Abstract 414: Translocation of Protein Phosphatase 1 with Inhibitor-2 from Sarcoplasmic Reticulum to Cytosol Augments Ca2+ Cycling in ardiomyocytes
Background: We and others have reported that myocardial protein phosphatase 1(PP1) activity in the end-stage heart failure (HF) is abnormally increased and contributes to the depressed cardiac function. In this regard, in vivo myocardial PP1 inhibition via endogenous cytosolic inhibitors such as inhibitor-2 (INH-2) has prevented HF progression by augmenting phospholamban (PLN) phosphorylation and thereby increasing cardiac function in the genetic cardiomyopathy model. However, it remained uncertain how INH-2, a cytosolic protein, exerts PP1 inhibition in the sarcoplasmic reticulum (SR). To this end, we investigated the molecular mechanism by which INH-2 inhibits PP1 and regulates contractility in cardiomyocytes.
Methods and Results: Cardiomyocytes were enzymatically isolated from six week-old male Wistar rats (n=10) and subjected to either adenoviral INH-2 infection or chemical PP1 inhibition by an application of 0.01–1microM of tautomycin (TM) and tautomycetin (TMC), followed by assessment of % cell shortening (%CS), Ca2+ transient, biochemical characteristics, and cell survival rate. Both adenoviral INH-2 delivery and chemical PP1 inhibitors induced a significant augmentation of %CS, Ca2+ transients, PLN phosphorylation at Ser 16. Although chemical PP1 inhibitors caused cardiomyocyte cell death within 24 hours after application in a dose dependent manner, INH-2 did not show any cytotoxities. Interestingly, INH-2 induced an increase in cytosolic PP1 catalytic subunit (PP1C) without accompanying by a corresponding increase in cytosolic PP1 activity, and resulted in a dramatic decrease in PP1C (approximately 50% control, p<0.05) in the SR preparation, leading to a significant decrease in SR-PP1 activity. Furthermore, in vitro application of recombinant INH-2 into the preparation of SR microvesicles caused a dose-dependent dissociation of PP1C from the SR, whereas TM and TMC did not show such effects.
Conclusion: These findings indicate that INH-2 promotes a translocation of PP1C from the SR to the cytosol, increases inactive cytosolic PP1, and decreases SR-PP1 activity, thereby increasing PLN phosphorylation. We propose that this is a novel regulatory mode of SR Ca2+ cycling via PP1 and endogenous inhibitors in cardiomyocytes.