Abstract 800: Protein Phosphatase 1 Beta is the Major Phospholamban Phosphatase that Regulates Cardiomyocyte Contractility.
Background: We previously reported that overactivation of protein phosphatase 1 (PP1) is responsible for the decrease in phospholamban (PLN) phosphorylation and cardiac contractility in the failing heart. Notably, PP1beta expression level is increased in various animal models and human heart failure. However, specific role of each PP1 isoform in cardiomyocytes is unclear. We therefore investigated the isoform specific role of PP1 in contractility and phosphorylation regulation using adenoviral RNA interference (RNAi) technique.
Methods: We created recombinant adenoviruses encoding short hairpin RNAs, to induce isoform-specific knockdown for PP1alpha (Ad.PP1 alpha-i), beta (Ad.PP1beta-i), and gamma (Ad.PP1gamma-i). Isolated cardiomyocytes from adult rats at 8 –10 weeks of age were transfected with each adenoviral vector at MOI of 20, 100, and 500, followed by the analysis for protein phosphorylation, cell shortening and intracellular calcium transient 72 hours after transfection.
Results: All adenoviral transfection induced approximately 90% decrease in mRNA expression and 60 to 80% decrease in protein levels of corresponding PP1 isoform without affecting other isoforms. Expression levels of major Ca2+ regulatory proteins including PLN, protein kinase A, sarcoendoplasmic reticulum ATPase, ryanodine receptor type 2 were not affected. Increase in PLN phosphorylation at Ser16 was observed in Ad.PP1alpha-i (1.5 folds, p<0.05 v.s. Control) and Ad.PP1beta-i (2 folds, P<0.05 v.s. Control), but not in Ad-PP1 gamma-i-transfected cardiomyocytes. Among the PP1 isoform-specific knockdown, PP1beta knockdown most effectively augmented the amplitude of Ca2+ transient and time constant of Ca2+ decay slope, and significantly increased %cell shortening at normal culture state and under the beta-adrenergic stimulated state.
Conclusion: Isoform-specific inhibition of PP1 may provide a novel therapeutic opportunity by circumventing the side effect of systemic PP1 inhibition. Because our data suggest that PP1beta is the major PLN phosphatase that regulates cardiomyocyte contractility, it may be a good therapeutic target for heart failure.