Published online before print October 30, 2006, doi: 10.1161/01.HYP.0000250468.02084.ce
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(Hypertension. 2007;49:193.)
© 2007 American Heart Association, Inc.
Original Articles |
Lack of Endothelial Nitric Oxide Synthase–Derived Nitric Oxide Formation Favors Hypertrophy in Adult Ventricular Cardiomyocytes
From the Justus-Liebig-Universität Gießen (S. Wenzel, C.R., S. Wingerning, K.-D.S.), Physiologisches Institut, Gießen, Germany; Justus-Liebig-Universität Gießen (J.R.), Institut für Veterinärphysiologie, Gießen, Germany; and Heinrich-Heine-Universität Düsseldorf (G.K.), Institut für Pharmakologie und Klinische Pharmakologie, Düsseldorf, Germany.
Correspondence to Klaus-Dieter Schlüter, Justus-Liebig-Universität Gießen, Physiologisches Institut, Aulweg 129, D-35392 Gießen, Germany. E-mail Klaus-Dieter.Schlueter{at}physiologie.med.uni-giessen.de
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Abstract |
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Reduced activity and expression of endothelial NO synthase (eNOS) is observed in cardiomyocytes from pressure-overloaded hearts with heart failure. The present study was aimed to investigate whether reduced eNOS-derived NO production contributes to the hypertrophic growth and phenotype of these cardiomyocytes. Cultured ventricular cardiomyocytes from adult rats were exposed to N
-nitro-L-arginine (L-NNA) to inhibit global NO formation, and cultured cardiomyocytes derived from eNOS-deficient mice were used as a model of genetic knockout of eNOS. Cell growth, formation of oxygen-derived radicals (reactive oxygen species [ROS]), activation of p38 mitogen-activated protein (MAP) kinase phosphorylation, and cytokine expression in cardiomyocytes were investigated. L-NNA caused a concentration-dependent acceleration of the rate of protein synthesis and an increase in cell size. This effect was sensitive to p38 MAP kinase inhibition or antioxidants. L-NNA induced a rapid increase in ROS formation, subsequent activation of p38 MAP kinase, and p38 MAP kinase–dependent increases in the expression of transforming growth factor-ß and tumor necrosis factor-
. Similar changes (increased ROS formation, p38 MAP kinase phosphorylation, and cytokine induction) were also observed in cardiomyocytes derived from eNOS+/+ mice when exposed to L-NNA. Cardiomyocytes from eNOS–/– mice displayed higher p38 MAP kinase phosphorylation and cytokine expression under basal conditions, but neither these 2 parameters nor ROS formation were increased in the presence of L-NNA. In conclusion, our data support the hypothesis that reduced eNOS activity in cardiomyocytes contributes to the onset of myocardial hypertrophy and increased cytokine expression, which are involved in the transition to heart failure.
Key Words: myocardial hypertrophy • cytokine • TNF- • TGF-ß
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Introduction |
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NO, initially identified as the endothelium-derived relaxation factor, is a key regulator of blood vessel tone. Endothelial cells generate NO by the endothelial isoform of NO synthase ([eNOS] encoded by the NOS-3 gene). eNOS-deficient mice develop a hypertensive phenotype and moderate cardiac hypertrophy.1 eNOS is also expressed in nonendothelial cells in concert with other NOS isoforms. In adult ventricular cardiomyocytes, eNOS and the neuronal NOS (nNOS encoded by the NOS-1 gene) are constitutively expressed. eNOS is located at the sarcolemmal and T-tubular caveolae where it is associated with caveolin-3. The activity of eNOS modifies ß-adrenoceptor signaling.2,3 nNOS seems to be localized at the sarcoplasmic reticulum, where it interferes with the activity of calcium-handling proteins.4 Interestingly, eNOS undergoes downregulation in cardiomyocytes under conditions of hypertrophic cardiomyopathy and heart failure, but nNOS undergoes upregulation.5–7
Under experimental conditions, inhibition of endogenous NO formation induces myocardial hypertrophy, but this is independent of its hypertensive effect.8 The underlying mechanisms by which reduced NO formation contributes to myocardial hypertrophy are unknown. In the present study we hypothesized that an inhibition of eNOS-derived NO disturbs the balance between NO and NAD(P)H oxidase–generated reactive oxygen species (ROS). As a possible signal transduction pathway, we focused on p38 MAP kinase activation and the previously identified p38 MAP kinase–dependent increase in protein synthesis and cytokine expression.9,10 To rule out possible interactions of other neurohumoral factors, we performed the experiments on isolated and cultured cardiomyocytes from adult rat hearts and from mice with a genetic knockout of eNOS.
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Methods |
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Cell Isolation and Cultivation
Ventricular cardiomyocytes were isolated from 200- to 250-g male Wistar rats as described before in great detail.11 Hearts were excised from anesthetized rats and immediately connected to a Langendorff perfusion system. Hearts were perfused with calcium-free Tyrode solution and collagenase for 20 minutes, then the ventricles were separated from the hearts and minced. Finally, the cell suspension was filtered through a Nylon mesh, and the remaining cell pellet was resuspended in Tyrode solution by a stepwise increase of the calcium concentration to 1.2 mmol/L. Then the cells were plated on cell culture dishes precoated with 4% (vol/vol) FCS and washed to remove round and nonattached cells after 4 hours. The basal culture medium (CCT) was modified medium 199 including Earl’s salts, 2 mmol/L of carnitine, 5 mmol/L of creatine, and 5 mmol/L of taurine. To prevent contaminations, 100 IU/mL of penicillin and 100 µg/mL of streptomycin were added. Cytosine-ß-D-arabinofuranoside (10 µmol/L) was added to inhibit growth of nonmyocytes. Where indicated, the procedure was performed in an identical way using hearts from spontaneously hypertensive rats.
Experiments on isolated ventricular cardiomyocytes from mice were performed in a similar way. Some modifications were performed to adapt the isolation protocol to mice as described before in detail.12 These modifications are related to the stepwise increase in calcium and the plating procedure. For mouse myocytes, laminin instead of FCS was used as an attachment substrate. Animal handling conformed with the Guide for the Care and Use of Laboratory Animals (National Institutes of Health publication 85-23).
Protein Synthesis and Cell Sizes
The rate of protein synthesis was determined by exposing the cultures to L-14C-phenylalanine (0.1 µCi/mL) for 24 hours. Incorporation of radioactivity into acid-insoluble cell mass was determined as described before13 and normalized to the precursor pool (acid soluble cell mass). Myocyte size was determined on microphotographs by a charge-coupled device camera as described previously.14
Immunoblotting
Cells were stimulated as described in the Results section. At the end of the incubation period, cells were lysed in lysis buffer. After sodium dodecyl sulfate–gel electrophoresis, proteins were transferred onto reinforced nitrocellulose by semidry blotting. The membranes were saturated with 2% (wt/vol) BSA and incubated with the first antibody for 2 hours. After the membranes were washed, alkaline phosphatase–labeled goat anti rabbit-IgG antibodies were added for another 2 hours. Bands were visualized by alkaline phosphatase activity.
Real-Time RT-PCR
The mRNA expression of transforming growth factor (TGF)-ß1 and tumor necrosis factor (TNF)- were quantified via real-time RT-PCR (see Reference 15 for further details of the RT-PCR protocol) using iQ SYBR Green supermix (BioRad). Hypoxanthine-phosphoribosyl-transferase was used as a housekeeping gene to normalize sample contents.
Detection of ROS
ROS generation in cells was assessed using the probe 2,7-dichlorofluorescein (DCF). The membrane-permeable diacetate from the dye (reduced DCF, DCF-DA, 10 µmol/L) was added to the cultures. Fluorescence intensity was measured in 
10 different cells per preparation. Fluorescence was analyzed using a fluorescence microscope combined with a video imaging system.
Quantification of TNF- Release
The release of TNF- into the supernatant of cultured cardiomyocytes was investigated as described before.16 Ten-microliter samples of the supernatant were used for a bioassay that used the cytotoxic effect of TNF- on the cell line WEHI. The assay revealed a linear correlation between 0.1 and 10 pg/mL of TNF-.
Statistical Analysis
Results are expressed as mean±SEM. Differences were analyzed by 1-way ANOVA, followed by Student–Neumann–Keul posthoc analysis. A value of P<0.05 was regarded as significant.
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Results |
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Influence of NOS Inhibition on Protein Synthesis of Rat Cardiomyocytes
To investigate whether endogenous NO production in ventricular rat cardiomyocytes is part of the regulatory process controlling protein turnover and cell size, we determined the rate of protein synthesis in the presence or the absence of N
-nitro-L-arginine (L-NNA), an inhibitor of constitutively expressed NOS, during 24-hour incubations. The influence of L-NNA on protein synthesis was compared with that of L-arginine, the natural substrate of NOS. In the presence of L-NNA, the rate of protein synthesis increased in a concentration-dependent way (Figure 1A). The effect of NO inhibition on protein synthesis reached the level of significance at 10 and 100 µmol/L. In all of the subsequent experiments, we used a concentration of 100 µmol/L of L-NNA. Addition of L-arginine (from 10 nmol/L to 100 µmol/L), however, had no influence on the basal rate of protein synthesis (Figure 1A). Figure 1B gives a representative example of cell sizes after 24 hours. L-NNA–treated rat cells (100 µmol/L) developed an increase in cell width, but cell lengths remained unchanged. On average, the calculated cell volumes increased from 18 788±782 to 28 385±1 173 µm3 in the presence of L-NNA (P<0.05 n=120 cells). The cross-sectional area increased from 27.23±1.08 to 42.46±1.75 µm2 (P<0.05; n=120 cells). These experiments suggest that NO inhibition increases protein synthesis and induces a concentric phenotype of hypertrophy.
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Inhibition of p38 MAP Kinase Activation Inhibits the Prohypertrophic Effect Evoked by L-NNA on Rat Cardiomyocytes
In the next set of experiments we blocked the p38 MAP kinase activation by addition of SB202190, the activation of the p42/p44 MAP kinase pathway by addition of PD98059, and the activation of the protein kinase C (PKC) pathway by addition of bisindolylmaleimide (also known as Gö5850). The concentrations used in this study are based on our previously performed concentration-response curves in the same system.10,17 In the present study we reconfirmed our previous findings that none of the 3 inhibitors alone attenuates the basal rate of protein synthesis (Figure 2). However, inhibition of either p38 MAP kinase or PKC activation, but not that of p42/p44 MAP kinase activation, attenuated the prohypertrophic stimulation caused by L-NNA (Figure 2). Inhibition of p38 MAP kinase did not attenuate the prohypertrophic stimulation of -adrenoceptor stimulation, but PKC inhibition attenuated the prohypertophic effects of L-NNA and phenylephrine (Figure 2). These data led us conclude that the activation of the stress-activated p38 MAP kinase is essential for the prohypertrophic effect of NOS inhibition.
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Inhibition of NO Formation and Generation of ROS in Rat Cardiomyocytes
As a first step to investigate possible upstream pathways involved in the aforementioned p38 MAP kinase–dependent effect of NOS inhibition on cell growth, we investigated the effect of NOS inhibition on ROS production. The fluorescence signal increased during a 15-minute time period constantly because of basal generation of ROS. As soon as L-NNA was added, the fluorescence signal increased more rapidly compared with basal production (Figure 3). On average, the fluorescence signal was 35±2% higher in L-NNA–treated cells compared nontreated cells after 30 minutes (P<0.05; 18 to 30 cells each). In contrast, no increase of the fluorescence signal was observed when the cells were analyzed in the presence of 4-amino-5-aminoethylaminopentyl-N-nitroguanidine, an nNOS-specific inhibitor (–6±7%; not significant versus untreated cells; n=18 to 22 cells each). The increase in ROS formation caused by L-NNA was attenuated in the presence of diphenylenium iodide (10 µmol/L) and apocynin (5 µmol/L): L-NNA at +43±9%; L-NNA+diphenylenium iodide at +15±12% (P<0.05 versus L-NNA); and L-NNA+apocynin at –10±14% (P<0.05 versus L-NNA).
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The causal relationship between ROS generation in the presence of L-NNA and the prohypertrophic stimulus evoked by L-NNA was investigated in additional experiments in which the increase in cell sizes because of L-NNA was attenuated by the presence of vitamin C (100 µmol/L) or N-acetyl cysteine (1 mmol/L), which were used as antioxidants. The concentrations used for these 2 antioxidants have recently been used to attenuate the angiotensin II- and ROS-dependent p38 MAP kinase activation in the same system.9 Representative single cell graphs are shown as online supplements (available at http://hyper.ahajournal.org). On average, in this set of experiments, the cell volume of cardiomyocytes increased by 21.6±2.2% in the presence of L-NNA compared with untreated control cultures (P<0.05; n=53 cells) but not in the presence of either vitamin C (+1.8±2.4%, not significant versus control; n=43 cells) or N-acetyl cysteine (+4.2±2.1%, not significant; n=51 cells). These data confirm a causal relationship between L-NNA–dependent increases in ROS generation and their contribution to cell growth.
Inhibition of NOS Activity and p38 MAP Kinase Activation in Rat Cardiomyocytes
As the experiments in the present study indicate that NOS inhibition leads to an acceleration of ROS production and a p38 MAP kinase–dependent prohypertrophic stimulation, we now examined whether NOS inhibition indeed activates p38 MAP kinase. In the presence of L-NNA, p38 MAP kinase phosphorylation was significantly increased (Figure 4A). On average, the ratio of the phosphorylated to the nonphosphorylated form of p38 MAP kinase increased by 71.4±15.8% in a time-dependent manner (Figure 4B). L-NNA may activate p38 MAP kinase via ROS generation or by its cGMP-lowering effect. Selective inhibition of soluble guanylyl cyclase in cardiomyocytes by 1-H-[1,2,4]oxaldiazolo[4,3-a]quinoxalin-1-1 (ODQ) produced less p38 MAP kinase phosphorylation compared with the values achieved by L-NNA (Figure 5).
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L-NNA and Cytokine Expression in Rat Cardiomyocytes
In addition to the L-NNA–dependent increase in cell growth, as indicated by a higher rate of protein synthesis and an increase in cell sizes, L-NNA–induced cytokine expression is another p38 MAP kinase–dependent phenomenon in cardiomyocytes accompanying prohypertrophic stimulation. The functional relevance of the observation that NOS inhibition leads to p38 MAP kinase activation is highlighted by the effects on cytokine expression. L-NNA significantly increased cellular TGF-ß1 expression in adult rat ventricular cardiomyocytes (Figure 6A). On average, TGF-ß1 expression in cardiomyocytes increased by 35.7±6.2%, and this effect was significantly attenuated in the presence of SB202190 to 13.7±3.3% (P<0.05 versus L-NNA; n=4). In parallel, the mean TNF- release into the supernatant was elevated (Figure 6B).
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Comparison of the Results to Cardiomyocytes Isolated From eNOS–/– Mice
The aforementioned experiments described the effect of global NOS inhibition on basal protein synthesis, ROS generation, p38 MAP kinase phosphorylation, and cytokine expression on rat ventricular cardiomyocytes. The lack of effect of the nNOS inhibitor (determined by ROS generation) suggests that the observed effects are specifically linked to eNOS-derived NO. To test this hypothesis, we investigated whether cardiomyocytes isolated from eNOS–/– mice develop a comparable phenotype. Ventricular cardiomyocytes were isolated from eNOS–/– mice or their corresponding eNOS+/+ littermates at the age of 12 to 16 weeks. At this time, hearts derived from eNOS–/– mice had developed a mild form of hypertrophy, as indicated by an increased heart weight/body weight ratio: 7.16±0.55 mg/g in eNOS+/+ mice and 9.70±0.41 mg/g in eNOS–/– mice (P<0.05; n=6). Lack of eNOS expression in cardiomyocytes from eNOS–/– mice was verified by immunoblot (Figure 7A). This went along with an increased TGF-ß1 expression and increased phosphorylation of p38 MAP kinase. On average, the amount of phosphorylated p38 MAP kinase in isolated adult ventricular cardiomyocytes from eNOS–/– mice was 
4-fold higher than that in isolated cardiomyocytes from eNOS–/– mice (Figure 7B).
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We next examined whether NOS inhibition in cardiomyocytes derived from eNOS–/– mice increases ROS formation. L-NNA increased ROS formation in cardiomyocytes derived from eNOS+/+ mice but not in those derived from eNOS–/– mice (Figure 8A). In contrast, angiotensin II (100 nmol/L) increased ROS production in both cell cultures by either 41.0±5.6% (eNOS+/+ mice) or 26.8±7.6% (eNOS–/– mice). As expected, L-NNA activated p38 MAP kinase in cardiomyocytes derived from eNOS+/+ mice but not in that derived from eNOS–/– mice (Figure 8B).
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) or eNOS–/– mice (
). Cells were treated with angiotensin II ([Ang-II] 100 nmol/L) or L-NNA for 30 minutes. Data are normalized to untreated control cells. Data are mean±SEM from n=4 to 8 cells. (B) Time-dependent induction of p38 MAP kinase phosphorylation by L-NNA in cardiomyocytes derived from eNOS+/+ mice (filled symbols) or eNOS–/– mice (open symbols). Data are mean±SEM from n=4 cultures. *P<0.05 vs eNOS+/+.Finally, we tested whether the lack of ROS formation in cardiomyocytes derived from eNOS–/– mice is caused by the mild hypertrophic phenotype itself. For this purpose we isolated cardiomyocytes from spontaneously hypertensive rats (SHRs). Cells were isolated from 6-month-old rats, because at this time the hearts from SHRs had developed a hypertrophic phenotype as expressed by a significantly higher heart weight/body weight ratio (6.76±1.11 versus 4.01±0.23 mg/g; n=8; P<0.05). L-NNA caused a 34±9% increase in the fluorescence signal because of ROS in cardiomyocytes derived from SHRs (n=16 to 22 cells; each P<0.05 versus untreated controls).
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Discussion |
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In the present study, the effect of chronic (24 hour) inhibition of NO production on basal protein synthesis, ROS formation, p38 MAP kinase phosphorylation, and cytokine expression was investigated. The main findings of this study are that an inhibition of eNOS-derived NO formation in rat ventricular cardiomyocytes induces cell growth, ROS formation, and p38 MAP kinase activation and increases cytokine expression. Similar results were obtained with isolated cardiomyocytes from eNOS+/+ mice treated with L-NNA and cardiomyocytes from eNOS–/– mice under basal conditions. This suggests a similar contribution of eNOS-derived NO to the pathways under investigation in both species. We conclude that reduced eNOS-derived NO formation in cardiomyocytes contributes to the ongoing process of hypertrophic cardiomyopathy. As mentioned in the introduction, reduced eNOS expression and function is a common finding in pressure-overloaded hearts.5–7 The observed effect of L-NNA on cytokine expression may be linked to processes involved in the transition of hypertrophy to heart failure. The question arises from our work of whether this relationship holds true under in vivo conditions and in the case of pathophysiological stages of hypertrophy. Experiments in which animals were fed with NG-nitro-L-arginine methyl ester and the blood pressure was normalized with hydralazine seem to support our hypothesis.8 One may argue that hydralazine acts via its antioxidative effect, but even this explanation is in agreement with our findings in which ROS formation was identified as a major trigger.
The effect of arginine derivates on basal NO production and cGMP content in isolated cardiomyocytes has been established previously.18–20 It is well known that an elevation of cellular NO/cGMP levels negatively regulates the rate of protein synthesis.21 In principle, the observed effect of eNOS inhibition on protein synthesis may be explained by the cGMP-lowering effect of such a treatment rather than by its direct effects on ROS production. However, our finding than an addition of antioxidants to the cells diminished the prohypertrophic effect of L-NNA is not in line with this possibility. Moreover, an inhibition of soluble guanylyl cyclase in the cells by ODQ did not produce such a strong p38 MAP kinase phosphorylation as that evoked by L-NNA. For these reasons we believe that the mechanism by which L-NNA increases protein synthesis is mainly independent from its effect on basal cGMP levels but linked to the ROS-dependent p38 MAP kinase activation. The observed inhibition of cell growth by addition of antioxidants is consistent with this.
Our data suggest a participation of p38 MAP kinase in the regulation of protein synthesis under such conditions but not that of p42/p44 MAP kinase. L-NNA does not activate p42/p44 MAP kinase, and our data confirmed the results of this study previously.22 p38 MAP kinase activation alone is not sufficient to stimulate the rate of protein synthesis, but under osmotic stress or reconstitution of mechanical activity, a p38 MAP kinase–dependent prohypertrophic effect was observed.10 Under such conditions, ROS formation is increased.23,24 Therefore, under all of the conditions that went along with an induction of ROS formation, p38 MAP kinase–dependent steps are involved in the prohypertrophic effect. Other prohypertrophic stimuli are nevertheless independent of p38 MAP kinase activation.25 We conclude from these results that p38 MAP kinase activation requires coactivation of further signaling steps to elicit hypertrophy. Such coactivated pathways may also be linked to ROS formation.
The strong myocardial p38 MAP kinase activation in cardiomyocytes from eNOS–/– mice found in this study is in contrast to the results of a former study.26 However, in this study, a very high expression of atrial natriuretic factor (ANF) was found, and ANF counterbalances the prohypertrophic effect of eNOS deficiency.26 In our hands, eNOS–/– mice developed a moderate hypertrophy in accordance with the observations made by Huang et al.1 A possible explanation for the difference may be a different level of ANF expression in the different strains. Finally, we found a coupling of the p38 MAP kinase–dependent prohypertrophic effect caused by L-NNA to PKC activation. Such a coupling of ROS formation and PKC activation has been described before and is related to either PKC-
or PKC-
.27,28 Both isoforms have been identified recently as responsible for the prohypertrophic growth response of adult ventricular rat cardiomyocytes.29
In a previous study we found that ROS-dependent p38 MAP kinase activation by angiotensin II is linked to an excessive induction of TGF-ß1. In SHRs, NOS inhibition causes end organ damage that could be blocked by p38 MAP kinase inhibition.30 We demonstrated in this study that NOS inhibition can increase ROS formation in cardiomyocytes from SHRs. In salt-sensitive rats, eNOS expression decreased when the animals were fed with an 8% NaCl diet for 5 weeks. Re-expression of eNOS by treatment with betaxolol reduced myocardial hypertrophy, p38 MAP kinase phosphorylation, and TGF-ß1 expression in these animals.31 Such an induction of TGF-ß1 is commonly found in all studies in which cardiac NO formation is inhibited, irrespective of whether this goes along with hypertrophic growth or not and independent of the effects on blood pressure.32 In addition to these studies on rats, blood pressure reduction in eNOS–/– mice was not sufficient to inhibit the onset of fibrosis and hypertrophy in these animals.33 It is in line with these findings that in rats undergoing protal hypertension associated with biliary cirrhosis, eNOS expression is upregulated but no fibrosis occurs even in the presence of hypertrophy.34
Our data strongly indicate a key role for p38 MAP kinase activation in the signaling events provoked by inhibition of NO formation. We did not further analyze the p38 MAP kinase isoform responsible for this effect. Rat cardiomyocytes express the main isoforms p38 MAP kinase- and p38 MAP kinase-ß.35 Isoproterenol activates p38 MAP kinase in rat cardiomyocytes.10 Recently, a p38 MAP kinase- inhibitor has been used to inhibit the isoproterenol-dependent loss of cardiac function in rats.36 Therefore, one may speculate that the p38 MAP kinase- isoform is the dominant isoform responsible for the effects shown here.
Perspectives
Is there a clinical relevance for our study? Downregulation of eNOS or reduced activity of eNOS has been shown before in patients with hypertensive hypertrophy, specifically in the elderly.37–39 Therefore, such an imbalance seems to contribute to the cardiac phenotype and hypertrophic responsiveness. We are aware of the fact that alterations in the NO system are not restricted to myocytes under such conditions. Vascular complications and cross-activities between endothelial cells and myocytes may also contribute. On the other hand, long-lasting application of low doses of NOS inhibitors leads to myocardial hypertrophy and fibrosis.40 Collectively, these data suggest that an endogenous NO production in cardiomyocytes contributes to the multiple alterations in the ageing, hypertensive heart. Restoring NO formation to its normal, restricted level in cardiomyocytes should, therefore, be an aim of all treatment strategies.
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Acknowledgments |
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Source of Funding
The study was supported by the Deutsche Forschungsgemeinschaft grant SCHL 324/5-2.
Disclosures
None.
Received June 14, 2006; first decision June 29, 2006; accepted October 9, 2006.
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