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Isolation of Atrial and Ventricular Cardiomyocytes for In Vitro Studies

  • Jelena Plačkić
  • Jens Kockskämper
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1816)

Abstract

High quality cardiomyocyte isolation is of critical importance for successful studies of myocardial function at the cellular and molecular level. Although previous work has established isolation procedures for various species, it still remains challenging to produce consistently a high yield of viable and healthy cardiomyocytes. The basis for the most successful and reproducible isolation of cardiomyocytes from intact hearts is the Langendorff retrograde perfusion technique. Here, we will illustrate in detail all practical aspects of the enzyme-based Langendorff isolation of rat atrial and ventricular cardiomyocytes. This includes a series of obligatory steps starting from quick aortic cannulation to rinse the heart from blood, short perfusion of the heart with Ca2+-free solution to dissociate cells at the level of intercalated discs, followed by longer perfusion with low Ca2+-containing enzyme solution in order to disrupt the extracellular matrix network, extraction of the released cardiomyocytes and gentle Ca2+ reintroduction to allow cells to return gradually to normal cytosolic Ca2+ levels. The average yield of intact viable ventricular myocytes that can be achieved with our protocol is ≈70% (range ≈50–90%). For atrial myocytes, in general, it is slightly (≈10%) lower than for ventricular myocytes. The yield depends on the age of the rat and the degree of cardiac remodeling such that digestion of older and more remodeled hearts (more fibrosis) usually results in lower yields. Isolated atrial and ventricular cardiomyocytes may be employed for studies of cardiomyocyte function (e.g., shortening/contraction, intracellular [Ca2+] transients) as well as for biochemical and molecular biological studies (e.g., immunoblotting, PCR).

Key words

Langendorff retrograde heart perfusion Cardiomyocyte isolation Collagenase digestion Atrial and ventricular cardiomyocytes 

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Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2018

Authors and Affiliations

  1. 1.Faculty of Pharmacy, Institute of Pharmacology and Clinical Pharmacy, Biochemical and Pharmacological Center (BPC) MarburgUniversity of MarburgMarburgGermany

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