Decoy peptides targeted to protein phosphatase 1 inhibit dephosphorylation of phospholamban in cardiomyocytes

Abstract

Cardiac sarcoplasmic reticulum Ca2+-ATPase (SERCA2a) plays a crucial role in Ca2+ handling in cardiomyocytes. Phospholamban (PLB) is an endogenous inhibitor of SERCA2a and its inhibitory activity is enhanced via dephosphorylation by protein phosphatase 1 (PP1). Therefore, the inhibition of PP1-mediated dephosphorylation of PLB might be an efficient strategy for the restoration of reduced SERCA2a activity in failing hearts. We sought to develop decoy peptides that would mimic phosphorylated PLB and thus competitively inhibit the PP1-mediated dephosphorylation of endogenous PLB. The phosphorylation sites Ser16 and Thr17 are located within the flexible loop region (amino adds 14-22) of PLB. We therefore synthesized a 9-mer peptide derived from this region (Psi PLB-wt) and two pseudo-phosphorylated peptides where Ser16 was replaced with Glu (Psi PLB-SE) or Thr17 was replaced with Glu (Psi PLB-TE). These peptides were coupled to the cell-permeable peptide TAT to facilitate cellular uptake. Treatment of adult rat cardiomyocytes with Psi PLB-SE or Psi PLB-TE, but not with Psi PLB-wt, significantly elevated the phosphorylation levels of PLB at Ser16 and Thr17. This increased phosphorylation of PLB correlated with an increase in contractile parameters in vitro. Furthermore, the perfusion of isolated rat hearts with Psi PLB-SE or Psi PLB-SE, but not with Psi PLB-wt, significantly improved left ventricular developed pressure that had been previously impaired by ischemia. These data indicate that Psi PLB-SE and Psi PLB-TE efficiently prevented dephosphorylation of PLB by serving as decoys for PP1. Therefore, these peptides may provide an effective modality to regulate SERCA2a activity in failing hearts. (C) 2012 Elsevier Ltd. All rights reserved.