Respirometric Oxidative Phosphorylation Assessment in Saponin-permeabilized Cardiac Fibers

The overall goal of this procedure is to prepare Saponin perme cardiac fibers for polaro graphic measurements. This is accomplished by first excising, the heart from the mouse. The second step of the procedure is to mechanically prepare cardiac fiber bundles containing a maximum of six to eight fibers loosely connected.

Next, the cardiac bundles are chemically perme blast with saponin. The cardiac bundles are then washed free of saponin in any remaining metabolites. Ultimately, results can be obtained that show insights you mitochondrial oxidative phosphorylation through polari graphic measurements.

Hi, I'm Curtis Huey from the laboratory of Dr.Jane Sheer in the Department of Biochemistry and Molecular Biology at the University of Calgary. Today we will show you a procedure for preparing saponin perme cardiac fibers. We use this procedure in our laboratory to assess in situ mitochondrial oxidative phosphorylation.

So let's get started. To begin this procedure, remove a marine heart and place it in a Petri dish containing the RP solution on ice. Using a dissection microscope, carefully remove any connective tissue and or fat.

Cut the heart in half along the septum excise 10 to 25 milligrams, wet weight of tissue by cutting from the endocardium surface of the left ventricle. Ensure that the incision is along the fiber orientation to minimize mechanical damage to the myocardium. Place the dissected subsample of tissue into a separate Petri dish containing the RP solution on ice.

Cut the heart into longitudinal strips along the fiber orientation with a diameter of one to 1.5 millimeters. Shorten the strips with a scalpel to length between two and four millimeters using extreme care and sharp forceps. Mechanically separate the fiber bundles.

Final fiber bundles should contain a maximum of six to eight fibers connected by small areas of contact and weighing between one and three milligrams wet weight. Visually, the cardiac tissue should change from the original red to a pale pink coloring. Begin cardiac fiber bundle preparation by placing a 12 well plate on ice.

Rinse the wells with RP solution to minimize calcium contamination. Add three milliliters of ice cold RP solution with 50 micrograms per milliliter saponin to one of the wells. Then transfer the mechanically prepared fiber bundles to the well.

Incubate the fiber bundles for 20 minutes with mild stirring on ice. Rinse a new well with mirror five solution pipette 10 milliliters of ice cold mirror five into the new. Well transfer the cardiac bundles to the well and incubate them for 10 minutes with mild stirring on ice.

Following incubation, repeat the mirror five. Wash two to three times with fresh mirror.Five. Transferring the cardiac bundles into 10 milliliters of ice cold mirror five each time.

The repetitive washing steps are to ensure the removal of saponin A-T-P-A-D-P and any remaining substrates from the fiber bundles directly prior to respiratory analysis. Obtained the wet weight of the individual fiber bundles by blotting them on an absorbent surface using forceps and holding the fiber to the surface for five seconds or until all moisture is wicked away. Using another absorbent surface, remove excess liquid from the forceps.

Tear the scale and place the fiber bundle on a plastic wabo for mass measurement. Transfer the cardiac fiber bundle to a new well with ice cold near five. The fiber bundle is now ready for oxygen consumption Assessment.

The following validation titrations are performed with a two chamber titration injection XI graph. These experiments may be performed as an individual protocol or included into a titration protocol depending on the experimental and diagnostic aim of the respir barometry studies. The ERO GRA 2K used here can measure oxygen concentration.

The device is connected to a computer equipped with DAT lab software, allowing changes in oxygen concentration to be reported as real-time oxygen consumption measurements throughout the duration of the experiment. Prior to placing the mirroring cardiac fibers into the XI graft chambers add 2.1 milliliters of mirror five to the chambers housing, the polari graphic oxygen sensors or POS. When the chambers are closed, two milliliters of mirror five will be present in the XI graft chamber.

Allow adequate time for air saturation and equilibration of the mirror five at 37 degrees Celsius. Once Equilibrated, place the cardiac fiber samples into the mirror five contained in the XI graft chambers stirred by poly odine. Fluoride coated stir bars.

Increase the oxygen concentration of the GRAT chambers to between 250 and 550 micromolar with an oxygen filled syringe. Allow five to 10 minutes for oxygen concentration stabilization using a 10 microliter Hamilton syringe. Add 10 microliters of two molar glutamate to the chamber for a final concentration of 10 millimolar.

Then use a separate Hamilton syringe to add five microliters of 800 millimolar malate for a final concentration of two millimolar in the chamber. This titration is used to determine the basal complex one supported respiration. Oxygen flux should stabilize within approximately five minutes of titration, allowing for the determination of the state two oxygen consumption, which is the oxygen consumption.

In the absence of a DP following two to five minutes of stable oxygen consumption, use a 25 microliter Hamilton micro syringe to add 20 microliters of 500 millimole A DP for a final saturating concentration of five millimolar. This concentration is used to reach greater than 90%saturation, allowing for the determination of the maximal state.Three. Mitochondrial respiration through complex one allow for an additional two to five minutes of stable oxygen consumption.

Then use a 10 microliter Hamilton micro syringe to add five microliters of four millimolar cytochrome C for a final concentration of 10 micromolar. The oxygen concentration at this point is used to perform a quality control analysis of outer mitochondrial membrane integrity. After another two to five minutes of stable oxygen consumption, add one microliter of four milligrams per milliliter ACIN to inhibit a TP synthase.

This titration step is used to validate the inner mitochondrial membrane intactness. After the repi metric oxphos assessment, remove the sample for dry weight or mitochondrial marker determination. Then remove mirror five from the glass chambers of the XI Grat.

Wash the chambers at least three times with distilled water. Subsequently wash the chambers at least three more times with 100%ethanol. To remove ethanol soluble inhibitors such as oligo mycin.

Follow this with two additional washes of 70%ethanol. Then add 70%ethanol and allow it to soak the XI graft chambers for 30 minutes to sterilize them. Finally, wash the chambers containing the POS at least five times with double distilled water oxygen consumption in properly prepared mirroring cardiac fibers is evaluated by the quality control protocol as shown the respiratory control ratio or RCR represents the coupling between oxygen consumption and oxidative phosphorylation in perme fiber preparations.

The RCR is the rate of respiration in state three relative to state two or alternatively state three over state four, well coupled perme mirroring cardiac preparations yield an RCR between three and six. The titration of cytochrome C is used as a validation of proper tissue preparation. Cytochrome C is a protein located in the intermembrane space at the mitochondrial inner membrane.

When the outer membrane of mitochondria is intact, the endogenous cytochrome C remains in the intermembrane space and the titration of exogenous cytochrome C has a negligible effect on respiration. Conversely, if the outer membrane of mitochondria is damaged, the endogenous cytochrome C can be released from the intermembrane space and will inhibit respiration until exogenous cytochrome C addition, allowing for the assessment of the pathological or experimental stressors influence on mitochondrial outer membrane intactness. The addition of CIN is used to assess alterations in leak respiration, which is oxygen consumption, not contributing to a DP.Phosphorylation control or wild type fibers should be sensitive to the addition of CIN and experience a significant reduction in oxygen flux.

A low RCR and reduced sensitivity to oligo mycin resulting in a relatively elevated oxygen flux, indicates damage to the inner mitochondrial membrane during preparation. Lack of response to substrate inhibitor uncoupling titrations as seen here may be indicative of prolonged incubation periods of sasin and skinned fiber bundles in RP and mirror five solutions. Subsequent efforts should minimize periods between animal sacrifice and oxygen consumption measurements.

We've just shown you how to prepare cardiac fiber bundles for polar graphic measurements. It's important to remember that when performing this procedure to use extreme care during the mechanical separation of the cardiac fibers. So that's it.

Thanks for watching and good luck with your experiments.