M. Fitzgerald et al., REDUCED RYANODINE RECEPTOR CONTENT IN ISOLATED NEONATAL CARDIOMYOCYTES COMPARED WITH THE INTACT TISSUE, Journal of Molecular and Cellular Cardiology, 26(10), 1994, pp. 1261-1265
The effects of isolation and culture of rat neonatal ventricular myocy
tes on the properties of the ryanodine receptor were investigated, [H-
3]-Ryanodine bound to a single class of sites in membranes prepared fr
om intact neonatal ventricle, with an affinity of 16.3 +/- 2.8 nM (mea
n +/- S.E.: n = 3) and a capacity of 546 +/- 64 fmol/mg protein (mean
+/- S.E.: n = 3). In contrast, no detectable displaceable binding of [
H-3]-ryanodine was observed when similar experiments were performed us
ing membranes prepared from isolated neonatal cardiomyocytes. The appa
rent absence of [H-3]-ryanodine binding in the neonatal cardiomyocytes
suggested either reduced ryanodine receptor protein or the conversion
of the receptors to a low affinity state. To distinguish between thes
e possibilities, the content of ryanodine receptor protein was measure
d using SDS-PAGE followed by western blotting. Membranes prepared from
neonatal ventricle contained substantial amounts of ryanodine recepto
r, as demonstrated by a dense band on western blots. However the corre
sponding band in preparations of isolated cells, while having similar
electrophoretic mobility, was barely detectable. It is concluded that
the ryanodine receptor protein is strongly expressed in intact neonata
l ventricle, but the level of expression is markedly reduced upon isol
ation of the cardiomyocytes. These findings demonstrate that ryanodine
receptor expression is significantly down-regulated when rat neonatal
ventricular myocytes are isolated and maintained in culture. This mar
ked decrease in expression of one of the key proteins required for exc
itation-contraction coupling suggests that regulatory factors present
in intact myocardium play an important role in maintaining expression
of elements of the contractile mechanism.