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An Improved Isolation Procedure for Adult Mouse Cardiomyocytes

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Abstract

Isolated adult mouse cardiomyocytes are an important tool in cardiovascular research, but are challenging to prepare. Because the energy supply determines cell function and viability, we compared total creatine ([Cr]) and [ATP] in isolated cardiomyocytes with the intact mouse heart. Isolated myocytes suffered severe losses of Cr (−70%) and ATP (−53%). Myocytes were not able to replete [Cr] during a 5 h incubation period in medium supplemented with 1 mM Cr. In contrast, adding 20 mM Cr to the digestion buffers was sufficient to maintain normal [Cr]. Supplementing buffers with 5 mM of inosine (Ino) and adenosine (Ado) to prevent loss of cellular nucleosides partially protected against loss of ATP. To test whether maintaining [ATP] and [Cr] improves contractile function, myocytes were challenged by varying pacing rate from 0.5 to 10 Hz and by adding isoproterenol (Iso) at 5 and 10 Hz. All groups performed well up to 5 Hz, showing a positive cell shortening–frequency relationship; however, only 16% of myocytes isolated under standard conditions were able to sustain pacing with Iso challenge at 10 Hz. In contrast, 30–50% of the myocytes with normal Cr levels were able to contract and maintain low diastolic [Ca2+]. Cell yield also improved in Cr and the Cr/Ino/Ado-treated groups (85–90% vs. 70–75% rod shaped in untreated myocytes). These data suggest that viability and performance of isolated myocytes are improved when they are protected from the severe loss of Cr and ATP during the isolation, making them an even better research tool.

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Acknowledgments

This project was supported by grants NIH HL52320 to JSI and UM, NIH HL 075619 to JSI, NIH HL 080127 to UM, and 0930260N AHA to IP and 5 P20 RR15555-10 (subproject 6) to IP from the National Center for Research Resources (NCRR), a component of the National Institutes of Health (NIH). Its contents are solely the responsibility of the authors and do not necessarily represent the official views of NCRR or NIH.

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Author notes

  1. Ming Zhu

    Present address: Pfizer, BioTherapeutics R&D, 200 Cambridge Park Drive, Cambridge, MA, 02140, USA

  2. Ulrike Mende

    Present address: Cardiovascular Research Center of Rhode Island Hospital and The Warren Alpert Medical School of Brown University, 1 Hoppin Street, Providence, RI, 02903, USA

Authors and Affiliations

  1. NMR Laboratory for Physiological Chemistry, Division of Cardiovascular Medicine, Department of Medicine, Brigham and Women’s Hospital, Harvard Medical School, Boston, MA, USA

    Ilka Pinz, Ming Zhu, Ulrike Mende & Joanne S. Ingwall

  2. Center for Molecular Medicine, Maine Medical Center Research Institute, 81 Research Drive, Scarborough, ME, 04074, USA

    Ilka Pinz

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  1. Ilka Pinz
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Correspondence to Ilka Pinz.

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Pinz, I., Zhu, M., Mende, U. et al. An Improved Isolation Procedure for Adult Mouse Cardiomyocytes. Cell Biochem Biophys 61, 93–101 (2011). https://doi.org/10.1007/s12013-011-9165-9

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