Characterization of inducible nitric oxide synthase expression in endotoxemic rat cardiac myocytes in vivo and following cytokine exposure in vitro.

Lipopolysaccharide (LPS) treatment results in widespread expression of the inducible isoform of nitric oxide (NO) synthase (iNOS). Although there is evidence for the expression of iNOS in heart tissue, regulation of myocardial iNOS expression is not known. To determine the time course and degree of iNOS induction in the adult heart, we examined iNOS mRNA expression and enzyme activity in (1) rat left ventricular tissue after LPS treatment in vivo, and (2) cultured, long-term rat cardiac myocytes maintained in serum and exposed to interleukin-1 beta, tumor necrosis factor-alpha, interferon-gamma, and/or LPS. iNOS mRNA was detected by Northern blot analysis and in situ hybridization. iNOS enzyme activity was measured in extracts of whole heart, and nitrate and nitrite (the stable end-products of NO) accumulation was quantified in cardiomyocyte culture media. iNOS mRNA was not detected in untreated hearts or cultured myocytes but was apparent within 3 h in both hearts obtained from LPS-treated animals and in cytokine-treated myocytes. In whole heart, iNOS mRNA expression peaked by 6 h after LPS and declined by 12 and 24 h. In situ hybridization demonstrated perinuclear localization of iNOS mRNA in both cardiac vascular smooth muscle and myocytes with maximal expression at 6 h after LPS injection. In cardiac myocytes, iNOS expression was maximal at 12 to 24 h, persisted through 48 h, and was partially inhibited by dexamethasone. Interferon-gamma was the most potent single cytokine with regards to myocyte iNOS induction. Nitric oxide release in cytokine-stimulated cardiac myocytes was largely in the form of nitrate and was associated with increased glucose uptake and lactate release; the former finding indicates that NO interacts with myocardial heme proteins and/or oxyradicals, while the latter suggests inhibition of oxidative metabolism. Although non-myocardial cells may significantly contribute to iNOS expression in whole heart tissue, significant iNOS expression and NO production also take place within the myocyte. Induced NO production may regulate myocardial perfusion and impair myocardial function and metabolism.