calibration of high-resolution respirometry instrument for measuring mitochondrial respiration in murine samples 

Real-Time Quantification of Mitochondrial Respiration in Muscle Fibers

Mitochondria are the cornerstone of cellular energy production, making them indispensable for maintaining optimal cellular and organismal homeostasis. These double-membraned organelles are primarily responsible for oxidative phosphorylation. This process efficiently converts nutrients, such as sugars and fatty acids, into adenosine triphosphate (ATP), the cellular currency for energy. Beyond their role in energy metabolism, mitochondria are also key regulators of various cellular processes, including apoptosis, calcium homeostasis, and reactive oxygen species (ROS)1,2. Because of their pivotal role in maintaining cellular and organismal homeostasis, disruptions in mitochondrial function often have detrimental effects on tissue and organismal health. In skeletal muscle, mitochondrial dysfunction is associated with numerous disease states, including metabolic disorders (e.g., obesity, diabetes, and cardiovascular disease), sarcopenia, and muscular dystrophy3,4,5,6,7,8.
Mitochondrial dysfunction can primarily manifest as altered mitochondrial content, number, and morphology, as well as disrupted metabolism. Thus, achieving a comprehensive understanding of mitochondrial dysfunction requires an integrative and holistic approach. Initial characterization studies involve examining expression levels of respiratory chain protein complexes as a readout of mitochondrial content, quantifying mitochondrial DNA and markers of biogenesis as a measure of mitochondrial biogenesis, and sophisticated electron microscopy imaging to assess mitochondrial morphology9,10. Additional assessments of mitochondrial function include evaluating cellular ROS and ATP production and mitochondrial membrane potential9.
Because mitochondria are essential for cellular energy production and homeostasis, a hallmark for assessing mitochondrial function is to measure mitochondrial oxidative phosphorylation (OXPHOS). High-resolution respirometry of permeabilized muscle fibers allows for the measurement of real-time changes in mitochondrial oxygen consumption rate as a readout for the dynamic changes in mitochondrial OXPHOS respiratory chain activity9,11,12. The application of selective chemical modulators and inhibitors allows one to measure the activity of different respiratory complexes directly and sequentially. Although isolated mitochondria may be used in respirometry, the use of fresh, permeabilized muscle fibers maintains endogenous mitochondrial networks and membrane integrity - thus allowing for more physiologically relevant measurements. Additionally, because different muscle fiber types have different substrate preferences and rates of respiration, this system allows one to measure changes in fuel preference and metabolic flexibility based on fiber type13.
Here, we describe a comprehensive protocol for mitochondrial OXPHOS measurements using either human or mouse skeletal muscle fibers in a high-resolution respirometer system. Included are methods to quantify mitochondrial oxygen respiration in oxidative or glycolytic fibers using either pyruvate or palmitoyl-carnitine as a substrate. This protocol allows for the use of other fuel substrates to address specific metabolic questions pertaining to defects in substrate utilization and fuel preference.

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

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

Our research focuses on the intersection of metabolism and stress responses in metabolic disease, a major theme of the Washington University NORD program. Through synergisms between our NORD cores, we are able to tackle complex questions in nutritional metabolism and disease through an interdisciplinary and collaborative approach. Traditional mitochondrial respiration assays examined isolated mitochondria.Isolating mitochondria is time-consuming and can compromise outer mitochondrial membrane integrity. Additionally, mitochondria do not perform alone, but are part of a dynamic network of cellular metabolic adaptation. Our protocol maintains this network to provide a physiologically relevant assessment of mitochondrial function.Our focus has extended to identifying regulators of metabolic tailoring that are important in health and disease. Key questions we are asking include, are these regulators, like site-1 protease, equally important across different tissue types? And what is their physiological significance?

Calibration of High-Resolution Respirometry Instrument for Measuring Mitochondrial Respiration in Murine Samples 

To begin, turn on the high-resolution respirometry and the vacuum system. Remove the stoppers, and then, using a vacuum system, remove the 70%ethanol storage solution. Refill the chamber with ultrapure molecular-grade water.After the final wash, add two milliliters of MiRO5 without creatine or blebbistatin to each chamber. Open the respirometry software. In the pop-up box, set the chamber stirrer speed to 750 rpm, temperature to 37 degrees Celsius, and data recording interval to two seconds.Set the gain to one and polarization voltage to 800 millivolts for the oxygen sensors. Click Connect to Oxygraph to establish communication with the instrument. In the prompted dialogue box, name and save the experimental file with the current data and calibration.Record oxygen concentration for at least 30 minutes to allow the chambers to warm up and to record signals for air calibration. At the end of the calibration, hold the shift key and the left mouse button and drag across a region on the timeline to mark a stable oxygen concentration region. Navigate to Oxygraph, followed by Oxygen Calibration.For air calibration, select the stable oxygen concentration mark and click Calibrate and Copy to Clipboard. Stop the air calibration recording and save the file.

calibration-high-resolution-respirometry-instrument-for-measuring

Research

JoVE Journal

Biology

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.

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 ...

Measuring Mitochondrial Oxidative Phosphorylation in Fresh Permeabilized Murine Skeletal Muscle Fibers

After calibration of the respirometry system, replace MiR05 from each chamber with fresh 2.1 milliliters of MiR05 solution. In the respirometry software, enter instrument settings. Set the file name and save settings.In the next dialogue box, enter the sample information, including the weight of each sample added to each chamber. Now, open the oxygen calibration window, click copy from file, and select the saved air calibration file. Then, click the calibrate and copy to clipboard button.Using fine forceps, carefully transfer muscle fiber bundles, previously isolated from the mouse, into the respiration solution. Place the stoppers on the chamber and push them about halfway to the bottom to semi-close the chamber. Once the O-rings on the stoppers engage with the chamber wall, use a twisting motion while pushing down to close.When the chamber is halfway closed, a small air bubble is observed at the top of the chamber. Fill a 10 milliliter plastic syringe with pure oxygen from an oxygen tank. Place the long, blunt needle on the syringe, then insert the needle in the first chamber and slowly deliver approximately one milliliter of oxygen.Monitor the chamber oxygen concentration. When the concentration reaches 350 to 400 nanomoles per milliliter, gently twist the stopper while pushing down to fully close the chamber. Observe the chamber and ensure that no air bubbles remain.To normalize oxygen flux data to the mass of tissue from the layout menu, select the layout 06 specific flux per unit sample layout. Ensure that oxygen concentration and oxygen flux have stabilized following oxygen addition to begin the respiration measurement. Using a 10 microliter glass syringe, add 2.5 microliters of 0.8 molar malate to each chamber.Press F4 to mark the timeline, and label the mark with M.Record stable oxygen flux for one to two minutes. For aerobic glycolytic, add 10 microliters of two molar glutamate and five microliters of two molar pyruvate to each chamber. Press F4 to mark the timeline, and label the mark with GP.Record stable oxygen flux for one to two minutes.Add 10 microliters of 2 molar glutamate and 10 microliters of 10 millimolar palmitoylcarnitine to each chamber for fatty acid substrates. Press F4 to mark the timeline, and label the mark with GPC. Record stable oxygen flux for one to two minutes.Using a 25 microliter glass syringe, add 20 microliters of 0.5 molar ADP to each chamber. Press F4 to mark the timeline, and label the mark with ADP. Record stable oxygen flux for one to two minutes.Now, add 20 microliters of one molar succinate to each chamber. Press F4 to mark the timeline, and label the mark with S.Record stable oxygen flux for one to two minutes. Using a 10 microliter glass syringe, add five microliters of four millimolar cytochrome c to each chamber.Press F4 to mark the timeline, and label the mark with cytochrome c. Record stable oxygen flux for one to two minutes. Titrate in 3 one microliter boluses of one millimolar FCCP, using a 10 microliter glass syringe.FCCP edition results in a brief decrease in oxygen flux levels. Wait for the oxygen flux to increase and stabilize before recording. Press F4 to mark the timeline, and label mark with FCCP.Record stable oxygen flux for one to two minutes. Once the assay is complete, gently twist and pull the stopper upward to remove it. Rinse the chamber thrice with ultrapure water, followed by three times with 70%ethanol.Place the stoppers in the chambers until resistance is felt, but do not close them completely. Then, cap the stoppers. Save the assay file and disconnect the instrument from the software.Finally, turn off the respirometer. In properly prepared murine samples, the addition of cytochrome c post-ADP did not affect oxygen flux, confirming the integrity of the outer mitochondrial membrane. However, when tissue samples were not prepared properly, adding cytochrome c post-ADP led to a 40%increase in the oxygen flux, indicating damage to the outer mitochondrial membrane.