REDUCED RYANODINE RECEPTOR CONTENT IN ISOLATED NEONATAL CARDIOMYOCYTES COMPARED WITH THE INTACT TISSUE

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.