The aim of this video is to describe a procedure for characterizing the mitochondrial permeability transition. Poor opening in mitochondria, isolated from mouse heart. First, mitochondria are isolated from mouse hearts.
Using the differential centrifugation method, the quality of the isolated mitochondria is then assessed using an oxy thermal oxygen electrode connected to a computer to measure the respiratory control ratio and to evaluate mitochondrial membrane integrity. Then using spectro fluoro metric instrumentation, simultaneous measurement of calcium handling, mitochondrial membrane potential and mitochondrial volume are used to determine permeability transition. Poor opening.
The resulting data showed differences in the calcium retention capacity and mitochondrial permeability transition. Poor sensitivity of isolated mitochondria, Opening of the mitochondrial permeability transition. Pore is a complex phenomenon that involves changes at several levels in mitochondrial physiology and anatomy.
The main advantage of this technique over existing methods is that the measurement of multiple parameters at the same time offers a more comprehensive picture of the phenomenon than the measurement of individual parameters alone, such as calcium, mobilization or swelling Prior to beginning the procedure, thaw the mitochondrial isolation buffer and check the pH, which should be 7.4. Then place all of the buffers, dishes, and tubes that will be used on ice. Note that all steps of the mitochondria isolation protocol must be performed on ice.
Place the hearts from euthanized mice on a Petri dish and rinse off any blood with cold mitochondria. Isolation buffer. Next, transfer the hearts to a new Petri dish using a blade, remove the fat and atria, then mince the hearts until a homogeneous product is obtained.
Transfer minced hearts to a 50 milliliter conical tube. Add 10 milliliters of mitochondria isolation buffer with 0.1 milligram per milliliter tripsin, and allow the sample to digest on ice for 10 minutes. Note that the use of trypsin increases the yield of isolated mitochondria and should be kept constant between mitochondrial preparation.
However, we recommend testing the functionality of isolated mitochondria on preparations obtained in the absence of trypsin to ensure that the enzyme does not interfere with their ions. After the incubation, add 10 milliliters of mitochondria isolation buffer containing 0.1%fatty acid-free, BS, a, and five milligrams trypsin inhibitor and mix by inverting the tube several times To neutralize the trypsin, recover the digested tissue by low to medium speed. Centrifugation for one minute of four degrees Celsius.
After the spin Resus, suspend the tissue in eight milliliters of mitochondria Isolation buffer with 0.1%fatty acid-free BSA, keeping the sample on ice. Use a motorized dance homogenizer with a Teflon pestle to homogenize the sample with four to six passes at 1, 200 to 1, 400 RPM. Transfer the homogenate to a 15 milliliter conical tube and centrifuge at 800 times G for 10 minutes of four degrees Celsius.
To pellet the nuclei and tissue debris. Following the spin, recover the supinate containing the mitochondria into micro centrifuge tubes and centrifuge them at 8, 000 times G for 15 minutes of four degrees Celsius. After the spin, the pellet will have a white layer on top of it.
Use a vacuum system to carefully aspirate and remove the white layer on top of the mitochondrial pellet. Then re resuspend the remaining darker pellet, which contains the mitochondria in one milliliter of mitochondria. Isolation buffer.
Next centrifuge the mitochondria at 8, 000 times G for 10 minutes of four degrees Celsius. Discard the supinate and resuspend the mitochondrial pellet. In 150 microliters of mitochondria, isolation buffer, keep the mitochondria on ice.
Respiration of isolated mitochondria is measured with an oxy thermal oxygen electrode connected to a computer and controlled by oxy. The plus software, the oxygen electrode should be calibrated on the day of the experiment. Add two milliliter mitochondria respiration buffer containing a final concentration of one micromolar rotenone to the oxy the chamber, seal the chamber and start recording the oxygen signal.
The signal recorded by the system and displayed on the computer screen is proportional to the oxygen concentration in the oxy therm chamber. Once the oxygen signal is stable, add 250 micrograms mitochondria and record basal respiration for one minute. The respiration at this point should be very low as mitochondria are aspiring on endogenous substrates.
This is state one respiration. Add succinate to a final concentration of five millimolar to initiate mitochondrial respiration at complex two and record the signal for one minute. Oxygen consumption should increase during state two respiration.
To induce state respiration, add a DP to a final concentration of 150 micromolar. At this point, oxygen consumption should increase due to a TP synthesis through oxidative phosphorylation. Record the signal until respiration slows indicating a DP consumption and transition to state four respiration record.
State four, respiration for at least one minute. Add CCCP to a final concentration of one micromolar and record the signal for one minute. At this point, uncoupled respiration should be maximal.
Calculate the respiratory control ratio by dividing oxygen consumption rate during state three. Respiration by the oxygen consumption rate during state four, respiration a target RCR should be greater than four. Next, to assess mitochondrial membrane integrity, induce state three respiration in a new sample of mitochondria by adding a DP to a final concentration of one millimolar.
Then record oxygen consumption for one minute, add 10 micromolar cytochrome C and record the oxygen consumption for two to three minutes. Since cytochrome C is impermeable to intact mitochondrial membranes in a good mitochondrial preparation, there will be no increase or a very small increase in respiration. An increase in respiration indicates broken or damaged mitochondrial membranes.
The instrumentation for the multi-parameter measurement of the mitochondrial permeability transition port opening consists of a quantum master dual emission spectro fluorimeter computer controlled by the Felix GX program. Using the Felix GX program, create a multidi acquisition protocol and enter the parameters for the measurement of calcium mitochondrial membrane potential and swelling for the ratio metric dyes, Forer and JC one generate the derived traces for a ratio and JC one ratio in a disposable four-sided meth ACRL vete mix one milliliter mitochondria assay buffer with a final concentration of one micromolar rotenone, five millimolar succinate and 800 nanomolar. For a FF, add 250 micrograms of mitochondria and place the sample in the sample compartment of the spectro fluorimeter.
Start recording the fluorescent signals after one minute, pause the acquisition, add 500 nanomolar JC one and then restart the acquisition. The JC one ratio signal should increase indicating die uptake by active mitochondria. Continue recording until JC one signal has reached a plateau.
Usually within five minutes, add calcium chloride to a final concentration of 20 micromolar. An instantaneous increase in Fluor FF ratio signal should be seen representing increased calcium presence in the assay buffer, followed by gradual decrease due to mitochondrial calcium uptake. The JC one ratio signal should exhibit a brief transient decrease indicative of slight mitochondrial membrane depolarization.
When the ER FF ratio signal returns to basal level, usually within one to 1.5 minutes at another 20 micromolar of calcium chloride, continue pulsing at fixed intervals until mitochondria cannot accumulate calcium and begin releasing calcium into the assay buffer. Mitochondrial permeability transition port opening is visualized by concurrent increase in the URA FF ratio signal due to calcium release. A decrease in the JC one signal ratio due to membrane potential collapse and a decrease in light scattering due to mitochondrial swelling mitochondrial while isolated from C 56 black six J mice.
As shown in this video, the oxygen consumption was then measured during state three respiration in the presence of succinate and a DP and during state four respiration after a DP consumption as shown here, a DP addition increases oxygen consumption due to oxidative phosphorylation and oxygen consumption slows. Once all a DP has been consumed, the response of isolated mitochondria to uncoupling is measured by the addition of CCCP in the presence of CCCP. The oxygen consumption rate is higher than during state three respiration to assess membrane integrity of the isolated mitochondria.
Oxygen consumption was measured in the presence of succinate and a DP and after the addition of cytochrome C.As seen here, the addition of cytochrome C does not significantly increase oxygen consumption with respect to a DP and succinate indicating intact mitochondrial membranes are not permeable to cytochrome C to assess mitochondrial permeability transition, poor opening in isolated heart mitochondria as an indicator of mitochondrial calcium retention capacity. Mitochondrial permeability transition. Poor opening was triggered by sequential additions of 20 micromolar.
Calcium chloride, extra mitochondrial calcium and membrane potential were measured with the ratio metric indicators. URA FF shown as the green trace and JC one shown as the red trace swelling of mitochondria was measured by recording light scattering at 525 nanometers shown by the black trace. Specificity of JC one and swelling signals were tested with one micromolar, CCCP and five microgram per milliliter of the microbial toxin ethin as can be seen here, calcium uptake by mitochondria is accompanied by a slight depolarization of the mitochondrial membrane.
After four calcium pulses, mitochondrial permeability transition pool opening is seen as an increase in URA FF indicating calcium release. A decrease in JC one indicating membrane potential collapse and an increase in mitochondrial matrix volume, which is seen as a reduction in light scattering as a marker of swelling to verify specificity of mitochondrial permeability transition port opening. The same measurements were performed in the presence of cyclosporine A, which binds to the mitochondrial permeability transition PO component cyclo fillin D.As shown in this figure, the presence of one micromolar cyclist boring A increases the number of calcium chloride pulses required to open the mitochondrial permeability transition PO versus the control sample.
After watching this video, you should have a good understanding of how to assess the opening of the mitochondrial permeability transition port in isolated heart mitochondria. The most difficult aspect of this procedure is obtaining good quality isolated mitochondria. Therefore, it is important to perform quality control measurements before using mitochondria for further experiments.
