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.
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.
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.
This study was approved by the Oklahoma State University Institutional Animal Care and Use Committee. Four Thoroughbred geldings (17.5 ± 1.3 years, 593 ± 45 kg) were used in this study to generate the representative results.
1. Obtaining skeletal muscle biopsy specimen
2. Setting up of the high-resolution respirometer
3. Measurement of mitochondrial membrane potential using TMRM
4. Measurement of ATP production using magnesium green (MgG)
5. Measurement of mitochondrial production of ROS using Amplex UltraRed (AmR)
6. Measurement of mitochondrial respiration
The proposed reference state is that of a healthy sedentary Thoroughbred (no increased fitness due to compulsory exercise) and a fresh muscle sample collected from the center of a postural muscle, containing a high percentage of mitochondria-rich type I skeletal muscle fibers and incubated under conditions approximating resting metabolism (i.e., 38 °C and pH 7.0). Under these conditions, the investigator can expect LN values of 2.71 ± 0.90, PN values of 62.40 ± 26.22, PN+S values of 93.67 ± 34.76, and PS values of 46.93 ± 14.58 pmol O2 x s-1 x mL-1 (Figure 3 and Figure 4). Respiration values of mitochondria incubated with TMRM are lower due to an inhibitory effect of that fluorophore (Figure 1). FCRLeak, FCRN, and FCRS are 0.05 ± 0.01, 0.66 ± 0.07, and 0.51 ± 0.07, respectively. The calculated oxidative phosphorylation efficiency is 0.97 ± 0.01. The absolute values for these respiratory states vary due to the individual subject and tissue oxidative capacity, but the relative pattern of these values is consistent with published results from permeabilized equine skeletal muscle fibers (i.e., increases in respiration with the addition of specific substrates, the combination of respiration through Complex I and Complex II being less than each of these electron sources individually due to saturation of the electron transfer system [ETS] downstream capacity). The calculated efficiency of isolated equine skeletal muscle mitochondria is consistent with the corresponding values reported for permeabilized equine skeletal muscle fibers11.
The rate of ATP synthesis is expected to be 438.59 ± 397.10 pM x s-1 for PN respiration, 383.18 ± 397.19 for PN+S respiration, and 172.07 ± 125.60 for PS respiration, per mg of source tissue used to isolate mitochondria. The decrease in ATP synthesis with the addition of succinate is likely due to competition at the quinone junction of the ETS by the two electron feeds, with the feed from Complex II (which does not contribute directly to mitochondrial membrane potential) reducing the flow of electrons through Complex I (which directly contributes to the mitochondrial membrane potential through the pumping of protons across the inner mitochondrial membrane).
The rate of H2O2 production is expected to be 0.079 ± 0.095 nmol.s-1 for LN respiration, 0.021 ± 0.043 for PN respiration, 0.026 ± 0.056 for PN+S respiration, and 0.237 ± 0.248 for PS respiration, per mg of source tissue used to isolate mitochondria (Figure 4). The relative rate of ROS production is consistent with the predicted magnitude of the mitochondrial membrane potential and the association between high membrane potential and ROS production. The exception to this relationship is the very high ROS production when respiration occurs through Complex II alone, due to the backward flow of electrons from the quinone junction to Complex I when it is blocked by rotenone.
Figure 1: Representative high-resolution respirometry trace of equine skeletal muscle mitochondrial respiration and relative membrane potential. The top window is the chamber oxygen concentration (left Y-axis) and oxygen flux (right Y-axis) over time (X-axis); the bottom window is chamber TMRM fluorescence (left Y-axis) and rate of fluorescence signal change (right Y-axis) over time (X-axis). Vertical marks indicate the addition of specific substrates to the chamber (mt: mitochondria suspension; P: pyruvate; G: glutamate; M: malate; D: ADP; S: succinate; R: rotenone; CCCP: Carbonyl cyanide m-chlorophenyl hydrazone). Please click here to view a larger version of this figure.
Figure 2: Representative high-resolution respirometry trace of calibration of chamber fluorescence signal for magnesium green and determination of Kd for magnesium and adenylates. Chamber MgG fluorescence (left Y-axis) and rate of fluorescence signal change (right Y-axis) over time (X-axis). The initial vertical marks indicate the addition of specific substrates to the chamber (MgG: Magnesium green; mt: mitochondria suspension; P: pyruvate; G: glutamate; M: malate; S: succinate; EDTA: ethylenediaminetetraacetic acid). This is followed by serial titration of MgCl2 using an automated titration pump (TIP) that increases the fluorescent signal, then serial titration of an adenylate (in this case ATP) that decreases the fluorescent signal. The titration of MgCl2 is also used at the beginning of each assay to calibrate the MgG signal, but is carried out prior to the addition of mitochondria and respirometry substrates such as P, G, M, and S. Please click here to view a larger version of this figure.
Figure 3: Representative high-resolution respirometry trace of equine skeletal muscle mitochondrial respiration and ATP synthesis. The top window is the chamber oxygen concentration (left Y-axis) and oxygen flux (right Y-axis) over time (X-axis); the bottom window is chamber MgG fluorescence (left Y-axis) and rate of fluorescence signal change (right Y-axis) over time (X-axis). Vertical marks indicate the addition of specific substrates to the chamber (mt: mitochondria suspension; P: pyruvate; G: glutamate; M: malate; D: ADP; S: succinate; R: rotenone). Please click here to view a larger version of this figure.
Figure 4: Representative high-resolution respirometry trace of equine skeletal muscle mitochondrial respiration and production of H2O2. The top window is the chamber oxygen concentration (left Y-axis) and oxygen flux (right Y-axis) over time (X-axis); the bottom window is chamber resorufin fluorescence (left Y-axis) and rate of fluorescence signal change (right Y-axis) over time (X-axis). Vertical marks indicate the addition of specific substrates to the chamber (mt: mitochondria suspension; P: pyruvate; G: glutamate; M: malate; D: ADP; S: succinate; R: rotenone). Also included are three intra-assay two-point calibrations of the fluorescent signal using H2O2. Please click here to view a larger version of this figure.
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 sufficiently reproducible from assay to assay for this approach. Therefore, each assay is calibrated with an automated MgCl2 titration, and the resulting calibration curve for calculating ATP synthesis for that individual assay. Furthermore, the Kd (dissociation constants) for magnesium and both ADP and ATP must be calculated for the specific conditions of the assay, including the specific tissue, incubation conditions, and substrates producing phosphorylating respiration, in order to ensure that the derivation of the ATP synthesis rate is accurate. The variability in the AmR assay is more widely recognized15,17, and it is common to insert several discrete recalibration points into the protocol at the investigator's discretion during the protocol. Historical data using equine skeletal muscle indicates that the typical responsiveness of the assay is approximately 0.646 V/mM of H2O2 at the first calibration point (prior to the addition of the sample), and may decrease 8%-15% over the span of the assay. Thus, frequent recalibration of the AmR assay is necessary for the detection of relatively small changes in the production of ROS, and the timing of the point calibrations within the protocol should be determined for each titration protocol.
Investigators must employ careful judgment throughout the assay performance in determining when to move to the next titration step. Ideally, the relevant signals being generated in real-time (O2 flux, fluorescence of TMRM, and the rate of change in MgG and resorufin fluorescent signal) should be stable, indicating a steady state within the chamber, but it is a judgment call on the part of the investigator as to what constitutes acceptable stability. At this time, there is no widely held standard for this decision; however, investigators must be acutely cognizant of the risks from both failing to wait long enough for a steady state to be established (resulting in data that imperfectly characterizes the intended respiratory state) and waiting so long that substrates become depleted, and the assay is no longer reliable. In the author's experience, failing to attain a stable signal within 30 min of any substrate titration is evidence of a sample (or machine) that is behaving in a non-biological manner, and the assay must be discarded.
The results using this approach to quantify mitochondrial physiology demonstrate considerable intersubject variability, with a typical coefficient of variation (CV) of 30%-40%. By contrast, analysis of permeabilized equine skeletal muscle fibers results in a CV of 19%-20%11, suggesting that the process of isolating mitochondria imposes substantial variability to the subsequent analysis in addition to the marked intersubject variability. Although the effect of the latter can be mitigated by using subjects as their own controls when possible, the former can only be minimized through careful attention to the isolation technique. If the experimental design does not allow for experimental replicates from a single biopsy, then the investigator may elect to express the respirometry data relative to a correction factor that reflects differences in mitochondrial content in different samples. The most common correction factors are the sample protein content and citrate synthase activity of the sample. However, as outlined in a recent consensus statement of scientists active in this field, there is no single method for performing this adjustment that is universally agreed upon18. Correcting for protein content and/or citrate synthase activity are the two most common approaches used for equine skeletal muscle tissue12,13, but both have their shortcomings. It may be necessary for different experimental designs to express data through both internal (i.e., flux control ratios) and external (protein, enzyme activity, other mitochondrial markers) in order to interpret mitochondrial function.
The described protocol builds upon the use of permeabilized muscle fibers, with several important differences. Permeabilized fiber assays require smaller biopsies, as high-resolution respirometry of equine skeletal muscle permeabilized fibers is typically performed with only ~2 mg of sample mass in each chamber6,7,8,9,10,11. In contrast, the protocol described here has the equivalent of nearly 10x that much fiber mass in each chamber. This difference highlights the inefficiency of mitochondrial isolation from fresh tissue. A possible reason for this inefficiency is the difficulty in recovering the skeletal muscle mitochondria that are located within the contractile protein network. The commercial kit for mitochondrial isolation used in this protocol includes treatment with a protease and the use of ionic media to help break down the contractile fibers, but it is likely that a substantial portion of the intermyofibrillar mitochondria is not recovered. This feature of the protocol is not only relevant for determining the amount of biopsy material needed, but also with regards to interpretation of the results. Isolated mitochondria are less susceptible to oxygen dependence, due to diffusion limitation compared to permeabilized fibers15; thus, hyperoxia is not needed with isolated mitochondria, and the respiration media can be sufficiently oxygenated (and re-oxygenated during the assay) by simply opening the chamber to room air. This aspect of oxygen delivery is particularly useful when measuring the production of H2O2 and other ROS, as ROS production is increased in a non-physiological manner during hyperoxic incubation19. Finally, the use of isolated mitochondria provides a more stable and reproducible fluorescent signal, due to the decreased non-specific protein binding of fluorophores compared to the use of fluorophores with permeabilized muscle fibers. Each sample preparation has advantages and disadvantages, and proficiency with both permeabilized fibers and isolated mitochondria provides the investigator with a robust set of assays for addressing a variety of questions related to mitochondrial physiology.
The authors have nothing to disclose.
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.
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 |