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Abstract: Eyes absent 2 (Eya2) is a transcription factor involved in a number of cellular and developmental processes. We have previously shown that Eya2 is up-regulated during regression of cardiac hypertrophy and blocks phenylephrine-induced development of cardiomyocyte hypertrophy in vitro, suggesting that Eya2 is a negative regulator of cardiac hypertrophy. In this study, we generated transgenic mice with cardiac-specific overexpression of Eya2 to elucidate the in vivo function of Eya2 in cardiac remodeling. Mild cardiac hypertrophy developed in Eya2 transgenic mice under baseline conditions with no obvious structural or functional defects. Eya2 overexpression inhibited development of cardiac hypertrophy as judged by the abrogation of increases in heart weight and cross-sectional cell surface areas and re-activation of fetal genes under pressure overload (4 weeks). Eya2 overexpression also prevented wall thinning, ventricular dilation, and deterioration of cardiac function as well as fibrosis and inflammation in the heart under long-term pressure overload (12 weeks). Gene expression profiling using the parametric analysis of gene set enrichment (PAGE) method revealed that gene sets related to mitochondrial biogenesis and metabolism were elevated in the Eya2 transgenic mice. We also observed that the PI3K/Akt/mTOR signaling cascade was preserved in the Eya2 transgenic mice, while it was significantly reduced in the wild type littermates under pressure overload. These results demonstrate that Eya2 prevents adverse cardiac remodeling under pressure overload partly through altering metabolic gene expression and preserving PI3K/Akt/mTOR signaling pathway. [Copyright &y& Elsevier]

Abstract: Cardiac sarcoplasmic reticulum Ca2+-ATPase (SERCA2a) plays a crucial role in Ca2+ handling in cardiomyocytes. Phospholamban (PLB) is an endogenous inhibitor of SERCA2a and its inhibitory activity is enhanced via dephosphorylation by protein phosphatase 1 (PP1). Therefore, the inhibition of PP1-mediated dephosphorylation of PLB might be an efficient strategy for the restoration of reduced SERCA2a activity in failing hearts. We sought to develop decoy peptides that would mimic phosphorylated PLB and thus competitively inhibit the PP1-mediated dephosphorylation of endogenous PLB. The phosphorylation sites Ser16 and Thr17 are located within the flexible loop region (amino acids 14–22) of PLB. We therefore synthesized a 9-mer peptide derived from this region (ΨPLB-wt) and two pseudo-phosphorylated peptides where Ser16 was replaced with Glu (ΨPLB-SE) or Thr17 was replaced with Glu (ΨPLB-TE). These peptides were coupled to the cell-permeable peptide TAT to facilitate cellular uptake. Treatment of adult rat cardiomyocytes with ΨPLB-SE or ΨPLB-TE, but not with ΨPLB-wt, significantly elevated the phosphorylation levels of PLB at Ser16 and Thr17. This increased phosphorylation of PLB correlated with an increase in contractile parameters in vitro. Furthermore, the perfusion of isolated rat hearts with ΨPLB-SE or ΨPLB-TE, but not with ΨPLB-wt, significantly improved left ventricular developed pressure that had been previously impaired by ischemia. These data indicate that ΨPLB-SE and ΨPLB-TE efficiently prevented dephosphorylation of PLB by serving as decoys for PP1. Therefore, these peptides may provide an effective modality to regulate SERCA2a activity in failing hearts. [Copyright &y& Elsevier]

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 PKCζ through its N-terminal thioredoxin-like domain. Purified PICOT protein inhibited the kinase activity of PKCζ in vitro, which indicated that PICOT is an endogenous inhibitor of PKCζ. The inhibition of PKCζ activity with a PKCζ-specific pseudosubstrate peptide inhibitor was sufficient to increase the cardiac contractility in vitro and ex vivo. Overexpression of PICOT or inhibition of PKCζ activity down-regulated PKCα 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 PKCζ 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 PKCζ/PP2A complex. In contrast to the overexpression of PICOT or inhibition of PKCζ, reduced PICOT expression resulted in up-regulation of PKCα 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 PKCα and PP2A activities through the inhibition of PKCζ activity. This finding provides a novel insight into the regulation of cardiac contractility. [Copyright &y& Elsevier]

Abstract: Despite its significant clinical implications, physiological hypertrophy remains poorly understood. In this study, the transcription coactivator Eya2 was shown to be up-regulated during physiological hypertrophy. Transgene- or adenovirus-mediated overexpression of Eya2 led to up-regulation of mTOR, a critical mediator of physiological hypertrophy. Luciferase reporter and chromatin immunoprecipitation assays revealed that Eya2 directly binds to and activates mTOR expression. The phosphorylation of mTOR downstream molecules was significantly enhanced in Eya2 transgenic (TG) hearts, implying that the Eya2-mediated induction of mTOR expression leads to an elevated mTOR activity. The transcription factor Six1 was also up-regulated during physiological hypertrophy and formed a complex with Eya2. Luciferase reporter and electrophoretic mobility shift assays revealed that the Eya2-Six1 complex binds to and enhances the expression of mTOR in a synergistic manner. Under pressure overload, Eya2 transgenic hearts developed hypertrophy which exhibited important molecular signatures of physiological hypertrophy, as assessed by gene expression profiling and measurements of expression levels of physiological hypertrophy-related genes by quantitative (q) RT-PCR. Examination of heart sections under electron microscopy revealed that the mitochondrial integrity remained largely intact in Eya2 transgenic mice, but not in wild-type littermates, under pressure overload. This finding was confirmed by measurements of mitochondrial DNA contents and the expression levels of mitochondrial function-related genes by qRT-PCR. These data suggest that Eya2 in a physical complex with Six1 plays a critical role in physiological hypertrophy. The cardioprotective effect of Eya2 appears to be due, at least in part, to its preservation of mitochondrial integrity upon pressure overload. [Copyright &y& Elsevier]

Abstract: CCN family members are matricellular proteins with diverse roles in cell function. The differential expression of CCN2 and CCN5 during cardiac remodeling suggests that these two members of the CCN family play opposing roles during the development of cardiac hypertrophy and fibrosis. We aimed to evaluate the role of CCN2 and CCN5 in the development of cardiac hypertrophy and fibrosis. In isolated cardiomyocytes, overexpression of CCN2 induced hypertrophic growth, whereas the overexpression of CCN5 inhibited both phenylephrine (PE)- and CCN2-induced hypertrophic responses. Deletion of the C-terminal (CT) domain of CCN2 transformed CCN2 into a CCN5-like dominant negative molecule. Fusion of the CT domain to the Carboxy-terminus of CCN5 transformed CCN5 into a CCN2-like pro-hypertrophic molecule. CCN2 transgenic (TG) mice did not develop cardiac hypertrophy at baseline but showed significantly increased fibrosis in response to pressure overload. In contrast, hypertrophy and fibrosis were both significantly inhibited in CCN5 TG mice. CCN2 TG mice showed an accelerated deterioration of cardiac function in response to pressure overload, whereas CCN5 TG mice showed conserved cardiac function. TGF-β-SMAD signaling was elevated in CCN2 TG mice, but was inhibited in CCN5 TG mice. CCN2 is pro-hypertrophic and -fibrotic, whereas CCN5 is anti-hypertrophic and -fibrotic. CCN5 lacking the CT domain acts as a dominant negative molecule. CCN5 may provide a novel therapeutic target for the treatment of cardiac hypertrophy and heart failure. [Copyright &y& Elsevier]

Abstract: PICOT (PKC-interacting cousin of thioredoxin) was previously shown to inhibit the development of cardiac hypertrophy, concomitant with an increase in cardiomyocyte contractility. To explore the physiological function of PICOT in the hearts, we generated a PICOT-deficient mouse line by using a gene trap approach. PICOT−/− mice were embryonic lethal indicating that PICOT plays an essential role during embryogenesis, whereas PICOT+/− mice were viable with no apparent morphological defects. The PICOT protein levels were reduced by about 50% in the hearts of PICOT+/− mice. Significantly exacerbated cardiac hypertrophy was induced by pressure overload in PICOT+/− mice relative to that seen in wild type littermates. In line with this observation, calcineurin-NFAT signaling was greatly enhanced by pressure overload in the hearts of PICOT+/− mice. Cardiomyocytes from PICOT+/− mice exhibited significantly reduced contractility, which may be due in part to hypophosphorylation of phospholamban and reduced SERCA activity. These data indicate that the precise PICOT protein level significantly affects the process of cardiac hypertrophy and cardiomyocyte contractility. We suggest that PICOT plays as a critical negative regulator of cardiac hypertrophy and a positive inotropic regulator. [Copyright &y& Elsevier]