Reduced ryanodine receptor content in isolated neonatal cardiomyocytes compared with the intact tissue.
The effects of isolation and culture of rat neonatal ventricular myocytes on the properties of the ryanodine receptor were investigated. [3H]-Ryanodine bound to a single class of sites in membranes prepared from intact neonatal ventricle, with an affinity of 16.3 +/- 2.8 nm (mean +/- 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 [3H]-ryanodine was observed when similar experiments were performed using membranes prepared from isolated neonatal cardiomyocytes. The apparent absence of [3H]-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 these possibilities, the content of ryanodine receptor protein was measured using SDS-PAGE followed by western blotting. Membranes prepared from neonatal ventricle contained substantial amounts of ryanodine receptor, as demonstrated by a dense band on western blots. However the corresponding 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 neonatal ventricle, but the level of expression is markedly reduced upon isolation 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.(ABSTRACT TRUNCATED AT 250 WORDS)
Fitzgerald
M
M
Cellular Biochemistry Laboratory, Baker Medical Research Institute, Prahran (Melbourne), Victoria, Australia.
Neylon
C B
CB
Marks
A R
AR
Woodcock
E A
EA
D000818:Animals; D000831:Animals, Newborn; D015153:Blotting, Western; D015220:Calcium Channels / Q000172:deficiency*; D002469:Cell Separation; D002478:Cells, Cultured; D015536:Down-Regulation; D006329:Heart Conduction System / Q000502:physiology; D006352:Heart Ventricles; D008861:Microsomes / Q000737:chemistry; D009124:Muscle Proteins / Q000172:deficiency*; D009200:Myocardial Contraction; D009206:Myocardium / Q000737:chemistry* / Q000166:cytology; D051381:Rats; D011921:Rats, Inbred WKY; D012433:Ryanodine / Q000378:metabolism; D019837:Ryanodine Receptor Calcium Release Channel
D015220:Calcium Channels; D009124:Muscle Proteins; D019837:Ryanodine Receptor Calcium Release Channel; D012433:Ryanodine