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 diseases^{6,7,8,9,10,11,12,13,14}. 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

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

1. Obtain skeletal muscle biopsies (follow sterile technique) from the center of the semitendinosus muscle (or other muscle of interest), using a 12 G University College Hospital (UCH) biopsy needle (see Table of Materials) while under light sedation, and using local anesthesia following a previously published report¹¹.
2. Transfer the biopsy specimens immediately into vials with ice-cold biopsy transport media solution (2.77 mM CaK₂-EGTA, 7.23 mM K₂-EGTA, 20 mM imidazole, 20 mM taurine, 50 mM K-MES, 0.5 mM dithiothreitol, 6.56 mM MgCl₂, 5.77 mM ATP, and 15 mM phosphocreatine, adjusted to pH 7.1; see Table of Materials). Transport to the laboratory for analysis.
   NOTE: Refer to Doerrier et al.¹⁵ for specific instructions on preparing the biopsy transport media.
3. Isolate mitochondria from the biopsy samples using a commercial kit, according to the manufacturer's instructions (see Table of Materials). The final storage buffer should not contain substrates such as ADP, ATP, and succinate, that are part of the respirometry assay.
4. Resuspend the final pellet of the isolated mitochondria using 80 µL of suspension media (225 mM mannitol, 75 mM sucrose, and 1 mM EGTA; see Table of Materials) per 100 mg of muscle used for mitochondria isolation.
5. Minimize the interval from the time of the biopsy procedure to the isolation of mitochondria, and keep the samples at 0-4 °C throughout the processing steps until added to the high-resolution respirometers.
   ​NOTE: Preliminary studies have found that suspensions of isolated mitochondria begin to lose functional capacity after approximately 2 h when maintained at 0-4 °C.

2. Setting up of the high-resolution respirometer

1. High-resolution respirometers are used to quantify mitochondrial respiration and associated processes. Use the software control program provided by the manufacturer (see Table of Materials) to control the respirometer, calibrate the sensors, and collect raw data. Analyze the samples in duplicate for each test condition.
   NOTE: In all cases, the recommended titrations and final reagent concentrations described in this protocol are based on a 2 mL respirometry chamber.
2. Determine the background O₂ flux and zero point of the O₂ sensor every 2-4 weeks through the serial titration of dithionite, following the manufacturer's instructions. Retain these calibration constants for subsequent assays until the calibration is repeated.
   NOTE: In contrast to previously published procedures using permeabilized muscle fibers^11,12,13,16, in which hyperoxia (250-500 µM) is necessary to avoid diffusion limitation of O₂ to the mitochondria, isolated mitochondria can be evaluated using a range of 50-200 µM O₂. This can be achieved simply by allowing the respiration media to equilibrate with room air prior to adding the mitochondrial suspension (i.e., following daily single-point calibration). Similarly, reoxygenation of the respiration chamber can be accomplished by simply opening the chamber until sufficient ambient oxygen has dissolved into the respirometry media to raise the oxygen concentration to the desired level.
3. Fill the respirometer chambers with magnesium-free media (0.5 mM EGTA, 60 mM K-lactobionate, 20 mM taurine, 10 mM KH₂PO₄, 20 mM HEPES, 110 mM sucrose, and 1 g/L bovine serum albumin [BSA] essentially fatty acid-free, adjusted to pH 7.1; see Table of Materials).
   NOTE: Refer to Komlodi et al.¹⁷ for specific instructions on preparing the respiration media.
   1. Set the instrument incubation temperature to 38 °C to represent the basal temperature of equine skeletal muscle, and set the mixing of the respiration media to 800 rpm using a magnetic stirrer turning in the bottom of the respirometer chamber.
   2. Turn chamber illumination off to avoid interference with fluorescent sensors.
   3. Energize the oxygen electrode with an 800 mV polarization voltage, and amplify the resulting signal with a gain setting of 1.
   4. Record the oxygen concentration every 2 s, calculate the oxygen flux as the negative slope of the oxygen measurement over the preceding 40 s (20 data points), and report as pmol x s^-1 x mL of incubation solution.
4. Calibrate the oxygen sensor by allowing the media to equilibrate with room air. Calculate the reference oxygen partial pressure, based on barometric pressure measured by the high-resolution respirometer and standard atmospheric oxygen concentration.

3. Measurement of mitochondrial membrane potential using TMRM

1. Use "Green" fluorescent sensors (530 nm dominant wavelength) to quantify the fluorescent signal from the respiration chamber. Energize the sensors at 400-500 mV; the resulting signal gets amplified with a gain of 1:1,000.
   NOTE: Specific settings must be optimized for individual instruments to capture the expected signal within the linear range of the sensor.
2. Add TMRM (4 µL of 1 mM solution for a final concentration of 2 µM; see Table of Materials) before the addition of mitochondria.
3. Calibrate the fluorescent signal using a simple two-point calibration of fluorescent signal (voltage) versus the amount of added fluorophore (mM), prior to the addition of the mitochondria.
   NOTE: Use the fluorescent signal calibration function in the machine control software to calibrate this signal. The raw signal from the fluorescent probe can require 30 min or more to stabilize in order to record the second point of the calibration.
4. Perform the final calibration of the TMRM signal after completion of the respirometry titration protocol by delivering several titrations of uncoupling agent (carbonyl cyanide m-chlorophenyl hydrazone; 2 µL per titration) until no further increases in TMRM fluorescent signal are observed, indicating the complete collapse of mitochondrial membrane potential (Figure 1).
   ​NOTE: This fluorescent signal value is considered equal to 0 mV transmembrane potential, and used as the reference point for relative membrane potential values recorded during the respirometry titration protocol.

4. Measurement of ATP production using magnesium green (MgG)

1. Use "Blue" fluorescent sensors (465 nm dominant wavelength) to quantify the fluorescent signal from the respiration chamber. Energize these sensors for individual instruments to capture the expected signal within the linear range of the sensor.
2. Perform the chemical setup and calibration of the MgG fluorescent signal after the addition of the mitochondria, but prior to the addition of any substrates. Add 8 µL of 2 mM ethylenediaminetetraacetic acid (EDTA) to the respirometry chamber to chelate cations (particularly Ca²⁺) that would compete with Mg²⁺ for binding to MgG, then add 4 µL of 1 mM MgG (1.1 µM) to the respiration chamber.
3. Calibrate the raw fluorescence signal with 10 x 2 µL sequential titrations of 100 mM MgCl₂, allowing 1 min between titrations for stabilization of the fluorescent signal (Figure 2).
   NOTE: This process, which is completed offline following completion of the assay and using templates provided by the manufacturer of the machine and associated software, produces a second-order curve of magnesium concentration to fluorescent signal (expected r² > 0.98), that will be used with a known amount of ADP added to the respirometry chamber and the previously determined K_d values to determine the corresponding concentration of ATP¹¹.
4. Determine the rate of ATP synthesis, which is the slope of the concentration of ATP over time throughout the protocol (Figure 3).

5. Measurement of mitochondrial production of ROS using Amplex UltraRed (AmR)

1. Use "Green" fluorescent sensors (530 nm dominant wavelength) to quantify the fluorescent signal from the respiration chamber. Energize the sensors at 300-400 mV; the resulting signal gets amplified with a gain of 1:1,000. Optimize specific settings for individual instruments to capture the expected signal within the linear range of the sensor.
   NOTE: If using respiration media without MgCl₂, then this should be added (20-60 µL of 100 mM MgCl₂ to produce 1-3 µM MgCl₂) prior to adding reagents for the AmR assay.
2. Perform the chemical setup and initial calibration of the AmR assay prior to the addition of the mitochondria. Add 30 µmoles of DTPA (6 µL of 5 mM solution) to chelate cations that might interfere with the reaction, then add superoxide dismutase (2 µL of a 5,000 U/mL stock solution to convert superoxide anions to H₂O₂ for a more comprehensive detection of reactive oxygen generation), horseradish peroxidase (5 µL of a 500 U/mL stock solution), and Amplex UltraRed (2 µL of a 10 mM stock solution) (see Table of Materials) to produce 5 U/mL, 1 U/mL, and 10 µM in the respirometry chamber, respectively.
3. Allow the fluorescent signal to stabilize, then add 0.2 µmoles of hydrogen peroxide (5 µL of a 40 µM solution made fresh daily) twice, approximately 5 min apart.
   NOTE: The fluorescent signal before and after the two titrations of H₂O₂ provides a three-point linear calibration curve of the system (expected r² > 0.95), the slope of which reflects the overall responsiveness of the system in reporting the relationship between the fluorescent signal and the production (or addition) of H₂O₂. Use the fluorescent signal calibration function in the machine control software to calibrate this signal.
4. Perform additional two-point calibrations (5 µL of a 40 µM solution made fresh daily) throughout the assay, to allow adjustment of the responsiveness of the assay as the chemistry of the respirometry changes throughout the assay, with the specific timing of these calibration points at the discretion of the investigator (Figure 4).

6. Measurement of mitochondrial respiration

1. Add 15 µL of isolated mitochondria suspension (step 1.4) to each 2 mL incubation chamber, so that the results represent the mitochondrial yield of 18.75 mg of muscle. Seal the incubation chamber. Vortex the sample between each titration to maintain a uniform suspension of the sample.
2. Measure the residual oxygen consumption (ROX) prior to the addition of any substrates. Subtract this value (typically less than 0.2 pmol O₂ x s^-1 x mL^-1) from the oxygen consumption values of each step in the substrate/uncoupler/inhibitor titration (SUIT) protocol¹¹ after completion of the protocol.
   NOTE: It is critical that the signals from both the oxygen sensor and the fluorescence sensor are allowed to stabilize for at least 1 min (as assessed by the stable calculated slope of the primary sensor signals), as the overall state of respiration is changed by the titrations in order to obtain reliable results. This applies to all titration steps.
3. Use a general-purpose SUIT that allows for the initial characterization of equine skeletal muscle mitochondrial function. Start with sequential titrations of pyruvate (5 µL of 2 M aqueous solution), glutamate (10 µL of 2 M aqueous solution), and malate (10 µL of 0.4 M aqueous solution) (see Table of Materials) into each chamber to produce Nicotinamide adenine dinucleotide (NADH) and stimulate non-phosphorylating (leak) respiration supported by NADH oxidized through Complex I (L_N) (Figure 1, Figure 3, and Figure 4).
4. Add ADP (20 µL of 500 mM aqueous solution; see Table of Materials) to stimulate phosphorylating respiration through Complex I (P_N).
5. Add succinate (20 µL of 1 M aqueous solution; see Table of Materials) to produce phosphorylating respiration through the combination of Complex I and Complex II (P_N+S).
   NOTE: With the combination of respiration through both Complex I and Complex II, oxygen consumption may be high enough to consume most of the oxygen dissolved in the incubation media. If the O₂ concentration decreases below 50 µM, reoxygenate the incubation media by unsealing the incubation chamber until the measured O₂ concentration has increased above 150 µM. Repeat this step as necessary to maintain sufficient O₂ for mitochondrial respiration.
6. Add rotenone (2 µL of 0.1 mM ethanol solution; see Table of Materials) to block Complex I. The resulting oxygen flux represents the capacity of Complex II to support mitochondrial oxygen consumption via the oxidation of succinate alone (P_S).
   CAUTION: Rotenone is a poisonous substance. Use standard laboratory safety and avoid ingestion or inhalation.
7. Calculate the mean value for a given experimental condition across individual respirometry chambers containing aliquots of a single biopsy.
   NOTE: Derived calculations, such as flux control ratios (FCRs), are valuable in identifying relative changes in different pathways, and include FCR_Leak (L_N/P_N), FCR_N (P_N/P_N+S), and FCR_S (P_S/P_N+S). Calculated oxidative phosphorylation efficiency (1-L_N/P_N+S) provides an estimate of the overall impact of leak respiration adjusted for total respiratory capacity.

Results

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 L_N values of 2.71 ± 0.90, P_N values of 62.40 ± 26.22, P_N+S ...

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...

Materials

Name	Company	Catalog Number	Comments
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