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thoracotomy was performed and hearts were cailrulated in situ, perfused and rapidly
excised. After the excision, hearts were mounted on a modified Langendorff perfusion
apparatus which allows switching between a single pass and recirculating perfusion at a
temperature of 370C. The perfusate consisted of calcium and a serum free medium
containing; 110 mM NaCl, 2.6 m]|dKCl, 1.2 rnM KHzPO+, 1.2 rnNI MgSOa, 25 mM
NaHCO3 and 11 mM Glucose (pH 7.4).The perfusion was then switched to recirculating
mode with a same buffer that now contained 25 pM calcium , 0 .lyo collagenase and 0.1Yo
bovine serum albumin for20 minutes.
The hearts were removed, cut into small pieces and disaggregated in the same
buffer. Disaggregation was achieved by gentle passing of the suspension through pipettes
with progressively smaller tip diameters. The suspension was filtered using a nylon mesh
(200pm) and re-suspended in medium M199 containing CaCl¡ After sedimentation (10
minutes), the cells were re-suspended in a serum-free medium Ml99 (Sigma-Aldrich,
Oakville, Ontario, Canada) and plated. Adult ventricular myocytes were then cultured
using a previously described method (Piper et al. 1988). Dishes were incubated with 4o/o
serum in Medium M199 (Sigma-Aldrich, Oakville, Ontario, Canada) 24 hours before the
plating. Serum containing medium was discarded and isolated myocytes were kept in a
primary culture, using serum free medium M199 (Sigma-Aldrich, Oakville, Ontario,
Canada).
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