Delayed preconditioning in cardiac myocytes with respect to development of a proinflammatory phenotype: role of SOD and NOS

OBJECTIVE: Both superoxide dismutase (SOD) and nitric oxide synthase (NOS) have been implicated in delayed preconditioning (DP) to ischemia/reperfusion (I/R) in the heart. We used isolated cardiac myocytes to test the hypothesis that SOD and NOS may interact in the development of DP. METHODS: Mouse neonatal cardiac myocytes were challenged with anoxia/reoxygenation (A/R; an in vitro counterpart to I/R) and normoxia/normoxia (N/N) served as the control. Two indices of inflammation were measured: oxidant stress (DHR oxidation) and polymorphonuclear leukocyte (PMN) transendothelial migration (cell culture inserts). The role of SOD was assessed using an antisense approach and the role of NOS was assessed using iNOS and eNOS deficient myocytes. RESULTS: Cardiac myocytes exposed to A/R (1) produced more oxidants (intracellular fluorescence emission from 2.0 +/- 0.1 for N/N to 3.0 +/- 0.3 for A/R; P<0.05) and (2) promoted PMN migration (% migration from 8.4 +/- 0.9 for N/N to 14.1 +/- 1.1 for A/R; P<0.05). DP occurred if the myocytes were pretreated with an A/R challenge 24 h earlier. That is, these A/R-induced responses were significantly reduced (fluorescence emission 1.9 +/- 0.1 and % migration 8.4 +/- 0.7; P<0.05 as compared to A/R with no pretreatment). Myocyte Mn-SOD, but not Cu/Zn-SOD, activity increased 24 h after the initial A/R challenge. A Mn-SOD antisense oligonucleotide prevented the development of DP. DP occurred in iNOS, but not eNOS, deficient myocytes. A/R increased mRNA for eNOS, but not iNOS, in wild-type myocytes. A/R increased Mn-SOD protein in both iNOS and eNOS deficient myocytes. However, Mn-SOD activity increased only in iNOS deficient myocytes. CONCLUSIONS: Collectively, these findings suggest that Mn-SOD and eNOS may act in concert in the development of DP in cardiac myocytes.     

Reduction of myocardial infarct size by inhibition of inducible nitric oxide synthase

The inducible nitric oxide synthase isoform (iNOS) is upregulated by cytokines and endotoxins in many types of cells, including cardiac myocytes. Nitric oxide (NO) induced by cytokines can be cytotoxic, and has been implicated in the pathophysiology of myocardial infarction, cardiomyopathy, and septic shock. To examine the role of iNOS in the ischemic myocardium, we studied: 1) the time course of expression of iNOS mRNA after myocardial infarction (MI) in male Sprague-Dawley rat hearts and expression of iNOS protein in the infarcted region; 2) whether hypoxia in vitro is a potential mediator of the induction of iNOS mRNA; and 3) whether inhibition of iNOS by two different selective inhibitors (aminoguanidine and S-methylisothiourea sulfate) in vivo influences infarct size. Myocardial infarction was induced by ligation of the left anterior descending coronary artery (LAD), and tissue was collected at selected times thereafter from both ligated and sham-operated rats. iNOS mRNA was induced in the infarcted region of the left ventricle for 7 days; iNOS protein was also detected in the infarcted area. We next tested whether hypoxia would induce iNOS in vitro. In cultured neonatal ventricular myocytes, iNOS mRNA was slightly induced by 6 to 24 h of hypoxia; however, iNOS protein was only detected when the cytokine interleukin-1β was present. To study whether iNOS activity contributed to myocardial damage (eg, infarct size), we administered the first dose of the NOS inhibitors 24 h before LAD occlusion and then a second dose after surgery. Inhibition of iNOS activity with aminoguanidine reduced infarct size by 20% but had no effect on infiltration by neutrophils, whereas the more selective inhibitor S-methylisothiourea sulfate reduced infarct size by 41%. These data suggest that NO derived from the iNOS isoform contributes to some of the myocardial injury following MI, possibly by causing myocardial cell death in areas bordering the ischemic region of the heart.     

Increased susceptibility to development of triggered activity in myocytes from mice with targeted disruption of endothelial nitric oxide synthase

Nitric oxide generated by cardiac myocytes or delivered by drugs has been shown to regulate cardiac contractile function and has been implicated in suppressing some cardiac arrhythmias, although this remains controversial. We examined the ability of the soluble cardiac glycoside, ouabain, to trigger arrhythmic contractions in ventricular myocytes isolated from mice lacking a functional endothelial nitric oxide synthase gene (eNOS(null)). Arrhythmic activity, defined as aftercontractions, was induced with ouabain (50 micromol/L) and recorded using a video-motion detector in isolated, electrically driven single ventricular myocytes from adult eNOS(null)or from their wild-type (WT) littermates. The rate of ouabain-induced arrhythmic contractions was significantly higher in eNOS(null)myocytes than in WT myocytes. Application of the NO donor S-nitroso-acetylcysteine (SNAC) significantly diminished the frequency of arrhythmic contractions in eNOS(null)myocytes. The antiarrhythmic effect of NO, whether generated by eNOS in WT cells or by SNAC, could be partially reversed by 1H-[1,2,4]oxadiazolo-[4, 3-a]- quinoxalin-1-one (ODQ), a specific soluble guanylyl cyclase inhibitor. Ouabain significantly increased intracellular cGMP in WT but not eNOS(null)hearts, and this cGMP response was blocked by ODQ. Since cardiac glycoside- induced aftercontractions are activated by the transient inward current (I(ti)), the role of NO in ouabain (100 micromol/L)- induced I(ti)was examined using the nystatin-perforated patch-clamp technique. The frequency of ouabain-induced I(ti)was significantly higher in eNOS(null)myocytes than in WT myocytes, and this could be suppressed by SNAC. These data demonstrate that NO derived from myocyte eNOS activation suppresses ouabain-induced arrhythmic contractions by a mechanism that might involve activation of guanylyl cyclase and elevation of cGMP.     

Cytokines in HIV-associated cardiomyopathy

Among the multiple cardiac manifestations occurring in HIV-infected patients, cardiomyopathy is one of the most challenging. Its incidence has only slightly decreased since the introduction of highly active antiretroviral therapy (HAART). Also, its pathogenesis remains relatively unclear. Although several studies demonstrated the presence of HIV genome in the heart of patients, more recent developments found that viral infection plays an indirect role only, as well as they recognized the contribution of proinflammatory cytokines in the progression of the disease. Experimental studies on animals and cultured myocytes have established the signalling pathway triggered by proinflammatory cytokines in heart failure and cardiomyopathy. Tumor necrosis factor-alpha (TNF-alpha), interleukin-1 (IL-1) and IL-6 promote expression of inducible nitric oxide synthase (iNOS) in cardiomyocytes through activation of p38 mitogen-activated protein kinase (p38 MAPK) and nuclear factor kappaB (NFkappaB). TNF-alpha and high concentrations of NO also induce cardiomyocyte apoptosis by TNF type 1 receptor activation. This biological framework, which is also involved in progression of cardiomyopathy in humans, is more pronounced in HIV-infected patients, in whom proinflammatory cytokines TNF-alpha, IL-1 and IL-6 are increased, resulting in an enhanced expression of cardiac iNOS, especially in patients with a low CD4 T cell count. This may account for the worse outcome of heart failure in HIV-infected patients. However, there are only few data today to support future therapeutic implications of cytokines antagonism in treatment of HIV-infected patients with cardiomyopathy. Whether modulation of TNF production or selective inhibition of p38 MAPK pathway could be useful approaches remains uncertain.     

Effects of immunosuppressive therapy on expression of inducible nitric oxide synthase (iNOS) during cardiac allograft rejection

Recent studies have indicated a role for nitric oxide (NO) in alloimmune responses and in allograft rejection. iNOS mRNA, protein and enzyme activity are induced in myocardium during cardiac allograft rejection. NO produced by iNOS is negatively inotropic and has the potential to be cytotoxic to cardiac myocytes. To investigate whether immunosuppressive agents would alter the expression of iNOS during cardiac allograft rejection, hearts from Wistar-Furth rats were transplanted into the abdomen of Lewis recipients. At day 5 allografts from treated and untreated animals were removed for pathological and biochemical examination. At day 5 the untreated allografts exhibited histological evidence of marked rejection (edema, infiltration with macrophages and lymphocytes, necrosis of cardiac muscle fibers). Abundant iNOS mRNA was apparent in Northern blots and iNOS enzyme activity was increased in ventricular homogenates and in cardiac myocytes purified from the untreated rejecting allografts. Incubation of isolated purified cardiac myocytes from normal rats for 24 h with cytokines known to be present during allograft rejection (IL-1 β, TNF- and IFN-γ) was also associated with increased iNOS mRNA and enzyme activity. When Wistar-Furth to Lewis allografts were treated from time of transplantation with FK 506, cyclosporine A, dexamethasone or a combination of all three drugs, histological evidence of rejection and the levels of iNOS mRNA and enzyme activity in ventricular homogenates were reduced significantly below those observed in the untreated allografts. The data in a rat model indicate that immunosuppressive drugs reduce myocardial iNOS mRNA and enzyme activity in rejecting cardiac allografts. The results are consistent with the hypothesis that the alloimmune response and cytokine release are involved in the expression of iNOS during cardiac transplantation rejection.     

脓毒症时各型一氧化氮合酶在大鼠心脏中的表达及其可能机制

目的研究脓毒症时各型一氧化氮合酶(NOS)在心脏中的损伤作用及其机制。方法成年雄性Wistar大鼠腹腔注射脂多糖(LPS)制备脓毒症模型。应用多导生理仪监测大鼠心功能变化;用分光光度计法测定大鼠心肌组织NOS的活性;用RT-PCR和Western blot对大鼠心肌组织各型NOS的表达进行半定量分析。结果给予LPS后6h大鼠心肌收缩和舒张功能受损下降,心肌中iNOS的活性明显升高,eNOS和nNOS(合称cNOS)活性减弱;RT-PCR和Western blot结果显示,给予LPS后cNOS的表达减少,给予LPS后iNOS表达量明显增加。结论脓毒症时,iNOS、nNOS和eNOS的表达和活性发生改变;心肌细胞上iNOS表达及活性升高,这些变化可能在心功能降低中发挥作用。     

Expression of Inducible Nitric Oxide Synthase in Experimental Viral Myocarditis

Nitric oxide (NO) is an important bioactive molecule with regulatory, cytotoxic or cytoprotective properties. In virus-induced myocarditis, NO mediates host defense mechanisms against the infection or causes cardiac dysfunctions. NO is synthesized from L-arginine by the enzyme nitric oxide synthase (NOS). The expression of the inducible form of the nitric oxide synthase (iNOS) is regulated by cytokines, involved in the complex myocardial immune response to enterovirus infections. The present study was undertaken to characterize the role of iNOS and NO in the murine model of viral myocarditis induced by coxsackievirus B3 (CVB3). In response to CVB3 infection we investigated the time course of iNOS induction in correlation with cytokine mRNA expression (TNF-alpha, IL-1 alpha, IFN-gamma, TGF-beta) in the heart of NMRI mice by RT-PCR. Positive PCR signals for viral RNA were found in the acute and chronic stage of disease by seminested PCR, indicating the persistence of viral genome. We found distinct expression of iNOS at all time points (1, 2, 3, 4, 7, 14, 28, 56, 98 days post infection [p.i.]). Higher iNOS mRNA levels were identified between days 4 until 28 p.i. in comparison to day 56 and 98 p.i. using densitometric values. The mRNA of the inflammatory cytokines TNF-alpha, IL-1 alpha, IFN-gamma appeared at days 1, 4, and 7 p.i., peaked at day 7 p.i. and persisted until day 98 p.i. Similar like the iNOS mRNA pattern was the expression profile of TGF-beta. Using in situ hybridization and immunohistochemistry iNOS was localized in infiltrates, vascular endothelial cells, smooth muscle cells, myocytes and throughout the interstitial spaces between myocardial fibers in the heart sections of NMRI mice. Increased levels of NO were measured as total nitrate/nitrite concentration in the sera of mice from day 7 until day 28 p.i.     

Ischemic preconditioning upregulates inducible nitric oxide synthase in cardiac myocyte.

Recent evidence has shown that the cardioprotection afforded by the late phase of ischemic preconditioning (PC) is mediated by upregulation of inducible nitric oxide synthase (iNOS). However, the specific cardiac cell type(s) that express(es) iNOS in response to ischemic PC remains unknown. Thus, mice underwent a sequence of six cycles of 4-min coronary occlusion/4-min reperfusion, which induces late PC, and tissue samples were collected at serial times for measurement of mRNA (Northern) and protein levels (Western). In addition, whole heart samples were cryosectioned for in situ hybridization and immunohistochemistry. The steady-state levels of iNOS mRNA in the ischemic regions started to increase at 1 h after ischemic PC, peaked at 3 h (201+/-31% of sham, n=5 P<0.01) and remained elevated at 24 h (177+/-22% of sham, n=5 P<0.01). In accordance with these data, iNOS protein expression was increased at 24 h (219+/-41% of sham, n=5 P<0.01). In contrast, neither endothelial nitric oxide synthase (eNOS) mRNA levels nor its protein expression changed at any time-point. The magnitude of iNOS upregulation after ischemic PC was mild compared with that noted 66 h after permanent coronary occlusion (360+/-53% of sham) or 8 h after endotoxin (3117+/-61% of control). After ischemic PC, diffuse iNOS signals were detected with in situ hybridization and immunohistochemistry in the cytoplasmic space of cardiac myocytes and, to a lesser degree, in the wall of large vessels, but were absent in smooth muscle and endothelium of small vessels and in fibroblasts. This pattern contrasted with that observed in mouse hearts subjected to permanent coronary occlusion where strong iNOS signals were concentrated in inflammatory cells but absent in cardiac myocytes. Thus, not only the degree of iNOS expression but also its cellular distribution were profoundly different in reversibly injured (preconditioned) v infarcted myocardium. We conclude that iNOS is rapidly upregulated after ischemic PC and that cardiac myocytes are the main source of ischemic PC-induced iNOS expression. This study demonstrates, for the first time, a differential pattern of iNOS expression in sublethal (PC) v lethal ischemia, which may have important implication for the role of iNOS in these two settings.     

The therapeutic effect of natriuretic peptides in heart failure; differential regulation of endothelial and inducible nitric oxide synthases

The abnormal regulation of nitric oxide synthase activity represents an underlying feature of heart failure. Increased peripheral vascular resistance, and decreased renal function may be in part related to impaired endothelium-dependent nitric oxide (NO) synthesis. Paradoxically, the chronic production of NO by inducible nitric oxide synthase (iNOS) in heart failure exerts deleterious effects on ventricular contractility, and circulatory function. Consequently, pharmacologically improving endothelium-dependent NO synthesis and the concomitant inhibition of iNOS activity would be therapeutically advantageous. Interestingly, natriuretic peptides have been shown to differentially regulate endothelial NOS (eNOS) and iNOS activity. Moreover, in both patients and animal models of heart failure, pharmacologically increasing plasma natriuretic peptide levels ameliorated vascular tone, renal function, and ventricular contractility. Based on these observations, the following review will explore whether the therapeutic benefit of the natriuretic peptide system in heart failure may occur in part via the amelioration of endothelium-dependent NO synthesis, and the concomitant inhibition of cytokine-mediated iNOS expression.     

Inducible nitric oxide synthase (iNOS) and endothelial nitric oxide synthase (eNOS) expression in fulminant hepatic failure

BACKGROUND/AIMS: Inducible nitric oxide synthase (iNOS) and endothelial nitric oxide synthase (eNOS) have important functions in inflammation and vasoregulation but their role in fulminant hepatic failure (FHF) is not well understood. METHODS: Intrahepatic in situ staining and semi-quantification of iNOS and eNOS by immunohistochemistry in 25 patients with FHF, in 40 patients with chronic liver diseases (CLD) and in ten normal controls (NC). RESULTS: Expression patterns of iNOS and eNOS differed. While in NC only faint iNOS expression was found in some Kupffer cells/macrophages and hepatocytes, eNOS was expressed constitutively in sinusoidal and vascular endothelial cells. In CLD, iNOS expression was induced in Kupffer cells/macrophages and hepatocytes, representing the main iNOS expressing cell types. Additionally, bile ducts, vascular endothelial cells and lymphocytes also expressed iNOS (P = 0.001). In contrast, no differences were found between eNOS expression in CLD and NC (P = 0.64). The same cell types expressed eNOS and iNOS in FHF but numbers of both were significantly enhanced, exceeding the levels seen in CLD (P < 0.001, P = 0.017). CONCLUSIONS: Our data demonstrate that iNOS and eNOS are differently regulated in physiologic conditions and in liver disease. While eNOS seems to be involved in the physiological regulation of hepatic perfusion, strong upregulation of iNOS might contribute to inflammatory processes in FHF.     

Inducible nitric oxide synthase deficiency protects the heart from systolic overload-induced ventricular hypertrophy and congestive heart failure

Inducible nitric oxide synthase (iNOS) protein is expressed in cardiac myocytes of patients and experimental animals with congestive heart failure (CHF). Here we show that iNOS expression plays a role in pressure overload-induced myocardial chamber dilation and hypertrophy. In wild-type mice, chronic transverse aortic constriction (TAC) resulted in myocardial iNOS expression, cardiac hypertrophy, ventricular dilation and dysfunction, and fibrosis, whereas iNOS-deficient mice displayed much less hypertrophy, dilation, fibrosis, and dysfunction. Consistent with these findings, TAC resulted in marked increases of myocardial atrial natriuretic peptide 4-hydroxy-2-nonenal (a marker of lipid peroxidation) and nitrotyrosine (a marker for peroxynitrite) in wild-type mice but not in iNOS-deficient mice. In response to TAC, myocardial endothelial NO synthase and iNOS was expressed as both monomer and dimer in wild-type mice, and this was associated with increased reactive oxygen species production, suggesting that iNOS monomer was a source for the increased oxidative stress. Moreover, systolic overload-induced Akt, mammalian target of rapamycin, and ribosomal protein S6 activation was significantly attenuated in iNOS-deficient mice. Furthermore, selective iNOS inhibition with 1400W (6 mg/kg per hour) significantly attenuated TAC induced myocardial hypertrophy and pulmonary congestion. These data implicate iNOS in the maladaptative response to systolic overload and suggest that selective iNOS inhibition or attenuation of iNOS monomer content might be effective for treatment of systolic overload-induced cardiac dysfunction.     

Effects of TCV-116 on expression of NOS and adrenomedullin in failing heart of Dahl salt-sensitive rats

We examined the effects of TCV-116, an angiotensin II type 1 receptor antagonist, on endothelial-cell nitric oxide synthase (eNOS), inducible NOS (iNOS), and adrenomedullin (ADM) expression in the left ventricle (LV) and evaluated these relation to myocardial remodeling in failing heart of Dahl salt-sensitive hypertensive rats (DS) fed a high-salt diet. TCV-116 (DSHF-T, 5 mg/kg/day, subdepressor dose) or vehicle (DSHF-V) were given from left ventricular hypertrophy to heart failure stage for 7 weeks. Markedly increased left ventricular end-diastolic diameter and reduced fractional shortening in DSHF-V was significantly ameliorated in DSHF-T. The eNOS mRNA and protein in the LV was significantly suppressed in DSHF-V compared with control rats (DR-C), and significantly increased in DSHF-T compared with DSHF-V. The iNOS mRNA and protein, ADM mRNA and immunoreactive ADM contents, and type I collagen mRNA in the LV were significantly increased in DSHF-V compared with DR-C, and significantly decreased in DSHF-T compared with DSHF-V. DSHF-V showed a significant increase of the wall-to-lumen ratio, perivascular fibrosis, and myocardial fibrosis, with all these parameters being significantly improved by TCV-116. In conclusion, myocardial remodeling and heart failure in DS rats fed a high-salt diet were significantly ameliorated by a subdepressor dose of TCV-116, which may be due to a increased in eNOS and a decreased in iNOS mRNA and protein expression in the LV. Moreover, the ADM mRNA and immunoreactive ADM contents are upregulated in failing heart of DS rats fed a high-salt diet, and increased ADM expression may have a role in the defense mechanism against further cardiac dysfunction and impaired myocardial remodeling.     

骨髓干细胞心肌内移植增加诱生型一氧化氮合酶的表达

目的:通过研究骨髓干细胞心肌内移植是否影响心脏血管内皮舒张功能相关的一氧化氮合酶(NOS)基因的表达,探讨骨髓干细胞心肌内移植对血管舒张功能的影响机制。 方法:对Lewis大鼠进行冠状动脉前降支结扎和骨髓干细胞梗死区域心肌内移植,术后梗死区域心脏取材。分为急性心肌梗死组、骨髓干细胞心肌内移植组各15只,及正常组3只。用逆转录多聚酶链反应(RT-PCR)方法检测诱生型一氧化氮合酶(iNOS)和内皮源性一氧化氮合酶(eNOS)的表达。 结果:急性心肌梗死组心肌组织中iNOS的表达在1天时明显增高,3天后迅速减低;骨髓干细胞心肌内移植组,心肌组织中iNOS的表达进一步增高,可维持1个月(P<0.01)。急性心肌梗死组以及骨髓干细胞心肌内移植组,心肌组织eNOS的表达没有增加(P>0.05)。 结论:骨髓干细胞心肌内移植增加心肌组织iNOS的表达,延长其表达时间,但不影响心肌组织eNOS的表达。     

Heterogeneous basal expression of nitric oxide synthase and superoxide dismutase isoforms in mammalian heart : implications for mechanisms governing indirect and direct nitric oxide-related effects.

The basal expression patterns of NO synthase (NOS; endothelial [eNOS], neuronal [nNOS], and cytokine-inducible [iNOS]) and superoxide dismutase (SOD; extracellular membrane bound [ECSOD], MnSOD, and CuZnSOD) isoforms in ferret heart (tissue sections and isolated myocytes) were determined by immunofluorescent localization. We demonstrate the following for the first time in the mammalian heart: (1) heterogeneous expression patterns of the 3 NOS and 3 SOD isoforms among different tissue and myocyte types; (2) colocalization of eNOS and ECSOD at both the tissue and myocyte levels; (3) a significant gradient of eNOS and ECSOD expression across the left ventricular (LV) wall, with both enzymes being highly expressed and colocalized in LV epicardial myocytes but markedly reduced in LV endocardial myocytes; and (4) specific subcellular localization patterns of eNOS and the 3 SOD isoforms. In particular, eNOS and ECSOD are demonstrated (electron and confocal microscopy) to be specifically localized to the sarcolemma of ventricular myocytes. Similar heterogeneous eNOS and ECSOD expression patterns were also obtained in human LV tissue sections, underscoring the general importance of these novel findings. Our data suggest a strong functional correlation between the activities of sarcolemmally localized myocyte eNOS and ECSOD in governing NO*/O(2-) interactions and suggest that NO-related modulatory effects on cardiac myocyte protein and/or ion channel function may be significantly more complex than is presently believed.     

Effect of tumor necrosis factor-alpha on endothelial and inducible nitric oxide synthase messenger ribonucleic acid expression and nitric oxide synthesis in ischemic and nonischemic isolated rat heart

OBJECTIVES: The present study aimed to investigate the influence of endogenous tumor necrosis factor-alpha (TNF-alpha) that was synthesized during ischemia and exogenous TNF-alpha on endothelial and inducible nitric oxide synthase (eNOS and iNOS) messenger ribonucleic acid (mRNA) expression and nitric oxide (NO) production in the isolated rat heart. BACKGROUND: Tumor necrosis factor-alpha is recognized as being a proinflammatory cytokine with a significant cardiodepressant effect. One of the proposed mechanisms for TNF-alpha-induced cardiac contractile dysfunction is increased NO production via iNOS mRNA upregulation, but the role of NO in TNF-alpha-induced myocardial dysfunction is highly controversial. METHODS: Isolated rat hearts studied by a modified Langendorff model were randomly divided into subgroups to investigate the effect of 1-h global cardioplegic ischemia or the effect of 1-h perfusion with exogenous TNF-alpha on the expression of eNOS mRNA and iNOS mRNA and on NO production. RESULTS: After 1 h of ischemia, there were significant increases in TNF levels in the effluent (from hearts), and eNOS mRNA expression had declined (from 0.91 +/- 0.08 to 0.68 +/- 0.19, p < 0.001); but there were no changes in iNOS mRNA expression, and NO was below detectable levels. Perfusion of isolated hearts with TNF-alpha had a cardiodepressant effect and decreased eNOS mRNA expression to 0.67 +/- 0.04 (p < 0.002). Inducible nitric oxide synthase mRNA was unchanged, and NO was below detectable levels. CONCLUSIONS: We believe this is the first study to directly show that TNF-alpha does not increase NO synthesis and release but does downregulate eNOS mRNA in the ischemic and nonischemic isolated rat heart.     

17 Beta-estradiol stimulates expression of endothelial and inducible NO synthase in rat myocardium in-vitro and in-vivo

OBJECTIVES: NO production has been attributed to play a major role in cardiac diseases such as cardiac hypertrophy and cardiac remodeling after myocardial infarction which display significant gender-based differences. Therefore we assessed the effect of 17 beta-estradiol (E2) on estrogen receptor (ER) alpha and beta and endothelial and inducible NO synthase in neonatal and adult rat cardiomyocytes. METHODS: The presence of ER alpha and ER beta was demonstrated by immunofluorescence and western blot analysis as well as the expression pattern of inducible NO synthase (iNOS) and endothelial NOS (eNOS) in isolated cardiomyocytes from neonatal and adult rats. Furthermore, regulation of myocardial iNOS and eNOS expression by estrogen was evaluated in the myocardium from ovariectomized or sham-operated adult Wistar-Kyoto rats. RESULTS: Incubation with E2 led to translocalization of the ER into the nucleus and increased receptor protein expression. E2 stimulated expression of iNOS and eNOS in both neonatal and adult cardiac myocytes. Coincubation with the pure anti-estrogen ICI 182,780 inhibited upregulation of ER and NOS expression. In ovariectomized rats myocardial iNOS and eNOS protein levels were significantly lower compared to sham-operated female animals. CONCLUSION: Taken together, these results show that E2 stimulates the expression of iNOS/eNOS in neonatal and adult cardiomyocytes in-vivo and in-vitro. These novel findings provide a potential mechanism of how estrogen may modulate NOS expression and NO formation in the myocardium.     

Hydrogen peroxide differentially modulates cardiac myocyte nitric oxide synthesis

Nitric oxide (NO) and hydrogen peroxide (H(2)O(2)) are synthesized within cardiac myocytes and play key roles in modulating cardiovascular signaling. Cardiac myocytes contain both the endothelial (eNOS) and neuronal (nNOS) NO synthases, but the differential roles of these NOS isoforms and the interplay of reactive oxygen species and reactive nitrogen species in cardiac signaling pathways are poorly understood. Using a recently developed NO chemical sensor [Cu(2)(FL2E)] to study adult cardiac myocytes from wild-type, eNOS(null), and nNOS(null) mice, we discovered that physiological concentrations of H(2)O(2) activate eNOS but not nNOS. H(2)O(2)-stimulated eNOS activation depends on phosphorylation of both the AMP-activated protein kinase and kinase Akt, and leads to the robust phosphorylation of eNOS. Cardiac myocytes isolated from mice infected with lentivirus expressing the recently developed H(2)O(2) biosensor HyPer2 show marked H(2)O(2) synthesis when stimulated by angiotensin II, but not following β-adrenergic receptor activation. We discovered that the angiotensin-II-promoted increase in cardiac myocyte contractility is dependent on H(2)O(2), whereas β-adrenergic contractile responses occur independently of H(2)O(2) signaling. These studies establish differential roles for H(2)O(2) in control of cardiac contractility and receptor-dependent NOS activation in the heart, and they identify new points for modulation of NO signaling responses by oxidant stress.