Inhibition of phospholamban phosphorylation by O-GlcNAcylation: implications for diabetic cardiomyopathy

Cardiac type sarco(endo)plasmic reticulum Ca-ATPase (SERCA2a) plays a major role in cardiac muscle contractility. Phospholamban (PLN) regulates the function via its Ser-phosphorylation. Since it has been proposed that the Ser/Thr residues on cytoplasmic and nuclear proteins are modified by O-linked N-acetylglucosamine (O-GlcNAc), we examined the effect of O-GlcNAcylation on PLN function in rat adult cardiomyocytes. The study with an enzymatic labeling and a co-immunoprecipitation using wild type and a series of mutants of PLN showed that PLN was O-GlcNAcylated and Ser 16 of PLN might be the site for O-GlcNAcylation. In cardiomyocytes treated with O-(2-acetamido-2-deoxy-D-glucopyranosylidene)amino-N-phenylcarbamate (PUGNAc), the O-GlcNAcylation was significantly increased compared to non-treated cells. Simultaneously, Ser-phosphorylation of PLN was reduced. In Chinese hamster ovary cells where PLN cDNA and O-GlcNAc transferase siRNA were co-transfected, the Ser-phosphorylation of PLN was significantly increased compared to controls. The same results were observed in heart homogenates from diabetic rats. In a co-immunoprecipitation of PLN with SERCA2a, the physical interaction between the two proteins was increased in PUGNAc-treated cardiomyocytes. Unlike non-treated cells, the activity of SERCA2a and the profiles of calcium transient in PUGNAc-treated cardiomyocytes were not significantly changed even after the treatment of catecholamine. These data suggest that PLN is O-GlcNAcylated to induce the inhibition of its