Isolation of Atrial and Ventricular Cardiomyocytes for In Vitro Studies

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


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 


  1. 1.
    Mitcheson JS, Hancox JC, Levi AJ (1998) Cultured adult cardiac myocytes: future applications, culture methods, morphological and electrophysiological properties. Cardiovasc Res 39(2):280–300CrossRefPubMedGoogle Scholar
  2. 2.
    Streckfuss-Bomeke K, Wolf F, Azizian A, Stauske M, Tiburcy M, Wagner S, Hubscher D, Dressel R, Chen S, Jende J, Wulf G, Lorenz V, Schon MP, Maier LS, Zimmermann WH, Hasenfuss G, Guan K (2013) Comparative study of human-induced pluripotent stem cells derived from bone marrow cells, hair keratinocytes, and skin fibroblasts. Eur Heart J 34(33):2618–2629. Scholar
  3. 3.
    Louch WE, Sheehan KA, Wolska BM (2011) Methods in cardiomyocyte isolation, culture, and gene transfer. J Mol Cell Cardiol 51(3):288–298. Scholar
  4. 4.
    Dow JW, Harding NG, Powell T (1981) Isolated cardiac myocytes. I. Preparation of adult myocytes and their homology with the intact tissue. Cardiovasc Res 15(9):483–514CrossRefPubMedGoogle Scholar
  5. 5.
    Bell RM, Mocanu MM, Yellon DM (2011) Retrograde heart perfusion: the Langendorff technique of isolated heart perfusion. J Mol Cell Cardiol 50(6):940–950. S0022-2828(11)00095-2 [pii]. Scholar
  6. 6.
    Isenberg G, Klockner U (1982) Calcium tolerant ventricular myocytes prepared by preincubation in a “KB medium”. Pflugers Arch 395(1):6–18CrossRefPubMedGoogle Scholar
  7. 7.
    Mitra R, Morad M (1985) A uniform enzymatic method for dissociation of myocytes from hearts and stomachs of vertebrates. Am J Phys 249(5 Pt 2):H1056–H1060Google Scholar
  8. 8.
    O’Connell TD, Rodrigo MC, Simpson PC (2007) Isolation and culture of adult mouse cardiac myocytes. Methods Mol Biol 357:271–296. Scholar
  9. 9.
    Pluteanu F, Hess J, Plackic J, Nikonova Y, Preisenberger J, Bukowska A, Schotten U, Rinne A, Kienitz MC, Schafer MK, Weihe E, Goette A, Kockskamper J (2015) Early subcellular Ca2+ remodelling and increased propensity for Ca2+ alternans in left atrial myocytes from hypertensive rats. Cardiovasc Res 106(1):87–97. Scholar
  10. 10.
    Plackic J, Preissl S, Nikonova Y, Pluteanu F, Hein L, Kockskamper J (2016) Enhanced nucleoplasmic Ca2+ signaling in ventricular myocytes from young hypertensive rats. J Mol Cell Cardiol 101:58–68. Scholar
  11. 11.
    Roth GM, Bader DM, Pfaltzgraff ER (2014) Isolation and physiological analysis of mouse cardiomyocytes. J Vis Exp 91:e51109. Scholar
  12. 12.
    van Deel ED, Najafi A, Fontoura D, Valent E, Goebel M, Kardux K, Falcao-Pires I, van der Velden J (2017) In vitro model to study the effects of matrix stiffening on Ca2+ handling and myofilament function in isolated adult rat cardiomyocytes. J Physiol 595(14):4597–4610. Scholar
  13. 13.
    Mulieri LA, Hasenfuss G, Ittleman F, Blanchard EM, Alpert NR (1989) Protection of human left ventricular myocardium from cutting injury with 2,3-butanedione monoxime. Circ Res 65(5):1441–1449CrossRefPubMedGoogle Scholar
  14. 14.
    Blanchard EM, Smith GL, Allen DG, Alpert NR (1990) The effects of 2,3-butanedione monoxime on initial heat, tension, and aequorin light output of ferret papillary muscles. Pflugers Arch 416(1–2):219–221CrossRefPubMedGoogle Scholar
  15. 15.
    Adams W, Trafford AW, Eisner DA (1998) 2,3-Butanedione monoxime (BDM) decreases sarcoplasmic reticulum Ca content by stimulating Ca release in isolated rat ventricular myocytes. Pflugers Arch 436(5):776–781CrossRefPubMedGoogle Scholar
  16. 16.
    Zimmerman AN, Hulsmann WC (1966) Paradoxical influence of calcium ions on the permeability of the cell membranes of the isolated rat heart. Nature 211(5049):646–647CrossRefPubMedGoogle Scholar
  17. 17.
    Piper HM (2000) The calcium paradox revisited: an artefact of great heuristic value. Cardiovasc Res 45(1):123–127CrossRefPubMedGoogle Scholar

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© 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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