Constitutive phosphorylation of inhibitor-1 at Ser67 and Thr75 depresses calcium cycling in cardiomyocytes and leads to remodeling upon aging

Abstract

The activity of protein phosphatase-1 (PP1) inhibitor-1 (I-1) is antithetically modulated by the cAMP-protein kinase A (PKA) and Ca(2+)-protein kinase C (PKC) signaling axes. β-adrenergic (β-AR) stimulation results in PKA-phosphorylation of I-1 at threonine 35 (Thr35) and depressed PP1 activity, while PKC phosphorylation at serine 67 (Ser67) and/or Thr75 increases PP1 activity. In heart failure, pThr35 is decreased while pSer67 and pThr75 are elevated. However, the role of Ser67/Thr75 phosphorylation in vivo and its effects on Ca(2+)-cycling are not known. Thus, our aim was to investigate the functional significance of Ser67 and Thr75 phosphorylation in intact hearts. We generated transgenic mice (TG) with cardiac-specific overexpression of constitutively phosphorylated I-1 at Ser67 and Thr75 (S67D/T75D) and evaluated cardiac function. The S67D/T75D cardiomyocytes exhibited significantly depressed Ca(2+)-kinetics and contractile parameters, compared with wild-type (WT) cells. The decreased Ca(2+)-cycling was associated with a 27% increase in PP1 activity, no alterations in PP2 activity and impaired phosphorylation of myosin-binding protein-C (MyBPC). Upon aging, there was cardiac remodeling associated with increases in systolic and diastolic left ventricular internal diameter dimensions (at 16months), compared with WTs. The results indicate that phosphorylation of I-1 at Ser67 and Thr75 is associated with increased PP1 activity and depressed cardiomyocyte Ca(2+)-cycling, which manifests in geometrical alterations over the long term. Thus, hyperphosphorylation of these sites in failing hearts may contribute to deteriorative remodeling.