Isolation of Atrial and Ventricular Cardiomyocytes for In Vitro Studies
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 wordsLangendorff retrograde heart perfusion Cardiomyocyte isolation Collagenase digestion Atrial and ventricular cardiomyocytes
- 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. https://doi.org/10.1093/eurheartj/ehs203CrossRefPubMedGoogle Scholar
- 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
- 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. https://doi.org/10.1093/cvr/cvv045CrossRefPubMedGoogle Scholar
- 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. https://doi.org/10.1113/JP274460CrossRefPubMedPubMedCentralGoogle Scholar