Acute inhibition of myoglobin impairs contractility and energy state of iNOS-overexpressing hearts

Elevated cardiac levels of nitric oxide (NO) generated by inducible nitric oxide synthase (iNOS) have been implicated in the development of heart failure. The surprisingly benign phenotype of recently generated mice with cardiac-specific iNOS overexpression (TGiNOS) provided the rationale to investigate whether NO scavenging by oxymyoglobin (MbO2) yielding nitrate and metmyoglobin (metMb) is involved in preservation of myocardial function in TGiNOS mice. 1H nuclear magnetic resonance (NMR) spectroscopy was used to monitor changes of cardiac myoglobin (Mb) metabolism in isolated hearts of wild-type (WT) and TGiNOS mice. NO formation by iNOS resulted in a significant decrease of the MbO2 signal and a concomitantly emerging metMb signal in spectra of TGiNOS hearts only (DeltaMbO2: -46.3+/-38.4 micromol/kg, DeltametMb: +41.4+/-17.6 micromol/kg, n=6; P<0.05) leaving contractility and energetics unaffected. Inhibition of the Mb-mediated NO degradation by carbon monoxide (20%) led to a deterioration of myocardial contractility in TGiNOS hearts (left ventricular developed pressure: 78.2+/-8.2% versus 96.7+/-4.6% of baseline, n=6; P<0.005), which was associated with a profound pertubation of cardiac energy state as assessed by 31P NMR spectroscopy (eg, phosphocreatine: 13.3+/-1.3 mmol/L (TGiNOS) versus 15.9+/-0.7 mmol/L (WT), n=6; P<0.005). These alterations could be fully antagonized by the NOS inhibitor S-ethylisothiourea. Our findings demonstrate that myoglobin serves as an important cytoplasmic buffer of iNOS-derived NO, which determines the functional consequences of iNOS overexpression.     

Contribution of caveolin protein abundance to augmented nitric oxide signaling in conscious dogs with pacing-induced heart failure

Myocardial NO signaling appears elevated in heart failure (HF). Whether this results from increased NO production, induction of the high-output NO synthase (NOS)2 isoform, or changes in NOS regulatory pathways (such as caveolae) remains controversial. We tested the hypothesis that increased abundance of caveolin-3 and/or sarcolemmal caveolae contribute to increased NO signaling in pacing-induced HF. Abundance of caveolin-3 (0.59+/-0.08 versus 0.29+/-0.08 arbitrary units, P = 0.01) but not caveolin-1 was increased in HF compared with control conditions, assessed by Western blot. Additionally, transmission electron microscopy revealed increased caveolae (2. 7+/-0.4 versus 1.3+/-0.3 per micrometer myocyte membrane, P<0.005). The association between caveolin-3 and NOS3 at the sarcolemma and T tubules was unchanged in HF compared with control myocytes. The impact of NOS inhibition with L-N(G)-methylarginine hydrochloride (L-NMMA) on beta-adrenergic inotropy was assessed in conscious dogs before and after HF. In control dogs, dobutamine (5 microg. kg(-1) x min(-1)) increased +dP/dt by 36+/-7%, and this was augmented to 66+/-24% by 20 mg/kg L-NMMA (P = 0.04 versus without L-NMMA, n = 8) but not affected by 10 mg/kg L-NMMA (34+/-10%, P = NS; n = 8). In HF, dobutamine +dP/dt response was depressed (P<0.001 versus control), and increased concentrations were required to match control inotropic responses (10 to 15 microg. kg(-1) x min(-1), 48+/-7%). L-NMMA enhanced +dP/dt responses similarly at 10 mg/kg (61+/-17%, P = 0.02; n = 4) and 20 mg/kg (54+/-7%, P = 0.04; n = 7). Caveolin-3 abundance positively correlated with L-NMMA augmentation of dobutamine inotropic responses in HF (r = 0.9, P = 0.03; n = 4). Thus, in canine pacing-induced HF, expression of caveolin-3 and of sarcolemmal caveolae is increased. This increase is associated with augmented agonist-stimulated NO signaling, likely via a compartmentation effect.     

LA419, a novel nitric oxide donor, prevents pathological cardiac remodeling in pressure-overloaded rats via endothelial nitric oxide synthase pathway regulation

Reduced endogenous NO production has been described in cardiovascular disorders as cardiac hypertrophy and heart failure. The therapy with conventional nitrates is limited by their adverse hemodynamic effects and drug tolerance. The novel NO donor LA419 has demonstrated important antithrombotic and anti-ischemic properties without those adverse effects. The aim of this study was to evaluate the effect of LA419 chronic treatment on cardiac hypertrophy development in a progressive model of left ventricular hypertrophy. Rats were randomly divided into 6 groups: sham and clip (euthanized 7 weeks after aortic stenosis), sham+vehicle, sham+LA419, clip+vehicle, and clip+LA419 (euthanized 14 weeks after the surgery and treated with vehicle or 30 mg/kg of LA419 once left ventricular hypertrophy was established). LA419 treatment for 7 weeks induced a marked reduction in the heart:body weight ratio (4.10+/-0.28 and 3.38+/-0.06 mg/g in clip+vehicle versus clip+LA419; P<0.001) and left ventricular diameter (11.96+/-0.25 and 9.90+/-0.20 mm in clip+vehicle versus clip+LA419; P<0.001) without modifying the high blood pressure observed in stenosed rats. Histological analysis revealed that LA419 attenuated myocardial and perivascular fibrosis observed in rats with pressure overload for 14 weeks. In addition, LA419 treatment restored endothelial NO synthase and caveolin-3 expression levels, enhanced the interaction between endothelial NO synthase and its positive regulator the heat shock protein 90, and re-established the normal cardiac content of cGMP in stenosed rats. Thus, LA419 prevented the progression to maladaptative cardiac hypertrophy in response to prolonged pressure overload through a mechanism that involved the re-establishment of the endothelial NO synthase signaling pathway.     

Comparison of the effects of omapatrilat and irbesartan/hydrochlorothiazide on endothelial function and cardiac hypertrophy in the stroke-prone spontaneously hypertensive rat: sex differences

OBJECTIVE: The novel antihypertensive agent, omapatrilat, is both an inhibitor of neutral endopeptidase and angiotensin-converting enzyme. This study investigated the effects of omapatrilat in comparison with an angiotensin I-receptor antagonist/diuretic combination on blood pressure, endothelial function and cardiac hypertrophy in stroke-prone spontaneously hypertensive rats (SHRSP). METHODS: Male and female SHRSP were treated orally with omapatrilat or irbesartan plus hydrochlorothiazide (I + H) or vehicle for 8 weeks. Systolic blood pressure was measured weekly by tail-cuff. Cardiac hypertrophy was monitored by echocardiography at 8, 12 and 16 weeks of age. Endothelial function [basal nitric oxide (NO) bioavailability and stimulated NO release] was examined in carotid arteries using organ bath pharmacology and in mesenteric resistance arteries using wire myography. RESULTS: Compared with untreated controls, omapatrilat and I + H significantly attenuated hypertension [male control, 198.3 +/- 6.9 mmHg versus omapatrilat, 149.6 +/- 3.8 mmHg (F = 8.63 P < 0.0001), versus I + H, 145.6 +/- 5.1 mmHg (F = 7.38 P < 0.0001); female control, 170.3 +/-8.3 mmHg versus omapatrilat, 120.0 +/- 4.6 mmHg (F = 8.36, P < 0.0001), versus I + H, 112.2 +/- 2.9 mmHg (F = 9.08, P < 0.0001)] and left ventricular hypertrophy [male + female controls, 3.02 +/- 0.38 mg/g versus omapatrilat, 2.47 +/- 0.26 mg/g (P < 0.0001; 95% confidence interval, 0.27, 0.83), versus I + H, 2.49 +/- 0.21 mg/g (P < 0.0001; 95% confidence interval, 0.25, 0.83)]. Both treatments also significantly increased male carotid artery basal NO bioavailability relative to control [control, 0.62 +/- 0.17 g/g versus omapatrilat, 1.95 +/- 0.17 g/g (P < 0.0001; 95% confidence interval, -1.83, -0.36), versus I + H, 1.57 +/- 0.21 g/g (P < 0.026; 95% confidence interval, -1.31, -0.12)]. However, stimulated NO (EC50) was only improved in omapatrilat-treated males [controls, 0.19 +/- 0.06 micromol/l versus omapatrilat, 0.05 +/- 0.01 micromol/l (P = 0.05; 95% confidence interval, -1.16, -0.03)]. CONCLUSIONS: Omapatrilat treatment significantly reduced left ventricular hypertrophy and improved endothelial function in carotid arteries from male SHRSP by NO-dependent mechanisms. Despite equivalent antihypertensive and antihypertrophic actions, a similar improvement in endothelial function, specifically stimulated NO release, was not observed after treatment with I + H.     

Altered calcium handling in left ventricular pressure-overload hypertrophy as detected with aequorin in the isolated, perfused ferret heart.

The purpose of this study was to test the hypothesis that systolic and diastolic dysfunction in left ventricular pressure-overload hypertrophy is caused by abnormal intracellular calcium handling. Experiments were performed with intact, buffer-perfused, isovolumic ferret hearts (n = 9 hypertrophied, n = 9 control) that were loaded with the bioluminescent indicator aequorin to monitor changes in cytoplasmic calcium. In each experiment, left ventricular pressure and intracellular calcium transients were simultaneously recorded. Compared with their age-matched controls, significant hypertrophy of the left ventricle developed 4 weeks after postvalvular aortic banding; at the time the animals were killed, the left ventricular weight/body weight ratio was increased in the banded animals (5.3 x 10(-3) versus 3.6 x 10(-3), p less than 0.001). As indicated by the diastolic pressure-volume relation, left ventricular distensibility was significantly diminished in the hypertrophied hearts. In comparison to the controls, the hypertrophied hearts demonstrated a prolonged duration of isovolumic contraction (time to 90% decline from peak: 278 +/- 5.4 versus 247 +/- 10.2 msec, p less than 0.05), but a marked decrease in peak systolic midwall stress (22.4 +/- 5.0 versus 38.6 +/- 5.7 g/cm2, p less than 0.05). The increased duration of isovolumic contraction correlated with a similar prolongation of the calcium transient (time to 90% decline from peak: 245 +/- 19.5 versus 127 +/- 13.2 msec, p less than 0.05), indicating that the rate of sequestration and perhaps release of calcium by the sarcoplasmic reticulum is decreased in hypertrophy.(ABSTRACT TRUNCATED AT 250 WORDS)     

Short-acting calcium antagonist clevidipine protects against reperfusion injury via local nitric oxide-related mechanisms in the jeopardised myocardium

BACKGROUND: Calcium antagonists may, in addition to their classical actions, release nitric oxide (NO) from coronary arteries. The aim of this study was to elucidate the possible interaction between the cardioprotective effect of a short-acting calcium antagonist and NO during myocardial ischaemia and reperfusion. METHODS: Anaesthetised pigs were subjected to 45 min ligation of the left anterior descending coronary artery (LAD) followed by 4 h of reperfusion. Five groups were given vehicle (n=9), clevidipine (n=8), the NO synthase inhibitor L-NMMA (n=6), clevidipine in combination with L-NMMA (n=6) or clevidipine in combination with L-NMMA and NO precursor L-arginine (n=6) into the LAD during the last 10 min of ischaemia and the first 5 min of reperfusion. RESULTS: There were no significant differences in LAD blood flow, mean arterial pressure, rate-pressure product or dP/dt between the groups before ischaemia or during reperfusion. The infarct size (IS) was 86+/-2% of the area at risk in the vehicle group. Clevidipine reduced the IS to 59+/-3% (P<0.001). When clevidipine was administered together with L-NMMA, the protective effect of clevidipine was abolished (IS, 87+/-3%; P<0.001 vs. clevidipine), whereas addition of L-arginine restored its cardioprotective effect (IS 60+/-3%; P<0.001 vs. vehicle). L-NMMA did not affect IS per se (88+/-5%). Endothelium-dependent coronary vasodilation induced by substance P was significantly larger in the clevidipine group than in the other groups. CONCLUSION: Local administration of a calcium antagonist during the late ischaemia and early reperfusion reduces IS and preserves coronary endothelial function. The cardioprotective effect of clevidipine is suggested to be dependent on maintained local bioavailability of NO.     

Extracellular ATP stimulates NO production in rat thick ascending limb

NO produced by NO synthase (NOS) 3 acts as an autacoid to regulate NaCl absorption in the thick ascending limb. ATP induces NO production by NOS 3 in endothelial cells. We hypothesized that extracellular ATP activates NOS in thick ascending limbs through P2 receptors. To test this, we measured intracellular NO production using the NO-selective fluorescent dye DAF-2 in suspensions of rat medullary thick ascending limbs. We found that ATP increased DAF-2 fluorescence in a concentration-dependent manner, reaching saturation at &200 micromol/L with an EC50 of 37 micromol/L. The increase was blunted by 74% by the nonselective NOS inhibitor L-omega-nitro-arginine-methyl-ester (2 mmol/L; 60+/-7 versus 16+/-6 arbitrary fluorescence units; P<0.02; n=5). In the presence of the P2 receptor antagonist suramin (300 micromol/L), ATP-induced NO production was reduced by 64% (101+/-11 versus 37+/-5 arbitrary fluorescence units; P<0.002; n=5). Blocking ATP hydrolysis with a 5'-ectonucleotidase inhibitor, ARL67156 (30 micromol/L) enhanced the response to ATP and shifted the EC(50) to 0.8 micromol/L. In the presence of ARL67156, the EC50 of the P2X-selective agonist beta,gamma-methylene-adenosine 5'-triphosphate was 4.8 micromol/L and the EC50 for the P2Y-selective agonist UTP was 40.4 micromol/L. The maximal responses for both agonists were similar. Taken together, these data indicate that ATP stimulates NO production in the thick ascending limb primarily through P2X receptor activation and that ATP hydrolysis may regulate NO production.     

Salt loading on plasma asymmetrical dimethylarginine and the protective role of potassium supplement in normotensive salt-sensitive asians

Asymmetrical dimethylarginine (ADMA) is an endogenous inhibitor of NO synthase. Because endothelial NO pathway is compromised in patients with salt-sensitive hypertension, we investigated whether the plasma ADMA can be modulated by chronic salt loading in normotensive salt-sensitive persons and its relationship with NO, and we further determined whether or not dietary potassium supplementation can reverse them. Sixty normotensive subjects (aged 20 to 60 years) were selected from a rural community of Northern China. All of the people were sequentially maintained on a low-salt diet for 7 days (3 g/day, NaCl), then a high-salt diet for 7 days (18 g/day), and high-salt diet with potassium supplementation for another 7 days (4.5 g/day, KCl). After salt loading, the plasma ADMA concentrations increased significantly in salt-sensitive subjects (0.89+/-0.02 micromol/L versus 0.51+/-0.02 micromol/L; P<0.05), whereas the plasma NOx levels reduced considerably (41.8+/-2.1 micromol/L versus 63.5+/-2.1 micromol/L; P<0.01). All of the abnormalities normalized when dietary potassium were supplemented (0.52+/-0.03 micromol/L versus 0.89+/-0.02 micromol/L for ADMA and 58.1+/-0.9 micromol/L versus 41.8+/-2.1 micromol/L for NOx). Statistically significant correlations were found among plasma ADMA level, the mean blood pressure, and the level of NO after salt loading in normotensive salt sensitive individuals. Our study indicates that high dietary potassium intake reduces blood pressure and ADMA levels while increasing NO bioactivity in normotensive salt-sensitive but not salt-resistant Asian subjects after salt loading.     

Close relationship of abnormal glucose tolerance with endothelial dysfunction in hypertension

Hypertension is frequently accompanied by left ventricular hypertrophy, endothelial dysfunction, and abnormal glucose metabolism. However, no study has examined the relative pathological significance of left ventricular hypertrophy and abnormal glucose metabolism on endothelial dysfunction in hypertension. This study was conducted to evaluate whether abnormal glucose tolerance assessed by 75-g oral glucose tolerance test or left ventricular hypertrophy is more closely associated with endothelial dysfunction in never-treated hypertensive patients without elevated fasting blood glucose. We studied 107 unmedicated hypertensive patients (mean age, 54+/-10 years) whose fasting blood glucose was <7.0 mmol/L. Endothelial function was assessed by change in brachial artery diameter in response to reactive hyperemia, and left ventricular mass index was determined by ultrasonography. Simple linear regression analysis demonstrated that endothelial function significantly correlated with left ventricular mass index and 2-hour blood glucose in 75-g oral glucose tolerance test, but not with fasting blood glucose. Multiple linear regression analysis revealed that endothelial function significantly correlated with 2-hour blood glucose (beta=-2.68, P<0.05) after we controlled for other clinical variables. Patients were divided into 3 groups according to 2-hour blood glucose levels. Endothelial function was more impaired in patients with diabetes (n=12; 4.7+/-1.8%) and in those with impaired glucose tolerance (n=31; 6.3+/-2.9%) than in those with normal glucose tolerance (n=64; 8.4+/-4.5%) (P<0.05), but left ventricular mass index was similar in these 3 groups. Abnormal glucose tolerance assessed by 75-g oral glucose tolerance test, rather than left ventricular hypertrophy, may have direct pathophysiological relevance to endothelial dysfunction in borderline to moderate hypertensive patients.     

Exercise capacity and blood pressure associations with left ventricular mass in prehypertensive individuals

Prehypertensive individuals are at increased risk for developing hypertension and cardiovascular disease compared with those with normal blood pressure. Early compromises in left ventricular structure may explain part of the increased risk. We assessed echocardiographic and exercise parameters in prehypertensive individuals (n=790) to determine associations between exercise blood pressure and left ventricular structure. The exercise systolic blood pressure at 5 metabolic equivalents (METs) and the change in blood pressure from rest to 5 METs were the strongest predictors of left ventricular hypertrophy. We identified the systolic blood pressure of 150 mm Hg at the exercise levels of 5 METs as the threshold for left ventricular hypertrophy. There was a 4-fold increase in the likelihood for left ventricular hypertrophy for every 10-mm Hg increment in systolic blood pressure beyond this threshold (OR: 1.15; 95% CI: 1.12 to 1.18). There was also a 42% reduction in the risk for left ventricular hypertrophy for every 1 MET increase in the workload (OR: 0.58; P<0.001). When compared with low-fit, moderate, and high-fit individuals exhibited significantly lower systolic blood pressure at an exercise workload of 5 METs (155+/-14 versus 146+/-10 versus 144+/-10; P<0.05), lower left ventricular mass index (48+/-12 versus 41+/-10 versus 41+/-9; P<0.05), and prevalence of left ventricular hypertrophy (48.3% versus 18.7% versus 21.6%; P<0.001). This suggests that moderate improvements in cardiorespiratory fitness achieved by moderate intensity physical activity can improve hemodynamics and cardiac performance in prehypertensive individuals and reduce the work of the left ventricle, ultimately resulting in lower left ventricular mass.     

Tetrahydrobiopterin and antioxidants reverse the coronary endothelial dysfunction associated with left ventricular hypertrophy in a porcine model

OBJECTIVE: Endothelium-dependent G-protein mediated relaxations of epicardial coronary arteries is impaired with left ventricular hypertrophy. The objective of this study was to assess the effect of L-arginine, BH(4) and the combination of two antioxidants, superoxide dismutase and catalase, on endothelium-dependent relaxations in a swine left ventricular hypertrophy model. METHODS: Aortic banding was performed 3 cm above the coronary ostia. Vascular reactivity studies were performed in standard organ chamber experiments to assess the NO pathway in the presence of methyltetrahydropterin (a BH(4) analogue), L-arginine, superoxide dismutase and catalase. RESULTS: There was a statistically significant increase in endothelium-dependent relaxation to serotonin and to bradykinin with methyltetrahydropterin and with superoxide dismutase plus catalase (P<0.05) but not with L-arginine compared to untreated coronary arteries from left ventricular hypertrophy animals. Plasma 3-nitrotyrosine level increased significantly from 918+/-122 to 1844+/-300 microM (P<0.05 vs. control) after 60 days of aortic banding. Endothelial dysfunction was not associated with a reduced expression of endothelial nitric oxide synthase 2 months after pressure overload left ventricular hypertrophy. CONCLUSIONS: Treatment with BH(4) and antioxidants constitutes an interesting approach for the prevention of endothelial dysfunction in epicardial coronary arteries associated with left ventricular hypertrophy.     

Role of cGMP-inhibited phosphodiesterase and sarcoplasmic calcium in mediating the increase in basal heart rate with nitric oxide donors

Nitric oxide (NO) donors increase heart rate (HR) through a guanylyl cyclase-dependent stimulation of the pacemaker current I(f), without affecting basal I(Ca-L). The activity of I(f)is known to be enhanced by cyclic nucleotides and by an increase in cytosolic Ca(2+). We examined the role of cGMP-dependent signaling pathways and intracellular Ca(2+)stores in mediating the positive chronotropic effect of NO donors. In isolated guinea pig atria, the increase in HR in response to 1-100 micromol/l 3-morpholino-sydnonimine (SIN-1; with superoxide dismutase, n=6) or diethylamine-NO (DEA-NO, n=8) was significantly attenuated by blockers of the cGMP-inhibited phosphodiesterase (PDE3; trequinsin, milrinone or Ro-13-6438, n=22). In addition, the rate response to DEA-NO or sodium nitroprusside (SNP) was significantly reduced following inhibition of PKA (KT5720 or H-89, n=15) but not PKG (KT5728 or Rp-8-pCPT-cGMPs, n=16). Suppression of sarcoplasmic (SR) Ca(2+)release by pretreatment of isolated atria with ryanodine or cyclopiazonic acid (2 micromol/l and 60 micromol/l, n=16) significantly reduced the chronotropic response to 1-100 micromol/l SIN-1 or DEA-NO. Moreover, in isolated guinea pig sinoatrial node cells 5 micromol/l SNP significantly increased diastolic and peak Ca(2+)fluorescence (+13+/-1% and +28+/-1%, n=6, P<0.05). Our findings are consistent with a functionally significant role of cAMP/PKA signaling (via cGMP inhibition of PDE3) and SR Ca(2+)in mediating the positive chronotropic effect of NO donors.     

Upregulation of the nitric oxide-cGMP pathway in aged myocardium: physiological response to l-arginine

Cardiovascular aging is associated with decreased endothelial vasoreactivity and prolonged diastolic relaxation. As diminished NO signaling contributes to age-associated endothelial dysfunction, we tested the hypothesis that impaired NO signaling or bioactivity also contributes to slowed ventricular relaxation with age. Accordingly, we measured myocardial NO synthase (NOS) enzyme activity, protein abundance, and cGMP production in old (22 to 25 months) and young adult (4 to 7 months) male Wistar rats. Both NOS3 protein abundance and calcium-dependent NOS activity were elevated in old compared with young adult hearts (7.2+/-1.1 versus 4.2+/-0.6 pmol/mg protein, respectively, P=0.03). However, NOS activity and protein abundance were similar in isolated myocytes, indicating that endothelial NOS likely explains the age difference. Cardiac effluent cGMP (enzyme immunoassay) was 4.8-fold higher (1794+/-373 fmol/min per mg heart tissue) in older versus younger hearts (P=0.003). To assess NO pathway responsiveness, we administered the NOS substrate l-arginine (100 micrometer) to isolated perfused rat hearts. Baseline isovolumic relaxation (tau) was prolonged in old (42.9+/-2.5 ms, n=16) versus young hearts (36.0+/-1.9 ms, n=11, P=0.03). l-Arginine decreased tau (P<0.001) and left ventricular end-diastolic pressure in both old and young hearts. Supporting an NO/cGMP-mediating mechanism, the NO donor sodium nitroprusside reduced tau (maximal effect, -14+/-2%, n=5, P<0.001), and this lusitropic effect was attenuated by the soluble guanylyl cyclase inhibitor 1H:-[1,2,4]oxadiazolo-[4,3,-a]quinoxalin-1-one (n=7, P<0.001). Thus, the NO-cGMP pathway is upregulated in the endothelial cells of aged hearts. l-Arginine, the NOS precursor, enhances ventricular relaxation in old and young hearts, indicating that the NOS pathway may be exploited to modulate diastolic function in aged myocardium.     

Attenuated in vitro coronary arteriolar vasorelaxation to insulin-like growth factor I in experimental hypercholesterolemia.

Insulin and insulin-like growth factor (IGF) 1 affect coronary vasoactivity. Experimental hypercholesterolemia is associated with coronary atherogenesis and altered vasomotor regulation. Because the IGF axis is altered during atherogenesis, we postulated that experimental hypercholesterolemia is associated with an altered coronary vasoactive response to IGF-1 in vitro. Coronary arteries and arterioles from pigs fed either a normal or high-cholesterol diet for 10 weeks were contracted with endothelin-1 and relaxed with cumulative concentrations of insulin or IGF-1 (10(-12) to 10(-7) mol/L). Control arterioles were also incubated with the nitric oxide synthase inhibitor 10(-4) mol/L N(G)-monomethyl-L-arginine (L-NMMA) or the potassium channel blocker 10(-2) mol/L tetraethylammonium (TEA), contracted with endothelin-1, and relaxed with insulin or IGF-1. Experimental hypercholesterolemia (1) increased serum cholesterol (9.5+/-1.0 versus 1.9+/-0.08 mmol/L; P<0.0001), (2) caused coronary arterial and arteriolar endothelial dysfunction in vitro (attenuated vasorelaxation to bradykinin), (3) did not alter the epicardial response to either insulin (P=0.80) or IGF-1 (P=0.12), and (4) significantly attenuated the arteriolar response to IGF-1 (maximal relaxation of 79+/-6% versus 42+/-8%; P=0.01) but not insulin (43+/-6% versus 53+/-7%; P=0.99). Control arteriolar vasorelaxation to IGF-1 was attenuated by both L-NMMA (P<0.001) and TEA (P=0.01), whereas only L-NMMA attenuated insulin (P<0.001). Staining for IGF-1 and IGF binding protein 2 was increased (P<0.05) in arterioles of cholesterol-fed pigs. IGF-1 and insulin are therefore coronary arteriolar vasorelaxants through different mechanisms. Experimental hypercholesterolemia is associated with resistance to the coronary arteriolar vasorelaxing effects of IGF-1 but not insulin, in conjunction with increased ligand and binding-protein expression. The IGF axis may contribute to the altered coronary vasoactivity in hypercholesterolemia.     

Late treatment with ramipril increases survival in old spontaneously hypertensive rats.

Spontaneously hypertensive rats (SHR) begin to die from cardiovascular complications at approximately 15 months of age. We tested whether chronic ACE-inhibitor treatment would extend the lifespan of such old animals. We also studied cardiac hypertrophy and function, endothelial function and expression, and activity of NO synthase (eNOS). One hundred 15-month-old SHR were randomized into 3 groups, control (n=10), placebo-treated (n=45), and ramipril-treated with an antihypertensive dose of 1 mg. kg(-1). d(-1) in drinking water (n=45). Ex vivo experiments were performed after 15 months (control) and 21 months, when approximately 80% of the placebo group had died. Late treatment with ramipril significantly extended lifespan of the animals from 21 to 30 months. Fully established cardiac hypertrophy, observed in placebo-treated animals and in controls, was significantly reversed by ramipril treatment. In isolated working hearts, a significantly improved function associated with increased cardiac eNOS expression was seen versus placebo and control hearts. Endothelial dysfunction in isolated aortic rings from control and placebo-treated SHR was significantly improved by ACE inhibition and associated with enhanced NO release. Late treatment of SHR with the ACE inhibitor ramipril extended lifespan from 21 to 30 months, which is comparable to the lifespan of untreated normotensive Wistar-Kyoto rats. This lifespan extension, probably due to blood pressure reduction, correlated with increased eNOS expression and activity followed by a regression of left ventricular hypertrophy and cardiac and vascular dysfunction.     

Endothelin-converting enzyme inhibition ameliorates angiotensin II-induced cardiac damage

We tested the hypothesis that endothelin-converting enzyme (ECE) inhibition ameliorates end-organ damage in rats harboring both human renin and human angiotensinogen genes (dTGR). Hypertension develops in the animals, and they die by age 7 weeks of heart and kidney failure. Three groups were studied: dTGR (n=12) receiving vehicle, dTGR receiving ECE inhibitor (RO0687629; 30 mg/kg by gavage; n=10), and Sprague-Dawley control rats (SD; n=10) receiving vehicle, all after week 4, with euthanasia at week 7. Systolic blood pressure was not reduced by ECE inhibitor compared with dTGR (205+/-6 versus 206+/-6 mm Hg at week 7, respectively). In contrast, ECE inhibitor treatment significantly reduced mortality rate to 20% (2 of 10), whereas untreated dTGR had a 52% mortality rate (7 of 12). ECE inhibitor treatment ameliorated cardiac damage and reduced left ventricular ECE activity below SD levels. Echocardiography at week 7 showed reduced cardiac hypertrophy (4.8+/-0.2 versus 5.7+/-0.2 mg/g, P<0.01) and increased left ventricular cavity diameter (5.5+/-0.3 versus 3.1+/-0.1 mm, P<0.001) and filling volume (0.42+/-0.04 versus 0.16+/-0.06 mL, P<0.05) after ECE inhibitor compared with untreated dTGR. ECE inhibitor treatment also reduced cardiac fibrosis, tissue factor expression, left ventricular basic fibroblast growth factor mRNA levels, and immunostaining in the vessel wall, independent of high blood pressure. In contrast, the ECE inhibitor treatment showed no renoprotective effect. These data are the first to show that ECE inhibition reduces angiotensin II-induced cardiac damage.     

Cardiac nitric oxide production due to angiotensin-converting enzyme inhibition decreases beta-adrenergic myocardial contractility in patients with dilated cardiomyopathy

OBJECTIVES: This study tested the hypothesis that angiotensin-converting enzyme (ACE) inhibitors attenuate beta-adrenergic contractility in patients with idiopathic dilated cardiomyopathy (DCM) through nitric oxide (NO) myocardial signaling. BACKGROUND: The ACE inhibitors increase bradykinin, an agonist of NO synthase (NOS). Nitric oxide inhibits beta-adrenergic myocardial contractility in patients with heart failure. METHODS: The study patients were given the angiotensin-1 (AT-1) receptor antagonist losartan for one week. The hemodynamic responses to intravenous dobutamine were determined before and during intracoronary infusion of enalaprilat (0.2 mg/min) with and without the NOS inhibitor N(G)-monomethyl-L-arginine (L-NMMA, 5 mg/min). RESULTS: In patients with DCM (n = 8), dobutamine increased the peak rate of rise of left ventricular pressure (+dP/dt) by 49 +/- 8% (p < 0.001) and ventricular elastance (Ecs) by 53 +/- 16% (p < 0.03). Co-infusion with enalaprilat decreased +dP/dt to 26 +/- 12% and Ecs to -2 +/- 17% above baseline (p < 0.05), and this anti-adrenergic effect was reversed by L-NMMA co-infusion (p < 0.05 vs. enalaprilat). In addition, intracoronary enalaprilat reduced left ventricular end-diastolic pressure (LVEDP), but not left ventricular end-diastolic volume, consistent with increased left ventricular distensibility. Infusion with L-NMMA before enalaprilat in patients with DCM (n = 5) prevented the reduction in +dP/dt, Ecs and LVEDP. In patients with normal left ventricular function (n = 5), enalaprilat did not inhibit contractility or reduce LVEDP during dobutamine infusion. CONCLUSIONS: Enalaprilat attenuates beta-adrenergic contractility and enhances left ventricular distensibility in patients with DCM, but not in subjects with normal left ventricular function. This response is NO modulated and occurs in the presence of angiotensin receptor blockade. These findings may have important clinical and pharmacologic implications for the use of ACE inhibitors, AT-1 receptor antagonists and their combination in the treatment of heart failure.     

Endothelial nitric oxide gene knockout mice: cardiac phenotypes and the effect of angiotensin-converting enzyme inhibitor on myocardial ischemia/reperfusion injury.

We tested the hypothesis that nitric oxide (NO) released by endothelial NO synthase (eNOS) is not only important in blood pressure regulation but also involved in cardiac function and remodeling and in the cardioprotective effect of angiotensin-converting enzyme inhibitors (ACEi). With the use of a 2D Doppler echocardiography system equipped with a 15-MHz linear transducer, we evaluated left ventricular (LV) morphology and function in conscious eNOS knockout mice (eNOS(-/-); n=15) and their wild-type littermates (eNOS(+/+); n=16). We also studied whether in eNOS(-/-) mice (1) myocardial ischemia/reperfusion injury is more severe and (2) the cardioprotective effect of ACEi is diminished or absent. In comparison with the wild type, eNOS(-/-) mice had significantly increased systolic blood pressure (128+/-3 versus 108+/-5 mm Hg; P<0.001) and decreased heart rate (531+/-22 versus 629+/-18 bpm; P<0.001) associated with increased LV posterior wall thickness (0.80+/-0.04 versus 0.64+/-0.02 mm; P<0.001) and LV mass (18.3+/-0.9 versus 13.1+/-0.5 mg/10 g body weight; P<0.01). Despite hypertension and LV hypertrophy, LV chamber dimension, shortening fraction and ejection fraction (indicators of LV contractility), and cardiac output did not differ between the 2 strains, which indicates that LV function in eNOS(-/-) mice is well compensated. We also found that in eNOS(+/+) mice, ACEi decreased the ratio of myocardial infarct size to area at risk from 62.7+/-3.9% to 36.3+/-1.6% (P<0. 001), whereas in eNOS(-/-) mice this effect of ACEi was almost abolished: the ratio of myocardial infarct size to area at risk was 67.2+/-2.9% in the vehicle-treated group and 62.7+/-3.9% in mice treated with ACEi. Moreover, infarct size in vehicle-treated eNOS(-/-) mice was not significantly different from eNOS(+/+) mice given the same treatment. We concluded that (1) endothelium-derived NO plays an important role in the regulation of blood pressure homeostasis; (2) NO released under basal conditions has no significant impact on cardiac function; and (3) ACEi protect the heart against ischemia/reperfusion injury in mice and that this effect is mediated in part by endothelium-derived NO.     

Effect of L—NAME on nitric oxide and gastrointestinal motility alterations in cirrhotic rats

AIM: To invsstigare the effect of L-NAME on nitric oxide andgastriubtestubal motility alterations in cirrhotic ratsMETHODS: Rats with cirrhosis induced by carbontetrachloride were randomly divided into two groups, one( n= 13) receiving 0. 5 mg@ kg-1 per clay of NG-nitro-L-argininemethyl ester (L-NAME), a nitric oxide synthase inhibitor,for 10 days, whereas the other group ( n = 13) and control( n = 10) rats were administrated the same volume of 9 g@ L-1saline.Half gastric emptying time and 2 h residual rate weremeasured by SPECT, using 99m Tc-DTPA-labeled bariumsuifate as test meal. Gastrointestinal transition time wasrecorded simultaneously. Serum concentration of nitrcoxide (NO) was determined by the kinetic cadmiunreduction and colorimetric methods. ImmunohistochemicalSABC method was used to observe the expression anddistribution of three types of nitric oxide synthase (NOS)isoforms in the mt gastrointestinal tract. Western blot wasused to detect expression of gastrointestinal NOS isoforms.RESULTS: Half gastric emptying time and trans-gastrointestinal time were significantly prolonged( 124.0 ± 26.4min; 33.7± 8.9min;72.1 ± 15.3 min; P＜0.01), (12.4±0.5h; 9.5±0.3 h; 8.2±0.8 h; P＜0.01), 2h residual rate wasraised in cirrhotic rots than in controls and cirrhotic ratstreated with L-NAME(54.9± 7.6 % ,13.7 ± 3.2 %, 34.9± 10.3%, P＜ 0.01). Serum concentration of NO was significantlyincreased in cirrhotic rots than in the other groups (8.20 ± 2.48)μmol@L-1, (5.94± 1.07) μmol@L-1 ,and control (5.66± 1.60) tμmol@L-1, P＜ 0.01. NOS staining intensities which weremainly located in the gastrointestinal tissues were markedlylower in cirrhotic rats than in the controls and cirrhotic ratsafter treated with L- NAME.CONCLUSION: Gastrointestinal motility was remarkablyinhibited in cirrhotic rats, which could he alleviated by L-NAME. Nitric oxide may play an important role in theinhibition of gastrointestinal motility in cirrhotic rats.     

Coronary microvascular endothelial cell redox state in left ventricular hypertrophy : the role of angiotensin II

Left ventricular hypertrophy (LVH) is associated with elevated plasma angiotensin II (Ang II) levels and endothelial dysfunction. The relationship between Ang II and endothelial dysfunction remains unknown, however, but it may involve an alteration in endothelial cell redox state. We therefore investigated the effect of Ang II on NADH/NADPH oxidase-mediated superoxide anion (O(2)(-)) production by cultured guinea pig coronary microvascular endothelial cells (CMVEs) and CMVEs freshly isolated from a guinea pig, pressure-overload model of LVH. Lucigenin chemiluminescence was used to measure O(2)(-) production in the particulate fraction of CMVE lysates. In cultured cells, incubation with Ang II (0.1 nmol/L to 1 micromol/L for 18 hours) resulted in significant (P<0.01) increases in both NADH- and NADPH-dependent O(2)(-) production, with a peak effect at 1 nmol/L. The latter was significantly (P<0.01) inhibited by the AT(1) receptor antagonist losartan (1 micromol/L for 18 hours). In contrast, the O(2)(-) response to Ang II (0.1 nmol/L to 1 micromol/L for 18 hours) was largely unaffected by concomitant exposure to the AT(2) antagonist PD 123319 (1 micromol/L). In freshly isolated CMVEs from nonoperated animals, NADH- and NADPH-dependent O(2)(-) production was not different from that in sham-operated animals but was significantly (P<0.05) elevated in the aortic-banded animals. Plasma Ang II levels were significantly (P<0.001) elevated in the aortic-banded (1.25+/-0.12 microg/L, n=12) compared with sham-operated animals (0.63+/-0.06 microg/L, n=12). These data suggest that the endothelial dysfunction associated with LVH may be due, at least in part, to the Ang II-induced upregulation of NADH/NADPH oxidase-dependent O(2)(-) production.