JoVE Logo
Faculty Resource Center

Sign In

Summary

Abstract

Introduction

Protocol

Representative Results

Discussion

Acknowledgements

Materials

References

Biology

Isolation, Culture and Transduction of Adult Mouse Cardiomyocytes

Published: August 28th, 2016

DOI:

10.3791/54012

1Cardiovascular Research Institute, University of California, San Francisco

This protocol describes a step-by-step method for the reproducible isolation and long-term culture of adult mouse cardiomyocytes with high yield, purity, and viability.

Cultured cardiomyocytes can be used to study cardiomyocyte biology using techniques that are complementary to in vivo systems. For example, the purity and accessibility of in vitro culture enables fine control over biochemical analyses, live imaging, and electrophysiology. Long-term culture of cardiomyocytes offers access to additional experimental approaches that cannot be completed in short term cultures. For example, the in vitro investigation of dedifferentiation, cell cycle re-entry, and cell division has thus far largely been restricted to rat cardiomyocytes, which appear to be more robust in long-term culture. However, the availability of a rich toolset of transgenic mouse lines and well-developed disease models make mouse systems attractive for cardiac research. Although several reports exist of adult mouse cardiomyocyte isolation, few studies demonstrate their long-term culture. Presented here, is a step-by-step method for the isolation and long-term culture of adult mouse cardiomyocytes. First, retrograde Langendorff perfusion is used to efficiently digest the heart with proteases, followed by gravity sedimentation purification. After a period of dedifferentiation following isolation, the cells gradually attach to the culture and can be cultured for weeks. Adenovirus cell lysate is used to efficiently transduce the isolated cardiomyocytes. These methods provide a simple, yet powerful model system to study cardiac biology.

Cultured cardiomyocytes are frequently used to monitor cell behavior in a well-controlled environment in vitro. For example, morphological, electrical, biochemical, or mechanical cell properties can be studied on engineered substrates,1,2 in defined media, and in response to small molecule drugs, peptides, gene regulation,3 or electrical stimulation.4 The cellular content can also be controlled using defined co-cultures.5 These in vitro experiments are useful in large drug or genetic screens and complement in vivo methods for various types of investigations involving cardiomyocyte biology.

Log in or to access full content. Learn more about your institution’s access to JoVE content here

All procedures outlined here have been approved by the Institutional Animal Use and Care Committee at the University of California, San Francisco.

NOTE: Briefly, after extracting the heart from the mouse thorax, coronary retrograde perfusion is used to efficiently digest the extracellular matrix with collagenase and protease XIV. The ventricles are then isolated, mechanically dissociated and filtered into a single cell suspension. Gravity sedimentation is performed to isolate .......

Log in or to access full content. Learn more about your institution’s access to JoVE content here

A wild type adult ICR (CD1) mouse heart typically yields 500,000 to 1 million cardiomyocytes from a successful isolation. Immediately after isolation, the cells maintain a mostly rod-shaped appearance (Figure 3A) with intact sarcomeres and can be used for functional studies involving cardiomyocyte contractility. A high percentage of rod-shaped cardiomyocytes (above 90%) is an indication of effective perfusion and digestion. Viable cardiomyocytes will be large (~ 100 - 200.......

Log in or to access full content. Learn more about your institution’s access to JoVE content here

The overall health of the isolated cardiomyocytes depends on several important aspects of this protocol. First, the time from heart extraction to perfusion is critical and should be performed in 5 min or less. Removal of calcium helps to dissociate cell-cell interactions, but can negatively impact cell health long-term.29-32 Thus, we find it sufficient to remove calcium during the initial few minutes of perfusion by EGTA (ethylene glycol tetraacetic acid) chelation,22 but quickly restore calciu.......

Log in or to access full content. Learn more about your institution’s access to JoVE content here

This project was funded by the UCSF Program for Breakthrough Biomedical Research (funded in part by the Sandler Foundation), the NIH Pathway to Independence Award (R00HL114738), and the Edward Mallinckrodt Jr. Foundation. JJ was supported by a postdoctoral fellowship from the NIH (T32HL007731). The authors are solely responsible for the contents of this work, which does not necessarily represent the official views of the NIH.

....

Log in or to access full content. Learn more about your institution’s access to JoVE content here

Name Company Catalog Number Comments
Equipment
Heated water jacket Radnoti 158831
Circulating heated water bath, Isotemp Fisher Scientific 3013
Laboratory pump Watson-Marlow 323
Hemostats Exelta 63042-090
Tissue forceps VWR 470128034
Dumont #7 curved forceps FST 91197-00
Dumont #5 fine forceps FST 11251-20
Small dissection scissors VWR 470128034
Extra fine bonn scissors FST 14084-08
Fine spring scissors FST 91500-09
Name Company Catalog Number Comments
Materials
NaCl Sigma S9888
KCl Sigma P9541
Na2HPO4-7H2O Fisher S25837
MgSO4-7H2O Fisher S25414
Taurine Sigma 86329
Butane dione monoxime (BDM) Sigma B0753
HEPES Fisher  BP310100
Glucose Sigma G-7021
Insulin Novo Nordisk 393153
EGTA Amresco 0732-288
Protease, type XIV Sigma P5147
Collagenase II Worthington LS004176
MEM Corning 15-010-CV
FBS, heat inactivated JRS 43613
Primocin Invitrogen NC9141851
Ethyl Carbamate Alfa Aesar AAA44804-18
215 micron mesh Component supply U-CMN-215-A
20 G blunt ended needle Becton Dickinson 305183
20 G beveled needle Becton Dickinson 305176
Lab tape VWR 89097-990
Surgical tape 3M 1527-0
Silk suture, 7-0 Teleflex 15B051000
Mouse anti-alpha-actinin antibody Sigma A7811
Alexa Fluor 488 goat anti-mouse IgG1 antibody Thermo Fisher A21121

  1. Patel, A. K., Celiz, A. D., et al. A defined synthetic substrate for serum free culture of human stem cell derived cardiomyocytes with improved functional maturity identified using combinatorial materials microarrays. Biomaterials. 61, 257-265 (2015).
  2. Mathur, A., Loskill, P., et al. Human iPSC-based Cardiac Microphysiological System For Drug Screening Applications. Sci Rep. 5, 8883 (2015).
  3. Mahmoud, A. I., Kocabas, F., et al. Meis1 regulates postnatal cardiomyocyte cell cycle arrest. Nature. 497 (7448), 249-253 (2013).
  4. Baumgartner, S., Halbach, M., et al. Electrophysiological and morphological maturation of murine fetal cardiomyocytes during electrical stimulation in vitro. J Cardiovasc Pharmacol Ther. 20 (1), 104-112 (2015).
  5. Ieda, M., Tsuchihashi, T., et al. Cardiac fibroblasts regulate myocardial proliferation through beta1 integrin signaling. Dev Cell. 16 (2), 233-244 (2009).
  6. Zhang, Y., Li, T. S., et al. Dedifferentiation and proliferation of mammalian cardiomyocytes. PLoS One. 5 (9), e12559 (2010).
  7. Engel, F. B., Schebesta, M., et al. p38 MAP kinase inhibition enables proliferation of adult mammalian cardiomyocytes. Gene Dev. 19 (10), 1175-1187 (2005).
  8. Sakurai, T., Lanahan, A., Woolls, M. J., Li, N., Tirziu, D., Murakami, M. Live cell imaging of primary rat neonatal cardiomyocytes following adenoviral and lentiviral transduction using confocal spinning disk microscopy. J Vis Exp. (88), e51666 (2014).
  9. Ahuja, P., Perriard, E., Perriard, J. C., Ehler, E. Sequential myofibrillar breakdown accompanies mitotic division of mammalian cardiomyocytes. J Cell Sci. 117 (Pt 15), 3295-3306 (2004).
  10. Kikuchi, K., Holdway, J. E., et al. Primary contribution to zebrafish heart regeneration by gata4(+) cardiomyocytes. Nature. 464 (7288), 601-605 (2010).
  11. Jopling, C., Sleep, E., Raya, M., Martì, M., Raya, A., Izpisúa Belmonte, J. C. Zebrafish heart regeneration occurs by cardiomyocyte dedifferentiation and proliferation. Nature. 464 (7288), 606-609 (2010).
  12. Porrello, E. R., Mahmoud, A. I., et al. Transient Regenerative Potential of the Neonatal Mouse Heart. Science. 331 (6020), 1078-1080 (2011).
  13. Heallen, T., Morikawa, Y., et al. Hippo signaling impedes adult heart regeneration. Development. 140 (23), 4683-4690 (2013).
  14. Lin, Z., Zhou, P., et al. Pi3kcb links Hippo-YAP and PI3K-AKT signaling pathways to promote cardiomyocyte proliferation and survival. Circ Res. 116 (1), 35-45 (2015).
  15. Zebrowski, D. C., Vergarajauregui, S., et al. Developmental alterations in centrosome integrity contribute to the post-mitotic state of mammalian cardiomyocytes. eLife. 4, e05563 (2015).
  16. Schluter, K. D., Piper, H. M. . Practical Methods in Cardiovascular Research. , (2005).
  17. Zhou, Y. Y., Wang, S. Q., et al. Culture and adenoviral infection of adult mouse cardiac myocytes: methods for cellular genetic physiology. Am J Physiol Heart Circ Phys. 279 (1), H429-H436 (2000).
  18. Kruppenbacher, J. P., May, T., Eggers, H. J., Piper, H. M. Cardiomyocytes of adult mice in long-term culture. Naturwissenschaften. 80 (3), 132-134 (1993).
  19. Fredj, S., Bescond, J., Louault, C., Potreau, D. Interactions between cardiac cells enhance cardiomyocyte hypertrophy and increase fibroblast proliferation. Journal of Cellular Physiology. 202 (3), 891-899 (2005).
  20. Li, Z., Sharma, R. V., Duan, D., Davisson, R. L. Adenovirus-mediated gene transfer to adult mouse cardiomyocytes is selectively influenced by culture medium. J Gene Med. 5 (9), 765-772 (2003).
  21. Luo, J., Deng, Z. L., et al. A protocol for rapid generation of recombinant adenoviruses using the AdEasy system. Nat Protoc. 2 (5), 1236-1247 (2007).
  22. Shioya, T. A simple technique for isolating healthy heart cells from mouse models. J Physiol Sci. 57 (6), 327-335 (2007).
  23. Li, D., Wu, J., Bai, Y., Zhao, X., Liu, L. Isolation and culture of adult mouse cardiomyocytes for cell signaling and in vitro cardiac hypertrophy. J Vis Exp. (87), e51357 (2014).
  24. Wolska, B. M., Solaro, R. J. Method for isolation of adult mouse cardiac myocytes for studies of contraction and microfluorimetry. Am J Physiol Heart Circ Phys. 271 (3), H1250-H1255 (1996).
  25. Pinz, I., Zhu, M., Mende, U., Ingwall, J. S. An improved isolation procedure for adult mouse cardiomyocytes. Cell Biochem Biophys. 61 (1), 93-101 (2011).
  26. Di Stefano, V., Giacca, M., Capogrossi, M. C., Crescenzi, M., Martelli, F. Knockdown of cyclin-dependent kinase inhibitors induces cardiomyocyte re-entry in the cell cycle. J Biol Chem. 286 (10), 8644-8654 (2011).
  27. Malouf, N. N., McMahon, D., Oakeley, A. E., Anderson, P. A. A cardiac troponin T epitope conserved across phyla. J Biol Chem. 267 (13), 9269-9274 (1992).
  28. Ehler, E., Moore-Morris, T., Lange, S. Isolation and culture of neonatal mouse cardiomyocytes. J Vis Exp. (79), e50154 (2013).
  29. Daly, M. J., Elz, J. S., Nayler, W. G. Contracture and the calcium paradox in the rat heart. Circ Res. 61 (4), 560-569 (1987).
  30. Piper, H. Culturing of calcium stable adult cardiac myocytes. J Mol Cell Cardiol. 14 (7), 397-412 (1982).
  31. Ashraf, M. Correlative studies on sarcolemmal ultrastructure, permeability, and loss of intracellular enzymes in the isolated heart perfused with calcium-free medium. Am J Pathol. 97 (2), 411-432 (1979).
  32. Piper, H. The calcium paradox revisited An artefact of great heuristic value. Cardiovasc Res. 45 (1), 123-127 (2000).
  33. Higuchi, H., Takemori, S. Butanedione monoxime suppresses contraction and ATPase activity of rabbit skeletal muscle. J Biochem. 105 (4), 638-643 (1989).
  34. Rother, J., Richter, C., et al. Crosstalk of cardiomyocytes and fibroblasts in co-cultures. Open biology. 5 (6), 150038 (2015).
  35. Fujio, Y., Nguyen, T., Wencker, D., Kitsis, R. N., Walsh, K. Akt Promotes Survival of Cardiomyocytes In Vitro and Protects Against Ischemia-Reperfusion Injury in Mouse Heart. Circulation. 101 (6), 660-667 (2000).
  36. Dambrot, C., Braam, S. R., Tertoolen, L. G. J., Birket, M., Atsma, D. E., Mummery, C. L. Serum supplemented culture medium masks hypertrophic phenotypes in human pluripotent stem cell derived cardiomyocytes. Journal of cellular and molecular medicine. 18 (8), 1509-1518 (2014).
  37. Karliner, J. S., Simpson, P. C., Taylor, J. E., Honbo, N., Woloszyn, W. Adrenergic receptor characteristics of cardiac myocytes cultured in serum-free medium: Comparison with serum-supplemented medium. Biochemical and Biophysical Research Communications. 128 (1), 376-382 (1985).
  38. Zheng, X., Baker, H., Hancock, W. S., Fawaz, F., McCaman, M., Pungor, E. Proteomic analysis for the assessment of different lots of fetal bovine serum as a raw material for cell culture. Part IV. Application of proteomics to the manufacture of biological drugs. Biotechnology progress. 22 (5), 1294-1300 (2006).
  39. Soonpaa, M. H., Kim, K. K., Pajak, L., Franklin, M., Field, L. J. Cardiomyocyte DNA synthesis and binucleation during murine development. Am J Physiol. 271 (5 Pt 2), H2183-H2189 (1996).
  40. Engel, F. B., Hsieh, P. C. H., Lee, R. T., Keating, M. T. FGF1/p38 MAP kinase inhibitor therapy induces cardiomyocyte mitosis, reduces scarring, and rescues function after myocardial infarction. P Natl Acad Sci USA. 103 (42), 15546-15551 (2006).
  41. Tian, Y., Liu, Y., et al. A microRNA-Hippo pathway that promotes cardiomyocyte proliferation and cardiac regeneration in mice. Sci Transl Med. 7 (279), 279ra38 (2015).
  42. Soonpaa, M. H., Koh, G. Y., et al. Cyclin D1 overexpression promotes cardiomyocyte DNA synthesis and multinucleation in transgenic mice. J Clin Invest. 99 (11), 2644-2654 (1997).
  43. Stewart, S., Stankunas, K. Limited dedifferentiation provides replacement tissue during zebrafish fin regeneration. Dev Biol. 365 (2), 339-349 (2012).
  44. Wu, C. H., Huang, T. Y., Chen, B. S., Chiou, L. L., Lee, H. S. Long-Duration Muscle Dedifferentiation during Limb Regeneration in Axolotls. PLoS One. 10 (2), e0116068 (2015).
  45. Nag, A. C., Lee, M. L., Kosiur, J. R. Adult cardiac muscle cells in long-term serum-free culture: myofibrillar organization and expression of myosin heavy chain isoforms. In vitro cellular & developmental biology journal of the Tissue Culture Association. 26 (5), 464-470 (1990).
  46. Lian, X., Hsiao, C., et al. Robust cardiomyocyte differentiation from human pluripotent stem cells via temporal modulation of canonical Wnt signaling. P Natl Acad Sci USA. 109 (27), E1848-E1857 (2012).
  47. Sohal, D. S., Nghiem, M., et al. Temporally Regulated and Tissue-Specific Gene Manipulations in the Adult and Embryonic Heart Using a Tamoxifen-Inducible Cre Protein. Circ Res. 89 (1), 20-25 (2001).
  48. Hsieh, P. C. H., Segers, V. F. M., et al. Evidence from a genetic fate-mapping study that stem cells refresh adult mammalian cardiomyocytes after injury. Nat Med. 13 (8), 970-974 (2007).

This article has been published

Video Coming Soon

JoVE Logo

Privacy

Terms of Use

Policies

Research

Education

ABOUT JoVE

Copyright © 2024 MyJoVE Corporation. All rights reserved