Isolation and Culture of Adult Mouse Cardiomyocytes for Cell Signaling and in vitro Cardiac Hypertrophy

Summary

We describe a reliable method for isolation of adult mouse cardiomyocytes. This protocol yields a consistent result for the culture of functional adult cardiomyocytes from a variety of genetically modified mice.

Abstract

Technological advances have made genetically modified mice, including transgenic and gene knockout mice, an essential tool in many research fields. Adult cardiomyocytes are widely accepted as a good model for cardiac cellular physiology and pathophysiology, as well as for pharmaceutical intervention. Genetically modified mice preclude the need for complicated cardiomyocyte infection processes to generate the desired genotype, which are inefficient due to cardiomyocytes’ terminal differentiation. Isolation and culture of high quantity and quality functional cardiomyocytes will dramatically benefit cardiovascular research and provide an important tool for cell signaling transduction research and drug development. Here, we describe a well-established method for isolation of adult mouse cardiomyocytes that can be implemented with little training. The mouse heart is excised and cannulated to an isolated heart system, then perfused with a calcium-free and high potassium buffer followed by type II collagenase digestion in Langendorff retrograde perfusion mode. This protocol yields a consistent result for the collection of functional adult mouse cardiomyocytes from a variety of genetically modified mice.

Introduction

Cardiomyocytes are not proliferative. There are some atrial cardiomyocyte cell lines, like HL-1 and AT-1 cells derived from mouse atrial tumors; however, there are no adult ventricular cardiomyocyte cell lines available for research. Primary cell cultures of adult mouse cardiomyocytes provide a powerful model for heart research at the cellular and molecular levels. To date, they have been used extensively for biochemical, physiological, and pharmacological research¹. Additionally, the frequent use of genetically modified mice has necessitated effective methods of cardiomyocyte isolation. Pure culture allows for conditions free from interaction with other organs and the systemic circulation, such as through endogenous neurohormonal and hormone-like factors^2,3. However, successful isolation of cardiomyocytes can be challenging.

The protocol we introduce herein is based on our experiences with adult rat cardiomyocytes and the method described by O'Connell et al^4,5. Especially in 2007⁵, they described detailed techniques from buffer preparations to cell culture and functional assay. Since mouse myocytes are highly susceptible to contracture compared to the rat cardiomyocytes, the buffers or media used for the perfusion, digestion, Ca²⁺ toleration, plating and culture in the mouse protocol are supplemented with a nonspecific excitation-contraction coupling inhibitor, 2,3-Butanedione monoxime (BDM) to inhibit their spontaneous contraction, hence the viability and yield of rod-shaped myocytes improve significantly. In the protocol introduced here, the myocytes are separated in a high potassium buffer from the isolated heart by modified Langendorff perfusion with type II collagenase. Collagenase II effectively breaks down the intercellular matrix and releases cells. The perfusion solution also keeps cellular metabolism at a low level⁶. In addition, the isolated heart system from Harvard Apparatus we used here is well-designed for precise temperature and constant pressure control⁷. This approach provides highly reproducible preparations and uniform populations of single cell type, which can be used in overnight culture for measuring signaling proteins or 2-3 day culture for cardiac hypertrophic assays.

Protocol

All research on mice was done according to procedures and guidelines of the National Institutes of Health, and the protocols were approved by the Institutional Animal Care and Use Committee of the University of Toledo, College of Medicine and Life Sciences.

1. Perfusion System Preparation

1. The day before isolation, fill the entire perfusion system (including reservoirs) with a solution of fast alkaline residue-free detergent. Allow the solution to soak into the system for a few hours or overnight.
2. The next morning, adjust the thermocycler temperature to 37 °C and allow the solution to warm up. Remove the detergent solution and flush the system thoroughly with sterilized distilled water. Do not forget all the side branches.
3. Fill the system with perfusion buffer via a constant peristaltic pump and draw any air bubbles out from the aorta chamber (bubble trap) with a side-mounted syringe to protect the heart from coronary occlusion. The flow rate of the peristaltic pump should be checked routinely.

2. Preparation of Buffers, Culture Media, and Dishes

1. Perfusion Buffer: Make 500 ml of 1x perfusion buffer prior to use. This calcium-free buffer contains 113 mM NaCl, 4.7 mM KCl, 0.6 mM KH₂PO₄, 0.6 mM Na₂HPO₄, 1.2 mM MgSO₄, 10 mM Na-HEPES, 12 mM NaHCO₃, 10 mM KHCO₃, 0.032 mM phenol red, 30 mM taurine, 10 mM BDM, and 5.5 mM glucose. Adjust the pH to 7.0 with 2 M HCl and filter through a 0.2 μm filter, store at 4 °C.
   Note: a) The perfusion solution should be prepared on the same day when it is used. In some cases, it is acceptable to store the solution for less than 3 days at 4 °C. b) This buffer has a high potassium concentration up to 15.3 mM, which can keep the cardiomyocytes quiescent, reduce ATP consumption and increase Ca²⁺-tolerant ability.
2. Digestion Buffer (300 U/ml, 25-50 ml/heart): prepare just prior to perfusion. Weigh 15,000 U of total activity of type II collagenase, dissolve in 50 ml perfusion buffer in culture hood. Add 25 μl of 100 mM CaCl₂ (stock solution, filter sterilized) to make the final calcium concentration 50 μM. Warm the buffer to 37 °C when ready for isolation. Unlike trypsin, collagenase works best in the presence of 50-100 μM Ca²⁺.
3. Stop Buffer (50 ml/heart): Operate in culture hood. Mix 45 ml of perfusion buffer with 5 ml sterile FBS.
   1. Ca²⁺ solution I (12.5 μM Ca²⁺): Add 2.5 μl of 100 mM CaCl₂ to 20 ml of stop buffer and mix.
   2. Ca²⁺ solution II (100 μM Ca²⁺): Add 10 μl of 100 mM CaCl₂ to 10 ml of stop buffer and mix.
   3. Ca²⁺ solution III (400 μM Ca²⁺): Add 40 μl of 100 mM CaCl₂ to 10 ml of stop buffer and mix.
   4. Ca²⁺ solution IV (900 μM Ca²⁺): Add 90 μl of 100 mM CaCl₂ to 10 ml of stop buffer and mix.
4. Plating medium (50 ml/heart): Transfer 43 ml of MEM containing 2 mM L-glutamine and 1.26 mM CaCl₂ into a sterile 50-ml conical tube in culture hood and add 5 ml of FBS, 1 ml of BDM stock solution (500 mM, filter sterilized), and 0.5 ml penicillin/streptomycin (10,000 U/ml). Mix and equilibrate for 2-3 hr prior to myocyte isolation in 2% CO₂, 37 °C incubator with the caps loose. Right before myocyte plating, add 0.5 ml of 200 mM ATP (pH 7.2, stock solution, filter sterilized) into the medium.
5. Culture medium (50 ml/heart): Transfer 48 ml of MEM containing 2 mM L-glutamine and 1.26 mM CaCl₂ into a sterile 50-ml conical tube in culture hood and add 1 ml of BDM stock solution (500 mM, filter sterilized), and 0.5 ml penicillin/streptomycin (10,000 U/ml). Mix and equilibrate in 2% CO₂, 37 °C incubator prior to myocyte isolation. Prior to use, add 0.5 ml of 100 mg/ml BSA (filter sterilized) into the medium.
   Note: Using a 2% CO₂ incubator facilitates maintaining the culture medium pH at 6.9-7.0, which helps the myoctyes maintain their rod-shaped morphology up to 24 hr.
6. Laminin (1-2 μg/cm²)-coated dishes or plates: Transfer 200 μl of 1 mg/ml natural mouse laminin to 20 ml of sterilized PBS (w/o calcium and magnesium) and mix. Add 1 ml to 35-mm dishes, 2.5 ml to 60-mm dishes, or 5 ml to 100-mm dishes, shake to evenly cover the surfaces, and place in 2% CO₂, 37 °C incubator until myocyte plating.
   Note: If the dishes or plates are not used on the day coated, they can be sealed with Parafilm and stored at 4°C overnight (Slow Coating). Sealing is required to prevent evaporation and contamination. Coated plates can be kept at 4 °C for up to 1 week.

3. Mouse Heart Removal and Cannulation

1. Soak the scissors and forceps in 70% ethanol for 30 min and let them dry.
2. Weigh the mouse to the nearest 0.1 gram. Record its body weight, strain, sex, and date of birth.
3. Inject 200 μl heparin (100 IU/mouse) i.p. 10 min prior to anesthesia to prevent coagulation of blood in the coronary arteries. Anesthetize the mouse with 200 mg/kg body weight of ketamine and 10 mg/kg body weight of xylazine by i.p. injection and wait for 5-10 min until the mouse stops responding to tail/toe pinches.
4. Secure the mouse in the supine position by gently fixing the forepaws and hindpaws to a pinnable work surface (i.e. styrofoam) on an animal surgery tray or table bench near the perfusion system.
5. Wipe the chest and abdomen with 70% ethanol. Make a midline skin incision from mid abdomen to the diaphragm with a surgical scissor.
6. Cut the diaphragm with another surgical scissor and hold the sternum with curved serrated forceps. Cut bilaterally and retroflect thoracic cage to expose the heart.
7. Lift the heart slightly using fine curved serrated and atraumatic forceps and dissect the heart out of the thoracic cavity as close as possible to the dorsal thoracic wall.
8. Transfer the heart to a 100-mm dish containing cold perfusion buffer. Carefully trim away the connective tissues such as lungs, thymus, and bronchi.
9. Identify the aorta and its cranial branches, which are usually hidden by thymus and fat pad. Cut the aorta below its first branch¹³, grasp the aortic wall, lift the heart, and slightly slip it on to the perfusion fluid-filled aortic cannula (1.0 mm OD stainless steel cannula with blunt/flat tip and notches 1 and 2 mm above the tip; or a 22 G reusable feeding needle) using two ultra fine-tip forceps.
   Note: a) The perfusion fluid should be dripping during heart cannulation to prevent gas bubbles from entering the coronary arteries; b) Keep the tip of the cannula just above the aortic valve.
10. Clamp the aorta to the cannula and ligate the aorta to the cannula with 6-0 surgical silk. The whole cannulation should take less than 120 sec.

4. Heart Perfusion and Digestion

1. Perfuse the heart with calcium-free perfusion buffer at flow rate of 4 ml/min about 4-5 min until the effluents become clear, then switch to digestion buffer containing 50 μM CaCl₂ and perfuse for 3.5-20 min depending on mouse strain, perfusion pressure and collagenase activity. If applicable, increase the aorta pressure to 70-80 mmHg when digesting. When necessary, enzyme perfusate can be recirculated for digestion. Typically, 300 U/ml enzyme buffer will digest a heart in 10-12 min at a flow rate of 4 ml/min; If applying afterload pressure at 70 mmHg, 300 U/ml will take only 3.5-5.5 min at a flow rate of 4 ml/min.
2. Stop digestion when the heart becomes slightly pale and flaccid. It should be spongy when gently pinched.

5. Cells dissociation and Calcium reintroduction

1. Pull the aorta from the cannula with 70% ethanol-soaked forceps and put the heart into a sterile 60-mm dish containing 2.5 ml digestion buffer. Move into the culture hood using sterile supplies, staying mindful of sterile techniques for this and follow-up steps.
2. Remove aorta, atria, and great vessels with fine surgical scissors. Gently tease the ventricle into 10-12 small pieces with two fine-tip forceps.
3. Gently pipette the heart pieces and cells up and down ~10x with a 10-ml transfer pipette and then transfer to a 15-ml polypropylene conical tube. Rinse the dish with 7.5 ml calcium solution I containing 12.5 μM CaCl₂ and transfer this into the tube, resulting in a final volume of 10 ml.
4. Gently pipette the cell suspension up and down using a fine-tip transfer pipette to disperse the large pieces of heart tissue.
5. Then centrifuge for 3 min at 20 x g to separate out small non-myocyte cells such as endothelial cells and fibroblasts.
6. Aspirate off the supernatant and resuspend the myocyte pellet in 10 ml calcium solution I with a supplement of 100 μl of 200 mM ATP (pH 7.2, stock solution, filter sterilized). Leave the tube in the hood for 3-5 min.
7. Transfer duplicate 10 μl aliquots to a hemacytometer to count rod-shaped and round myocytes. Calculate the total number of myocytes and calculate the percentage of rod-shaped myocytes.
8. Centrifuge the myocytes for 3 min at 20 x g. Remove the supernatant and resuspend the pellets in 10 ml calcium solution II containing 100 μM CaCl₂. Disperse the myocytes with the fine-tip transfer pipette by pipetting up and down.
9. Repeat the above step (5.8) using calcium solution III (400 μM CaCl₂) and IV (900 μM CaCl₂), respectively.
10. For mouse myocyte primary culture, resuspend the final myocyte pellet in 5 ml of plating medium. Count the total number of myocytes and calculate the percentage of rod-shaped myocytes.

6. Cell Culture

1. Adjust the concentration of rod-shaped myocytes to 25,000 rod-shaped myocytes/ml in a 50-ml tube. Gently pipette the myocytes using a 10-ml pipette to suspend them well. Avoid cell sedimentation in the pipette and tube to ensure equal cell density for each dish or plate.
2. After aspirating off the laminin coating solution, plate the myocytes into those dishes or plates. Gently slide the dishes or plates forward and backward and side-to-side in a cross-like pattern three to four times on the surface of the culture hood.
3. Place the dishes or plates in a 2% CO₂ incubator at 37 °C. Incubate for 1-3 hr to allow myocyte attachment at a rate of about 80%.
4. After attachment, gently aspirate off the medium containing unattached myocytes and cell debris. Gently add culture medium to the sides of the dishes or plates. Perform this medium change one by one. Immediately return the dishes or plates to the incubator.
5. After O/N culture, the myocytes can be changed to the same medium without BDM before studies of short term cell signaling. Keep the BDM in long term culture for hypertrophic or apoptosis assays.
   Note: BDM (2,3-butanedione monoxime) can inhibit the myosin-ATPase and prevent contraction. Its inhibition of myosin-based contraction is readily reversible. It is reported that BDM may have some potential side effects, such as acting as a non-specific phosphatase, increasing calcium release from SR, and alteration of free calcium concentration and cellular electrical properties, so before the treatment, the BDM should be removed. Moreover, in some cases, a specific myosin II inhibitor Blebbistatin could be an alternative drug to inhibit cardiomyocyte contraction^8,9.  However, in our cases, BDM is better than Blebbistatin for the sake of the quality and quantity of the cardiomyocytes.

Results

1. Successful Isolation Quantification

Two criteria are used to quantify the success of the isolation: first, the total number of cardiomyocytes isolated, and second, the ratio of rod-shaped calcium-tolerant to round non-tolerant myocytes. Generally this protocol takes around 75-90 min from heart removal to myocyte plating and yields around 1 million rod-shaped cardiomyocytes (70-90% of total myocytes harvested) from one adult mouse heart. This may vary with mouse body weight and strain. Typ...

Discussion

For the best preparation, the most critical steps are: 1) promptly hooking up the mouse heart to the cannula after excision; 2) appropriate heart perfusion. Also, note that water quality, perfusion temperature, pH of buffer, sterilization, chemical purity, and clean, non-contaminated tubing and chambers are also important factors. 18.2 mΩ molecular biology-grade H₂O is highly recommended for buffer preparation. The pH of 200 mM ATP stock solution should be adjusted to 7.2, a step which is easily missed.

It is e...

Materials

Name	Company	Catalog Number	Comments
Isolated heart system for small rodent	Harvard Apparatus	IHSR-mouse 73-4019
Aortic Cannula, OD 1.0 mm	Harvard Apparatus	73-2816
Dumont #4 Forceps	Fine science tools	11294-00
Straight sharp serrated scissors	Fine science tools	14070-12
Serrated Graefe forceps	Fine science tools	11050-10
Curved, serrated Graefe forceps	Fine science tools	11052-10
Straight Mini Serrefines clamps	Fine science tools	18054-28
MEM	Gibco	11575-032
Collagenase II	Worthington	4176
Mucosal universal detergent	Sigma	Z637181	Mucasol is a fast alkaline residue-free detergent.