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

Summary

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.

Abstract

Mitochondria and oxidative metabolism are critical for maintaining cardiac muscle function. Research has shown that mitochondrial dysfunction is an important contributing factor to impaired cardiac function found in heart failure. By contrast, restoring defective mitochondrial function may have beneficial effects to improve cardiac function in the failing heart. Therefore, studying the regulatory mechanisms and identifying novel regulators for mitochondrial function could provide insight which could be used to develop new therapeutic targets for treating heart disease. Here, cardiac myocyte mitochondrial respiration is analyzed using a unique cell culture system. First, a protocol has been optimized to rapidly isolate and culture high viability neonatal mouse cardiomyocytes. Then, a 96-well format extracellular flux analyzer is used to assess the oxygen consumption rate of these cardiomyocytes. For this protocol, we optimized seeding conditions and demonstrated that neonatal mouse cardiomyocytes oxygen consumption rate can be easily assessed in an extracellular flux analyzer. Finally, we note that our protocol can be applied to a larger culture size and other studies, such as intracellular signaling and contractile function analysis.

Introduction

To sustain a continuous cardiac contractile function, cardiomyocytes must maintain a constant supply of cellular energy primarily in the form of ATP¹. In the heart, approximately 95% of ATP is generated by mitochondria, mainly through oxidative phosphorylation, showing that mitochondria play a crucial bioenergetic role in cardiac function^2,3. Supporting this notion is that dysregulation of mitochondrial function can lead to cardiomyopathy and heart failure^4,5. Conversely, restoring mitochondrial function has been shown to improve cardiac function of the failing heart^6,7. Therefore, studying the mechanism of mitochondrial bioenergetics and identifying novel regulators of mitochondrial function in cardiomyocytes will not only reveal mechanistic insights of cardiac energy production but also could provide insight that will lead to development of new therapeutic targets to treat heart diseases^6,8.

Compared to the whole heart, which contains a mixture of myocytes and non-myocytes⁹, cardiomyocyte cultures are extremely pure, with minimal contamination of non-myocytes from the heart, such as fibroblasts and endothelial cells¹⁰. In addition, isolating cardiomyocytes from neonatal pups enables culturing a large number of cells in a small amount of time, compared to isolating cells from adult hearts^10,11. Most importantly, primary cultured adult mouse cardiomyocytes have short survival times (e.g. 24 h) and at longer time points de-differentiate. Neonatal mouse cardiomyocytes can survive and be manipulated for upwards of 7 days in culture, making them ideal for testing the effects of drug compounds and gene manipulation on the function of mitochondria in cardiomyocytes¹⁰. Of course, there are significant biological differences between the adult and neonatal cells, but the longer duration available for culture of neonatal cells makes them appropriate for many different types of studies, including those of mitochondrial function.

To date, primary cultured neonatal mouse and rat cardiomyocytes have been used as models to study cardiac bioenergetics^12,13. In recent years, studies used an extracellular flux analyzer to measure oxygen consumption rate (OCR) and evaluate oxidative capacity in mouse and rat neonatal cardiomyocytes^14,15. While compared to rats, the cell viability of mouse neonatal cardiomyocytes is lower and has greater variability¹⁶. Also, the ability to study cells from genetically engineered mouse models makes the mouse cell model very important. Given that OCR studies are so sensitive to cell number and seeding density, development of a reproducible, reliable, and simple protocol to achieve consistent cell yield and viability is needed.

Here, we report an optimized protocol that has been developed which uses cultured mouse neonatal cardiomyocytes along with a 96-well-format extracellular flux analyzer for OCR analysis. This protocol greatly increases reproducibility of the assay. In addition, the protocol not only provides a novel and reproducible method for OCR analysis, but also could be adapted to a larger size culture for other experimental purposes, such as that which may be needed to study myofibrillar functions and intracellular signaling pathways.

In particular, this protocol describes a one-day procedure for isolation and culture of neonatal mouse cardiomyocytes in a 96-well cell culture plate. In addition, it describes the procedure to measure oxygen consumption using an extracellular flux analyzer. All solutions used are sterile or sterile filtered. All tools are sterilized by 75% ethanol. We provide a Table of Materials for various parts of the procedure. For culturing cardiomyocytes, all procedures and steps are performed in a standard cell culture hood. This protocol is developed for the isolation of neonatal mouse hearts from one litter (approximately 8-10 pups). However, the protocol can also be adapted for isolating cardiomyocytes from multiple litters.

Protocol

For work with neonatal mice, please refer to local university/institute guidelines set forth by the animal care programs and adhere to one’s institutional and other appropriate regulations. All methods described in this protocol have been approved by the UC San Diego Institutional Animal Care and Use Committee (IACUC) and adhere to federal and state regulations.

1. Preparation of Reagents

1. Prepare 25 mL of pre-digestion solution: HBSS (without Ca²⁺ and Mg²⁺) supplemented with trypsin (0.5 mg/mL). Sterilize the solution using a 0.22 µm filter and keep on ice until use. Make pre-digestion solution on the day of experiment.
   NOTE: It is critical to use HBSS without Ca²⁺ and Mg²⁺, as Ca²⁺ and Mg²⁺ will cause myocyte contraction and subsequent cell death during isolation.
2. Prepare 30 mL of collagenase digestion buffer: collagenase (0.8 mg/mL, approximately 350 U/mL) dissolved in HBSS (without Ca²⁺ and Mg²⁺) buffer. Sterilize the solution using a 0.22 µm filter and keep on ice until use. Make collagenase digestion solution on the day of experiment.
3. Prepare 500 mL of cardiomyocyte culture media (growth media): Mix 375 mL of DMEM, 125 mL of M-199, 25 mL of Horse serum, and 12.5 mL of FBS. Supplement with 1% penicillin and 1% streptomycin solution.
4. Prepare mitochondrial stress test medium: Make 200 mL of DMEM based stress test medium (DMEM without NaHCO₃, see Table of Materials) supplemented with 1 mM sodium pyruvate, 2 mM L-glutamine, and 10 mM glucose, and 2 mM Hepes.
   NOTE: Make 1 L of medium with DMEM without sodium pyruvate, L-glutamine, glucose, and Hepes, filer sterile, and store in 4 °C. Prepare the stress test medium on the day of the assay by adding other reagents. Using DMEM medium without NaHCO₃ is critical.
5. Adjust pH of mitochondrial stress test media to 7.4 on the day of use. Warm media to 37 °C before use.
6. Prepare oligomycin: Prepare 5 mL of a 5 mM stock solution in DMSO, make 250 µL aliquots, and store at -20 °C.
7. Prepare FCCP: Prepare 5 mL of a 5 mM stock solution in DMSO, make 250 µL aliquots, and store at -20 °C.
8. Prepare antimycin A: Prepare 5 mL of a 5 mM stock solution in DMSO, make 250 µL aliquots, and store at -20 °C.
9. Prepare rotenone: Prepare 5 mL of a 5 mM stock solution in DMSO, make 250 µL aliquots, and store at -20 °C.
   NOTE: All reagents and solutions used in this protocol are listed in Table 1.

2. Harvesting and Pre-digestion of Hearts from Neonatal Mice (Day 1)

1. Autoclave scissors, forceps, and a Moria spoon to sterilize.
2. Perform all steps in the cell culture hood for sterility.
3. Aliquot 5 mL of HBSS (without Ca²⁺, Mg²⁺) to each well of a 6-well cell culture plate; place on ice. Aliquot 10 mL of HBSS into a 10 cm cell culture dish.
4. Prepare 20 mL of trypsin pre-digestion solution in a 50 mL sterile conical tube. Keep all solutions on ice.
5. Quickly dip newborn (day 0) mice in 70% ethanol solution for sterilization.
6. Decapitate pups using sterile scissors (straight) without anesthesia, and then open chest along the sternum to allow access to the chest cavity and the heart. (Figure 1A)
   NOTE: 1) It is critical to use P0 neonatal mice to achieve high cell viability. 2) This euthanasia method is permitted for neonates in accordance with NIH and American Veterinary Medical Association guidelines ¹⁷.
7. Extract hearts from the body with a fine scissors and transfer immediately into the sterile cell culture dish containing HBSS (without Ca²⁺, Mg²⁺) (Figure 1B).
8. Remove any residual lung tissue, larger vessels, etc. (and atria, if desired). Wash hearts in the HBSS solution using gentle agitation.
9. Cut each heart with a fine scissors into 8 pieces and transfer the all heart tissue with forceps into one well of a 6-well cell culture plate with HBSS (Figure 1C and 1D).
10. Wash the hearts by transferring the hearts from well to well in the 6-well plate filled with HBSS, using a Moria spoon (Figure 1D).
    NOTE: Transferring the hearts from well to well is enough to wash out the blood. Since blood interferes with enzymatic digestion it is important to wash the hearts with HBSS and remove blood.
11. Transfer the hearts with a Moria spoon into a conical tube containing 20 mL of trypsin (0.5 mg/mL) and incubate with gentle agitation at 4°C for 4 h (Figure 1E).

3. Prepare a 96-well Culture Plate (Day 1)

1. Prepare 5 mL of coating solution: PBS containing 0.5% gelatin (autoclave before use) and 1% fibronectin solution. (e.g. 5 mL gelatin solution plus 50 µL of fibronectin solution)
2. Aliquot 50 µL of coating solution into each well of the 96-well cell culture plate (see Table of Materials). If bubbles are present, remove them by using a 20 µL pipette to suck bubbles out.
   NOTE: It is important to cover all the surface area of each well with coating solution.
3. Incubate the plate in a 37 °C cell culture incubator for 1 h or more to allow drying of the matrix coating.
4. Aspirate any residual coating solution before seeding cardiomyocytes.

4. Enzymatic Digestion and Plating of Cells (Day 1)

1. Pre-warm collagenase digestion solution in a 37 °C water bath.
   NOTE: This is step is important to achieve efficient enzymatic digestion.
2. Move the conical tube containing hearts and pre-digestion solution from 4 °C to a cell culture hood. (Figure 1F)
3. Let the hearts sink to the bottom of the tube and remove the pre-digestion solution by using a 10 mL serological pipette (1 to 2 mL of the isolation medium may remain in the tube).
4. Add 10 mL of HBSS into the tube. Re-suspend the hearts with HBSS 2-3 times to wash out trypsin using a 10 mL serological pipette. Aspirate HBSS (1 to 2 mL may remain in the tube).
5. Add 10 mL of pre-warmed collagenase digestion solution into the tube with hearts. (Figure 1G)
6. Incubate the tube with hearts in a 37 °C water bath for 10 min without agitation (1st digestion).
7. After 1st digestion, move the tube to the cell culture hood. Gently triturate hearts by re-suspending the hearts within the tube gently 10 times using a 10 mL serological pipette. This will allow hearts to disperse and cells to be released from heart tissue. (Figure 1H)
   NOTE: Since cardiomyocytes are fragile, gentle trituration is important to achieve high viability.
8. Let the undigested tissue sink, transfer digested solution enriched in cardiomyocytes (approximately 9 - 10 mL) to a new conical tube, and immediately add an equal amount of cell culture media to stop the collagenase digestion.
9. Add 10 mL of collagenase digestion solution into the tube containing the remaining undigested heart tissue.
10. Incubate the tube with heart tissue in a 37 °C water bath for 10 min (2nd digestion).
11. Repeat procedure 4.7 and 4.8.
    NOTE: If there is still much undigested tissue, repeat digestion one more time. However, in most cases, two digestions are enough to disperse most of the cells from the heart tissue.
12. Place a sterile cell-strainer (100 µm nylon mesh) in a new sterile 50 mL conical tube. Pre-wet the cell strainer with 2-3 mL of cell culture media and pass cells through the cell-strainer. Rinse the cell-strainer with 2-3 mL of cell culture media. (Figure 1I)
13. Centrifuge conical tube containing cardiomyocytes for 5 min at 180 x g (Figure 1J). Aspirate the supernatant (Figure 1K), which will contain cell tissue debris and re-suspend the cell pellet in 10 mL of cell culture media (Figure 1L).
14. Gently resuspend the cells and plate cells onto a 10 cm cell culture dish (plastic without any type of coating) and incubate for 1 h in a cell culture incubator (1st pre-plating) (Figure 1M). This pre-plating step allows non-cardiomyocytes, such as fibroblasts and endothelial cells, to adhere to the uncoated cell-culture dish.
    NOTE: At this point, cardiomyocytes are typically a round shape and appear shiny under the microscope. (Figure 1N).
15. After the 1 h incubation, gently agitate the plate, wash non-adherent cells (enriched in cardiomyocytes) from the 10 cm culture dish, and re-suspend cells by repeatedly pipetting the cell culture medium over the dish using a 10 mL serological pipette. Then, transfer non-adherent cells (enriched in cardiomyocytes) into a new 10 cm cell culture dish (plastic without any coating) and incubate for an additional 1 h in a cell culture incubator (2nd pre-plating).
    NOTE: Cells that attach to the non-coated plate are dominantly non-cardiomyocytes: fibroblasts and endothelial cells, that can be visualized under a microscope (Figure 1O).
16. After 2nd pre-plating, gently agitate the plate, wash non-adherent cells (cardiomyocytes) from the 10 cm culture dish, then transfer the cardiomyocytes into a new 50 mL conical tube.

5. Counting Cells and Plating Cells into a 96-well Cell Culture Plate (Day 1)

1. Count the cells using a hemocytometer.
2. Plate the cells into an extracellular matrix coated 96-well cell culture plate at a density between 10–30 x 10³ cells/well by using a multi-channel pipette in a final volume of 200 µL (Figure 1P). Use wells A1, A12, H1, and H12 for background: add 200 µL of culture medium as other wells in these wells (no cells). Incubate the plate in a 37 °C cell culture incubator.
   NOTE: In this study, oxygen consumption was tested by using different cell densities such as 10 x 10³, 20 x 10³, or 30 x 10³ cells/well. (Figure 2A). Also, as above, cardiomyocytes immediately after isolation are typically a round shape and appear shiny under the microscope. Viable cells will flatten out within 16-24 h of culture.

6. Oxygen Consumption Assay using a 96-well-format Extracellular Flux Analyzer (Day 2)

NOTE: Oxygen consumption assay can be carried out one day after plating the cells or later. Neonatal cardiomyocytes cultured using this protocol can survive up to 7 days post isolation.

1. Hydrate a flux analyzer sensor cartridge (see Table of Materials) for at least 3 hours, but ideally for a full day, before the assay. Add 200 µL of Calibrant solution (see Table of Materials) into the each well of the utility plate, put the sensor cartridge back onto the utility plate, and incubate in a 37°C incubator without CO₂ or O₂ supplementation.
2. Change cell culture media to mitochondria stress test medium one hour prior to the assay. Cardiomyocytes are fragile. Therefore, gently remove the cell culture media by using a multi-channel pipette and wash the cells with 200 µL of pre-warmed mitochondrial stress test media twice. After second wash, add 175 µL of pre-warmed mitochondria stress test media and culture the cells in a 37°C incubator without CO₂ or O₂ supplementation.
3. Prepare concentrated test compounds. For mitochondria stress test, prepare 3.0 mL each of 16 µM oligomycin, 9 µM FCCP, and a mixture of 20 µM rotenone and 20 µM antimycin A, all in mitochondrial stress test medium.
   NOTE: Each compound at the concentration described has been tested. However, titrating the concentration of each compound in one’s own laboratory is necessary.
4. Load 25 µL of each compound into the injector ports of the sensor cartridge using a multichannel pipette (Figure 1Q). The volume and final concentration are described in Table 2.
5. Set up extracellular flux assay protocol. The program is described in Table 3.
6. Start the program. First, put the sensor cartridge into the machine for calibration (Figure 1R). Replace the calibrant for the assay plate once the calibration step is done.
   NOTE: Using the software provided by manufacturer, indicate groups of wells and each compound and port.
7. If desired, after the assay, carefully discard all assay medium by using a multi-channel pipette and store the cell culture microplate at -20 °C for future cell normalization using protein assay.
8. Measure protein content. Add 50 µL of standard RIPA cell lysis solution (see Table of Materials). Incubate the plate on ice for 30 min to fully lyse cells. Transfer all material to a new clear flat bottom 96 well assay plate.
9. Measure protein concentration by BCA assay according to manufacturer’s protocol.
   NOTE: Coating the well with extracellular matrix results in a high protein concentration in each well. Therefore, subtract the amount of the background well(s) (no cells) to get actual cell protein concentration. The protein concentration derived from cells is low in a 96-well culture plate. In addition, protein concentration can vary from well-to-well due to extracellular matrix coating. Therefore, using cell number to normalize the OCR is suggested.

Results

By using the protocol described, hearts were isolated from day 0 neonatal pups. 5 x 10⁵ cells/pup were obtained, and cardiomyocytes were seeded at densities of 10 x 10³, 20 x 10³, or 30 x 10³ cells/well, in 96 well plates (Figure 2A). After overnight culture, cardiomyocytes were found well-attached to the coated plastic surface and there were very few unattached cells (the unattached cells will still appear as round...

Discussion

In this study, we have established a simple protocol for isolating and culturing mouse neonatal cardiomyocytes. By using these cardiomyocytes, we also optimized the conditions to measure oxygen consumption rate by using an extracellular flux analyzer system. The protocol allows one to use mouse neonatal cardiomyocytes as a model system to examine how various factors can alter oxygen consumption in the principal working cells of the heart, akin to what would be measured in the intact organ. Our protocol is different from ...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Antimycin A	SIGMA	A8674	Inhibits complex III of the mitochondria
Cell strainer 100 μm pores	FALCON	352360	To capture undigested tissue
Collagenase type II	Worthington	LS004176	To make collagenase digestion solution
D-Glucose	SIGMA	75351	To make mitochodnrial stress test medium
DMEM high glucose	Life technologies	11965-092	To make cell culture medium
DMEM without NaHCO3, Glucose, pyruvate, glumanine, and Hepes	SIGMA	D5030-10X1L	To make mitochodnrial stress test medium
FCCP (Carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone)	SIGMA	C2920	Uncouples mitochondrial respiration
Fetal bovine serum (FBS)	Life technologies	26140-079	To make cell culture medium
Fibronectin from bovine plasma	SIGMA	F1141-5MG	To make coating solution for tissue culture plates
Fine scissors	Fine Sciences Tools	14060-10	For dissection of hearts
Gelatin from porcine skin	SIGMA	G-1890	To make coating solution for tissue culture plates
HBSS (Hank's balanced salt solution, without Ca2+, Mg2+)	Cellgro	21-022-CV	To wash hearts and make pre-digestion and collagnase digestion solution
HEPES (1 M)	Fisher scientific	15630080	To make mitochodnrial stress test medium
Horse serum	Life technologies	26050-088	To make cell culture medium
L-Glutamine	SIGMA	G-3126	To make mitochodnrial stress test medium
M-199	Cellgro	10-060-CV	To make cell culture medium
Moria spoon	Fisher scientific	NC9190356	To wash hearts
Oligomycin	SIGMA	75351-5MG	Inhibits mitochondrial ATP synthase
RIPA buffer	Fisher scientific	89900	To lyse the cells for protein assay
Rotenone	SIGMA	R8875	Inhibits complex I of the mitochondria
Seahorse XFe96 Extracellular Flux Analyzer	Agilent		Device used to analyze oxygen consumption rate
Seahorse XFe96 FluxPak	Agilent	102601-100	Package of flux analyzer culture plates, sensor cartridges, and calibrant
Sodium pyruvate	SIGMA	P2256	To make mitochodnrial stress test medium
Straight scissors	Fine Sciences Tools	91401-12	For dissection of hearts
Syringe filter 0.2 μm size			For sterile filtration of digestion medium
Trypsin	USB Corporation	22715 25GM	To make pre-digestion solution