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J Am Coll Cardiol, 1998; 32:1179-1186 © 1998 by the American College of Cardiology Foundation |
* Institüt für Experimentelle und Klinische Pharmakologie ünd Toxikologie, Abteilung Pharmakologie, Universitäts-Krankenhaus Eppendorf, Universität Hamburg, Hamburg, Germany
Pharmakologisches Institut, Universität Johannes-Gutenberg, Mainz, Germany
Manuscript received September 23, 1997; revised manuscript received June 15, 1998, accepted June 22, 1998.
Address for correspondence: Dr. Birgitt Stein, Institüt für Experimentelle und Klinische Pharmakologie ünd Toxikologie, Abteilung Allgemeine Pharmakologie, Universitäts-Krankenhaus Eppendorf, Universität Hamburg, Martinistraße 52, 20246 Hamburg, Germany
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Background. In heart failure, plasma levels of nitrates were found to be elevated. However, data on myocardial NOS expression in heart failure are conflicting.
Methods. Using RNase protection analysis and Western blotting, the expression of NOS III and NOS II was investigated in ventricular myocardium from nonfailing (NF) hearts (n = 5) and from failing hearts of patients with idiopathic dilated cardiomyopathy (dCMP, n = 14), ischemic cardiomyopathy (iCMP, n = 9) or postmyocarditis cardiomyopathy (mCMP, n = 7). Furthermore, immunohistochemical studies were performed to localize NOS III and NOS II within the ventricular myocardium.
Results. In failing human hearts, NOS III mRNA levels were increased to 180% in dCMP, 200% in iCMP and to 210% in mCMP as compared to NF hearts. Similarly, in Western blots (using constitutively expressed beta-tubulin as a reference) NOS III protein expression was increased about twofold in failing compared to NF hearts. Immunohistochemical studies with a selective antibody to NOS III showed no obvious differences in the staining of the endothelium of cardiac blood vessels from NF and failing human hearts. However, NOS III-immunoreactivity in cardiomyocytes was significantly more intense in failing compared to NF hearts. Low expression of NOS II mRNA was detected in only 2 of 30 failing human hearts and was not found in NF hearts. Inducible-type NOS protein was undetectable in either group.
Conclusions. We conclude that the increased NOS III expression in the ventricular myocardium of failing human hearts may contribute to the contractile dysfunction observed in heart failure and/or may play a role in morphologic alterations such as hypertrophy and apoptosis of cardiomyocytes.
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There is evidence that NO reduces myocardial contractility by different mechanisms (2–14, reviewed in 15). This has been shown for high concentrations of NO, as produced by the inducible isoform of NOS (NOS II). Interestingly, also physiologic concentrations of NO, as produced by Ca2+-dependent isoforms of NOS, seem to be sufficient for this effect (15). Nevertheless, the physiologic as well as the pathophysiologic role of L-arginine-derived NO in regulating myocardial contractility remain controversial (16–20, reviewed in 21).
One uncertainty refers to heart failure. Congestive heart failure is characterized by contractile dysfunctions, for example, diminished contractile response to beta-adrenergic agonists (22), attenuated force-frequency-relationship (23) and various morphologic as well as cellular abnormalities, for example, hypertrophy (24), apoptosis (25) and changes in calcium handling (26). Some of these defects have been proposed to be associated with a stimulation of the L-arginine-NO system (11,27). However, there is controversy as to whether NOS II is expressed in the myocardium of failing human hearts. De Belder et al. (28,29) reported increased enzyme activity of a Ca2+-independent NOS in the myocardium of patients with dilated cardiomyopathy, myocarditis and postpartum cardiomyopathy, but not in ischemic or valvular heart disease. Others demonstrated NOS II mRNA and immunoreactivity in hearts from patients with dilated cardiomyopathy (30–32), but also in ischemic and valvular heart disease (30). In previous studies we have found NOS II immunoreactive protein only in septic hearts, but not in other forms of cardiomyopathy (33). In the current study we confirm the lack of significant expression of NOS II mRNA and protein in human hearts with different forms of cardiomyopathy. Since there are no reports concerning the expression of NOS III in ventricular myocardium from patients with heart failure, we investigated additionally NOS III gene expression. Nitric oxide synthase protein has been demonstrated in atrial tissue from nonfailing (NF) human hearts (34) and rat ventricular cardiomyocytes (9,10). Recent studies demonstrated that in vivo gene transfection of NOS III in cardiomyocytes caused apoptosislike cell death, thereby mimicking the morphologic features of acute myocarditis or ischemic injury (27). Moreover, given the modulating activity of NOS III-derived NO on the inotropic effects of beta-blockers and cholinergic agonists (8–12) as well as the force-frequency-relation (13,14), we hypothesized that an increased expression of the endothelial-type NOS III may occur in human heart failure and contribute to the contractile and/or morphologic abnormalities.
Here we report an increased expression of NOS III mRNA and protein in various forms of human cardiomyopathy, using RNase protection analysis, Western blotting and immunohistochemistry.
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RNA preparation and RNase protection assay.
Total RNA was extracted from left ventricular tissue samples (0.5 to 1.0 g) by acid guanidium thiocyanate-phenol-chloroform extraction, as described previously (35). RNA yields were 0.76 ± 0.08 (NF), 0.68 ± 0.04 (dCMP), 0.68 ± 0.05 (iCMP) and 0.67 ± 0.05 (mCMP) µg/mg tissue. The average OD260/280 was 1.95 (n = 35). All RNA preparations were checked in 1% formaldehyde containing agarose gels. RNase protection assays were performed with RPA II kit (Ambion Inc., Austin, Texas) according to the manufacturer’s protocol with minor modifications. Plasmids (pBluescript) containing human NOS II or NOS III cDNA-fragments were gifts from D.A. Geller (36; Department of Medicine, University of Pittsburgh, Pittsburgh, Pennsylvania) and P.A. Marsden (37; Department of Surgery, University of Toronto, Toronto, Ontario, Canada). Antisense-RNA probes for NOS II, NOS III and Gs
were generated by in vitro transcription of 0.5 µg linearized plasmid DNA (restriction of NOS II with Nae I and NOS III with Sma I) using T3 or T7 RNA polymerase. The reaction was performed in 1x transcription buffer (Ambion Inc.) containing ATP, GTP, CTP (20 nmol/L, each), 50 µCi 32P-UTP (800 Ci/mM; NEN, Dreieich, Germany), RNase inhibitor (40 U; Promega, Madison, Wisconsin) and dithiothreitol (10 mmol/L) in a total volume of 20 µl for for 2 h at 25°C. After transcription, the reaction mix was treated with RNAse-free DNase I (20 U, Boehringer, Mannheim, Germany) for 15 min at 37°C. Subsequently, the antisense-RNA probes were purified by 8M urea gel electrophoresis (Long Ranger Gel, AT Biochem, Malvern, Pennsylvania). The respective bands were cut out with sterile razor blades under autoradiographic control, and RNA-probes were eluted by overnight incubation in elution buffer (Ambion Inc.) at 37°C. 32P-labeled NOS II and NOS III antisense-RNA probes (50,000 disintegrations per minute [dpm], each) and Gs antisense-RNA probe (7,500 dpm) were hybridized (hybridization buffer from Ambion Inc.) with 10 to 30 µg of total RNA at 45°C overnight. The reaction mix was digested with RNase A/T1 (5 µg/ml and 100 U/ml, respectively) for 30 min at 37°C. Incubations of 32P-labeled antisense-probes (50,000 dpm each) with tRNA were treated with RNases and served as negative controls, others (2,000 dpm each) were treated with solution Dx (Ambion Inc.) and served as positive controls. After addition of solution Dx, the samples were incubated for 30 min at –80°C and then centrifuged at 15,000 x g (15 min, 4°C). The pellets were resuspended in formamide containing loading buffer (Ambion Inc.), denatured for 5 min at 95°C and loaded onto a 5% polyacrylamide gel containing 8M urea. Autoradiography of dried gels was performed at –20°C for 5 to 10 days.
Densitometric analyses of RNase protection assays were performed using a Phosphoimager (ZERO-Dscan, Scanalytics, Billerica, Massachusetts). Resulting densities and pixel values were analyzed by ZERO-Dscan software. Five-point calibration curves were constructed with specific sense-orientated RNAs (0.5 to 20 pg) for NOS II and NOS III that were supplemented with 20 µg tRNA (Fig. 1). Sense-RNAs were produced by in vitro transcription in the presence of trace amounts (1:10,000) of 32P-UTP to monitor the incorporation efficiency. Sizes of NOS II and NOS III sense-RNA were 2,121 nt for NOS II (2,060 nt cDNA plus 61 nt multiple cloning site [MCS]) and 2,282 nt for NOS III (2,180 nt cDNA plus 102 nt MCS), respectively. The reaction usually yielded between 5 and 10 µg RNA that were purified by gel filtration on Sephadex G-50, diluted to 100 pg/ml in TE/0.5% SDS and stored at –80°C.
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mRNA which has been demonstrated to be unchanged in human heart failure (38). To obtain comparable counts we used labeled antisense probes of 7,500 dpm for Gs (specific activity 
1 x 108 dpm/µg) compared to 50,000 dpm for NOS II or NOS III (specific activity 2 x 109 dpm/µg). The mean of all Gs pixel values on one gel was divided by the individual Gs pixel value, yielding a factor that expressed deviations from the average and thereby loading differences. This factor was multiplied with the respective NOS-mRNA value. Factors were 1.10 ± 0.12 (NF, n = 5), 1.19 ± 0.08 (dCMP, n = 14), 1.14 ± 0.11 (iCMP, n = 9) and 1.16 ± 0.16 (mCMP, n = 7). Correction by this procedure did not alter the magnitude or the direction of the results, but lowered the standard deviation. Protein extraction and Western blotting. Tissue samples from human heart transplants were homogenized in three volumes of an ice-cold Tris-buffer (50 mmol/L Tris-HCl pH 7.5, 0.5 mmol/L EGTA, 0.5 mmol/L EDTA, 2 mmol/L dithiothreitol, 7 mmol/L glutathione, 10% [v/v] glycerol, 10 µg/ml pepstatin A, 20 U/l aprotinin, 10 µg/ml leupeptin and 0.2 mmol/L PMSF) using an Ultra-Turrax (Kinematica A6, Littau-Luzern, Suisse). For solubilization of membrane-bound proteins, homogenates were treated with the detergent 3-[(3-cholamidopropyl)dimethylammonio]-1-propane-sulfonate (CHAPS) at a final concentration of 20 mmol/L (rotating shaker, 20 min at 4°). The CHAPS extracts were centrifuged at 160,000 x g. Protein contents were measured in the resulting supernatants with the micro-adapted Bradford (39) assay. Proteins were separated by denaturing discontinuous polyacrylamide gel electrophoresis using 7.5% resolving gels and blotted onto nitrocellulose membranes (Schleicher & Schuell, Dassel, Germany) in a semi-dry electrophoretic transfer cell (Trans-Blot, Bio-Rad, Munich, Germany). Blots were blocked for 60 min with 3% (w/v) bovine serum albumin and 0.05% (w/v) Tween 20 in TBS (10 mmol/L Tris/HCl, pH 7.4, in 0.9%, w/v, NaCl). They were then incubated for 90 min at room temperature with the primary antibody in TBS containing 0.5% (w/v) gelatin and 0.05% (w/v) Tween 20. The following primary antibodies were used: a monoclonal anti-NOS II antibody (1:1,500) and a polyclonal anti-NOS III antibody (1:500), purchased from Transduction Laboratories (Lexington, Kentucky), and a monoclonal anti-beta-tubulin antibody (1:500, Sigma Bio Sciences, Deisenhofen, Germany). Blots were washed in TBS/gelatin/Tween and immunoreactive proteins were visualized with NBT/X-phosphate (4-nitroblue tetrazolium chloride/5-bromo-4-chloro-3-indolyl-phosphate) after a 60-min incubation with the secondary antibody conjugated to alkaline phosphatase.
NOS III immunohistochemistry. Ten-micrometer sections were prepared using a cryostat. Sections were thaw-mounted onto poly-L-lysine-coated glass slides (Sigma), air-dried and slightly fixed in ice-cold acetone for 5 to 10 s. First, sections were washed in PBS for 10 min, and endogenous peroxidase was blocked by incubation for 30 min in a freshly prepared solution of 40% (v/v) methanol and 0.6% (v/v) H2O2 in PBS. Blocking of endogenous biotin or biotin-binding proteins was performed with a commercially available avidin/biotin-kit (Camon, Wiesbaden, Germany). Thereafter, sections were washed for 10 min in incubation medium (4% dry-milk powder in PBS) and incubated with a 10% solution of normal serum (Camon) from the species in which the secondary antibody was generated. Incubation with the primary polyclonal anti-NOS III antibody (Transduction Laboratories, 1:50) was performed overnight at 4°C; sections were washed in incubation medium and exposed to the secondary biotinylated antibody (Camon, 1:100) for 60 min at room temperature. This was followed by at least two washes in PBS and incubation with a 1:100 dilution of the streptavidin-biotinylated horseradish peroxidase complex (Amersham, Buchler, Germany) in PBS for 20 min. After repeated washing with PBS, the chromogen-reaction was performed by the use of 3.3'-diaminobenzidine (Sigma) according to the instructions of the manufacturer. The sections were washed in distilled water, counterstained with Mayer’s acid hemalaum, mounted in glycerol gelatin and coverslipped.
Statistical analysis. All values presented are means ± SEM. For statistical significance analysis of variance and Student t test were used except for NOS III mRNA expression. Secondary to its nonparametric distribution we used Kruskal–Wallis’ rank test and Mann–Whitney U-test to compare NOS III expression between NF and failing hearts. Bonferoni’s test values < 0.05 were regarded as significant.
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(403 nt) RNAs (Fig. 2). Hybridization of these probes with the respective sense-RNAs gave protected fragments of 587 nt for NOS II (570 + 17 nt MCS) and 462 nt for NOS III (433 + 29 nt MCS; Fig. 2, lane 6). Hybridization with total RNA from ventricular tissue of NF or failing human hearts resulted in protected fragments of 570 nt for NOS II, 433 nt for NOS III and two fragments of 353 nt and 338 nt for Gs (Fig. 2, lanes 8 to 22). As both antisense-probes and sense-RNAs contained portions of the multiple cloning site, they differed in their size from the respective protected fragments of total RNA.
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180% (0.77 ± 0.09, n = 14; dCMP), 200% (0.83 ± 0.19, n = 9; iCMP) and 210% (0.88 ± 0.04, n = 7; mCMP; Fig. 3A). Nitric oxide synthase-mRNA levels tended to be increased also in iCMP, but this effect did not reach significance. This is due to the unequal distribution (nonparametric distribution) of the individual values as demonstrated in Fig. 3B. Inducible-type NO-mRNA expression was detected only in 7% (two out of 30, one dCMP and one mCMP) of failing human hearts and was not detected in NF hearts. On the other hand, in stimulated human hepatocytes (300 ng/ml LPS and 3 U/ml IFN-
, 18 h) the NOS II antisense-probe hybridized to a single band of the expected size for the NOS II, indicating that NOS II-mRNA expression was detectable with the methods (Fig. 2, lane 23).
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NOS II expression. Alterations in NOS II expression have been reported in human heart failure (30–32). In the present study, only 2 of 30 failing human hearts showed low expression of NOS II mRNA as detected by quantitative RNase protection analysis. In NF hearts NOS II mRNA was totally absent. Similarly, in Western blots, NOS II immunoreactivity was detected neither in failing nor in NF hearts. This confirms and extends our recent findings on different types of heart failure demonstrating significant NOS II protein expression exclusively in failing hearts from patients with sepsis (33). In contrast, others found expression of NOS II in other types of human heart failure (30–32). De Belder et al. (28,29) reported significant Ca2+-independent NOS activity in endomyocardial biopsies from patients with dCMP, mCMP and postpartum cardiomyopathy (suggesting the presence of NOS II), but not in patients with ischemic or valvular heart disease. However, measurement of the Ca2+-dependence of NOS enzymatic activity does not allow an unequivocal isoform identification. Previous immunohistochemical studies have detected strong immunoreactivity for NOS II in myocytes from dCMP hearts, but only moderately intense staining in myocytes from patients with iCMP and no signal in hypertrophic or NF myocardium (31,32). Haywood et al. (30), performing reverse transcription-polymerase chain reaction (RT-PCR), described that NOS II occurred in 67% of patients with dCMP, 59% of patients with iCMP and in 100% of patients with valvular heart disease. Similarly, Satoh et al. (32) observed NOS II mRNA in 54% of dCMP patients, but not in patients with hypertrophic cardiomyopathy also using RT-PCR. These data indicate that NOS II is not expressed in all patients with the same etiology and in addition varies between different types of heart failure. Thus, the apparent differences between the present findings and previous findings of others can be explained by the different methods used. Reverse transcription-polymerase chain reaction is a sensitive method to detect trace amounts of mRNA (1 mole), whereas RNase protection analysis allows the measurement of biologically relevant concentrations of mRNA (in the range of 0.1 pg). Hence, trace amounts of NOS II mRNA present in some failing hearts and detected by RT-PCR may have escaped our RNase protection assays. Furthermore, it is conceivable that NOS II expression is different in right and left ventricular tissue. In the present study we used mainly (26 of 30 failing hearts) left ventricular tissue, whereas others (28–32) investigated NOS II expression in right ventricular tissue.
NOS III expression.
We now demonstrate NOS III mRNA and protein both in failing and NF myocardium. We found NOS III mRNA levels increased in failing hearts: dCMP 180%, iCMP 200% and mCMP 210%, compared with NF hearts (100%). Accordingly, on Western blots increased expression of NOS III protein was detected in failing hearts as compared to NF hearts. One may hypothesize that such an increase results from an enhanced expression in endothelial cells. However, the immunohistochemical results argue against this interpretation. We found similar NOS III staining intensities in the endothelium of blood vessels from failing and NF hearts. In contrast, NOS III immunoreactivity in normal myocardium was clearly intensified in the cardiomyocytes from failing hearts. Thus, the recently reported elevated plasma nitrate levels in patients with heart failure (44) may be explained in part by the increased NOS III expression in cardiomyocytes. However, the finding of elevated plasma levels is not undisputed, since others demonstrated decreased NO production in coronary microvessels from failing human hearts (45). Furthermore, in studying a dog model for heart failure, Zaho et al. (46) reported reduced expression of NOS III mRNA and protein in the aorta of these dogs.
Functional role of NOS III upregulation. Nitric oxide production by NOS III within the cardiomyocyte could exert an effect on myocardial contractility. Recent reports demonstrated that NO formed by NOS III reduced inotropic responsiveness to beta-blockers (8,9,11) and force-frequency relation (13,14) in the heart, probably by acting via the cGMP signal cascade to modulate L-type calcium channels (9,47–49). Moreover, it has been shown that the beta-adrenergic hyporesponsiveness mediated by NO is augmented in an animal model of heart failure (50). Thus, it is conceivable that an increased NO-formation due to elevated NOS III expression in the failing myocardium is involved in the diminished responsiveness to beta-blockers and/or the attenuated myocardial force-frequency relation in human heart failure. However, considerable efforts by others failed to support the abovementioned conclusions (17–21). The present finding of an increased NOS III expression in cardiomyocytes from failing hearts may be of pathophysiologic relevance. Recently, it has been demonstrated that in vivo gene transfection of NOS III into cardiomyocytes caused apoptosislike cell death and that these morphologic features mimicked acute myocarditis or ischemic injury (27). Therefore, the increased NOS III expression in failing hearts may contribute to the morphologic alterations seen under these conditions.
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1 During publication process the following related paper has been published by Vejlstrup NG, Bouloumie A, Boesgaard S, Andersen CB, Nielsen-Kudsk JE, Mortensen SA, Kent JD, Harrison DG, Busse R, Alsershvile J. Inducible nitric oxide synthase (iNOS) in the human heart: Expression and localization in congestive heart failure. J Mol Cell Cardiol 1998;30:1215–23. 
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. Br J Pharmacol. 1995;114:27–34[Medline]
-subunit Gi2 in human end-stage heart failure. Circ Res. 1992;70:688–696This article has been cited by other articles:
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