Expression of Inducible Nitric Oxide Synthase in Human Heart Failure
Background There is increasing evidence that alterations in nitric oxide synthesis are of pathophysiological importance in heart failure. A number of studies have shown altered nitric oxide production by the endothelial constitutive isoform of nitric oxide synthase (NOS), but there is very little information on the role of the inducible isoform.
Methods and Results We analyzed inducible NOS (iNOS) expression in ventricular myocardium taken from 11 control subjects (who had died suddenly from noncardiac causes), from 10 donor hearts before implantation, and from 51 patients with heart failure (24 with dilated cardiomyopathy [DCM], 17 with ischemic heart disease [IHD], and 10 with valvular heart disease [VHD]). Reverse transcription–polymerase chain reaction was used to confirm the presence of intact mRNA and to detect expression of iNOS and atrial natriuretic peptide (ANP). ANP was used as a molecular phenotypic marker of ventricular failure. iNOS was expressed in 36 of 51 biopsies (71%) from patients with heart failure and in none of the control patients (P<.0001). iNOS expression could also be detected in 50% of the donor hearts. All samples that expressed iNOS also expressed ANP. iNOS gene expression occurred in 67% of patients with DCM, 59% of patients with IHD, and 100% of patients with VHD. To determine whether iNOS protein was expressed in failing ventricles, immunohistochemistry was performed on three donor hearts and nine failing hearts with iNOS mRNA expression. Staining for iNOS was almost undetectable in the donor myocardium and in control sections, but all failing hearts showed diffuse cytoplasmic staining in cardiac myocytes. Expression of iNOS could be observed in all four chambers. Western blot analysis with the same primary antibody showed a specific positive band for iNOS protein in the heart failure specimens; minimal iNOS protein expression was seen in donor heart samples.
Conclusions iNOS expression occurs in failing human cardiac myocytes and may be involved in the pathophysiology of DCM, IHD, and VHD.
It is becoming apparent that NO is one of the most important regulatory factors in physiological processes throughout the body.1 It can be generated by three different isoforms of the enzyme NOS: brain (type I), ecNOS (type III), and iNOS (type II). Recent research in heart failure has focused on alterations in NO production by ecNOS2 3 4 ; far less is known about the behavior of the inducible isoform in this condition. iNOS is expressed in a wide range of tissues in response to stimulation by lipopolysaccharide.5 6 In adult rats in vivo, injection of lipopolysaccharide results in expression of mRNA by myocytes cultured from the ventricular myocardium7 and in increased calcium-independent NOS activity in homogenized ventricular myocardium.8 In humans, iNOS has been cloned from cultured hepatocytes stimulated with lipopolysaccharide combined with the cytokines IL-1β, TNF-α, and IFN-γ9 and from cultured chondrocytes stimulated with IL-1β.10 There also is evidence that IL-2 infusions in patients receiving cancer chemotherapy can cause expression of iNOS,11 and the enzyme has been proposed as the mediator of the catecholamine-resistant hypotension characteristic of septic shock.12
Because circulating cytokine13 14 and total nitrate15 levels are elevated in some patients with heart failure, the question has been raised of whether iNOS can be induced in failing myocardium. Initial reports have suggested that iNOS is induced in the ventricular myocardium of patients with dilated cardiomyopathy16 and with “focal healing myocarditis.”17 It has been suggested that inflammatory processes in the myocardium of such patients may be responsible for the induction of iNOS. However, levels of circulating cytokines such as TNF-α are elevated in patients with end-stage heart failure regardless of origin14 and in myocardial infarction.18 Recently, a wider role has been proposed for the pathophysiological effects of cytokines beyond what are usually considered to be inflammatory cardiac diseases.19 Thus, there may be mechanisms that result in the induction of iNOS in the failing ventricle other than local inflammation.
Interest in whether iNOS is expressed in failing myocardium has been heightened by observations that NO can exert negative inotropic20 21 22 and cytolytic23 effects on myocytes. Beyond the effects on intracellular second messengers such as cGMP, NO is known to bind to iron-containing proteins such as those in the respiratory chain and to complex with oxygen free radicals to form the highly toxic peroxynitrite radical.24 25 iNOS generates higher and more sustained levels of NO than ecNOS, and it appears likely that high local concentrations of NO in cardiac myocytes exert important pathophysiological effects.
The aim of the present study was to determine whether there was evidence for iNOS mRNA and protein expression in the ventricular myocardium of patients with heart failure secondary to idiopathic dilated cardiomyopathy, ischemic heart disease, and valvular heart disease and to compare the findings with control tissue.
Right ventricular myocardium from the endocardial surface of the intraventricular septum was obtained at the time of cardiac transplantation or at diagnostic endomyocardial biopsy from patients being treated for heart failure. In addition, samples were taken during cardiopulmonary bypass from the interventricular septum of patients undergoing valve replacement surgery who were being treated for mild-to-moderate heart failure. Control myocardium was obtained from two sources: donor hearts before implantation (donor hearts) and after autopsies performed on victims of sudden death from a noncardiac cause within 6 hours of the time of death (controls).
In a subgroup of patients with end-stage heart failure undergoing cardiac transplantation, myocardial tissue was taken from all four cardiac chambers. The sites sampled were the right and left atrial appendages, the left ventricular free wall, and the right side of the interventricular septum.
Clinical information on the subjects studied is shown in Tables 1⇓ and 2⇓. All living patients who underwent biopsy gave informed consent before the procedure in accordance with the hospitals’ human subjects review boards.
In all cases, the myocardial tissue obtained was immediately frozen in liquid nitrogen and stored at −80°C. Total RNA was isolated from tissue samples according to the method of Chomozynski and Sacchi26 with Trizol reagent (GIBCO-BRL Life Technologies Inc). RNA was extracted with the use of chloroform and precipitated by isopropanol. The RNA was washed in 75% ethanol, dried, and stored under 100% ethanol at −70°C until required. RNA was quantified by spectrophotometry with the A260/280 method. Before use, samples were centrifuged at 4°C and resuspended in RNAse-free water. First-strand cDNA was synthesized from total RNA with monkey Moloney leukemia virus RT (Perkin-Elmer Cetus) and random hexamers. Amplification by PCR was carried out in a total reaction volume of 50 μL. The sequences of the primers and the conditions used are shown in Table 3⇓. Three microliters of cDNA from the 30-μL product of the RT step was added to each reaction mix. Thermal cycling was performed with a PTC 100 thermal cycler (MJ Research). Electrophoresis of the amplified products was performed on 1.5% agarose gel containing Tris acetate/EDTA and ethidium bromide. A HaeIII digest of φ174 DNA (GIBCO-BRL Life Technologies) was used as a molecular size standard. Gels were visualized with UV irradiation and photographed.
Amplification of β-actin was used to demonstrate the presence of intact mRNA in each total RNA sample and to help demonstrate approximate equivalence of mRNA loading in each RT-PCR. Atrial natriuretic peptide gene expression is normally undetectable in healthy ventricular myocardium but is induced under the conditions associated with heart failure27 and was used as a sensitive molecular marker of disturbed gene expression in human ventricular myocardium.
RT-PCR Control Experiments
Absence of genomic contamination in the cDNA samples was confirmed by the use of a PCR reaction with primers that amplify the promoter region of apolipoprotein (a)–related gene C, a nontranscribed region of genomic DNA.28 These primers were designed to sensitively and specifically amplify genomic DNA only, and the technique was validated against serial dilutions of genomic DNA and total RNA with or without RNAse pretreatment before RT-PCR (G.A. Haywood, C.D. Byrne, unpublished data, 1994). This method allows screening of cDNA for genomic contamination without the need to use RT-negative RNA controls, thus saving on the consumption of total RNA. If any genomic contamination were present, the samples were also tested with PCR for human skeletal α-actin to ensure that a false-positive result had not been obtained by amplification of an intronless β-actin pseudogene. Any false-positive results were excluded from the analysis. The ANP, human skeletal α-actin, and iNOS primers each spanned intron sequences29 30 31 to enable identification of amplification of cDNA from any genomic DNA amplification by the size of the product. Selected samples were also pretreated with RNAse before RT-PCR to confirm complete elimination of the product bands. In addition, a number of samples were treated with DNAse 1 before RT and PCR to ensure that normalization to equivalent β-actin band intensity had not been affected by the amplification of any genomic contamination. Amplification of mRNA for β-actin and human skeletal α-actin was confirmed in all control samples.
Western Blot Analysis
Total protein was extracted from ventricular myocardium with the Trizol method according to manufacturer’s instructions (GIBCO-BRL Life Technologies Inc) solubilized with 1% SDS and separated on 8% denaturing SDS polyacrylamide gels (15 to 25 μg/lane). Proteins were then transferred onto a nitrocellulose membrane (Hybond-ECL, Amersham) by wet electroblotting for 4 hours. Blots were blocked for 1 hour at room temperature with 5% nonfat dry milk in TBS-T (20 mmol/L Tris-HCl, 200 mmol/L NaCl, 0.1% Tween-20) before incubation with mouse anti-macrophage NOS monoclonal IgG (Transduction Laboratories). Incubation with the primary antibody was at a dilution of 1:500 for 1 hour at room temperature and, after washing, with the second antibody (horseradish peroxidase–conjugated sheep anti-mouse immunoglobulin antibody [Amersham]) at 1:1500 for 1 hour. Specific proteins (131 kD) were detected by enhanced chemiluminescence (Amersham) according to the manufacturer’s instructions. Prestained protein markers (Sigma) were used for molecular mass determinations. Two micrograms of mouse macrophage cell lysate stimulated with IFN-γ (10 ng/mL) and lipopolysaccharide (1 μg/mL) for 12 hours (Transduction Laboratories) were used as a positive control.
Fresh, unfixed tissue frozen in OCT (Miles Inc) was sectioned with a cryostat. Adjacent sections were placed on duplicate slides. Sections were fixed with acetone at 4°C for 10 minutes, air-dried at room temperature for 20 minutes, and rehydrated in PBS for 15 minutes. They were then treated with 3% H2O2 for 10 minutes and washed again in PBS for 15 minutes. Sections were then blocked with 5% rabbit serum for 15 minutes. Detection of iNOS was performed with the same primary antibody as used for Western blotting in a 1:10 dilution. Adjacent sections were treated with a 1:20 dilution of an anti-desmin mouse monoclonal primary antibody (Sigma) to allow colocalization to cardiac myocytes. The sections were then incubated for 1 hour at room temperature and then washed in PBS for 15 minutes. Control sections for each patient were incubated with mouse ascites (Sigma). Secondary antibody (rabbit anti-mouse, biotinylated; DAKO) was applied as a 1:50 dilution, incubated at room temperature for 30 minutes, and washed for 15 minutes in PBS. Streptavidin-peroxidase conjugate was applied for 15 minutes, followed by AEC chromagen. Counterstaining was done with hematoxylin.
In addition, a subgroup of donor heart and left ventricular failing heart sections were analyzed with a fluorescent double-staining technique. Sections were fixed in acetone for 10 minutes at 4°C, air-dried, and rehydrated with PBS. They were then blocked with 10% horse serum in 0.3% Tween-20 and 0.85% NaCl in 10 mmol/L phosphate buffer at room temperature for 15 minutes. Sections were then incubated with 1:10 mouse anti-macrophage NOS monoclonal IgG (Transduction Laboratories) in a 1:5 dilution of blocking solution in PBS at room temperature for 1 hour. After washes with PBS, the sections were incubated with biotinylated anti-mouse IgG 1:200 (Vector Labs) and rabbit polyclonal anti-desmin 1:20 (Sigma) for 1 hour. After being washed again in PBS, they were then incubated with fluorescein avidin D 1:250 (Vector Laboratories) and goat anti-rabbit Texas red 1:100 (Vector Laboratories) in PBS, pH 8.2. Sections were then washed, dried, and mounted.
Statistical analysis was performed with the statistical software package statview (Abacus) on an Apple Macintosh computer. Age differences between the experimental groups were compared with ANOVA, and posthoc testing was done with Scheffé’s F test. The frequency of positive PCR results from the different groups was compared with the use of multiple-comparison contingency table analysis with the χ2 test.
The age, method of tissue sampling, and severity of heart failure of the patients in the different etiological groups are shown in Tables 1⇑ and 2⇑. The age of the control subjects was not significantly different than that of the other groups (F=6.563 and P=.013; P=.517, P=.972, P=.117, and P=.132 for donors, dilated cardiomyopathy, ischemic heart disease, and valvular heart disease, respectively). However, heart donors were significantly younger than patients with ischemic heart disease (P=.002) or with valvular heart disease (P=.004), whereas not different from those with dilated cardiomyopathy (P=.175).
The frequency of gene expression for iNOS and ANP in the different patient groups analyzed by RT-PCR amplification to 35 cycles is shown in Fig 1⇓. All patients who expressed iNOS also expressed ANP. The presence of intact mRNA was confirmed in all samples. The difference in the frequency of expression of iNOS between the control patients’ biopsies and the explanted hearts was highly significant overall and for each of the etiological subgroups (dilated cardiomyopathy, ischemic heart disease, and valvular heart disease; P<.0001 overall and for each pairwise comparison). A representative electrophoretic gel for patients from the different subgroups is shown in Fig 2⇓.
Amplification of cDNA from the control patients to 40 cycles still failed to result in any detectable iNOS PCR product bands. In contrast cDNA from nonfailing donor hearts showed amplification bands at 35 cycles in 5 of 10 patients.
No iNOS PCR product bands could be detected when RT-PCR was performed on total RNA from unstimulated cultured human monocytes (THP1 cells, American Type Culture Collection). Similarly, iNOS message was undetectable in a human heart cDNA library taken from four normal hearts and with characteristics indicating that the library is a suitable source for the detection of low-abundance mRNAs (Clontech Laboratories).
Frequency of iNOS Expression Compared With NYHA Class
When patients with heart failure from the different etiological groups were pooled, iNOS expression was present in all 11 patients in NYHA class II, in 17 of 24 patients in NYHA class III, and in 8 of 14 patients in NYHA class IV. Thus, iNOS expression appeared to be significantly more common in NYHA class II heart failure patients than in either class III (P<.05) or class IV (P<.02) heart failure patients.
Regional Expression of iNOS
Total RNA was analyzed from each of the four chambers of the hearts of six patients with end-stage heart failure and positive iNOS expression in the right ventricle. PCR product bands specific for iNOS were detectable in all four chambers at varying intensities. In some cases, iNOS mRNA expression appeared to be most prominent in the left ventricle.
Sections were analyzed from the right ventricle of three nonfailing donor hearts and from three patients with dilated cardiomyopathy, three patients with ischemic heart disease, and one patient with valvular heart disease. Myocardial sections were also taken from all four chambers of the heart of one patient from each of the etiological groups. Representative sections with staining for iNOS and for desmin (to allow myocyte localization) are shown in Fig 3⇓. In Fig 3A⇓ through Fig 3D⇓, pinkish-brown staining indicates binding of the primary antibody-secondary antibody/chromagen complex. In Fig E and Fig F, the green fluorescence indicates iNOS, and the red fluorescence indicates desmin. Detection of iNOS protein could be clearly colocalized, with desmin indicating iNOS expression in the cytoplasm of cardiac myocytes. Only minimal positive staining was observed on sections from nonfailing donor hearts, including that from tissue that had been positive for iNOS mRNA expression. To test for nonspecific staining by the secondary antibody/chromagen complex, mouse ascites was substituted for the primary antibody on control sections from patients with failing ventricular myocardium. No significant nonspecific staining was observed with the chromagen complex; background levels of fluorescence were also markedly lower than the levels of fluorescence seen in the sections that stained positive for either iNOS or desmin. No obvious differences were observed in the pattern of staining among the subjects in each etiological group, and there were no clear differences in the pattern or intensity of staining present in myocardium from the different cardiac chambers. All hearts from patients with heart failure that were analyzed showed positive staining.
Western Blot Analysis
Protein was extracted from two of the donor hearts and four of the failing hearts examined by immunohistochemistry. The primary antibody used for immunohistochemical staining specifically recognized the 130-kD iNOS protein in the heart failure samples. A mouse stimulated-macrophage cell lysate was used as a positive control. Stronger bands were seen in failing hearts positive for iNOS mRNA by RT-PCR than in positive donor hearts (Fig 4⇓).
Results of the present study provide evidence that heart failure is associated with induction of iNOS gene expression in the ventricular myocardium and that iNOS protein is present in ventricular myocytes from patients with end-stage heart failure secondary to dilated cardiomyopathy, ischemic heart disease, or valvular heart disease.
Unlike the brain and ec isoforms of NOS, iNOS is not considered to be expressed in healthy tissues.12 The finding that iNOS gene expression was undetectable in control samples was therefore anticipated. It appears, however, that iNOS mRNA expression parallels induction of atrial natriuretic peptide gene expression in ventricular myocardium and, like atrial natriuretic peptide,27 32 is associated with early as well as end-stage heart failure. It has been suggested that inducible iNOS is part of a primitive but evolutionarily conserved inflammatory response.9 The patients in this study did not have systemic sepsis but may have had chronically elevated levels of proinflammatory cytokines. Alternatively, it may be that other abnormal stimuli, such as altered mechanical stresses on myocytes, are capable of inducing expression of iNOS independent of cytokine activation.
Not all ventricular samples from failing hearts analyzed in the present study were positive by RT-PCR. This may reflect that gene expression for iNOS is triggered by factors or conditions that are not universally present in end-stage heart failure. The group with the highest frequency of detectable expression was the patients with valvular heart disease, most of whom had mild-to-moderate (NYHA class II) heart failure. It may be that iNOS mRNA expression is phasic during the progression of heart failure and that analysis of samples taken at a single time point in the course of the disease underestimates the overall frequency of gene expression. Other explanations for the nonuniformity of iNOS gene expression in end-stage heart failure are that there may be negative feedback on gene transcription or that there may be negative regulation at the transcriptional or pretranslational level by cellular factors such as TGF-β.33 34
One of the difficulties in studying diseased human myocardium is the absence of a readily obtainable source of normal control tissue. We overcame this problem by using tissue obtained at autopsy from patients without cardiac disease who had died suddenly from a noncardiac cause. Autopsies were performed close to the time of death, while good-quality mRNA was still present. There must still be a theoretical possibility that iNOS mRNA undergoes more rapid degradation than mRNA for β-actin or human skeletal α-actin and that this could have resulted in the absence of iNOS amplification from these control samples. The absence of iNOS mRNA in the control ventricular myocardium is, however, in accord with the more general observation of a lack of iNOS expression in healthy tissues in mammals, and iNOS expression could not be detected in a normal human heart cDNA library. Insufficient quantities of control subjects’ myocardium were available for protein analysis in addition to RT-PCR, and we therefore used donor heart myocardium for the control tissue in our analysis of protein expression. Nonfailing donor heart tissue is by far the most common source of control myocardium reported in the literature by groups studying human heart failure. It was, however, of interest to note that iNOS mRNA expression was detectable in some donors (50%). This may have been due to the abnormal conditions associated with brain death, ventilation, explanation of the donor heart, and transportation. The particular component of these events that results in induction of the iNOS gene cannot be identified from this study. The expression of iNOS mRNA in donor myocardium did not, however, result in significant levels of protein expression. The explanation for this may be quantitative, with too little iNOS protein synthesized by the donor hearts to be detected by immunohistochemical staining. Alternatively, it may be that iNOS gene expression occurs late in the sequence of abnormal events to which donor hearts are subjected, resulting in insufficient time for significant protein translation to occur. It is also possible that abnormal gene expression can occur under the metabolic conditions that are present during transportation of the donor heart but that these conditions prevent translation of the protein.
One concern in interpreting the results is that the process of cardiothoracic surgery or explantation might be sufficient to account for the induction of iNOS in the explanted failing and nonfailing hearts. It seems unlikely, however, that this accounts for the positive results in the failing hearts because endomyocardial biopsies removed at cardiac catheterization from patients with end-stage heart failure, in which the interval between biopsy and snap-freezing in liquid nitrogen was 10 to 15 seconds, were as positive for iNOS gene expression as the explanted failing hearts.
It could also be argued that the absence of iNOS protein in the donor hearts was a function of the younger age of this group, but this would be contrary to the considerable literature indicating that iNOS is not constitutively expressed. Furthermore, some patients with failing ventricles expressing iNOS were younger than some donors of hearts that were negative for iNOS protein.
The observations arising from this study showed some differences compared with previous reports.16 17 With the arginine-citrulline activity assay, these investigators inferred the relative contributions to total NO synthesis from ecNOS and iNOS by measuring the effect of calcium chelators. They concluded that iNOS was induced in dilated cardiomyopathy and “focal healing myocarditis” but not in ischemic heart disease or aortic regurgitation. The enzymatic activity of the murine equivalent of iNOS, mouse mac-NOS, shows almost complete calcium independence, and the effect on NO and citrulline generation of chelating agents such as EDTA allows effective discrimination between iNOS and ecNOS. However, it has been demonstrated that transfection of embryonic kidney cells with a human iNOS construct resulted in NOS catalytic activity that was lowered by 70% in the presence of calcium-chelating agents. A murine mac-NOS construct transfected in the same cells produced NOS activity that showed classic calcium independence.9 It is therefore possible that the interpretation of whether iNOS or ecNOS was responsible for NO production in the different etiological groups in the studies by De Belder and colleagues16 17 may have been misleading. It seems unlikely that the calcium sensitivity displayed by human iNOS expressed in embryonic kidney cells would differ substantially from human iNOS expression in cardiac myocytes; the human iNOS gene is a single copy gene,31 and there is no evidence to suggest that different iNOS proteins are synthesized in different organs.
We did not attempt to measure or correlate the presence of iNOS with the severity of ventricular dysfunction in the patients with heart failure. NO donors have been shown to reduce isolated guinea pig myocyte contraction amplitude at concentrations of 3×10−5 mol/L22 and to reduce developed tension in human atrial muscle strips at 10−3 mol/L.21 These effects may be mediated via cGMP or by cytotoxic effects on iron-containing enzymes of the respiratory chain.35 The concentration of NO that is present in the cytosol of human ventricular myocytes in failing hearts is unknown and cannot be directly determined, and correlations in patients between total NO synthesis, calcium-independent NO synthesis, cGMP levels, and ventricular function will be subject to large numbers of uncontrolled variables. It is also possible that chronic elevation of intracellular levels of NO results in cell death through cytolysis or apoptosis,36 37 resulting in left ventricular dysfunction through depletion of the total myocyte population. Studies of the functional significance of the induction of iNOS in failing myocardium may require the development of animal models and the chronic use of specific iNOS inhibitors such as aminoguanidine.38
Evidence of iNOS gene expression was present in ventricular myocardium of patients with both mild-to-moderate and severe heart failure regardless of etiology but was virtually undetectable in nondiseased myocardium. Expression of iNOS mRNA appears to be an early event paralleling expression of atrial natriuretic peptide. These observations raise the possibility that autocrine and paracrine actions of iNOS may be of pathophysiological importance in patients with failing ventricles.
Selected Abbreviations and Acronyms
|ecNOS||=||endothelial constitutive isoform of nitric oxide synthase|
|iNOS||=||inducible nitric oxide synthase|
|NOS||=||nitric oxide synthase|
|RT-PCR||=||reverse transcription–polymerase chain reaction|
|SDS||=||sodium dodecyl sulfate|
|TGF||=||transforming growth factor|
We are most grateful to Dr Margaret Billingham for discussion and advice related to the immunohistochemical data, Barbara Sato for expert technical assistance with the fluorescent double-staining technique, and Dr Byron W. Brown, Jr, for statistical advice. We also thank the surgical staff at Stanford, London, and Hanover, who assisted us in the collection of the myocardial samples, and in particular we thank Hermann Reichenspurner and John Stevens.
- Received April 17, 1995.
- Revision received September 7, 1995.
- Accepted September 10, 1995.
- Copyright © 1996 by American Heart Association
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