Role of phospholamban in cyclic GMP mediated signaling in cardiac myocytes

Abstract

We tested the hypothesis that the negative functional effects of cyclic GMP on cardiac myocytes were mediated through phospholamban (PLB) and activation of sarcoplasmic reticulum Ca 2+ -ATPase. Using ventricular myocytes from wild type (WT, n=10) and PLB knockout (PLB-KO, n=10) mouse hearts, functional changes were measured using a video edge detector at baseline and after 10 -6 , 10 -5 M 8-bromo-cyclic GMP (cGMP), 10 -8 , 10 -7 M C-type natriuretic peptide (CNP), or 10 -6 , 10 -5 M S-nitroso-N-acetyl-penicillamine (SNAP, nitric oxide donor). Changes in cytosolic Ca 2+ concentration were assessed in fura 2-loaded WT and PLB-KO myocytes. Cyclic GMP dependent phosphorylation analysis was also performed in WT and PLB-KO myocytes. 8-bromo-cGMP 10 -5 M caused a significant decrease in %shortening (3.6±0.2% to 2.3±0.1%) in WT, but little change in PLB-KO myocytes (3.4±0.1% to 3.2±0.2%). Similarly, CNP and SNAP reduced %shortening of WT, but not PLB-KO myocyte. Changes in other contractile parameters such as maximum rate of shortening and relaxation were consistent with the changes in % shortening. Intracellular Ca 2+ transients changed similarly to cell contractility in WT and PLB-KO myocytes treated with cGMP and CNP; i.e. Ca 2+ transients decreased with cGMP or CNP in WT myocytes, but were unchanged in PLB-KO myocytes. cGMP dependent phosphorylation analysis showed that some proteins were phosphorylated by cGMP to a lesser extent in PLB-KO compared with WT myocytes, suggesting impaired cGMP-kinase function in PLB-KO cardiac myocytes. These results indicated that cGMP-induced reductions in cardiac myocyte function were at least partially mediated through the action of phospholamban. Copyright © 2007 S. Karger AG.