Chemokines, nitric oxide, and myocardial depression

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Infiltrating neutrophils exacerbate ischemia-reperfusion injury by releasing free radicals, proteolytic enzymes, proinflammatory cytokines, and by plugging microcapillaries. Certain chemokines, such as IL-8 in humans, play key roles in attracting neutrophils to sites of injury or inflammation. We have recently described the temporal expression of three neutrophil chemoattractant CXC chemokines - LIX (lipopolysachharide-induced CXC chemokine), KC (cytokine-induced neutrophil chemoattractant) and MIP-2 (macrophage inflammatory protein-2) - during myocardial ischemia-reperfusion injury in the rat. In addition, we found that administration of LIX induces NO production and depresses contractile performance in isolated cardiomyocytes, and depresses myocardial contractile function in vivo. These observations suggest that LIX-induced myocardial dysfunction may be mediated by a direct, neutrophil-independent, effect on myocardial production of NO and/or free radicals. The specific aims of this proposal are as follows. (1) We will determine whether LIX depresses myocardial function in vivo via NO generation, and whether pretreatment with L-NIL, a selective inhibitor of iNOS, will reduce NO generation and prevent LIX- induced LV dysfunction. LIX will be administered in vivo either alone or after pre-treatment with L-NIL, and myocardial depression will be assessed by echocardiography. Expression and localization of nitric oxide synthases, measurement of reactive oxygen and nitrogen intermediates and peroxynitrite will be measured. (2) We will determine whether LIX-induced myocardial dysfunction in vivo is independent on neutrophil infiltration. Neutrophils will be depleted by anti-neutrophil antiserum prior to administration of LIX. (3) We will determine the cellular signaling mechanisms responsible for LIX-mediated iNOS induction in isolated cardiomyocytes. The activity of iNOS promoter-driven reporter gene constructs will be assessed in vitro in transfected adult rat cardiomyocytes. The results from these studies will further our knowledge of chemokine biology and lead to new therapeutic strategies to prevent or treat myocardial dysfunction in ischemia-reperfusion injury.