CLINICAL STUDIES
Expression, activity and functional significance of inducible nitric oxide synthase in the failing human heart
Helmut Drexler, MDa,
Stephanie Kästner, BSa,
Armin Strobel, BSa,
Roland Studer, PhDa,
Otto E. Brodde, PhD* and
Gerd Hasenfuß, MDa
a Department of Cardiology, Medical University of Hannover, Hannover, Germany
* Department of Pharmacology, University of Halle, Halle, Germany
Manuscript received February 19, 1998;
revised manuscript received May 27, 1998,
accepted June 12, 1998.
Address for correspondence: Dr. Helmut Drexler, Medizinische Hochschule Hannover, Abteilung Kardiologie, Carl-Neuberg-Strasse 1, 30625 Hannover, Germany
drexler.helmut{at}mh-hannover.de
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Abstract
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Objectives. The study was designed to evaluate the functional impact of nitric oxide (NO) generation within the myocardium on cardiac contraction in the failing human heart.
Background. Heart failure is associated with activation of cytokines and expression of inducible nitric oxide synthase (NOS II), which generates NO from L-arginine. Nitric oxide has been shown to modulate myocardial performance, raising the possibility that cardiac generation of NO by NOS II modulates cardiac contraction in the failing human heart.
Methods. Left ventricular (LV) tissue of 24 patients with end-stage heart failure was obtained during cardiac transplantation. Gene expression of NOS II and endothelial NO-synthase (NOS III) was quantified by competitive reverse transcription-polymerase chain reaction and compared to tissues of five nonfailing donor hearts. Nitric oxide synthase II activity was determined by citrulline assay and related to changes in force of contraction induced by the ß-adrenergic agonist isoproterenol, NO-donors and/or N-mono-methyl-L-arginine (L-NMMA), an inhibitor of NOS.
Results. While NOS III mRNA was reduced in failing hearts, NOS II mRNA was increased in failing LV tissue and correlated with NOS II activity. High NOS II activity was associated with early relaxation and impaired responsiveness to ß-adrenergic stimulation, that is, the inotropic response to isoproterenol in failing hearts was inversely related to NOS II activity (r = 0.61, p < 0.005). Nitric oxide donors or L-NMMA did not affect myocardial performance in failing hearts at baseline. However, L-NMMA enhanced the positive inotropic response to ß-adrenergic stimulation in failing hearts with high NOS II activity. Nitric oxide donors attenuated the isoproterenol-induced increase in force of contraction of failing hearts.
Conclusions. Cardiac production of NO by NOS II attenuates the positive inotropic effects of ß-adrenergic stimulation and hastens relaxation in failing human hearts.
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Abbreviations and Acronyms
| | L-NMMA | = N-mono-methyl-L-arginine | | LV | = left ventricular | | NO | = nitric oxide | | NOS II | = inducible nitric oxide synthase | | NOS III | = endothelial NO-synthase | | PCR | = polymerase chain reaction | | RT | = reverse transcription | | SNAP | = S-nitroso-N-acetyl-penicillamine | | SNP | = sodium nitroprusside |
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Nitric oxide (NO) is recognized as an important signal molecule between and within cells (1) and is synthesized from L-arginine by three different isoforms of the enzyme nitric oxide synthase (NOS). Two isoforms are constitutively expressed while one isoform is induced in response to cytokines and endotoxin (for review see reference 2). While the pivotal role of endothelium-derived NO in regulation of vasomotor tone is now established, the impact of NO on cardiac function has only recently been recognized. Removal of endocardium or endothelium has been shown to modulate cardiac contraction (3). Moreover, exposure of cardiac muscle to cytokines impairs cardiac contractility, an effect that is mediated by NO (4). In fact, the expression of an inducible NOS (NOS II) within cardiomyocytes appears to be responsible for the NO-mediated cardiodepressant effect of cytokines (5,6). In addition, both basal and cytokine-induced generation of NO in isolated cardiomyocytes attenuates the myocardial inotropic response to ß-adrenergic stimulation (6), extending experimental and clinical observations that cytokine activation and/or septic shock are associated with an impaired responsiveness to ß-agonists (7,8).
Advanced stages of chronic heart failure are often associated with systemic and cardiac cytokine activation (9,10) which, in turn, may stimulate the expression of the NOS II in the failing heart (11,12). Clinical studies have revealed that NO can modulate cardiac contraction in humans (13) and impair the ß-adrenergic stimulation in patients with left ventricular (LV) function (14). These observations raise the possibility that the cardiac synthesis of NO modulates cardiac contraction and the positive inotropic effects of ß-adrenergic stimulation in the failing human heart. Accordingly, we examined the gene expression, activity and functional significance the NOS II in failing human hearts.
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Methods
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Patients.
Failing human hearts (age 52 ± 3 years) were obtained from patients undergoing heart transplantation (dilated cardiomyopathy n = 11, coronary artery disease n = 13). All patients were in New York Heart Association functional class IV heart failure and demonstrated abnormal pretransplant hemodynamics (cardiac index 2.1 ± 0.2 L/min m2, ejection fraction 24% ± 3%, pulmonary wedge pressure 23 ± 2 mm Hg). Twenty patients were treated with digoxin, 22 with an angiotensin-converting enzyme inhibitor, 23 with diuretics and 13 with various additional vasodilators; 6 patients were on assist-devices. Five nonfailing control hearts (46 ± 4 years) were obtained from organ donors whose hearts could not be used for transplantation because of the lack of a suitable recipient. Tissue samples of the LV free wall were taken at the time of explantation, frozen in liquid nitrogen and stored at –80°C until use or submerged in a protective solution at room temperature and oxygenated by bubbling with 95% oxygen 5% carbon dioxide (15). Care was taken not to take scarred, fibrotic or adipose tissue or endocardium, epicardium or great vessels. This study was approved by the Ethical Committee of the University of Freiburg.
Quantitative reverse transcription (RT) polymerase chain reaction (PCR).
Total cellular RNA was isolated from 100 to 200 mg of frozen LV tissue (16). The amount of RNA was evaluated by spectrophotometry. The integrity of the RNA was checked by gel electrophoresis. Because of the low expression of NOS II and endothethial NO-synthase (NOS III), quantification of NOS II and NOS III mRNA was performed by competitive RT-PCR in the presence of a defined concentration of a shortened NOS III or NOS II competitor RNA that served as an internal standard, as previously described (17). The NOS III competitor template was obtained from a 891 bp (NOS II: 419 bp) cDNA fragment using the human NOS III (18) and NOS II cDNA (19) using appropriate restriction enzymes (NOS III: 766 bp, NOS II: 314 bp). Equal amounts of total RNA (2 µg) were mixed with increasing quantities of NOS II (12.5 to 6.25 x 104 molecules) or NOS III (10 to 1 x 106 molecules). Random hexanucleotide primers and Moloney murine leukemia virus reverse transcriptase were used to synthesize complementary DNA. The RT reactions (total volume 25 µl) were performed by incubation at 42°C for 60 min. Duplicate samples of PCR were performed (18). Denaturing, annealing and extension reactions proceeded 36 (NOS II) or 30 (NOS III) times at 94°C for 1 min, 62°C (NOS II) or 60°C (NOS III) for 2 min and 72°C for 3 min. As a negative control no amplification product occurred if reverse transcriptase or total RNA was omitted in the first-strand cDNA reaction. The PCR products of NOS III and NOS II mRNA had the expected size, as shown by gel electrophoresis, and the specificity of the amplified products was confirmed by restriction enzyme analysis and by hybridization with specific internal oligonucleotide probes. After electrophoresis of the PCR products and staining with ethidium bromide, the bands were photographed under ultraviolet transillumination. Densitometric analysis was performed with photographic negatives (17). To correct for differences in size of target and competitor PCR products, the band densities of the respective competitor PCR products were multiplied by the specific factor (NOS II: 1.33; NOS III: 1.16). The ratio of competitor to target products was plotted against the number of competitor molecules on a log scale. At the competition equivalence point (log ratio = 0) the original number of target mRNAs corresponds to the initial number of competitor RNA molecules used (Fig. 1, a and b) . In control experiments, the optimal amount of total RNA and PCR cycle profile was determined. Sense and antisense primer oligonucleotides were selected from the human cDNA sequences of NOS II (18) (sense: 1614–1633, 5'-GGGAGCATCACCCCCGTGTT-3', antisense primer: 2012–2033; 5'-GAGCGATTTCTTCAGTTTCTCT-3' and NOS III (19) (sense: 827–847, 5'-CCCAGCCAACGTGGAGATCAC-3', antisense primer: 1694–1715, 5'-GGACACCACGTCATACTCATCC-3').
NOS activity.
Samples (200 mg) were homogenized in ice-cold buffer containing 320 mM sucrose; 10 mM HEPES, 1 mM DL-dithiothreitol, 10 µg/ml leupeptin, 100 µg/ml PMSF, 10 µg/ml soybean trypsin inhibitor and 2 µg/ml aprotinin. The homogenate was centrifuged at 100 000 g (30 min at 4°C), the soluble fraction used for the measurement of NOS activity. Ice-cold tissue extract (40 µl) was added to 100 µl of buffer, consisting of 60 mM potassium phosphate, pH 7.2, 72 mM L-valine, 1.2 mM NADPH, 12 µM tetrahydrobiopterin, 12 pg/ml calmodulin, 30 µM FAD, 30 µM FMN, 1.2 mM L-citrulline, 20 µM L-arginine and L-[3H]arginine (Amersham, Dreieich, Germany, 0.15 µCi), 1.2 mM MgCl2 and 1.25µM CaCl2. Duplicate incubations for 30 min at 37°C were performed for each sample in the presence or absence of either EGTA (1 mM) or EGTA plus N-mono-methyl-L-arginine (L-NMMA) (1 mM) to determine the level of the Ca2+-dependent (constitutive = NOS III) and Ca2+-independent (inducible = NOS II) NOS activities, respectively. The reaction was terminated by removal of substrate and by addition of 1:1 (v/v) H2O/Dowex AF 50W 1 x 8 mesh 200 to 400, 8% cross linked (Sigma, Deisenhofen, Germany) + 10 mM EGTA, followed by centrifugation at 16,000 g; supernatant was removed and examined for the presence of [3H]-citrulline by liquid-scintillation counting, as described recently (20).
Radioligand binding studies.
Crude membrane fractions from tissues were prepared by standard homogenization and centrifugation procedures (21). Protein content was determined by the method of Bradford (22) using bovine immunoglobulin G as a standard. Radioligand binding experiments were performed with (–) –[125I]-iodocyanopindolol (ICYP, spec. activity 2, 200 Ci/mmole, New England Nuclear, Dreieich, Germany) at 37°C for 90 min in 10 mM Tris-HCL, 154 mM NaCl buffer pH 7.4 containing 0.5 mM ascorbic acid (21); nonspecific binding was defined by 1 µM (±) – CGP 12177. The relative amount of ß1- and ß2-adrenoceptors in the cardiac membranes was determined from competition curves of the highly selective ß1-adrenoceptor antagonist CGP 20712 A (23) with ICYP (100 pM) binding, as recently described (22). CGP 20712 A competition curves were analyzed by the iterative curve fitting program InPlot (GraphPad Software, San Diego, California). Statistical analysis was performed using the F-ratio test to measure the goodness of fit of the competition curves for either one of the two sites.
Muscle strip preparation.
Left ventricular thin trabeculae of all 24 failing hearts were dissected from the endocardial surface of the heart, as recently described (24), and transferred to the muscle chamber and connected to the force gauge by using fine steel hooks. The muscle was submerged in normal oxygenated Krebs–Ringer solution to wash out the protective solution (37°C). The solution contained (mmol/l) Na+ 152; K+ 3.6; Cl– 135; HCO3– 25; Mg2+ 0.6; H2PO4– 1.3; SO42– 0.6; Ca2+ 2.5; Hepes 5; glucose 11.2; insulin 10 IU/L. Isometric twitches were evoked at 1-s intervals with stimulation voltage 20% above threshold voltage (duration 5 ms). After an equilibration period of 30 to 60 min, muscles were stretched gradually (0.05 to 0.1 mm steps) to the length at which maximum steady-state twitch force was reached (1max). Isometric contractions in muscle strips of each failing heart were recorded before and after isoproterenol (10–7 mol/L). Thereafter, experimental protocols were performed as described below. Cross-sectional area for normalization of force values was calculated as the ratio of blotted weight to muscle length (average cross-sectional area of muscle strips: 0.57 ± 0.02 mm). Isometric contractions were recorded at 1max, stimulation frequency 60 min–1, 37°C. Isometric force, rates of force rise and fall and timing parameters were measured from the recordings. Active force values are given throughout this report (amplitude of the force signal between diastolic force at the end of the stimulus interval and peak systolic force).
Protocol I. Influence of NOS II activity and exogenous NO on isometric contractions.
Isometric contractions were recorded before and 10 min after sodium nitroprusside (SNP; 10–5, 10–4 and 10–3 mol/L) or S-nitroso-N-acetyl-penicillamine (SNAP; 10–6, 10–5, 10–4 mol/L). After washout of SNP (SNAP), isoproterenol was applied (10–7 mol/L), SNP or SNAP was applied again in the presence of isoproterenol. Experiments were performed in 11 muscle strip preparations from 8 patients (SNAP n = 5/4). Control experiments were performed in muscle preparations from the same hearts without SNP or SNAP (n = 10) and did not reveal changes in systolic or diastolic force.
Protocol II. Influence of L-NMMA on isometric contraction.
Isometric force was recorded before and 90 min after L-NMMA (10–4 mol/L). Experiments were performed in eight muscle strip preparations from eight patients. Control experiments were performed in paired muscles strip preparations from the same hearts using Krebs–Ringer solution but without L-NMMA (n = 8), which did not significantly alter systolic or diastolic function.
Statistical analysis.
All data are presented as mean ± SEM. Statistical differences of two groups were calculated using the unpaired Student’s t-test while three or more groups were analyzed by analysis of variance (ANOVA). Multiple measurements at different concentrations or before and after isoprenaline/L-NMMA were analyzed by ANOVA and repeated measures followed by Student–Newman–Keuls test. Correlations were examined by linear regression analysis. Significance was accepted at the level of p < 0.05.
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Results
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Expression and activity of NOS III and NOS II.
The gene expression of NOS III was significantly reduced in failing hearts as compared to nonfailing hearts (Fig. 1, c). Inducible nitric oxide synthase gene expression was absent or low in nonfailing hearts but detectable in all failing hearts (Fig. 1, c). There were no significant differences in NOS III or NOS II gene expression between dilated (n = 11) and ischemic cardiomyopathy (n = 13) (NOS III: 5.8 ± 1.2 vs. 5.7 ± 0.8 transcripts 106/µRNA; NOS II: 4.8 ± 0.4 vs. 6.1 ± 1.1 transcripts 104/µ RNA). Similarly, there were no significant differences in total or NOS II activity between dilated and ischemic cardiomyopathy (Table 1). Inducible nitric oxide synthase gene expression was related to NOS II activity (n = 24; r = 0.54, p < 0.01) in the failing heart.
NOS II activity and response to isoproterenol.
The isoproterenol-induced increase in force of contraction and in +dT/dt varied widely in failing hearts (force: +5.6% to 294%; +dT/dt: +13% to 496%) and both were inversely related to cardiac NOS II activity (force: Fig. 2) (+dT/dt: r = –0.48, p < 0.02) but not to total NOS activity. When patients were divided into two groups according to their NOS II activity values above or below the median (n = 12 each), early relaxation (RT 50) and time to peak tension at baseline were reduced in patients with high NOS II activity (Table 2). Force of contraction (Fig. 3) and +dt/dt (Table 2) were not different for patients with low vs. high NOS II activities. However, the isoproterenol-induced increase in force of contraction (Fig. 3) and +dT/dt (Table 2) was reduced in patients with NOS II activities above the median.
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Figure 2 Relationship between LV NOS II-(iNOS) activity and the percent increase in twitch-tension (force of contraction) induced by isoproterenol (10–7 M).
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Figure 3 Left ventricular tissues of patients were divided into two groups (n = 12 each) according to the median values of their NOS II-(iNOS) activity (individual data are depicted in Figure 2). (Left panel) Mean values (± SEM) for NOS II-(iNOS) activities of these two groups (statistically significant by definition). (Middle panel) Force of contraction at baseline (B) and following isoproterenol (ISO) for both groups, both absolute values and percent increase with isoproterenol are depicted. (Right panel) ß-Adrenoceptor density (Bmax) of both groups. White bars = patients with iNOS-activity > 3,120; solid bars = patients with iNOS-activity < 3,120.
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Effect of L-NMMA on isoproterenol-induced force of contraction.
N-mono-methyl-L arginine did not affect contractile force at baseline, but enhanced the isoproterenol-induced increases in force of contraction. The latter effect varied between +1% and 48% and was positively related to cardiac NOS II activity (Fig. 4).
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Figure 4 Relationship between NOS II-(iNOS) activity and the improvement of isoproterenol-induced twitch-tension (force of contraction) by L-NMMA (%). L-NMMA enhanced the isoproterenol-induced force of contraction in patients with high baseline NOS II-(iNOS) activity.
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Effect of exogenous NO on force of contraction.
There was a modest decrease in force of contraction at baseline with relatively high doses of SNP (Table 3), an effect that was not observed with moderate doses of SNAP (Fig. 5). The isoproterenol-induced increase of force of contraction was attenuated with both SNP (Table 3) and SNAP (Fig. 5). Indices of relaxation were not significantly altered by both NO-donors (for SNP see Table 3).
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Figure 5 Dose-dependent effect of the NO donor SNAP on twitch-tension (force of contraction) before and after administration of isoproterenol (10–7 M) in five muscle strip preparations from four patients. Data (mean ± SEM) are depicted in percent twitch-tension as compared to control values.
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ß-receptor density.
The ß1-adrenergic receptor density is decreased in failing hearts which, in part, could explain the attenuated effect of the ß-adrenergic stimulating agent isoproterenol in failing hearts. However, while the isoproterenol-induced positive-inotropic effect varied widely in failing hearts, no significant relationship to ß-adrenergic receptor density was observed. Patients with a blunted response to isoproterenol and high cardiac NOS II activities had similar ß1- and ß2-adrenergic receptor densities as compared to those with marked positive inotropic response to isoproterenol and low NOS II activities (ß1: 9.2 ± 1.4 vs. 10.5 ± 1.8; ß2: 4.1 ± 0.4 vs. 3.3 ± 0.5 fmol ICYP/mg protein). The KD was not statistically different in these groups (14.8 ± 3.9 vs. 8.0 ± 1.2 pM).
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Discussion
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We found that heart failure is associated with expression of the NOS II in the LV myocardium and that NO generated by this enzyme modulates cardiac relaxation and contraction in the failing heart. High cardiac NOS II activity in failing left ventricles was associated with shortening of relaxation and an attenuated positive inotropic response to ß-adrenergic stimulation. Vice versa, inhibition of cardiac NOS II activity by L-NMMA improved the ß-agonist-induced positive inotropic effect, suggesting that NO generated within the failing heart by NOS II attenuates the ß-agonist-induced positive inotropic action. This blunted effect of isoproterenol cannot be explained by a downregulation of ß1-adrenergic receptors since the ß1-adrenergic receptor density was similar in patients with high and low NOS II activity and the effect of the ß-adrenergic stimulation was modulated by the inhibition of synthesis of NO with L-NMMA.
The expression of NOS II in failing hearts did not differ between patients with dilated and ischemic cardiomyopathy, which is consistent with a recent report (12). However, de Belder et al. (25) reported appreciable NOS II activities only in patients with dilated or peripartum cardiomyopathy, that is, conditions with presumed cytokine activation or inflammation. The lack of NOS II activity in their patients with coronary artery disease may be related to the relatively stable clinical condition of these patients and determination in right ventricular biopsy specimens. We and Haywood et al. (12) examined LV tissue from patients with end-stage heart failure obtained during cardiac transplantation, thus, at a more advanced stage of the disease, when systemic cytokine activation is more likely to occur.
Demonstration of NOS II mRNA and activity in tissue homogenates cannot identify the cell type within the myocardium expressing NOS II. However, the expression of NOS II in cardiac myocytes of failing human hearts has recently been demonstrated by immunohistochemistry (12,26), which is consistent with experimental observations (27). Notably, the extent and cellular distribution of NOS II expression within the myocardium may differ between ischemic and dilated cardiomyopathy (26). In contrast to the NOS II, whose expression in failing hearts is most likely related to cytokines such as tumor necrosis factor, interferon gamma, interleukin-1 and -6, the gene expression of NOS III was reduced in failing hearts, both in dilated and ischemic cardiomyopathy, which is consistent with recent observations that NO production in coronary microvessels from failing hearts is reduced (28). Conceivably, the reduced expression of NOS III in failing hearts, both in patients with dilated and ischemic cardiomyopathy, may explain, in part, the presence of endothelial dysfunction in the coronary microvasculature observed in these disorders (29,30). Since NO is involved in coronary blood flow regulation, the impaired expression of NOS III may have functional implications for the coronary blood flow regulation in these patients with severe chronic heart failure.
Comparison with previous studies.
Our finding that endogenous generation of NO within the myocardium modulates cardiac relaxation and attenuates the ß-agonist-induced, positive inotropic action is consistent with previous observations. There is evidence that exogenous NO and stimulation of endothelial release of NO elicit a myocardial relaxant effect (13,31), probably by reducing the myofilament response to calcium (32). There is experimental evidence that interleukin-1 and tumor necrosis factor inhibit myocyte ß-adrenergic responsiveness (7). In addition, both basal and cytokine-induced NO can attenuate the myocardial inotropic response to ß-adrenergic stimulation (6). Moreover, NO has been shown to inhibit the positive inotropic response to ß-adrenergic stimulation in humans with LV dysfunction in vivo (14) and myocardial NOS II gene expression in the human allograft influences the LV contractile response to ß-adrenergic stimulation (33). The present observations are consistent with these reports. Our results suggest that the response to ß-adrenergic stimulation is closely related to the endogenous cardiac generation of NO by NOS II. Thus, cardiac NOS II-activity in patients with severe heart failure appears to blunt the responsiveness to ß-adrenergic stimulation. The negative inotropic effect of NO during ß-adrenergic stimulation has been related to cGMP-mediated inhibition of Ca2+ influx via L-type sarcolemmal voltage-dependent Ca2+ channels (34). Thus, besides reduced ß1-receptor density and increased levels of Gi-proteins (for review see reference 35), cardiac generation of NO appears to contribute to the attenuated response elicited by ß-adrenergic stimulation in the failing heart, that is, by acting via a cGMP-dependent mechanism. It should be noted that NO can act via a number of additional mechanisms, such as programmed cell death (36), cytotoxic effects (37) or by modulation of myocyte oxygen consumption (38), all of which may result in impaired cardiac function and prognosis in patients with chronic heart failure.
Study limitations.
A limitation of this study is that insufficient LV tissue from normal donors was available for the assessment of NOS II activity and force of contraction. However, the wide range of NOS II expression and activity in failing hearts provided a solid basis for the evaluation of the functional consequences of NOS II activity in failing hearts. Similarly, ß-adrenoceptor number and subtype distribution were measured only in failing hearts. However, the values compare favorably with recent data from our laboratory (39) and we were markedly reduced as compared to normal values (72.6 + 9.4 fmol/mg protein). Since the ß-receptor density did not differ in both groups, the impaired response in group II cannot be explained by the reduced ß-receptor density. We applied the citrulline assay to NOS activity as established by Moncada’s group (11). It should be noted that this approach to differentiate constitutive and NOS II activity has its limitations. However, NOS II rather than total cardiac NOS activity was related to changes in force of contraction. This observation suggests that the emergence of NOS II expression and activity in the left ventricle has functional implications. While both SNP and SNAP affected the positive inotropic effects of ß-adrenergic stimulation, only SNP (in relatively high doses) modestly reduced basal contraction, an effect which may be related to an inhibition of the cGMP-inhibited cAMP phosphodiesterase (40). Alternatively, this depressor effect of SNP is related to an unspecific effect of the high dose used since SNAP (in moderate doses) failed to affect basal contraction.
Conclusion.
Taken together, our results indicate that NO modulates LV function in patients with chronic heart failure and, therefore, the cardiac synthesis of NO by the NOS II and/or the underlying cytokine activation may represent a target for intervention in these patients.
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Footnotes
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Supported in part by the Deutsche Forschungsgemeinschaft (Dr 148/6-3 and 148/7-2) and Landesministerium für Wissenschaft und Forschung, Stuttgart, Germany.
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Top
Abstract
Methods
5%). The PCR products, indicated as angiotensin-converting enzyme target (643 bp) and NOS III competitor (518 bp), were separated by gel electrophoresis, stained with ethidium bromide and visualized by UV irridiation. On the right-hand side, a molecular weight DNA standard (M) was loaded. (Bottom) The band densities of the NOS III target and competitor DNA were evaluated using a laserdensitometer and a computer-based imaging system. The mean values of duplicate samples were plotted as logarithm of the ratio of competitor to gene target PCR products vs. the logarithm of the known number of competitor molecules. At the competition equivalence point (log ratio = 0) the original number of target mRNAs corresponds to the initial number of competitor RNA molecules used. (B) Restriction analysis of PCR NOS II products, showing specific NOS II fragments. (C) Quantification of NOS III (eNOS) and NOS II (iNOS) gene expression by competitive RT-PCR in nonfailing (NF; n = 5) and failing (CHF; n = 24) LV tissues. The number of transcripts (representing mRNA molecules per microgram total RNA) is depicted.