Rui F D S Mesquita

King's College London, Londinium, England, United Kingdom

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Publications (2)10.72 Total impact

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    ABSTRACT: The pathways which regulate resolution of inflammation and contribute to positive remodelling of the myocardium following injury are poorly understood. Here we show that protein kinase C epsilon (PKCε) co-operates with the phosphatase calcineurin (CN) to potentiate induction of cardioprotective gene expression whilst suppressing expression of fibrosis markers. This was achieved by detailed analysis of the regulation of cyclooxygenase-2 (COX-2) expression as a marker gene and by using gene expression profiling to identify genes regulated by co-expression of CN-Aα/PKCε in adult rat cardiac myofibroblasts (ARVFs) on a larger scale. Gene-chip analysis of CN-Aα/PKCε co-expressing ARVFs showed that COX-2 provides a signature for wound healing and is associated with downregulation of fibrosis markers including connective tissue growth factor (CTGF), fibronectin and collagens Col1a1, Col3a1, Col6a3, Col11a1, Col12a1 and Col14a1 with concomitant upregulation of cardioprotection markers including COX-2 itself, lipocalin-2 (LCN-2), tissue inhibitor of metalloproteinase-1 (TIMP-1), interleukin-6 (IL-6) and inducible nitric oxide synthase (iNOS). In primary rat cardiomyocyte cultures TLR4-agonist- or PKCε/CN-dependent COX-2 induction occurred in co-resident fibroblasts and was blocked by selective inhibition of CN or PKC α/ε or elimination of fibroblasts. Furthermore, ectopic expression of PKCε and CN in ARVFs showed that the effects are mediated by specific NFAT sites within the COX-2 promoter as confirmed by site directed mutagenesis and chromatin immunoprecipitation (ChIP). Therefore, PKCε may negatively regulate adverse myocardial remodelling by co-operating with CN to downregulate fibrosis and induce transcription of cardioprotective wound healing genes including COX-2.
    Molecular and Cellular Biology 12/2013; 34(4). DOI:10.1128/MCB.01098-13 · 4.78 Impact Factor
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    ABSTRACT: To determine the role of calcineurin and Src tyrosine kinase in the regulation of inducible nitric oxide synthase (iNOS) expression and protection in cardiomyocytes. iNOS expression was studied in isolated neonatal rat ventricular myocyte cultures in response to bacterial lipopolysaccharide (LPS) or following transfection with constitutively active calcineurin or Src and in hearts isolated from wild-type or calcineruin Abeta knockout mice. Cell injury in response to simulated ischemia-reperfusion was studied following overexpression of active calcineurin. Regulation of the iNOS gene promoter by calcineurin was studied using promoter-luciferase reporter and chromatin immunoprecipitation assays. Overexpression of constitutively active Src co-operated with [Ca2+]c elevation to induce iNOS expression, and LPS-induced iNOS expression was abrogated by pharmacological inhibition of calcineurin or tyrosine kinase. LPS also induced tyrosine kinase-dependent but calcineurin-independent phosphorylation of Src Tyr418. LPS induced myocardial iNOS expression in wild-type but not calcineurin Abeta knockout mice. Overexpression of constitutively active calcinuerin in isolated cardiomyocytes caused dephosphorylation and nuclear accumulation of the c1 isoform of nuclear factor of activated T-cells (NFATc1), induced strong iNOS expression, and induced NOS-dependent protection against simulated ischemia-reperfusion prior to cardiomyocyte hypertrophy. Co-transfection of a mouse iNOS promoter-luciferase reporter in combination with active calcineurin and wild-type or dominant negative Src confirmed that constitutive activation of calcineurin was sufficient for transactivation. Chromatin immunoprecipitation confirmed calcineurin-dependent in vivo binding of NFATc1 to consensus sites within the iNOS promoter. These results support a cardioprotective role for calcineurin mediated by NFAT-dependent induction of iNOS expression and co-operativity between calcineurin and Src.
    Cardiovascular Research 10/2006; 71(4):672-83. DOI:10.1016/j.cardiores.2006.05.026 · 5.94 Impact Factor