. Thus, equine subjects can not only provide sufficient tissue for the complete characterization of mitochondrial function, but also serve as longitudinal controls for high-quality, mechanistic studies into mitochondrial physiology. For this reason, additional assays to quantify mitochondrial membrane potential, ATP synthesis, and the production of ROS that complement the measurement of oxygen consumption in this tissue have been developed, in order to provide a more robust characterization of mitochondrial physiology in equine skeletal muscle.
Protocol
Representative Results
Discussion
The addition of fluorescent signals to the standard output of the high-resolution respirometer provides valuable information regarding mitochondrial physiology, but meticulous calibration of the fluorescent signal is critical for quality data. The original protocols for the use of MgG suggest that the calibration curves generated while calculating magnesium-adenylate dissociation constants could be applied to subsequent assays4; however, the fluorescent signal from the MgG may not be not sufficien…
Declarações
The authors have nothing to disclose.
Acknowledgements
The authors would like to acknowledge the generous support of the John and Debbie Oxley Endowed Chair for Equine Sports Medicine and the Grayson Jockey Club Research Foundation.
Materials
| ADP | Sigma-Aldrich (MilliporeSigma) | A5285 | |
| Amplex UltraRed | Life Technologies | A36006 | |
| ATP | Sigma-Aldrich (MilliporeSigma) | A2383 | |
| BSA | Sigma-Aldrich (MilliporeSigma) | A6003 | |
| Calcium carbonate | Sigma-Aldrich (MilliporeSigma) | C4830 | |
| CCCP | Sigma-Aldrich (MilliporeSigma) | C2759 | |
| DatLab 7.0 | Oroboros Inc | Software to operate O2K fluororespirometer | |
| Dithiothreitol | Sigma-Aldrich (MilliporeSigma) | D0632 | |
| DTPA | Sigma-Aldrich (MilliporeSigma) | D1133 | |
| EGTA | Sigma-Aldrich (MilliporeSigma) | E4378 | |
| Glutamate | Sigma-Aldrich (MilliporeSigma) | G1626 | |
| HEPES | Sigma-Aldrich (MilliporeSigma) | H7523 | |
| Horseradish peroxidase | Sigma-Aldrich (MilliporeSigma) | P8250 | |
| Hydrogen peroxide | Sigma-Aldrich (MilliporeSigma) | 516813 | Must be made fresh daily prior to assay |
| Imidazole | Sigma-Aldrich (MilliporeSigma) | I2399 | |
| K-MES | Sigma-Aldrich (MilliporeSigma) | M8250 | |
| Magnesium chloride hexahydrate | Sigma-Aldrich (MilliporeSigma) | M9272 | |
| Magnesium Green | Thermo Fisher Scientific | M3733 | |
| Malate | Sigma-Aldrich (MilliporeSigma) | M1000 | |
| Mannitol | Sigma-Aldrich (MilliporeSigma) | M9647 | |
| Mitochondrial isolation kit | Sigma-Aldrich (MilliporeSigma) | MITOISO1 | |
| O2K fluororespirometer | Oroboros Inc | Multiple units required to run full spectrum of assays concurrently. | |
| Phosphocreatine | Sigma-Aldrich (MilliporeSigma) | P7936 | |
| Potassium hydroxide | Sigma-Aldrich (MilliporeSigma) | P1767 | |
| Potassium lactobionate | Sigma-Aldrich (MilliporeSigma) | L2398 | |
| Potassium phosphate | Sigma-Aldrich (MilliporeSigma) | P0662 | |
| Pyruvate | Sigma-Aldrich (MilliporeSigma) | P2256 | Must be made fresh daily prior to assay |
| Rotenone | Sigma-Aldrich (MilliporeSigma) | R8875 | |
| Succinate | Sigma-Aldrich (MilliporeSigma) | S2378 | |
| Sucrose | Sigma-Aldrich (MilliporeSigma) | 84097 | |
| Superoxide dismutase | Sigma-Aldrich (MilliporeSigma) | S8160 | |
| Taurine | Sigma-Aldrich (MilliporeSigma) | T0625 | |
| Titration pump | Oroboros Inc | ||
| Titration syringes | Oroboros Inc | ||
| TMRM | Sigma-Aldrich (MilliporeSigma) | T5428 | |
| UCH biopsy needle | Millenium Surgical Corp | 72-238067 | Available in a range of sizes |
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