Name: high-resolution fluoro-respirometry of equine skeletal muscle
Uploaded: 2023-02-03T14:40:00.000+00:00
Duration: 7 min 39 s
Description: Watch this Scientific Journal Video about High-Resolution Fluoro-Respirometry of Equine Skeletal Muscle at JoVE.com

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.

This study was approved by the Oklahoma State University Institutional Animal Care and Use Committee. Four Thoroughbred geldings (17.5 &#177; 1.3 years, 593 &#177; 45 kg) were used in this study to generate the representative results.
1. Obtaining skeletal muscle biopsy specimen
<ol>
	<li>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.</li>
	<li>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&#160;for specific instructions on preparing the biopsy transport media.</li>
	<li>Isolate mitochondria from the biopsy samples using a commercial kit, according to the manufacturer&#39;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.</li>
	<li>Resuspend the final pellet of the isolated mitochondria using 80 &#181;L 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.</li>
	<li>Minimize the interval from the time of the biopsy procedure to the isolation of mitochondria, and keep the samples at 0-4 &#176;C throughout the processing steps until added to the high-resolution respirometers. 
	&#8203;NOTE: Preliminary studies have found that suspensions of isolated mitochondria begin to lose functional capacity after approximately 2 h when maintained at 0-4 &#176;C.</li>
</ol>
2. Setting up of the high-resolution respirometer
<ol>
	<li>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.</li>
	<li>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&#39;s instructions. Retain these calibration constants for subsequent assays until the calibration is repeated. 
	NOTE: In contrast to previously published procedures using permeabilized muscle fibers11,12,13,16, in which hyperoxia (250-500 &#181;M) is necessary to avoid diffusion limitation of O2 to the mitochondria, isolated mitochondria can be evaluated using a range of 50-200 &#181;M 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.</li>
	<li>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.
	<ol>
		<li>Set the instrument incubation temperature to 38 &#176;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.</li>
		<li>Turn chamber illumination off to avoid interference with fluorescent sensors.</li>
		<li>Energize the oxygen electrode with an 800 mV polarization voltage, and amplify the resulting signal with a gain setting of 1.</li>
		<li>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.</li>
	</ol>
	</li>
	<li>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.</li>
</ol>
3. Measurement of mitochondrial membrane potential using TMRM
<ol>
	<li>Use &#34;Green&#34; 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.</li>
	<li>Add TMRM (4 &#181;L of 1 mM solution for a final concentration of 2 &#181;M; see Table of Materials) before the addition of mitochondria.</li>
	<li>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.</li>
	<li>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 &#181;L per titration) until no further increases in TMRM fluorescent signal are observed, indicating the complete collapse of mitochondrial membrane potential (Figure 1). 
	&#8203;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.</li>
</ol>
4. Measurement of ATP production using magnesium green (MgG)
<ol>
	<li>Use &#34;Blue&#34; 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.</li>
	<li>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 &#181;L 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 &#181;L of 1 mM MgG (1.1 &#181;M) to the respiration chamber.</li>
	<li>Calibrate the raw fluorescence signal with 10 x 2 &#181;L 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 &#62; 0.98), that will be used with a known amount of ADP added to the respirometry chamber and the previously determined Kd values to determine the corresponding concentration of ATP11.</li>
	<li>Determine the rate of ATP synthesis, which is the slope of the concentration of ATP over time throughout the protocol (Figure 3).</li>
</ol>
5. Measurement of mitochondrial production of ROS&#160;using Amplex UltraRed (AmR)
<ol>
	<li>Use &#34;Green&#34; 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 &#181;L of 100 mM MgCl2 to produce 1-3 &#181;M MgCl2) prior to adding reagents for the AmR assay.</li>
	<li>Perform the chemical setup and initial calibration of the AmR assay prior to the addition of the mitochondria. Add 30 &#181;moles of DTPA (6 &#181;L of 5 mM solution) to chelate cations that might interfere with the reaction, then add superoxide dismutase (2 &#181;L 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 &#181;L of a 500 U/mL stock solution), and Amplex UltraRed (2 &#181;L of a 10 mM stock solution) (see Table of Materials) to produce 5 U/mL, 1 U/mL, and 10 &#181;M in the respirometry chamber, respectively.</li>
	<li>Allow the fluorescent signal to stabilize, then add 0.2 &#181;moles of hydrogen peroxide (5 &#181;L of a 40 &#181;M 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 &#62; 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.</li>
	<li>Perform additional two-point calibrations (5 &#181;L of a 40 &#181;M 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).</li>
</ol>
6. Measurement of mitochondrial respiration
<ol>
	<li>Add 15 &#181;L 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.</li>
	<li>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)&#160;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.</li>
	<li>Use a general-purpose SUIT that allows for the initial characterization of equine skeletal muscle mitochondrial function. Start with sequential titrations of pyruvate (5 &#181;L of 2 M aqueous solution), glutamate (10 &#181;L of 2 M aqueous solution), and malate (10 &#181;L 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 (LN) (Figure 1, Figure 3, and Figure 4).</li>
	<li>Add ADP (20 &#181;L of 500 mM aqueous solution; see Table of Materials) to stimulate phosphorylating respiration through Complex I (PN).</li>
	<li>Add succinate (20 &#181;L of 1 M aqueous solution; see Table of Materials) to produce phosphorylating respiration through the combination of Complex I and Complex II (PN+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 &#181;M, reoxygenate the incubation media by unsealing the incubation chamber until the measured O2 concentration has increased above 150 &#181;M. Repeat this step as necessary to maintain sufficient O2 for mitochondrial respiration.</li>
	<li>Add rotenone (2 &#181;L 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 (PS). 
	CAUTION: Rotenone is a poisonous substance. Use standard laboratory safety and avoid ingestion or inhalation.</li>
	<li>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 FCRLeak (LN/PN), FCRN (PN/PN+S), and FCRS (PS/PN+S). Calculated oxidative phosphorylation efficiency (1-LN/PN+S) provides an estimate of the overall impact of leak respiration adjusted for total respiratory capacity.</li>
</ol>

<ol>
	<li>Wilson, D. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Energy+metabolism+in+muscle+approaching+maximal+rates+of+oxygen+utilization.">Energy metabolism in muscle approaching maximal rates of oxygen utilization.</a> Medicine and Science in Sports and Exercise. 27 (1), 54-59 (1995).</li><li>Gnaiger, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Mitochondrial Pathways and Respiratory Control. An Introduction to OXPHOS Analysis. 4th edn. , (2014).</li><li>Ehrenberg, B., Montana, V., Wei, M. D., Wuskell, J. P., Loew, L. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Membrane+potential+can+be+determined+in+individual+cells+from+the+nernstian+distribution+of+cationic+dyes.">Membrane potential can be determined in individual cells from the nernstian distribution of cationic dyes.</a> Biophysical Journal. 53 (5), 785-794 (1988).</li><li>Chinopoulos, C., Kiss, G., Kawamata, H., Starkov, A. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measurement+of+ADP-ATP+exchange+in+relation+to+mitochondrial+transmembrane+potential+and+oxygen+consumption.">Measurement of ADP-ATP exchange in relation to mitochondrial transmembrane potential and oxygen consumption.</a> Methods in Enzymology. 542, 333-348 (2014).</li><li>Krumschnabel, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Simultaneous+high-resolution+measurement+of+mitochondrial+respiration+and+hydrogen+peroxide+production.">Simultaneous high-resolution measurement of mitochondrial respiration and hydrogen peroxide production.</a> Methods in Molecular Biology. 1264, 245-261 (2015).</li><li>Lemieux, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitochondrial+function+is+altered+in+horse+atypical+myopathy.">Mitochondrial function is altered in horse atypical myopathy.</a> Mitochondrion. 30, 35-41 (2016).</li><li>Houben, R., Leleu, C., Fraipont, A., Serteyn, D., Votion, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Determination+of+muscle+mitochondrial+respiratory+capacity+in+Standardbred+racehorses+as+an+aid+to+predicting+exertional+rhabdomyolysis.">Determination of muscle mitochondrial respiratory capacity in Standardbred racehorses as an aid to predicting exertional rhabdomyolysis.</a> Mitochondrion. 24, 99-104 (2015).</li><li>Votion, D. M., Gnaiger, E., Lemieux, H., Mouithys-Mickalad, A., Serteyn, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Physical+fitness+and+mitochondrial+respiratory+capacity+in+horse+skeletal+muscle.">Physical fitness and mitochondrial respiratory capacity in horse skeletal muscle.</a> PLoS One. 7 (4), 34890 (2012).</li><li>Votion, D. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Alterations+in+mitochondrial+respiratory+function+in+response+to+endurance+training+and+endurance+racing.">Alterations in mitochondrial respiratory function in response to endurance training and endurance racing.</a> Equine Veterinary Journal Supplement. (38), 268-274 (2010).</li><li>Tosi, I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Altered+mitochondrial+oxidative+phosphorylation+capacity+in+horses+suffering+from+polysaccharide+storage+myopathy.">Altered mitochondrial oxidative phosphorylation capacity in horses suffering from polysaccharide storage myopathy.</a> Journal of Bioenergetics and Biomembranes. 50 (5), 379-390 (2018).</li><li>Davis, M. S., Fulton, M. R., Popken, A. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+hyperthermia+and+acidosis+on+equine+skeletal+muscle+mitochondrial+oxygen+consumption.">Effect of hyperthermia and acidosis on equine skeletal muscle mitochondrial oxygen consumption.</a> Comparative Exercise Physiology. 17 (2), 171-179 (2021).</li><li>Latham, C. M., Fenger, C. K., White, S. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RAPID+COMMUNICATION:+Differential+skeletal+muscle+mitochondrial+characteristics+of+weanling+racing-bred+horses1.">RAPID COMMUNICATION: Differential skeletal muscle mitochondrial characteristics of weanling racing-bred horses1.</a> Journal of Animal Science. , (2019).</li><li>White, S. H., Warren, L. K., Li, C., Wohlgemuth, S. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Submaximal+exercise+training+improves+mitochondrial+efficiency+in+the+gluteus+medius+but+not+in+the+triceps+brachii+of+young+equine+athletes.">Submaximal exercise training improves mitochondrial efficiency in the gluteus medius but not in the triceps brachii of young equine athletes.</a> Scientific Reports. 7 (1), 14389 (2017).</li><li>White, S. H., Wohlgemuth, S., Li, C., Warren, L. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+communication:+Dietary+selenium+improves+skeletal+muscle+mitochondrial+biogenesis+in+young+equine+athletes.">Rapid communication: Dietary selenium improves skeletal muscle mitochondrial biogenesis in young equine athletes.</a> Journal of Animal Science. 95 (9), 4078-4084 (2017).</li><li>Doerrier, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-resolution+FluoRespirometry+and+OXPHOS+protocols+for+human+cells,+permeabilized+fibers+from+small+biopsies+of+muscle,+and+isolated+mitochondria.">High-resolution FluoRespirometry and OXPHOS protocols for human cells, permeabilized fibers from small biopsies of muscle, and isolated mitochondria.</a> Methods in Molecular Biology. 1782, 31-70 (2018).</li><li>Li, C., White, S. H., Warren, L. K., Wohlgemuth, S. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+aging+on+mitochondrial+function+in+skeletal+muscle+of+American+American+Quarter+Horses.">Effects of aging on mitochondrial function in skeletal muscle of American American Quarter Horses.</a> Journal of Applied Physiology. 121 (1), 299-311 (2016).</li><li>Komlodi, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+mitochondrial+incubation+media+for+measurement+of+respiration+and+hydrogen+peroxide+production.">Comparison of mitochondrial incubation media for measurement of respiration and hydrogen peroxide production.</a> Methods in Molecular Biology. 1782, 137-155 (2018).</li><li>Gnaiger, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitochondrial+physiology.">Mitochondrial physiology.</a> Bioenergetic Communications. , (2020).</li><li>Li Puma, L. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Experimental+oxygen+concentration+influences+rates+of+mitochondrial+hydrogen+peroxide+release+from+cardiac+and+skeletal+muscle+preparations.">Experimental oxygen concentration influences rates of mitochondrial hydrogen peroxide release from cardiac and skeletal muscle preparations.</a> American Journal of Physiology: Regulatory, Integrated, and Comparative Physiology. 318 (5), 972-980 (2020).</li></ol>

The authors have no conflicts of interest related to this manuscript.

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...

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&#160;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&#160;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.

<table><tbody><tr><td>ADP</td><td>Sigma-Aldrich (MilliporeSigma)</td><td>A5285</td><td></td></tr><tr><td>Amplex UltraRed</td><td>Life Technologies</td><td>A36006</td><td></td></tr><tr><td>ATP</td><td>Sigma-Aldrich (MilliporeSigma)</td><td>A2383</td><td></td></tr><tr><td>BSA</td><td>Sigma-Aldrich (MilliporeSigma)</td><td>A6003</td><td></td></tr><tr><td>Calcium carbonate</td><td>Sigma-Aldrich (MilliporeSigma)</td><td>C4830</td><td></td></tr><tr><td>CCCP</td><td>Sigma-Aldrich (MilliporeSigma)</td><td>C2759</td><td></td></tr><tr><td>DatLab 7.0</td><td>Oroboros Inc</td><td></td><td>Software to operate O&lt;sub&gt;2&lt;/sub&gt;K fluororespirometer</td></tr><tr><td>Dithiothreitol</td><td>Sigma-Aldrich (MilliporeSigma)</td><td>D0632</td><td></td></tr><tr><td>DTPA</td><td>Sigma-Aldrich (MilliporeSigma)</td><td>D1133</td><td></td></tr><tr><td>EGTA</td><td>Sigma-Aldrich (MilliporeSigma)</td><td>E4378</td><td></td></tr><tr><td>Glutamate</td><td>Sigma-Aldrich (MilliporeSigma)</td><td>G1626</td><td></td></tr><tr><td>HEPES</td><td>Sigma-Aldrich (MilliporeSigma)</td><td>H7523</td><td></td></tr><tr><td>Horseradish peroxidase</td><td>Sigma-Aldrich (MilliporeSigma)</td><td>P8250</td><td></td></tr><tr><td>Hydrogen peroxide</td><td>Sigma-Aldrich (MilliporeSigma)</td><td>516813</td><td>Must be made fresh daily prior to assay</td></tr><tr><td>Imidazole</td><td>Sigma-Aldrich (MilliporeSigma)</td><td>I2399</td><td></td></tr><tr><td>K-MES</td><td>Sigma-Aldrich (MilliporeSigma)</td><td>M8250</td><td></td></tr><tr><td>Magnesium chloride hexahydrate</td><td>Sigma-Aldrich (MilliporeSigma)</td><td>M9272</td><td></td></tr><tr><td>Magnesium Green</td><td>Thermo Fisher Scientific</td><td>M3733</td><td></td></tr><tr><td>Malate</td><td>Sigma-Aldrich (MilliporeSigma)</td><td>M1000</td><td></td></tr><tr><td>Mannitol</td><td>Sigma-Aldrich (MilliporeSigma)</td><td>M9647</td><td></td></tr><tr><td>Mitochondrial isolation kit</td><td>Sigma-Aldrich (MilliporeSigma)</td><td>MITOISO1</td><td></td></tr><tr><td>O&lt;sub&gt;2&lt;/sub&gt;K fluororespirometer</td><td>Oroboros Inc</td><td></td><td>Multiple units required to run full spectrum of assays concurrently.</td></tr><tr><td>Phosphocreatine</td><td>Sigma-Aldrich (MilliporeSigma)</td><td>P7936</td><td></td></tr><tr><td>Potassium hydroxide</td><td>Sigma-Aldrich (MilliporeSigma)</td><td>P1767</td><td></td></tr><tr><td>Potassium lactobionate</td><td>Sigma-Aldrich (MilliporeSigma)</td><td>L2398</td><td></td></tr><tr><td>Potassium phosphate</td><td>Sigma-Aldrich (MilliporeSigma)</td><td>P0662</td><td></td></tr><tr><td>Pyruvate</td><td>Sigma-Aldrich (MilliporeSigma)</td><td>P2256</td><td>Must be made fresh daily prior to assay</td></tr><tr><td>Rotenone</td><td>Sigma-Aldrich (MilliporeSigma)</td><td>R8875</td><td></td></tr><tr><td>Succinate</td><td>Sigma-Aldrich (MilliporeSigma)</td><td>S2378</td><td></td></tr><tr><td>Sucrose</td><td>Sigma-Aldrich (MilliporeSigma)</td><td>84097</td><td></td></tr><tr><td>Superoxide dismutase</td><td>Sigma-Aldrich (MilliporeSigma)</td><td>S8160</td><td></td></tr><tr><td>Taurine</td><td>Sigma-Aldrich (MilliporeSigma)</td><td>T0625</td><td></td></tr><tr><td>Titration pump</td><td>Oroboros Inc</td><td></td><td></td></tr><tr><td>Titration syringes</td><td>Oroboros Inc</td><td></td><td></td></tr><tr><td>TMRM</td><td>Sigma-Aldrich (MilliporeSigma)</td><td>T5428</td><td></td></tr><tr><td>UCH biopsy needle</td><td>Millenium Surgical Corp</td><td>72-238067</td><td>Available in a range of sizes</td></tr></tbody></table>

high-resolution fluoro-respirometry of 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 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 &#176;C and pH 7.0). Under these conditions, the investigator can expect LN values of 2.71 &#177; 0.90, PN values of 62.40 &#177; 26.22, PN+S ...

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High-Resolution Fluoro-Respirometry of Equine Skeletal Muscle

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 ...

high-resolution-fluoro-respirometry-of-equine-skeletal-muscle

Research

JoVE Journal

Biology

1.2K Views.  Oklahoma State University. Deze protocollen stellen de onderzoekers in staat om verder te gaan dan alleen het meten van mitochondriaal zuurstofverbruik, en in plaats daarvan de meting van belangrijke mitochondriale eindproducten, ATP en reactieve zuurstofsoorten mogelijk te maken. Het belangrijkste voordeel van deze techniek is dat de onderzoeker de mitochondriale efficiëntie kan meten door de snelheid van zuurstofverbruik direct te vergelijken met de snelheid van de productie van het eindproduct. Of het eindproduct n...

Video: High-Resolution Fluoro-Respirometry of Equine Skeletal Muscle

Using Isolated Mitochondria from Minimal Quantities of Mouse Skeletal Muscle for High throughput Microplate Respiratory Measurements

Skeletal muscle mitochondria play a specific role in many disease pathologies. As such, the measurement of oxygen consumption as an indicator of mitochondrial function in this tissue has become more prevalent. Although many technologies and assays exist that measure mitochondrial respiratory pathways in a variety of cells, tissue and species, there is currently a void in the literature in regards to the compilation of these assays using isolated mitochondria from mouse skeletal muscle for use in microplate based technologies. Importantly, the use of microplate based respirometric assays is growing among mitochondrial biologists as it allows for high throughput measurements using minimal quantities of isolated mitochondria. Therefore, a collection of microplate based respirometric assays were developed that are able to assess mechanistic changes/adaptations in oxygen consumption in a commonly used animal model. The methods presented herein provide step-by-step instructions to perform these assays with an optimal amount of mitochondrial protein and reagents, and high precision as evidenced by the minimal variance across the dynamic range of each assay.

The methods presented provide step-by-step instructions for the performance of a collection of microplate based respirometric assays using isolated mitochondria from minimal quantities of mouse skeletal muscle. These assays are able to measure mechanistic changes/adaptations in mitochondrial oxygen consumption in a commonly used animal model.

Skeletal muscle mitochondria play a specific role in many disease pathologies. As such, the measurement of oxygen consumption as an indicator of ...

Developmental Biology

Preparation and Respirometric Assessment of Mitochondria Isolated from Skeletal Muscle Tissue Obtained by Percutaneous Needle Biopsy

Respirometric profiling of isolated mitochondria is commonly used to investigate electron transport chain function. We describe a method for obtaining samples of human Vastus lateralis, isolating mitochondria from minimal amounts of skeletal muscle tissue, and plate based respirometric profiling using an extracellular flux (XF) analyzer. Comparison of respirometric profiles obtained using 1.0, 2.5 and 5.0 &#956;g of mitochondria indicate that 1.0 &#956;g is sufficient to measure respiration and that 5.0 &#956;g provides most consistent results based on comparison of standard errors. Western blot analysis of isolated mitochondria for mitochondrial marker COX IV and non-mitochondrial tissue marker GAPDH indicate that there is limited non-mitochondrial contamination using this protocol. The ability to study mitochondrial respirometry in as little as 20 mg of muscle tissue allows users to utilize individual biopsies for multiple study endpoints in clinical research projects.

Methods for biopsy of Vastus lateralis, preparation of purified mitochondria, and respirometric profiling are described. The use of small muscle volume makes this technique suitable for clinical research applications.

Respirometric profiling of isolated mitochondria is commonly used to investigate electron transport chain function. We describe a method for obtaining ...

Medicine

Adult and Embryonic Skeletal Muscle Microexplant Culture and Isolation of Skeletal Muscle Stem Cells

Cultured embryonic and adult skeletal muscle cells have a number of different uses. The micro-dissected explants technique described in this chapter is a robust and reliable method for isolating relatively large numbers of proliferative skeletal muscle cells from juvenile, adult or embryonic muscles as a source of skeletal muscle stem cells. The authors have used micro-dissected explant cultures to analyse the growth characteristics of skeletal muscle cells in wild-type and dystrophic muscles. Each of the components of tissue growth, namely cell survival, proliferation, senescence and differentiation can be analysed separately using the methods described here. The net effect of all components of growth can be established by means of measuring explant outgrowth rates. The micro-explant method can be used to establish primary cultures from a wide range of different muscle types and ages and, as described here, has been adapted by the authors to enable the isolation of embryonic skeletal muscle precursors.
Uniquely, micro-explant cultures have been used to derive clonal (single cell origin) skeletal muscle stem cell (SMSc) lines which can be expanded and used for in vivo transplantation. In vivo transplanted SMSc behave as functional, tissue-specific, satellite cells which contribute to skeletal muscle fibre regeneration but which are also retained (in the satellite cell niche) as a small pool of undifferentiated stem cells which can be re-isolated into culture using the micro-explant method.

The micro-dissected explants technique is a robust and reliable method for isolating proliferative skeletal muscle cells from juvenile, adult or embryonic muscles as a source of skeletal muscle stem cells. Uniquely, these cells have been clonally derived to produce skeletal muscle stem cell lines used for in vivo transplantation.

Cultured embryonic and adult skeletal muscle cells have a number of different uses. The micro-dissected explants technique described in this chapter is a ...

Simultaneous Brightfield, Fluorescence, and Optical Coherence Tomographic Imaging of Contracting Cardiac Trabeculae Ex Vivo

In cardiac muscle, intracellular Ca2+ transients activate contractile myofilaments, causing contraction, macroscopic shortening, and geometric&#160;deformation. Our understanding of the internal relationships between these events has been limited because we can neither &#39;see&#39; inside the muscle nor precisely track the spatio-temporal nature of excitation-contraction dynamics. To resolve these problems, we have constructed a device that combines a suite of imaging modalities. Specifically, it integrates a brightfield microscope to measure local changes of sarcomere length and tissue strain, a fluorescence microscope to visualize the Ca2+ transient, and an optical coherence tomograph to capture the tissue&#39;s geometric&#160;changes throughout the time-course of a cardiac cycle. We present here the imaging infrastructure and associated data collection framework. Data are collected from isolated rod-like tissue structures known as trabeculae carneae. In our instrument, a pair of position-controlled platinum hooks hold each end of an ex vivo muscle sample while it is continuously superfused with nutrient-rich saline solution. The hooks are under independent control, permitting real-time control of muscle length and force. Lengthwise translation enables the piecewise scanning of the sample, overcoming limitations associated with the relative size of the microscope imaging window (540 &#181;m by 540 &#181;m) and the length of a typical trabecula (&#62;2000 &#181;m). Platinum electrodes at either end of the muscle chamber stimulate the trabecula at a user-defined rate. We exploit the stimulation signal as a trigger for synchronizing the data from each imaging window to reconstruct the entire sample twitching under steady-state conditions. Applying image-processing techniques to these brightfield imaging data provides tissue displacement and sarcomere length maps. Such a collection of data, when incorporated into an experiment-modeling pipeline, will provide a deeper understanding of muscle contractile homogeneity and heterogeneity in physiology and pathophysiology.

Simultaneous Brightfield, Fluorescence, and Optical Coherence Tomographic Imaging of Contracting Cardiac Trabeculae Ex Vivo

This protocol presents a collection of sarcomere, calcium, and macroscopic geometric&#160;data from an actively contracting cardiac trabecula ex vivo. These simultaneous measurements are made possible by the integration of three imaging modalities.

In cardiac muscle, intracellular Ca2+ transients activate contractile myofilaments, causing contraction, macroscopic shortening, and ...

Bioengineering

Confocal Imaging of Single Mitochondrial Superoxide Flashes in Intact Heart or In Vivo

Mitochondrion is a critical intracellular organelle responsible for energy production and intracellular signaling in eukaryotic systems. Mitochondrial dysfunction often accompanies and contributes to human disease. Majority of the approaches that have been developed to evaluate mitochondrial function and dysfunction are based on in vitro or ex vivo measurements. Results from these experiments have limited ability in determining mitochondrial function in vivo. Here, we describe a novel approach that utilizes confocal scanning microscopy for the imaging of intact tissues in live aminals, which allows the evaluation of single mitochondrial function in a real-time manner in vivo. First, we generate transgenic mice expressing the mitochondrial targeted superoxide indicator, circularly permuted yellow fluorescent protein (mt-cpYFP). Anesthetized mt-cpYFP mouse is fixed on a custom-made stage adaptor and time-lapse images are taken from the exposed skeletal muscles of the hindlimb. The mouse is subsequently sacrificed and the heart is set up for Langendorff perfusion with physiological solutions at 37 &deg;C. The perfused heart is positioned in a special chamber on the confocal microscope stage and gentle pressure is applied to immobilize the heart and suppress heart beat induced motion artifact. Superoxide flashes are detected by real-time 2D confocal imaging at a frequency of one frame per second. The perfusion solution can be modified to contain different respiration substrates or other fluorescent indicators. The perfusion can also be adjusted to produce disease models such as ischemia and reperfusion. This technique is a unique approach for determining the function of single mitochondrion in intact tissues and in vivo.

Confocal Imaging of Single Mitochondrial Superoxide Flashes in Intact Heart or In Vivo

Confocal scanning microscopy is applied for imaging single mitochondrial events in perfused heart or skeletal muscles in live animal. Real-time monitoring of single mitochondrial processes such as superoxide flashes and membrane potential fluctuations enables the evaluation of mitochondrial function in a physiologically relevant context and during pathological perturbations.

Mitochondrion is a critical intracellular organelle responsible for energy production and intracellular signaling in eukaryotic systems. Mitochondrial ...

High-resolution Respirometry to Assess Mitochondrial Function in Permeabilized and Intact Cells

A high-resolution oxygraph is a device for measuring cellular oxygen consumption in a closed-chamber system with very high resolution and sensitivity in biological samples (intact and permeabilized cells, tissues or isolated mitochondria). The high-resolution oxygraph device is equipped with two chambers and uses polarographic oxygen sensors to measure oxygen concentration and calculate oxygen consumption within each chamber. Oxygen consumption rates are calculated using software and expressed as picomoles per second per number of cells. Each high-resolution oxygraph chamber contains a stopper with injection ports, which makes it ideal for substrate-uncoupler-inhibitor titrations or detergent titration protocols for determining effective and optimum concentrations for plasma membrane permeabilization. The technique can be applied to measure respiration in a wide range of cell types and also provides information on mitochondrial quality and integrity, and maximal mitochondrial respiratory electron transport system capacity.

High-resolution respirometry is used to determine mitochondrial oxygen consumption. This is a straightforward technique to determine mitochondrial respiratory chain complexes&#39; (I-IV) respiratory rates, maximal mitochondrial electron transport system capacity, and mitochondrial outer membrane integrity.

A high-resolution oxygraph is a device for measuring cellular oxygen consumption in a closed-chamber system with very high resolution and sensitivity in ...

Isolation of Intact Mitochondria from Skeletal Muscle by Differential Centrifugation for High-resolution Respirometry Measurements

Mitochondria are involved in cellular energy metabolism and use oxygen to produce energy in the form of adenosine triphosphate (ATP). Differential centrifugation at low- and high-speed is commonly used to isolate mitochondria from tissues and cultured cells. Crude mitochondrial fractions obtained by differential centrifugation are used for respirometry measurements. The differential centrifugation technique is based on the separation of organelles according to their size and sedimentation velocity. The isolation of mitochondria is performed immediately after tissue harvesting. The tissue is immersed in an ice-cold homogenization medium, minced using scissors and homogenized in a glass homogenizer with a loose-fitting pestle. The differential centrifugation technique is efficient, fast and inexpensive and the mitochondria obtained by differential centrifugation are pure enough for respirometry assays. Some of the limitations and disadvantages of isolated mitochondria, based on differential centrifugation, are that the mitochondria can be damaged during the homogenization and isolation procedure and that large amounts of the tissue biopsy or cultured cells are required for the mitochondrial isolation.

Here, a quadriceps muscle specimen is taken from an anaesthetized pig and mitochondria are isolated by differential centrifugation. Then, the respiratory rates of mitochondrial respiratory chain complexes I, II and IV are determined using high-resolution respirometry.

Mitochondria are involved in cellular energy metabolism and use oxygen to produce energy in the form of adenosine triphosphate (ATP). Differential ...

High-Resolution Respirometry to Assess Bioenergetics in Cells and Tissues Using Chamber- and Plate-Based Respirometers

High-resolution respirometry (HRR) allows monitoring oxidative phosphorylation in real-time for analysis of individual cellular energy states and assessment of respiratory complexes using diversified substrate-uncoupler-inhibitor titration (SUIT) protocols. Here, the usage of two high-resolution respirometry devices is demonstrated, and a basic collection of protocols applicable for the analysis of cultured cells, skeletal and heart muscle fibers, and soft tissues such as the brain and liver are presented. Protocols for cultured cells and tissues are provided for a chamber-based respirometer and cultured cells for a microplate-based respirometer, both encompassing standard respiration protocols. For comparative purposes, CRISPR-engineered HEK293 cells deficient in mitochondrial translation causing multiple respiratory system deficiency are used with both devices to demonstrate cellular defects in respiration. Both respirometers allow for comprehensive measurement of cellular respiration with their respective technical merits and suitability dependent on the research question and model under study.

Assessing oxidative phosphorylation using high-resolution respirometers has become an integral part of the functional analysis of mitochondria and cellular energy metabolism. Here, we present protocols for the analysis of cellular energy metabolism using chamber and microplate-based high-resolution respirometers and discuss the key benefits of each device.

High-resolution respirometry (HRR) allows monitoring oxidative phosphorylation in real-time for analysis of individual cellular energy states and ...

Functional Site-Directed Fluorometry in Native Skeletal Muscle Cells

Functional Site-Directed Fluorometry in Native Cells to Study Skeletal Muscle Excitability

Functional site-directed fluorometry has been the technique of choice to investigate the structure-function relationship of numerous membrane proteins, including voltage-gated ion channels. This approach has been used primarily in heterologous expression systems to simultaneously measure membrane currents, the electrical manifestation of the channels&#39; activity, and fluorescence measurements, reporting local domain rearrangements. Functional site-directed fluorometry combines electrophysiology, molecular biology, chemistry, and fluorescence into a single wide-ranging technique that permits the study of real-time structural rearrangements and function through fluorescence and electrophysiology, respectively. Typically, this approach requires an engineered voltage-gated membrane channel that contains a cysteine that can be tested by a thiol-reactive fluorescent dye. Until recently, the thiol-reactive chemistry used for the site-directed fluorescent labeling of proteins was carried out exclusively in Xenopus oocytes and cell lines, restricting the scope of the approach to primary non-excitable cells. This report describes the applicability of functional site-directed fluorometry in adult skeletal muscle cells to study the early steps of excitation-contraction coupling, the process by which muscle fiber electrical depolarization is linked to the activation of muscle contraction. The present protocol describes the methodologies to design and transfect cysteine-engineered voltage-gated Ca2+ channels (CaV1.1) into muscle fibers of the flexor digitorum brevis of adult mice using in vivo electroporation and the subsequent steps required for functional site-directed fluorometry measurements. This approach can be adapted to study other ion channels and proteins. The use of functional site-directed fluorometry of mammalian muscle is particularly relevant to studying basic mechanisms of excitability.

Author Spotlight: Functional Site-Directed Fluorometry in Native Cells to Study Skeletal Muscle Excitability  

Functional site-directed fluorometry is a method to study protein domain motions in real time. Modification of this technique for its application in native cells now allows the detection and tracking of single voltage-sensor motions from voltage-gated Ca2+ channels in murine isolated skeletal muscle fibers.

Functional site-directed fluorometry has been the technique of choice to investigate the structure-function relationship of numerous membrane proteins, ...

Real-Time Quantification of Mitochondrial Respiration in Muscle Fibers

Measurement of Mitochondrial Respiration in Human and Mouse Skeletal Muscle Fibers by High-Resolution Respirometry

Mitochondrial function, a cornerstone of cellular energy production, is critical for maintaining metabolic homeostasis. Its dysfunction in skeletal muscle is linked to prevalent metabolic disorders (e.g., diabetes and obesity), muscular dystrophies, and sarcopenia. While there are many techniques to evaluate mitochondrial content and morphology, the hallmark method to assess mitochondrial function is the measurement of mitochondrial oxidative phosphorylation (OXPHOS) by respirometry. Quantification of mitochondrial OXPHOS provides insight into the efficiency of mitochondrial oxidative energy production and cellular bioenergetics. A high-resolution respirometer provides highly sensitive, robust measurements of mitochondrial OXPHOS in permeabilized muscle fibers by measuring real-time changes in mitochondrial oxygen consumption rate. The use of permeabilized muscle fibers, as opposed to isolated mitochondria, preserves mitochondrial networks, maintains mitochondrial membrane integrity, and ultimately allows for more physiologically relevant measurements. This system also allows for the measurement of fuel preference and metabolic flexibility - dynamic aspects of muscle energy metabolism. Here, we provide a comprehensive guide for mitochondrial OXPHOS measurements in human and mouse skeletal muscle fibers using a high-resolution respirometer. Skeletal muscle groups are composed of different fiber types that vary in their mitochondrial fuel preference and bioenergetics. Using a high-resolution respirometer, we describe methods for evaluating both aerobic glycolytic and fatty acid substrates to assess fuel preference and metabolic flexibility in a fiber-type-dependent manner. The protocol is versatile and applicable to both human and rodent muscle fibers. The goal is to enhance the reproducibility and accuracy of mitochondrial function assessments, which will improve our understanding of an organelle important to muscle health.

Author Spotlight: Unveiling Mitochondrial Function and Cellular Metabolic Adaptation in Metabolic Diseases

Here, we describe a comprehensive method for measuring mitochondrial oxidative phosphorylation in fresh permeabilized skeletal muscle fibers from either human or mouse muscle. This method allows for the real-time quantification of mitochondrial respiration and the assessment of fuel preference and metabolic flexibility while preserving existing mitochondrial networks and membrane integrity.

Mitochondrial function, a cornerstone of cellular energy production, is critical for maintaining metabolic homeostasis. Its dysfunction in skeletal muscle ...

High-Resolution Fluoro-Respirometry of Equine Skeletal Muscle

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