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.
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.
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 factors2,3. 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 al4,5. 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.
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
2. Preparation of Buffers, Culture Media, and Dishes
3. Mouse Heart Removal and Cannulation
4. Heart Perfusion and Digestion
5. Cells dissociation and Calcium reintroduction
6. Cell Culture
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. Typically, 0.7-1.0 million rod-shaped myocytes can be collected from a ~25 g C57BL/6J mouse, 1.2-1.6 million rod-shaped myocytes from a ~35 g black Swiss mouse, and 0.7-1.2 million rod-shaped myocytes can be collected from a ~30 g Na/K-ATPase heterozygous (α1S/R)10 or ~22 g cardiac specific NCX-KO mouse11. Isolated myocytes normally have a distinct rod-shape with rectangular ends and clear cross-striations, shown in Figure 1.
2. Cell Function Identification
Our previous data have clearly shown that in cultured neonatal rat cardiomyocytes, 100 μM ouabain can activate PI3K-dependent Akt phosphorylation and induce hypertrophy 12. To further confirm these results in adult mouse cardiomyocytes, adult C57BL/6J mouse cardiomyocytes were cultured and treated with ouabain. Figures 2 and 3 clearly show that ouabain can increase Akt phosphorylation in a dose-dependent manner and induce [3H]-leucine incorporation during protein synthesis.
Figure 1. Normal rod-shaped cardiomyocytes from Black Swiss mouse after overnight culture. Myocytes were photographed under phase contrast microscopy using photo software.
Figure 2. Activation of Akt by ouabain in cultured adult C57BL/6J mouse cardiomyocytes. Myocytes were exposed to 0-50 μM ouabain for 5 min, and lysates were assayed for phosphorylated (Ser473) Akt (p-Akt) and Akt by western blots. Activation was quantified as ratio of phosphorylated to total Akt (n=5-10). *P < 0.05 vs. control, **P < 0.01 vs. control.
Figure 3. Ouabain stimulates protein synthesis in cultured adult C57BL/6J mouse cardiomyocytes. After 4 hr serum deprivation, myocytes were treated with the indicated conditions in the presence or absence of ouabain or 100 nM ET-1 (positive control) together with [3H]-leucine (1 μCi/ml) for 12 hr. Cellular lysates were precipitated with 5% TCA and resuspended in 0.2 M NaOH/0.1% SDS, and the radioactivity was counted in a scintillation counter. The protein synthesis per sample was calculated to cpm/mg protein and normalized as compared to the control.
Solution/Buffer | Perfusion Buffer | Digestion Buffer | Stop buffer | Ca2+ Solution I | Ca2+ Solution II | Ca2+ Solution III | Ca2+ Solution IV |
NaCl, mM | 113 | 113 | 113 | 113 | 113 | 113 | 113 |
KCl, mM | 4.7 | 4.7 | 4.7 | 4.7 | 4.7 | 4.7 | 4.7 |
KH2PO4, mM | 0.6 | 0.6 | 0.6 | 0.6 | 0.6 | 0.6 | 0.6 |
Na2HPO4, mM | 0.6 | 0.6 | 0.6 | 0.6 | 0.6 | 0.6 | 0.6 |
MgSO4, mM | 1.2 | 1.2 | 1.2 | 1.2 | 1.2 | 1.2 | 1.2 |
Na-HEPES, mM | 10 | 10 | 10 | 10 | 10 | 10 | 10 |
NaHCO3, mM | 12 | 12 | 12 | 12 | 12 | 12 | 12 |
KHCO3, mM | 10 | 10 | 10 | 10 | 10 | 10 | 10 |
Phenol red, μM | 32 | 32 | 32 | 32 | 32 | 32 | 32 |
Taurine, mM | 30 | 30 | 30 | 30 | 30 | 30 | 30 |
BDM, mM | 10 | 10 | 10 | 10 | 10 | 10 | 10 |
Glucose, mM | 5.5 | 5.5 | 5.5 | 5.5 | 5.5 | 5.5 | 5.5 |
FBS, % (v/v) | 0 | 0 | 10 | 10 | 10 | 10 | 10 |
Collagenase II, mg/ml | 0 | 1 | 0 | 0 | 0 | 0 | 0 |
CaCl2, μM | 0 | 50 | 0 | 12.5 | 100 | 400 | 900 |
Table 1. Solutions/buffers for adult mouse cardiomyocytes isolation.
Name | Company | Catalog # | Preparation | Storage |
Fetal Bovine Serum (FBS) | Atlanta Biolgoicals | S11550 | N/A | 25 ml aliquots in sterile 50 ml tubes at -20 °C. |
Bovine serum albumin (BSA) 100 mg/ml, 100x | Sigma | A7906 | 5 g in 50 ml diH2O, sterile filter through 0.22 μm sterile syringe filter | 5 ml aliquots in sterile 15 ml tubes at -20 °C. |
CaCl2 100 mM, 100x | Sigma | C4901 | 0.555 g in 50 ml diH2O, and sterile filter through 0.22 μm sterile syringe filter | 10 ml aliquots in sterile 15 ml tubes at room temperature. |
Laminin 1 mg/ml, 100x | Life technologies | 23017-015 | N/A | 200 μl aliquots in sterile 0.5 ml microcentrifuge tubes at -20 °C. |
2,3-Butanedione monoxime (BDM) 500 mM, 50x | Sigma | B0753 | 1.01 g BDM in 20 ml diH2O, sterilize the solution by filtering through 0.22 μm filter in hood. | store at 4 °C. |
Adenosine-5’-triphosphate disodium salt (Na2-ATP) 200 mM, 100x | Sigma | A6419 | Add 5 ml diH2O to dissolve 1 g Na2-ATP in 50 ml centrifuge tube, then use 2 mol/l NaOH to adjust pH to 7.2 and bring the final volume to 9 ml with diH2O. sterilize the solution through 0.22 μm syringe filter. | 0.5 ml aliquots in 1.5 ml sterile microcentrifuge tubes at -20 °C. |
NaCl 3.77 M, 33.3x | Sigma | S7653 | 66 g in 300 ml diH2O | store at 4 °C. |
KCl 470 mM, 100x | Fisher Scientific | P217 | 3.5 g in 100 ml diH2O | store at 4 °C. |
KH2PO4 60 mM, 100x | Sigma | P5379 | 0.82 g in 100 ml diH2O | store at 4 °C. |
Na2HPO4 60 mM, 100x | Sigma | S0876 | 0.85 g in 100 ml diH2O | store at 4 °C. |
MgSO4 120 mM, 100x | Sigma | M-1880 | 3 g in 100 ml diH2O | store at 4 °C. |
HEPES 1 M, 100x | Life technologies | 15630-080 | N/A | store at 4 °C. |
NaHCO3 600 mM, 50x | Sigma | S6014 | 10.1 g in 200 ml diH2O | store at 4 °C. |
KHCO3 1 M, 100x | Fisher Scientific | P235 | 10.1 g in 100 ml diH2O | store at 4 °C. |
Phenol red 3.2 mM, 100x | Sigma | P5530 | 0.12 g in 100 ml diH2O | store at room temperature. |
Table 2. Stock solution preparation and storage for adult mouse cardiomyocytes isolation and culture.
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 essential to quickly identify the aorta and to cut just below its first branch as shown by Flynn13. For efficient hook-up, a suitably-sized stainless steel cannula with notches and small serrated cross clamp are recommended. After ligation, the tip of the cannula should be above the aortic valve in order to help the collagenase fully interact with heart tissue during coronary circulation. Sometimes, the cell yield seems large initially but many cells are round-shaped after the calcium toleration step. One explanation is that coagulated blood in the vessels may not be completely removed from the heart, which leads to inadequate digestion. Another possibility is over digestion of the heart. Prolonged exposure to collagenase reduces myocyte calcium-tolerance2. During dissociation and calcium reintroduction, it is also important to handle the cardiomyocytes as gently as possible to maintain myocyte viability.
In summary, adult cardiomyocyte culture from genetically modified mice is a powerful tool for cardiac research. However, future work should keep in mind the genetic difference between humans and rodents when interpreting results.
The authors have nothing to disclose.
This work was supported by National Heart, Lung, and Blood Institute Grant HL-36573. We thank Mr. David Sowa for editing this manuscript.
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. |