PICOT increases cardiac contractility by inhibiting PKC zeta activity

Abstract

Protein kinase C (PKC)-interacting cousin of thioredoxin (PICOT) has distinct anti-hypertrophic and inotropic functions. We have previously shown that PICOT exerts its anti-hypertrophic effect by inhibiting calcineurin-NFAT signaling through its C-terminal glutaredoxin domain. However, the mechanism underlying the inotropic effect of PICOT is unknown. The results of protein pull-down experiments showed that PICOT directly binds to the catalytic domain of PKC zeta through its N-terminal thioredoxin-like domain. Purified PICOT protein inhibited the kinase activity of PKC zeta in vitro, which indicated that PICOT is an endogenous inhibitor of PKC zeta. The inhibition of PKC zeta activity with a PKC zeta-specific pseudosubstrate peptide inhibitor was sufficient to increase the cardiac contractility in vitro and ex vivo. Overexpression of PICOT or inhibition of PKC zeta activity down-regulated PKC alpha activity, which led to the elevation of sarcoplasmic reticulum Ca2+-ATPase (SERCA) 2a activity, concomitant with the increased phosphorylation of phospholamban (PLB). Overexpression of PICOT or inhibition of PKC zeta activity also down-regulated protein phosphatase (PP) 2A activity, which subsequently resulted in the increased phosphorylation of troponin (Tn) I and T, key myofilament proteins associated with the regulation of contractility. PICOT appeared to inhibit PP2A activity through the disruption of the functional PKC zeta/PP2A complex. In contrast to the overexpression of PICOT or inhibition of PKC zeta, reduced PICOT expression resulted in up-regulation of PKC alpha and PP2A activities, followed by decreased phosphorylation of PLB, and TnI and T, respectively, supporting the physiological relevance of these events. Transgene- or adeno-associated virus (AAV)-mediated overexpression of PICOT restored the impaired contractility and prevented further morphological and functional deterioration of the failing hearts. Taken together, the results of the present study suggest that PICOT exerts its inotropic effect by negatively regulating PKC alpha and PP2A activities through the inhibition of PKC zeta activity. This finding provides a novel insight into the regulation of cardiac contractility. (c) 2012 Elsevier Ltd. All rights reserved.