Background — Inducible nitric oxide synthase (iNOS) is expressed in the myocardium after myocardial infarction (MI) and in heart failure. Its pathophysiological role in these conditions, however, is not clear. We hypothesized that increased NO production from iNOS expression causes myocardial dysfunction and results in higher mortality after MI. Methods and Results — MI was induced by left coronary artery ligation in iNOS ^−/− mutant and wild-type mice. Mortality was followed up for 30 days. MI resulted in a significant increase in mortality in both iNOS ^−/− and wild-type mice compared with sham operation ( P <0.01). Mortality was significantly decreased and LV myocardial contractility was increased, however, in iNOS ^−/− mice compared with the wild-type mice ( P <0.05). Five days after MI, myocardial iNOS mRNA expression, plasma nitrate and nitrite concentrations, and myocardial and plasma nitrotyrosine levels were significantly increased in wild-type compared with iNOS ^−/− mutant mice ( P <0.05). Both basal LV +dP/dt and its response to dobutamine were significantly increased in iNOS ^−/− compared with the wild-type mice ( P <0.05). Conclusions — Increased NO production from iNOS expression contributes to myocardial dysfunction and mortality after MI in mice.

Background— Nitric oxide (NO) produced by endothelial NO synthase (eNOS) plays an important role in the regulation of cell growth, apoptosis, and tissue perfusion. Recent studies showed that mice deficient in eNOS developed abnormal aortic bicuspid valves. The aim of the present study was to additionally investigate the role of eNOS in heart development. Methods and Results— We examined postnatal mortality, cardiac function, and septum defects in eNOS ^−/− , eNOS ^+/− , and wild-type mice. Postnatal mortality was significantly increased in eNOS ^−/− (85.1%) and eNOS ^+/− (38.3%) compared with wild-type mice (13.3%, P <0.001). Postmortem examination found severe pulmonary congestion with focal alveolar edema in mice deficient in eNOS. Heart shortening determined by ultrasound crystals was significantly decreased in eNOS ^−/− compared with wild-type mice ( P <0.05). Congenital atrial and ventricular septal defects were found in neonatal hearts. The incidence of atrial or ventricular septal defects was significantly increased in eNOS ^−/− (75%) and eNOS ^+/− (32.4%) neonates compared with those of the wild-type mice (4.9%). At embryonic days 12.5 and 15.5, cardiomyocyte apoptosis and myocardial caspase-3 activity were increased in the myocardium of eNOS ^−/− compared with wild-type embryos ( P <0.01), and increases in apoptosis persisted to neonatal stage in eNOS ^−/− mice. Conclusions— Deficiency in eNOS results in heart failure and congenital septal defects during cardiac development, which is associated with increases in cardiomyocyte apoptosis. Our data demonstrate that eNOS plays an important role in normal heart development.

Background— Lipopolysaccharide (LPS) induces cardiomyocyte tumor necrosis factor-α (TNF-α) production, which is responsible for myocardial depression during sepsis. The aim of this study was to investigate the role of gp91 ^phox -containing NADH oxidase signaling in cardiomyocyte TNF-α expression and myocardial dysfunction induced by LPS. Methods and Results— In cultured mouse neonatal cardiomyocytes, LPS increased NADH oxidase (gp91 ^phox subunit) expression and superoxide generation. Deficiency of gp91 ^phox or inhibition of NADH oxidase blocked TNF-α expression stimulated by LPS. TNF-α induction was also inhibited by tempol, N -acetylcysteine, or 1,3-dimethyl-2-thiourea. NADH oxidase activation by LPS increased ERK1/2 and p38 phosphorylation, and inhibition of ERK1/2 and p38 phosphorylation blocked the effect of NADH oxidase on TNF-α expression. Isolated mouse hearts were perfused with LPS (5 μg/mL) alone or in the presence of apocynin for 1 hour. Myocardial TNF-α production was decreased in gp91 ^phox -deficient or apocynin-treated hearts compared with those of wild type ( P <0.05). To investigate the role of gp91 ^phox -containing NADH oxidase in endotoxemia, mice were treated with LPS (4 mg/kg IP) for 4 and 24 hours, and their heart function was measured with a Langendorff system. Deficiency of gp91 ^phox significantly attenuated LPS-induced myocardial depression ( P <0.05). Conclusions— gp91 ^phox -Containing NADH oxidase is pivotal in LPS-induced TNF-α expression and cardiac depression. Effects of NADH oxidase activation are mediated by ERK1/2 and p38 MAPK pathway. The present results suggest that gp91 ^phox -containing NADH oxidase may represent a potential therapeutic target for myocardial dysfunction in sepsis.

Pregestational diabetes is a major risk factor of congenital heart defects (CHDs). Glutathione is depleted and reactive oxygen species (ROS) production is elevated in diabetes. In the present study, we aimed to examine whether treatment with N-acetylcysteine (NAC), which increases glutathione synthesis and inhibits ROS production, prevents CHDs induced by pregestational diabetes.

OBJECTIVE: The role of NOX2-containing NADPH oxidase in the development of diabetes is not fully understood. We hypothesized that NOX2 deficiency decreases reactive oxygen species (ROS) production and immune response and protects against streptozotocin (STZ)-induced β-cell destruction and development of diabetes in mice. RESEARCH DESIGN AND METHODS: Five groups of mice—wild-type (WT), NOX2−/−, WT treated with apocynin, and WT adoptively transferred with NOX2−/− or WT splenocytes—were treated with multiple-low-dose STZ. Blood glucose and insulin levels were monitored, and an intraperitoneal glucose tolerance test was performed. Isolated WT and NOX2−/− pancreatic islets were treated with cytokines for 48 h. RESULTS: Significantly lower blood glucose levels, higher insulin levels, and better glucose tolerance was observed in NOX2−/− mice and in WT mice adoptively transferred with NOX2−/− splenocytes compared with the respective control groups after STZ treatment. Compared with WT, β-cell apoptosis, as determined by TUNEL staining, and insulitis were significantly decreased, whereas β-cell mass was significantly increased in NOX2−/− mice. In response to cytokine stimulation, ROS production was significantly decreased, and insulin secretion was preserved in NOX2−/− compared with WT islets. Furthermore, proinflammatory cytokine release induced by concanavalin A was significantly decreased in NOX2−/− compared with WT splenocytes. CONCLUSIONS: NOX2 deficiency decreases β-cell destruction and preserves islet function in STZ-induced diabetes by reducing ROS production, immune response, and β-cell apoptosis.

The purpose of this study was to investigate the role of endothelial nitric-oxide synthase (eNOS), cAMP, and p38 MAPK in tumor necrosis factor-α (TNF-α) expression induced by lipopolysaccharide (LPS). LPS dose- and time-dependently induced phosphorylation of p38 MAPK and TNF-α expression in neonatal mouse cardiomyocytes. TNF-α expression was preceded by p38 MAPK phosphorylation, and selective inhibition of p38 MAPK abrogated LPS-induced TNF-α expression. Deficiency in eNOS decreased basal and LPS-stimulated TNF-α expression in cardiomyocytes. NOS inhibitorl-NAME attenuated LPS-induced p38 MAPK phosphorylation and TNF-α production in wild-type cardiomyocytes, whereas NO donor 2,2′-(hydroxynitrosohydrazono)bis-ethanamine (DETA-NO) (2 μm) or overexpression of eNOS by adenoviral gene transfer restored the response of eNOS^−/−cardiomyocytes to LPS. These effects of NO were mediated through cAMP-dependent pathway based on the following facts. First, deficiency in eNOS decreased basal levels of intracellular cAMP, and DETA-NO elevated intracellular cAMP levels in eNOS^−/−cardiomyocytes. Second, a cAMP analogue 8-Br-cAMP mimicked the effect of NO in eNOS^−/− cardiomyocytes. Third, either inhibition of cAMP or cAMP-dependent protein kinase attenuated LPS-stimulated p38 MAPK phosphorylation and TNF-α production in wild-type cardiomyocytes. In conclusion, eNOS enhances LPS-stimulated TNF-α expression in cardiomyocytes. Activation of p38 MAPK is essential in LPS-stimulated TNF-α expression. Moreover, the effects of NO on LPS-stimulated TNF-α expression are mediated through cAMP/cAMP-dependent protein kinase-dependent p38 MAPK pathway in neonatal cardiomyocytes.