CLINICAL STUDIES
Inducible nitric oxide synthase in skeletal muscle of patients with chronic heart failure
Urs N. Riede, MD*,
Ulrich Förstermann, MD ,
Helmut Drexler, MD with the technical assistance of Brigitte Freudenberg-Plessow
* Department of Pathology, University of Freiburg, Freiburg, Germany
Department of Pharmacology, Johannes-Gutenberg-University, Mainz, Germany
Department of Medicine, Hannover Medical University, Hannover, Germany
Manuscript received March 4, 1998;
revised manuscript received May 20, 1998,
accepted June 4, 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 expression and localization of inducible nitric oxide (NO) synthase (NOS II) was evaluated as a source of NO which has been shown to affect muscle contraction.
Background. Advanced stages of chronic heart failure are associated with systemic activation of cytokines which have been shown to stimulate the expression of NOS II in various cell types, including myocytes. We hypothesized that systemic cytokine activation could lead to expression of NOS II in skeletal muscle of patients with chronic heart failure.
Methods. Skeletal muscle specimens were obtained by percutaneous needle biopsy in six normal volunteers and eight patients with heart failure (New York Heart Association class III). Electron microscopy immunocytochemistry (immunogold labeling) with specific anti-NOS antibodies was utilized to elucidate the intracellular localization of NOS II and neuronal NO synthase (NOS I) in myocytes of skeletal muscle. Reverse transcriptase, competitive polymerase chain reaction (PCR) was applied to quantify NOS II mRNA in skeletal muscle.
Results. Inducible nitric oxide synthase was readily expressed in the cytosol of skeletal muscle myocytes; NOS I expression was sparse. Polymerase chain reaction results indicated that NOS II gene expression is increased in patients with chronic heart failure.
Conclusions. Inducible NO synthase is expressed in human skeletal muscle and its gene expression is increased in patients with severe heart failure. Given the experimental evidence that NO can attenuate contractile performance of skeletal muscle and can mediate muscle wasting, an increased local production of NO in skeletal muscle by NOS II may have important implications for patients with severe heart failure.
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Abbreviations and Acronyms
| | CHF | = chronic heart failure | | EM | = electron microscopy | | NO | = nitric oxide | | NOS | = NO synthase | | NOS I | = neuronal nitric oxide synthase | | NOS II | = inducible nitric oxide synthase | | PCR | = polymerase chain reaction | | RT | = reverse transcriptase | | TNF | = tumor necrosis factor |
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Nitric oxide (NO) is an important signaling molecule that mediates a variety of essential physiologic processes, including neurotransmission and neuromodulation, vasodilatation and host cell defense (1). Alterations in NO production have been implicated in various pathophysiologic processes, such as atherosclerosis, heart failure, hypotension and organ failure associated with septic shock (1,2).
Nitric oxide is synthesized from L-arginine by NO synthases (NOS), a family of isoenzymes with distinct functional, biochemical and regulatory properties (1,3). Both the neuronal NOS I and the endothelial NOS III are constitutive isoforms and are discretely expressed in specific tissues. They rapidly transduce signaling events in a calcium-dependent manner. While NOS I occurs in a variety of cell types, including neurons, epithelial cells, mesangial cells and skeletal muscle cells, NOS III is expressed predominantly in the endothelium and its activity accounts for endothelium-dependent vascular relaxation. In contrast to these calcium-dependent enzymes, the inducible calcium-independent NOS II is expressed in immunologically activated cells, that is, in response to cytokines such as tumor necrosis factor (TNF)- , interferon gamma and certain interleukins.
Advanced stages of chronic heart failure (CHF) are often associated with systemic and cardiac cytokine activation (4) and expression of NOS II in the myocardium (5). Recently, it has been demonstrated that skeletal muscle expresses NOS II and that this expression can be stimulated with endotoxin (6). Therefore, it is hypothesized that systemic cytokine activation could stimulate the expression of NOS II in skeletal muscle of patients with CHF. Accordingly, the present study examined the expression of NOS II in skeletal muscle of healthy volunteers and patients with CHF.
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Methods
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Patients.
Human skeletal muscle was obtained from six healthy volunteers (age 44 ± 4 years) and eight patients with CHF (age 52 ± 7) after informed consent. The study was approved by the Ethical Committee of the University of Freiburg. Volunteers had normal physical examination and peak oxygen consumption (33 ± 2 ml/min/kg) and did not take any medication. All patients were in New York Heart Association functional class III heart failure and demonstrated impaired exercise capacity (peak oxygen consumption 14 ± 2 ml/min/kg) and ejection fraction (25% ± 3%). All patients were treated with digoxin, diuretics and angiotensin-converting enzyme (ACE)-inhibitors. Percutaneous needle biopsies were obtained from the middle part of the vastus lateralis muscle under local anesthesia using the Bergström technique (7) as established in our laboratory (8).
Reverse transcriptase (RT) polymerase chain reaction (PCR).
Total cellular RNA was isolated from frozen tissue (9). The amount of RNA was evaluated by spectrophotometry. The integrity of the RNA was verified by gel electrophoresis. Quantification of NOS II mRNA was performed by competitive RT-PCR in the presence of a defined concentration of a shortened NOS II competitor RNA that served as an internal standard, as previously described (10). The NOS II competitor template (314 bp) was obtained from a 419-bp cDNA fragment of the human NOS II cDNA (11) using appropriate restriction enzymes. Equal amounts of total RNA (2 µg) were mixed with increasing quantities of competitor (12.5 to 6.25 x 104 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 at 42°C for 60 min. Sense and antisense primer oligonucleotides were selected from the human cDNA sequences of NOS II (11) (sense positions: 1614 to 1633, 5'-GGGAGCATCACCCCCGTGTT-3', antisense primer positions: 2012 to 2033; 5'-GAGCGATTTCTTCAGTTTCTCT-3'. Duplicate samples of PCR reaction were performed. Denaturing, annealing and extension reactions proceeded 36 times at 94°C for 1 min, 62°C for 2 min and 72°C for 3 min. As a negative control, no amplification product occurred if reverse transcriptase or total RNA were omitted in the first-strand cDNA reaction. The PCR products of 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. 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 (1.33). 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 (see Fig. 1). In control experiments the optimal amount of total RNA and PCR cycle profile was determined. In one volunteer and one patient each, only duplicate determinations of NOS II mRNA were performed because the amount of extracted RNA did not allow multiple RT-PCRs with different concentrations of internal standard.
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Figure 1 NOS II mRNA quantification by competitive RNA-PCR. A constant amount of total RNA was mixed with an increasing number of NOS II-competitor RNA molecules, reverse transcribed into cDNA and amplified in duplicate samples by PCR. The PCR products, indicated as NOS II target (419 bp) and NOS II competitor (315 bp), were separated by gel electrophoresis, stained with ethidium bromide and visualized by UV irradiation. The band densities of NOS II target and competitor DNA were determined. The mean value 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.
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Electron microscopy (EM) immunocytochemistry.
Muscle biopsies were fixed in PLP (0.5% glutaraldehyde, 5% paraformaldehyde in 0.1M Na-phosphate buffer, 0.2M Lysin-HCl, 0.01 M Na-metaperjodate, pH 7.3) at 4°C for 30 min and then minced into 1 mm pieces and postfixed for 3.5 h in PLP, followed by 3x rinsing in PBS-buffer and 4 x 15 min incubation in NH4Cl 50 mM as detergent. After 10x rinsing in PBS + 2% saccharose overnight, specimens were dehydrated in dimethylformamid and embedded in Lowicryl using the quick method of Altman et al. (12). This allows the detection of immunohistochemical reaction products without dislocation due to dehydration. The immunohistochemical analysis was performed on ultrathin sections mounted on formvar-coated nickel grids. For the immunohistochemical incubation, an anti-NOS I polyclonal antibody (13) (diluted 1:20) or an anti-NOS II polyclonal antibody (14) (diluted 1:7.5) and a rabbit nonimmune IgG (2.5 mg/ml) as a control were used, each in PBS + 0.5% BSA + 1.5% normal goat serum and BSAc (Aurion) 1:10. Western blot data (15) have demonstrated that the anti-NOS I antibody recognizes both the essential NOS I and the recently described splice variant NOS I-µ (16) that is primarily expressed in skeletal muscle. Sections were incubated in blocking serum (PBS + 0.5% BSA + 5% normal goat serum + BSAc 1:10) for 25 min. Incubation of grids was performed in the first antibody solution overnight at 4°C, then 1 h at room temperature followed by intense rinsing with incubation buffer. The secondary antibody (IgG gold-goat-anti rabbit 1:100) containing Tween 20 (1 µl Tween + 2 ml buffer) was incubated for 2 h at room temperature followed by intense rinsing. Tissues were fixed for 5 min in 2% glutaraldehyde in PBS, followed by rinsing with PBS. Staining was performed for 30 min in 2% uranyl acetate dissolved in water and lead citrate.
Statistical analysis.
Data are mean ± SEM. Comparisons between controls and patients with heart failure (mRNA transcripts) were performed by unpaired t-test.
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Results
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NOS II mRNA in human skeletal muscle.
Reverse transcription-polymerase chain reaction analysis demonstrated low levels of NOS II mRNA in skeletal muscle of normal volunteers. In patients with heart failure, NOS II mRNA levels were increased (Fig. 2). The increase in NOS II mRNA was observed both in patients with dilated (n = 5) and ischemic (n = 3) cardiomyopathy and the expression levels did not correlate with duration of heart failure; however, the number of patients was too small to exclude that these and other factors may affect NOS II gene expression. Angiotensin-converting enzyme mRNA in these biopsy specimens was determined using a previously established competitive PCR (10) and did not reveal differences between normal volunteers and patients with heart failure. (27.4 ± 12.7 104 vs. 26.2 ± 15.6 transcripts per 100 ng total RNA).
EM immunocytochemical localization of NOS II and NOS I in human skeletal muscle.
Anti-NOS II antibody-coupled immunogold particles were localized in the sarcoplasma of skeletal muscle cells, predominantly over myofibrils (Fig. 3A). Anti-NOS I antibody-coupled immunogold particles were observed scarcely over the sarcolemma of skeletal muscle cells (Fig. 3B). In control sections (using canine IgG instead of primary antibody) no immunogold particles were found (Fig. 3C). Immunogold staining did not allow quantification of NOS II protein. Therefore, significant differences between NOS II expression between normal volunteers and patients with heart failure could not be established. The relative abundance of NOS II may vary according to the skeletal muscle fiber type studied (6) which cannot be identified by immunogold EM staining. Only skeletal muscle cells and capillary endothelium was visualized in our sections (rather than arterioles). Endothelial cells of capillaries did not show any immunogold staining with the anti-NOS II of anti-NOS I antibodies.
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Figure 3 Electron microscopy immunogold-labeling using anti-NOS II antibody (A), anti-NOS I antibody (B) and control (C) of skeletal muscle in a patient with heart failure. Note the numerous small black dots grouped over myofibrils using anti-NOS II antibody while staining with these dots is scarce with anti-NOS I antibody and absent in control.
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Discussion
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The present study demonstrates expression of NOS II mRNA and protein in human skeletal muscle.
While the EM immunocytochemical localization of NOS II in human skeletal muscle did not allow quantification of NOS II expression within skeletal muscle, the RT-PCR results suggest that NOS II gene expression is increased in patients with severe CHF, at an advanced stage of the disease, when systemic cytokine activation is likely to occur (4). These observations extend previous findings demonstrating NOS II expression in the myocardium of patients with heart failure (5). Thus, it appears that the systemic and/or local cytokine activation in patients with severe heart failure is associated with cardiac and increased peripheral expression of NOS II. This NOS II expression is likely to be a specific event because mRNA level for ACE in human skeletal muscle showed no difference between normal subjects and CHF patients.
Recent evidence suggests the expression of NOS II also in response to signals that are noninflammatory, nonimmunologic or unapparent (17–19). However, in contrast to NOS II expression in normal lung epithelial cells or nephron cells (18,19), exposure of skeletal muscle myocytes to bacteria appears unlikely. The present finding that NOS II is constitutively expressed in human skeletal muscle of control subjects is consistent with recent observations by Park et al. (20). Similar observations have been made in specific pathogen-free guinea pigs and have been associated with functional consequences in these animals (6). The underlying mechanisms for the expression of NOS II in normal skeletal muscle remains to be determined. However, evidence has recently been presented that normal human skeletal muscle expresses TNF- (21). Whether other cytokines are expressed by normal human skeletal muscle is unknown. Nevertheless, this observation raises the possibility that TNF- may be involved in the expression of NOS II in normal skeletal muscle.
We did not detect NOS II expression in endothelial cells of capillaries. Due to the small size of tissues prepared for EM (containing only skeletal muscle tissue), no arterioles with vascular smooth muscle cells or macrophages were visualized in the sections investigated. Thus, we cannot exclude the possibility that NOS II expression occurs in these cell types, particularly in patients with heart failure. Indeed, the enhanced vasoconstrictor response following the inhibition of NO-synthesis by N-methyl-L-arginine would be consistent with the notion that severe heart failure is associated with expression of NOS II in the vascular wall of peripheral resistance vessels (22).
Neuronal nitric oxide synthase was the first isoform to be identified in skeletal muscle (23). Its distribution within muscle tissue is still somewhat controversial. Kobzik et al. (23) reported that NOS I in rat skeletal muscle was restricted to type II fibers (fast-twitch) whereas type I fibers (slow-twitch intermediate fibers) were NOS I negative. However, in a recent study by Brennan et al. (24), fibers from the quadriceps muscle of normal mice were all NOS I positive, whereas no staining was seen in muscle of NOS I knockout mice. We have demonstrated previously that, by far, the highest expression of NOS I in guinea pig skeletal muscle is in the neuromuscular endplate (6). This regional distribution of NOS I may explain the low abundance of immunogold staining in our EM immunocytochemical analysis which cannot identify neuromuscular structures. Endothelial nitric oxide synthase expression has also been demonstrated in skeletal muscle (25), although the expression level seems to be relatively low (6). Our present observation together with previous experimental studies (6,23,25) suggest that all three isoforms of NOS may be present in distinct structures and/or fiber types of skeletal muscle.
Limitations of the present study.
Some limitations need to be addressed. First, the skeletal muscle biopsy may have contained trace amounts of blood vessels and blood cells which could have contributed to the increased NOS II mRNA levels observed in CHF patients. The present approach cannot exclude the contribution of NOS II gene expression in vascular smooth muscle cells. However, provided that the increased NOS II mRNA levels in CHF are translated into increased NOS activity, the enhanced NO derived from either source (smooth muscle cell or skeletal muscle) may affect both vasomotor tone and skeletal muscle function in a autocrine or paracrine fashion. Trace amounts of erythrocytes, platelets and/or leukocytes are likely to be included in skeletal muscle biopsies. However, it appears unlikely that these "contaminating cells" account for the fourfold to fivefold increase in NOS II mRNA levels in CHF. We did not detect NOS II mRNA in leukocytes obtained from two CHF patients who expressed NOS II mRNA in their skeletal muscle specimens. Furthermore human neutrophils have recently been shown to express NOS I but no NOS II (26). Furthermore NOS II protein has been detected in skeletal muscle of CHF patients (Fig. 3A), suggesting that the NOS II mRNA is likely to derive from the same cellular source.
Potential functional implications.
The expression of NOS II in skeletal muscle may have important functional consequences. Nitric oxide rapidly and reversibly inhibits the steady-state turnover of isolated cytochrome oxidase at submicromolar concentrations. The inhibition of cytochrome oxidase is competitive with oxygen, and NO binds with high affinity to the oxygen-binding site of cytochrome oxidase when its site is reduced (27). Inhibition of this enzyme may cause decreased ATP production and increases the cellular levels of ADP, AMP, GDP and Pi, which regulate a large range of cellular processes, including muscle contraction. Indeed, NO has been shown to attenuate the contractile performance of skeletal muscle (6,23) and to inhibit oxygen consumption (28).
Beyond the regulation of contractile force, NO may have important long-term implications in skeletal muscle. There is evidence that oxidative stress pathways interact with NO to modulate cytoprotective or cytotoxic effects (29). More recently, Buck and Chojkier (30) demonstrated that NOS activity of skeletal muscle mediates effects of oxidative stress on muscle dedifferentiation and muscle wasting. These observations may have important implications given the notion that NOS II expression is increased in patients with severe CHF and the evidence for muscle wasting in this setting (31), that is, enhanced local production of NO by NOS II following cytokine activation may be involved in muscle wasting of patients with advanced stages of heart failure and cachexia.
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Footnotes
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Supported, in part, by the Deutsche Forschungsgemeinschaft (Dr 148/7-2, Fo 144/3-1, 3-2).
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Abstract
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