Abstract
Objective: To determine the role of calcineurin and Src tyrosine kinase in the regulation of inducible nitric oxide synthase (iNOS) expression and protection in cardiomyocytes. Methods: 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 Aβ 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. Results: 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 Aβ knockout mice. Overexpression of constitutively active calcinuerin in isolated cardiomyocytes caused deposphorylation and nuclear accumulation of the c1 isoform of nuclear factor of activated T-cells (NFATc1), induced strong iNOS expression, and induced NOSdependent 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. Conclusions: These results support a cardioprotective role for calcineurin mediated by NFAT-dependent induction of iNOS expression and co-operativity between calcineurin and Src.