Influence of different culture conditions on sarcoplasmic reticular calcium transport in isolated neonatal rat cardiomyocytes

摘要

This study investigates sarcoplasmic reticulum (SR) calcium-(Ca~(2+)) transport ATPase (SERCA2a) and phospholamban (PLB) in cultured spontaneously contracting neonatal rat cardiomyocytes (CM) to ascertain the function of both SR proteins under various culture conditions. The two major SR proteins were readily detectable in cultured CM by immunofluorescent microscopy using specific anti-SERCA2 and anti-PLB antibodies. Double labeling technique revealed that PLB-positive CM also labeled with anti-SERCA2. Coexpression of SERCA2 and PLB in CM was supported by measurement of cell homogenate oxalate-supported Ca~(2+) uptake which was completely inhibited by thapsigargin and stimulated by protein kinase. A-catalyzed phosphorylation. Under serum-free conditions, incubation of CM with the SERCA2a expression modulator 3,3 ,5-triiodo-L-thyronine (100 nM, 72 h) resulted in elevated Ca~(2+) uptake of +33%. Specific Ca~(2+) uptake activity was not altered if insulin was omitted from the serum-free cluture medium but total SR Ca~(2+) transport activity was reduced under this culture condition. The results indicate that primary culture of spontaneously contracting neonatal rat CM can be employed as a useful model system for investigating both short- and long-term mechanisms determining the Ca~(2+) re-uptake function of the SR under defined culture conditions.