Abstract 1533: Protein Phosphatase 1 Beta is Most Abundant Isoform in the Longitudinal Sarcoplasmic Reticulum and Regulates Phospholamban Phosphorylation in Cardiomyocytes
We and others previously reported that overactivation of protein phosphatase 1 (PP1) is responsible for inefficient Ca2+ handling via sarcoplasmic reticulum (SR) in the failing myocardium. PP1 catalytic unit includes three distinct genes, namely PPP1CA (PP1a), PPP1CB (PP1b), and PPP1CC (PP1g), each of which is not well characterized in terms of effects on Ca2+ cycling and localization in the subcellular microdomain. We investigated the isoform specific role of PP1 on SR-mediated Ca2+ cycling and correlated its characteristics with subcellular distribution. Adenoviral RNAi technique was employed to induce isoform-specific knockdown for PP1a, PP1b, and PP1g in adult rat cardiomyocytes, followed by analysis of cell shortening, Ca2+ transients, and phosphorylation of key molecules. Subcellular localization of each isoform was characterized by imunoblotting against canine junctional and longitudinal SR fractions which are purified by sucrose gradient ultracentrifuge. In addition, molecular association between PLN and PP1 was determined in the 1% digitonin-solubilized SR. All adenoviral RNAi transfection induced approximately 90% decrease in mRNA expression and 60 to 80% decrease in protein levels of corresponding PP1 isoform without affecting expressions of other isoforms. Among the PP1 isoform-specific knockdown, PP1b knockdown most effectively augmented the amplitude and decay time constant of Ca2+ transient with increased PLN phosphorylation, and significantly increased %cell shortening at baseline and under adrenergic stimulation. PP1b was most abundantly enriched in the longitudinal SR fraction from canine SR among the PP1 isoforms. On the other hand, PP1g was enriched in the junctional SR fraction. PLN and PP1b were co-immunoprecipitated together with sarcoendoplasmic Ca2+ ATPase 2a (SERCA) and BiP, a luminal endoplasmic reticulum chaperone protein. These data indicate that PP1b is the major PLN phosphatase and regulates SR Ca2+ uptake in the longitudinal SR of cardiomyocytes. The physical interaction of PP1 with BiP in addition to that with SERCA and PLN suggests that protein dephosphorylation by PP1b is important for protein folding as in the function of endoplasmic reticulum.