These protocols allow the investigators to go beyond simply measuring mitochondrial oxygen consumption, and instead permit the measurement of key mitochondrial end products, ATP and reactive oxygen species. The main advantage of this technique is that the investigator can measure mitochondrial efficiency by directly comparing the rate of oxygen consumption to the rate of end product production. Whether the end product is ATP or reactive oxygen.
The techniques require very careful attention to detail, particularly in avoiding bubbles. Whether they're in the respiration chamber or titration syringes, bubbles are your enemy because they make the measurements less precise. After obtaining the skeletal muscle biopsies.
fill the respirometer chambers with magnesium-free media and seal the incubation chamber. Set the instrument incubation temperature to 38 degrees Celsius to represent the basal temperature of equine skeletal muscle. And set the mixing of the respiration media to 750 rpm using a magnetic stir turning in the bottom of the respirometer chamber.
Next, turn off the chamber illumination to avoid interference with fluorescent sensors. Energize the oxygen electrode with an 800 millivolts polarization voltage and amplify the resulting signal with a gain setting of one. Record the oxygen concentration every two seconds and calculate the oxygen flux as the negative slope of the oxygen measurement over the proceeding 40 seconds.
Then 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. Use green fluorescent sensors to quantify the fluorescent signal from the respiration chamber.
Energize the sensors at 400 to 500 millivolts and the resulting signal gets amplified with a gain of 1 to 1, 000. Next, add TMRM to the respiration chamber before adding mitochondria and calibrate the fluorescent signal using a simple two point calibration of the fluorescent signal versus the amount of added fluorophore before the addition of the mitochondria. Perform the final calibration of the TMRM signal after completion of the respirometry titration protocol by delivering several titrations of uncoupling agent until no further increases in TMRM fluorescent signal are observed indicating the complete collapse of mitochondrial membrane potential.
Use blue fluorescent sensors to quantify the fluorescent signal from the respiration chamber and energize these sensors for individual instruments to capture the expected signal within the linear range of the sensor. Add eight microliters of two millimolar EDTA to the respiratory chamber to chelate cations that would compete with magnesium ions for binding to magnesium green. Then, add four microliters of one millimolar magnesium green to the respiration chamber.
Calibrate the raw fluorescent signal with 10 by 2 microliters sequential titrations of 100 millimolar magnesium chloride, allowing one minute between titrations to stabilize the fluorescent signal. Determine the ATTP synthesis rate which is the slope of the concentration of ATP over time throughout the protocol. Use green fluorescent sensors to quantify the fluorescent signal from the respiration chamber.
Energize the sensors at 300 to 400 millivolts. And the resulting signal gets amplified with a gain of 1 to 1, 000. Optimize specific settings for individual instruments to capture the expected signal within the linear range of the sensor.
Before adding the mitochondria perform the chemical setup and initial calibration of the Amplex UltraRed Assay. Add 30 micromoles of DTPA to chelate cations that might interfere with the reaction. Then add superoxide dismutase or serratus peroxidase and Amplex UltraRed in the respirometry chamber.
Allow the fluorescent signal to stabilize. Then add 0.2 micromoles of hydrogen peroxide twice with a gap of five minutes. Perform additional two point calibrations 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 investigator's discretion.
Vortex the sample to maintain a uniform sample suspension and add 15 microliters of isolated mitochondria suspension to each two milliliter incubation chamber so that the results represent the mitochondrial yield of 18.75 milligrams of muscle. Before adding any substrates, measure the residual oxygen consumption and subtract this value from the oxygen consumption values of each step in the substrate uncoupled inhibitor titration or SUIT protocol after completion. Use a general purpose SUIT that allows for the initial characterization of equine skeletal muscle mitochondrial function.
Start with sequential titrations of pyruvate, glutamate, and malate into each other into each chamber to produce nicotinamide adenine dinucleotide and stimulate nonphosphorylating respiration supported by NADH oxidized through complex one-based leak. Then add ADP to stimulate phosphorylating respiration through complex one phosphorylating respiration. Add succinate to produce phosphorylating respiration by combining complex one and complex two phosphorylating respiration Add rotenone to block complex one.
The resulting oxygen flux represents the capacity of complex two to support mitochondrial oxygen consumption by the oxidation of succinate alone. A high resolution respirometry trace of equine skeletal muscle mitochondrial respiration and relative membrane potential is shown. Respiration values of mitochondria incubated with TMRM are lower due to an inhibitory effect of that fluorophore.
Mitochondrial respiration and ATTP synthesis of equine skeletal muscle containing a high percentage of mitochondria rich type one skeletal muscle fibers and incubated under conditions approximating resting metabolism are shown. Respirometry trace of equine skeletal muscle mitochondrial respiration and production of hydrogen peroxide is shown. High resolution respirometry requires a lot of patience.
The person running the assay will have numerous time points at which they need to make a judgment call regarding whether a steady state has been reached and the next step can occur. We've only demonstrated a single titration protocol. There are dozens more protocols that can be applied in the same way to address specific questions regarding mitochondrial metabolism.
