Isolation of LAMs
Single LAMs were enzymatically isolated from New Zealand White rabbits
(weight 1.5-2.0 kg) as described in a previous study11. In brief, hearts were removed rapidly and perfused
with Ca2+-free Tyrode solution containing the
following compounds
(in
mM; purchased from Sigma-Aldrich, MA, USA):
135 NaCl, 5.4 KCl, 1.0
MgCl2, 10 glucose, 0.33
NaH2PO4, and 10 HEPES) at pH=7.4 with
NaOH for 5-10 minutes to washout blood. Then, the hearts were perfused
with Ca2+-free Tyrode solution containing collagenase
type I (0.5 g/L) and bovine serum albumin (BSA, 1.0 g/L) for 30-40
minutes before being perfused with KB solution, which contained the
following compounds (in mM): 70 KOH, 40 KCl, 20
KH2PO4, 50, glutamic acid, 20 taurine,
0.5 EGTA, 10 glucose, 10 HEPES, and 3.0 MgSO4) at pH=7.4
titrated with KOH for another 5-10 minutes. After perfusion, the left
atrium was collected and cut into small pieces gently in KB solution.
LAMs were filtered through a cell strainer (mesh number 200) and stored
in KB solution at room temperature. All solutions used in this study
were saturated with 100% O2 and were maintained at 37
°C.