Inhibition of endogenous cardiac phosphatase activity and measurement of sarcoplasmic reticulum calcium uptake: A possible role of phospholamban phosphorylation in the hypertrophied myocardium

Abstract

The activity of the sarcoplasmic reticulum (SR) CaATPase in cardiac muscle is regulated by phospholamban via its ability to be phosphorylated. It is unclear what role phospholamban phosphorylation plays in cardiac adaptation and disease. The study of the native phospholamban phosphorylation in tissue has been technically difficult because of the presence of endogenous enzymes. Using mobility shifts on SDS PAGE gels we have demonstrated that significant dephosphorylation of phospholamban occurs during tissue homogenisation in the absence of phosphatase inhibitors. Endogenous kinases do not appear to alter phospholamban phosphorylation. When 10 mM NaF (a phosphatase inhibitor) was used in the preparation of crude SR homogenates. CaATPase activity (measured by oxalate stimulated calcium uptake) was stimulated almost 2 fold, p < 0.01. Increased CaATPase activity in NaF was associated with increased phospholamban phosphorylation. Phosphatase inhibitors were used in tissue homogenisation to determine phospholamban phosphorylation in normal hearts and in cardiac hypertrophy induced by abdominal aortic constriction. In 50 mM NaF which completely inhibits endogenous phosphatases, phospholamban from hypertrophied hearts had a slower mobility compared with normal hearts. This suggests that phospholamban was more highly phosphorylated in cardiac hypertrophy. Increased phospholamban phosphorylation following cardiac hypertrophy may enable the myocardium to compensate functionally in the early stages of adaptation.