Cellular and Molecular Life Sciences
Functional characterization of the human α-cardiac actin mutations Y166C and
M305L involved in hypertrophic cardiomyopathy
Mirco Müller, Antonina Joanna Mazur, Elmar Behrmann, Ralph P. Diensthuber, Michael B. Radke, Zheng Qu,
Christoph Littwitz, Stefan Raunser, Cora-Ann Schoenenberger, Dietmar J. Manstein, and Hans Georg Mannherz
Corresponding author: Dr. Hans Georg Mannherz; hans.g.mannherz@rub.de
Online Resource 1: Isolation of adult and neonatal rat cardiomyocytes
Isolation of adult rat cardiomyocytes (ARCs) – Adult Wistar Kyoto rat cardiomyocytes were isolated as
originally described [1]. Urethane (1g/kg) anaesthetized rats were killed in accordance with the guidelines of the
European community (86/609/EEC). The aorta was cannulated to a sterile Langendorff-perfusion system. The
hearts were placed in a perfusion chamber and perfused with 1.6 mg/ml liberase blendzyme 4 in HBS buffer (10
mM Hepes, pH 7.4, 140 mM NaCl, 5.4 mM KCl, 1 mM MgCl2). When the hearts became swollen, the atria were
separated from the ventricles; which were placed in separate dishes. Further digestion proceeded until single
cells were observed microscopically. Then the tissue was placed in a stop solution (100 mM NaCl, 45 mM KCl,
1 mM MgCl2, 10 mM Hepes, 3 mM N-acetyl-L-cystein, 5 mM sodium pyruvate) and subsequently in a stop
solution containing bovine serum albumin (BSA) (1 mg/ml) and DNase I (1 mg/ml) with increasing Ca2+
concentration (0.02 mM – 1.0 mM) to prevent the occurrence of the Ca2+ paradox [2]. 105 ventricular
cardiomyocytes were plated per dish in 35 mm culture dish and incubated in serum-free M199 medium
supplemented with 25 µg/ml gentamycin, 25 µg/ml kanamycin and an 1% (w/v) insulin-transferrin-selenium mix
(Sigma-Aldrich, Germany) at 37 °C and 1% CO2 for up to 3 weeks.
Isolation of neonatal rat cardiomyocytes (NRCs) – Cardiomyocytes from 1-5 days old rats were isolated
according to the modified protocol described by Przygodzki et al. [3]. Neonatal rats were killed in accordance
with the guidelines of the European community (86/609/EEC). Briefly, dissected heart tissue in buffer A (20
mM Hepes, pH 7.4, 120 mM NaCl, 1 mM NaH2PO4, 5.5 mM glucose, 5.4 mM KCl, 0.8 mM MgSO4) containing
heparin (19.5 U/mg) was digested at 37 °C for 1 h in buffer A supplemented with the enzymes collagenase type
II (80 U/ml) and pancreatinin (0.6 mg/ml). Every 10 min the supernatants containing released cells were taken
and centrifuged at 327 x g and room temperature for 8 min and the cell pellet was resuspended in 2 ml of culture
medium [DMEM/M199 4:1, 10% horse serum, 5% fetal calf serum, 1% penicillin/streptomycin/neomycinmixture, 1% glutamax (all obtained from Invitrogen)]. Meanwhile new portions of enzymes were added to the
tissue. After the digestion was repeated 5 times, the suspended cells were pooled and filtered through a nylon
cell strainer with 100 µm pores. Subsequently the cells were washed once more to remove the remaining
proteases and placed in a 100 x 20 mm tissue culture dish for preplating that lasted for 2 h in an incubator (37
°C). During this step fibroblasts and other non-cardiomyocyte cells attach to the bottom of the dish, while the
cardiomyocytes remain in the supernatant [4]. Then the cardiomyocytes were collected by gentle centrifugation
and seeded in 35 mm tissue culture dishes coated with 10 µg/ml laminin and 10 µg/ml fibronectin to a final
density of 3.5 x 105 cells. The next day, the medium was changed and fresh medium containing cytosine β-Darabinofuranoside (10 µg/ml) was added to the cells. The NRCs were incubated in this medium for 48 h in order
to inhibit the growth of the remaining mesenchymal cells [5]. 48 h later the medium was changed again to
DMEM/M199 without cytosine arabinoside. Contamination of isolated NRCs with other cells was around 5-10%
as estimated by immunofluorescence analysis by staining with a mouse anti-α-cardiac actin antibody and
Hoechst 33432. It was possible to maintain viable neonatal cardiomyocytes for up to 3 weeks when incubated in
DMEM/M199 at 37 °C and 5% CO2.
1. Bechem M, Pott L, Rennebaum H (1983) Atrial muscle cells from hearts of adult guinea-pigs in culture: a new
preparation for cardiac cellular electrophysiology. Eur J Cell Biol 31:366-369
2. Chapman RA, Tunstall J (1987) The calcium paradox of the heart. Prog Biophys Mol Biol 50:67-96.
doi:0079-6107(87)90004-6 [pii]
3. Przygodzki T, Sokal A, Bryszewska M (2005) Calcium ionophore A23187 action on cardiac myocytes is
accompanied by enhanced production of reactive oxygen species. Biochim Biophys Acta 1740:481-488.
doi:10.1016/j.bbadis.2005.03.009
4. Marengo FD, Wang SY, Wang B, Langer GA (1998) Dependence of cardiac cell Ca2+ permeability on sialic
acid-containing sarcolemmal gangliosides. J Mol Cell Cardiol 30:127-137. doi:10.1006/jmcc.1997.0579
5. Tanaka M, Ito H, Adachi S, Akimoto H, Nishikawa T, Kasajima T, Marumo F, Hiroe M (1994) Hypoxia
induces apoptosis with enhanced expression of Fas antigen messenger RNA in cultured neonatal rat
cardiomyocytes. Circ Res 75:426-433