Analyzing Oxygen Consumption Rate in Primary Cultured Mouse Neonatal Cardiomyocytes Using an Extracellular Flux Analyzer

The goal of this protocol is to illustrate how to use mouse neonatal cardiomyocytes as a model system to examine how various factors can alter oxygen consumption in the heart.

This protocol allows us to isolate and culture high bi-BBT, known only to mouse cardiomyocytes. To evaluate mitochondrial function, oxygen consumption rate of cardiomyocytes can be analyzed by an actual silver flux analyzer. One of the advantages of this technique is that oxygen consumption of cardiomyocytes can be easily measured in their adherent state in 96-well format, which enables testing of multiple conditions with a large number of replicates.

This vessel provides important insights into the research area of cardiology. In addition to oxygen consumption, we can also study other metabolic parameters, such as greygrices and peracid epoxidation. Achieving high variability of cardiomyocytes is critical for this experiment.

This requires efficient digestion of hearts. Since neo-natal cardiomyocytes are very fragile, gentle handling of cardiomyocytes is also important. In the cell culture hood, aliquot 5ml of HBSS to each well of a 6-well cell culture plate and place it on ice.

In addition, aliquot 10ml of HBSS to a 10cm cell culture dish, then, prepare 20 ml of Trypsin Predigestion solution in a 50ml sterile conical tube. Keep all the solutions on ice. Next, extract the heart and transfer it immediately to the sterile cell culture dish containing HBSS.

Remove any residual lung tissue and larger vessels. Wash the heart in HBSS using gentle agitation. Subsequently, cut the heart with fine scissors into 8 pieces and transfer the heart tissue with forceps into one well of a 6-well cell culture plate with HBSS.

Using a moria spoon, wash the heart tissue by transferring it from well to well in the 6-well plate filled with HBSS. Then transfer the heart tissue into a conical tube containing 20ml of Trypsin and incubate with gentle agitation at 4 degree celsius for 4 hours. In this procedure, prewarm the collagenase digestion solution in a water bath at 37 degrees celsius.

Transfer the conical tube containing heart tissue and predigestion solution from 4 degrees celsius to a cell culture hood. Let the heart tissue sink to the bottom of the tube and remove the predigestion solution by using a 10ml serological pipette. Next, add 10ml of HBSS into the tube.

Resuspend the heart tissue with HBSS 2 to 3 times to wash out Trypsin using a 10ml serological pipette. Aspirate HBSS and add 10ml of prewarmed collagenase digestion solution into the tube with heart tissue. For the first digestion, incubate the tube with heart tissue in a water bath at 37 degrees celsius for 10 minutes without agitation.

Afterward, transfer the tube to the cell culture hood. Triturate the tissue by resuspending it gently 10 times using a 10ml serological pipette. This will allow the heart to disperse and the cells to be released from the heart tissue.

Let the undigested tissue sink. Transfer the digested solution enriched in cardiomyocytes to a new conical tube and immediately add an equal amount of cell culture medium to stop the collagenase digestion. Then add 10ml of collagenase digestion solution into the tube containing the remaining undigested heart tissue.

For the second digestion, incubate the tube with heart tissue in a 37 degree celsius water bath for 10 minutes. Repeat the procedures of the first digestion and transfer the digested solution enriched in cardiomyocytes to a new conical tube before stopping the collagenase digestion. Next, place a sterile cell strainer in a new sterile 50ml conical tube.

Pre-wet the cell strainer with 2 to 3 ml of cell culture medium and pass the cells through the cell strainer. Subsequently, rinse the cell strainer with cell culture medium. Then, centrifuge the conical tube containing cardiomyocytes for 5 minutes at 180 times gravity.

After 5 minutes, aspirate the supernatant and resuspend the cell pellet in 10ml of cell culture medium. Plate the cells onto a 10cm cell culture dish and incubate them for one hour. Afterward, gently agitate the plate.

Wash off the non-adherent cells and resuspend the cells by repeatedly pipetting the cell culture medium over the dish using a 10ml serological pipette. Then, transfer the non-adherent cells into a new 10cm cell culture dish and incubate them for another hour. After an hour, gently agitate the plate and wash off the non-adherent cells.

Transfer the cardiomyocytes into a new 50ml conical tube. Next, prepare a 96-well culture plate by aliquoting 50 microliters of coating solution in each well of the 96-well cell culture plate. If bubbles are present, remove them using a 20 microliter pipette.

Incubate the plate at 37 degrees celsius for at least one hour to allow drying of the matrix coating. Then, count the cells using a hemocytometer. Plate the cells into the extra-cellular matrix coated 96-well cell culture plate at a density between 10 to 30 thousand cells per well.

And place the cells in the incubater. To preform the oxygen consumption assay, hydrate a flux analyzer sensor cartridge for at least 3 hours, add 200 microliters of calibrant solution into each well of the utility plate. Place the sensor cartridge back onto the utility plate and incubate in 37 degrees celsius without CO2 or O2 supplementation for at least 3 hours.

One hour prior to the assay, gently remove the cell culture medium and wash the cells with 200 microliters of prewarmed mitochondrial stress test medium twice. After the second wash, add 175 microliters of prewarmed mitochondrial stress test medium. Then culture the cells at 37 degrees celsius without carbon dioxide or oxygen supplementation.

Prepare 3ml of each of the test compounds in mitochondrial stress test medium. Load 25 microliters of each compound into the injector ports of the sensor cartridge using a multi-channel pipette. State of the cells and any pretreatments can affect maximum restoration elicited by injection of uncoupler FCCP.

Cell sensitivity to the uncoupler can also change, therefore, it is critical to optimize the working concentration of FCCP for each particular treatment. Subsequently, set up extra-cellular flux assay protocol and start the program. First, put the sensor cartiridge into the machine for calibration.

Replace the calibrant for the assay plate once calibration step is done. If desired, after the assay, carefully discard all assay medium using a multi-channel pipette. And store the cell culture mircoplate at negative 20 degrees celsius for future cell normalization using protein assay.

By using the protocol described, hearts were isolated from day 0 neo-natal pups and cardiomyocytes were seeded at densities of 10, 20 or 30 thousand cells per well in 96-well plates. After overnight culture, cardiomyocytes were found well attached to the coated plastic surface and there were very few unattached cells. At this point, spontaneously contracting cardiomyocytes were easily visible.

A seeding density of 30, 000 cells per well showed confluence one day after seeding as the cells spread. Cardiomyocytes were immunostained with an antibody against sarcomeric alpha actinin, a cardiomyocyte specific marker. As shown here, most of the cells showed positive staining of alpha actinin indicating the high purity of the cardiomyocyte isolation.

A scheme of one typical mitochondrial stress test is shown here. The mitochondrial stress test starts with a base line measurement of the oxygen consumption rate, this is followed by the injection of Oligo myosin which inhibits ATPA's. Then, the uncoupling agent FCCP was injected to measure maximum oxygen consumption rate.

Finally, with the injection of two electron transport complex inhibitors, mitochondrial respiration completely stops and OCR decreases to its lowest level. To obtain consistent and reproducible results. It is critical to achieve high survivablity.

Therefore, it is important to use newborn neonates and gently handled heart tissues in cardiomyocytes. By using new or existing genetically manipulated mouse lines, this method can be easily translated to data that may lead to understanding new mechanisms for bioenergetic regulation in the heart. Mitochondrial play key roles in heart function.

We expect this method to help identifying novel regulators and pathways regulating oxidating metabolism and finding new therapeutic targets for treating heart failure.