Abstract 17164: The Pentameric Form of Phospholamban is Important for the Regulation of Phospholamban Phosphorylation and Function
Phospholamban (PLN) regulates myocyte calcium cycling by inhibiting the Ca2+ATPase SERCA2a. Protein kinase A (PKA) mediated phosphorylation attenuates PLN activity leading to enhanced calcium uptake rates and accelerated cardiac relaxation. In vivo, PLN is present in monomeric and pentameric form. It is believed that PKA primarily targets the PLN monomer. However, we found that a R9C mutant of PLN dominantly inhibits PLN phosphorylation only within the pentamer suggesting a significant role of the pentamer in determining phosphorylation and, thus, PLN activity.
To investigate the role of the pentamer in PLN phosphorylation and function, the sensitivity, kinetics and stoichiometry of phosphorylation were analyzed in monomeric and pentameric PLN mutants expressed in a human cell line (HEK293AD). We found an independent increase of phosphorylation for monomer and pentamer upon forskolin stimulation, both in a concentration and time-dependent manner. Intriguingly, phosphorylation signals of PLN pentamers increased first. However, phosphorylation of monomers was accelerated in the absence of pentamers. Western blot analyses using Phostag®acrylamide gels demonstrated pentamers that carried between 0 and 5 phosphate residues indicating that pentamer phosphorylation occurs stepwise without preference for specific stoichiometries. Cross-linking experiments using transfected HEK cells and mouse heart lysates, respectively, confirmed the direct interaction of pentameric as well as monomeric PLN with the Ca2+-ATPase. To evaluate the functional significance of the PLN pentamer in vivo, transgenic mice with exclusive expression of monomeric PLN were generated and compared to mice that expressed both, monomers and pentamers. Following induction of cardiac stress by transverse aortic banding, mice lacking PLN pentamers developed higher heart weight, a blunted response to adrenergic stimulation and impaired survival.
Our data show that both PLN pentamers and monomers are suitable PKA targets and interact with SERCA2a. Mouse hearts lacking PLN pentamers showed a decreased tolerance to left ventricular pressure overload. These results indicate an important role of the PLN pentamer in the regulation of PLN activity and function.
- © 2013 by American Heart Association, Inc.