Calcineurin regulates NFAT-dependent iNOS expression and protection of cardiomyocytes: Co-operation with Src tyrosine kinase

Objective: To determine the role of calcineurin and Src tyrosine kinase in the regulation of inducible nitric oxide synthase (NOS) expression and protection in cardiomyocytes. Methods: NOS 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 A beta knockout mice. Cell injury in response to simulated ischemia-reperfusion was studied following overexpression of active calcineurin. Regulation of the NOS gene promoter by calcineurin was studied using promoter-luciferase reporter and chromatin immunoprecipitation assays. Results: Overexpression of constitutively active Src co-operated with [Ca2+](c) elevation to induce NOS expression, and LPS-induced NOS expression was abrogated by pharmacological inhibition of calcineurin or tyrosine kinase. LPS also induced tyrosine kinase-dependent but calcineurin-independent phosphorylation of Src Tyr(418). LPS induced myocardial NOS expression in wild-type but not calcineurin A knockout mice. Overexpression of constitutively active calcinuerin in isolated cardiomyocytes caused deposphorylation and nuclear accumulation of the cl isoform of nuclear factor of activated T-cells (NFATc1), induced strong NOS expression, and induced NOS-dependent protection against simulated ischemia-reperfusion prior to cardiomyocyte hypertrophy. Co-transfection of a mouse NOS 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 NOS promoter. Conclusions: These results support a cardioprotective role for calcineurin mediated by NFAT-dependent induction of NOS expression and co-operativity between calcineurin and Src. (c) 2006 European Society of Cardiology. Published by Elsevier B.V. All rights reserved.