Mechanical strain suppresses inducible nitric-oxide synthase in cardiac myocytes.

We investigated the effects of precisely controlled mechanical strain on nitric-oxide synthase activity in cultured neonatal rat cardiac myocytes. Incubation of cardiac myocytes for 24 h with 4 ng/ml interleukin-1beta and 100 units/ml interferon-gamma stimulated an increase in nitric oxide production, inducible nitric-oxide synthase (iNOS) mRNA, and iNOS protein. Mechanical strain suppressed nitric oxide production, iNOS mRNA, and iNOS protein stimulated by cytokines in an amplitude-dependent manner. Losartan (1 microM), an angiotensin II type 1 receptor antagonist, weakly inhibited the effect of strain, suggesting that paracrine angiotensin II is not the mediator of the strain effect. In addition, cycloheximide (10 microM), a protein synthesis inhibitor, inhibited the effect of strain by 46%. Transforming growth factor-beta (1 ng/ml) suppressed iNOS mRNA expression, but anti-transforming growth factor-beta antibody (30 microg/ml) did not block the effect of strain. In contrast, staurosporine (100 nM; a nonselective protein kinase inhibitor), calphostin C (1 microM; a selective protein kinase C inhibitor), and pretreatment with phorbol 12-myristate 13-acetate abolished the effect of strain. Genistein (100 microM), a tyrosine kinase inhibitor, partially inhibited the effect of strain. Thus, cyclic mechanical deformation suppresses cytokine-induced iNOS expression in cardiac myocytes, and this effect is mediated at least partially via activation of protein kinase C.

Nitric oxide (NO) 1 plays an important role as an intercellular messenger in most or all mammalian tissues (1)(2)(3)(4). Three genes encode nitric-oxide synthase of two biochemical types. Type I neuronal and type III endothelial nitric-oxide synthases are constitutive but dormant until activated briefly by Ca 2ϩ transients that sustain the binding of calmodulin. A third NOS, inducible nitric-oxide synthase (iNOS, or type II nitric-oxide synthase), is expressed only after transcriptional induction (5).
iNOS is up-regulated in several tissues by lipopolysaccharide and cytokine stimulation, where it generates the sustained release of large amounts of NO (6).
In cardiac diseases, iNOS has been identified in endothelial cells, vascular smooth muscle cells, infiltrating macrophages and lymphocytes, and cardiac myocytes (7)(8)(9). Several studies have shown that NO can modulate myocardial contractility (10 -12). Hosenpud et al. (10) reported that interleukin-1␤ (IL-1␤) exhibits a negative inotropic effect on canine heart in vivo through NO production. Recently, Haywood et al. (13) and Habib et al. (14) reported that iNOS gene expression occurs frequently in failing human cardiac myocytes of patients with dilated cardiomyopathy, ischemic heart disease, or valvular heart disease.
In cardiac myocytes, mechanical strain induces hypertrophy through activation of the phosphorylation cascade of protein kinases including protein kinase C, Raf-1 kinase, the extracellular signal-regulated kinase, and the stress-activated protein kinases, followed by increases in specific gene expression and protein synthesis (15)(16)(17)(18)(19). The early molecular response to mechanical strain in cardiac myocytes includes transcription of immediate-early response genes such as c-fos, c-myc, c-jun, and egr-1 (20,21). Many growth factors can induce hypertrophy and specific gene expression in cardiac myocytes (22)(23)(24). This indicates that paracrine and autocrine mechanisms may exist for initiation of the hypertrophic response.
Few previous studies have investigated the relationship between NO and mechanical forces (25)(26)(27)(28)(29). Increased levels of NO are released by endothelial cells when stimulated by shear stress or cyclic strain (25)(26)(27)(28)(29). However, it remains unknown whether mechanical strain modulates NO synthesis in cardiac myocytes. Using a mechanical deformation device that applies a highly uniform biaxial strain field over the culture substrate, we investigated the effects of mechanical strain on nitric-oxide synthase activity in cardiac myocytes.
Culture of Neonatal Rat Ventricular Myocytes (NRVM)-NRVM from 1-day-old Sprague-Dawley rats were isolated by previously described methods (30). The ventricles were cut into several pieces and incubated overnight at 4°C in a 1 mg/ml concentration of 1:300 trypsin (Life Technologies, Inc.) in Hanks' balanced salt solution (Life Technologies, Inc.). The ventricular tissue was then digested with 1 mg/ml collagenase type II (239 units/mg; Worthington) in Hanks' balanced salt solution and centrifuged twice at 50 ϫ g to remove less dense cells such as fibroblasts. The cells were cultured at 37°C and 5% CO 2 in Dulbecco's modified Eagle's medium (DMEM; BioWhittaker, Inc., Walkersville, MD) containing 7% fetal calf serum (FCS; Life Technologies, Inc.), 50 units/ml penicillin, and 50 g/ml streptomycin (Life Technologies, Inc.).
Mechanical Strain Device and Preparation of Cells-Mechanical deformation was applied to a thin and transparent membrane on which cells were cultured, an approach that produces controlled cellular strain as well as visualization of cells. The device used in this study provides a nearly homogeneous and uniform biaxial strain profile, i.e. strains that are equal at all locations on the membrane and in all directions. This approach eliminates locations on the substrate that have very high strains (20 -30%) in one direction. We have previously measured membrane strains with high-resolution video (31); the cams used for this study gave strains of 1, 4, 9, and 14%.
NRVM were plated on a fibronectin-coated (2 g/ml) membrane dish at a density of 3,000,000 cells/dish in 13 ml of DMEM containing 7% FCS and incubated for 48 h. Approximate cell confluence was 85-90%. NRVM were then made quiescent by washing four times with 10 ml of Hanks' balanced salt solution and incubating with 10 ml of DMEM containing 1% insulin/transferrin/selenium media supplement (ITS, Sigma), 50 units/ml penicillin, and 50 g/ml streptomycin. All experiments were performed on NRVM that had been serum-starved for 24 h. Mechanical strain was then applied at a specified constant frequency and amplitude, and control dishes received no mechanical strain.
Measurement of Nitrite Concentration-The formation of nitrite was used as an indicator of NO release by NRVM. Nitrite accumulation was measured by the Griess reaction as described previously (32). Briefly, the medium was centrifuged at 3000 rpm for 10 min at 4°C to remove cellular debris. The nitrite content in the supernatant was measured by combining 150 l of medium with 900 l of Griess reagent (0.75% sulfanilamide in 0.5 N HCl and 0.075% naphthylethylenediamine dihydrochloride), and the concentration of the resultant chromophore was determined spectrometrically at 543 nm. Nitrite concentration was calculated from known concentrations of sodium nitrite within the linear range of the assay (0.5-20 M).
Western Blot of iNOS Protein-NRVM were lysed directly in each dish by application of 2ϫ sample buffer containing 250 mM Tris-HCl (pH 6.8), 10% glycerol, 4% SDS, and 2% ␤-mercaptoethanol, and the mixture was boiled for 5 min. The lysate was centrifuged, and the supernatant was collected. Total protein concentration was measured by the Bradford method (Bio-Rad), and equal quantities of total protein were loaded on a 10% SDS-polyacrylamide gel and transferred to a nitrocellulose membrane in 25 mM Tris base (pH 8.5), 0.2 M glycine, and 20% methanol. The nitrocellulose membrane was blocked with 5% nonfat dried milk in Tris-buffered saline washing buffer containing 20 mM Tris base (pH 7.6), 137 mM NaCl, and 0.1% Tween 20 for 2 h. For the detection of iNOS, the membrane was incubated with 1:1000 diluted mouse monoclonal anti-iNOS antibody (Transduction Laboratories, Lexington, KY) for 1 h at 37°C and washed with Tris-buffered saline washing buffer for 30 min. After application of goat anti-mouse IgG coupled to peroxidase (Bio-Rad), the membrane was developed by the enhanced chemiluminescence method (ECL, Amersham Pharmacia Biotech).
Northern Blots-Total RNA was isolated by the guanidium thiocyanate/phenol chloroform method (33). A 217-base pair rat iNOS cDNA, which was prepared by reverse transcriptase-polymerase chain reaction (34), was radiolabeled by the random priming method with [␣-32 P]dCTP and the Klenow fragment of DNA polymerase (Stratagene, La Jolla, CA). For Northern blotting, 15 g of total RNA was loaded on a 1.0% formaldehyde gel (2.0 M), transferred to a nylon membrane (Stratagene), and UV-cross-linked with a UV Stratalinker (Stratagene). The probe was hybridized with QuikHyb solution (Stratagene) at 68°C for 1 h. The washed membrane was exposed to x-ray film overnight at Ϫ80°C. Normalization of RNA for equal loading was carried out by rehybridizing the blots with a glyceraldehyde-3-phosphate dehydrogenase (GAPDH) cDNA probe (CLONTECH, Palo Alto, CA).
Statistical Analysis-Data are expressed as the means Ϯ S.E. of four samples, which represented at least three separate experiments. Differences were analyzed by one-way analysis of variance combined with Sheffé's test, and p values Ͻ 0.05 were considered to be statistically significant.

Effects of Cytokines and Mechanical Strain on Nitrite
Production by NRVM-We first investigated the effect of the combination of IL-1␤ and IFN-␥ on NO production by cultured cardiac myocytes. As shown in Fig. 1, treatment of NRVM with IL-1␤ (4 ng/ml) and IFN-␥ (100 units/ml) caused a marked accumulation of nitrite in the culture medium in a time-dependent manner. However, surprisingly, this cytokine-induced nitrite accumulation in NRVM was significantly suppressed by 9% cyclic mechanical strain at 1 Hz. 9% cyclic mechanical strain by itself did not affect the basal levels of nitrite production by NRVM.
Effects of Mechanical Strain on iNOS Protein and mRNA Accumulations-We next investigated whether mechanical strain suppressed increases in iNOS protein and mRNA levels in cytokine-stimulated NRVM. The expression of iNOS protein was analyzed by immunoblotting with anti-iNOS antibody. No immunoreactive iNOS was detected in unstimulated and stretched NRVM (Fig. 2). The iNOS protein band with a molecular mass of 130 kDa appeared after exposure to the combination of IL-1␤ and IFN-␥ in a time-dependent manner, and these iNOS protein accumulations were markedly suppressed by 9% cyclic mechanical strain at 1 Hz. Furthermore, as shown in Fig. 3, unstimulated and stretched NRVM expressed no detectable iNOS mRNA, whereas exposure to the combination of IL-1␤ and IFN-␥ clearly induced iNOS mRNA accumulations in a time-dependent manner. 9% cyclic mechanical strain at 1 Hz suppressed the cytokine-stimulated increase in iNOS mRNA accumulation. In seven separate experiments, strain suppressed iNOS mRNA by 61 Ϯ 4% (p Ͻ 0.001). In addition, when NRVM were subjected to cyclic strains of 1, 4, 9, and 14% at 1 Hz for 8 h, the effect of mechanical strain on iNOS mRNA expression was amplitude-dependent (Fig. 4). In these studies, no morphologic changes in cardiac myocytes were detected following strains of 1-14%.

Effects of Angiotensin II Type 1 Receptor Antagonism on Suppression of iNOS mRNA Expression by Mechanical
Strain-Mechanical strain can cause release of angiotensin II from cardiac myocytes, which acts as a mediator of the stretchinduced hypertrophic response via the angiotensin II type 1 receptor (23). To explore the mechanism of the suppressive effects of mechanical strain on NO synthesis, we investigated whether the suppressive effect of cyclic mechanical strain on iNOS mRNA expression in cytokine-stimulated NRVM is angiotensin II-dependent. Cytokine-stimulated NRVM were stretched using 9% cyclic strain at 1 Hz for 8 h in the presence or absence of angiotensin II type 1 receptor antagonist. As shown in Fig. 5, losartan (1 M), an angiotensin II type 1 receptor antagonist, weakly inhibited (11% reduction by den- FIG. 1. Effects of cytokines and mechanical strain on nitrite production by NRVM. NRVM were plated on 2 g/ml fibronectin in DMEM containing 7% FCS for 48 h. After serum deprivation for 24 h, myocytes were exposed to vehicle (E), 9% cyclic mechanical strain at 1 Hz (q), IL-1␤ (4 ng/ml) ϩ IFN-␥ (100 units/ml) (Ⅺ), and IL-1␤ ϩ IFN-␥ ϩ mechanical strain (f) for 24 h. Nitrite accumulation in the culture medium was measured using Griess reagent. Data are the means Ϯ S.E. of four samples. * and **, p Ͻ 0.05 and p Ͻ 0.01 compared with control cells exposed to IL-1␤ ϩ IFN-␥, respectively. sitometry) the suppression of iNOS mRNA expression by cyclic mechanical strain in cytokine-stimulated NRVM. Thus, it is unlikely that angiotensin II mediates the suppressive effect of cyclic mechanical strain on iNOS mRNA expression in cytokine-stimulated NRVM.
Effects of Cycloheximide on Suppression of iNOS mRNA Expression by Mechanical Strain-Mechanical strain may induce many proteins including growth factors, vasoconstrictors, and other unknown proteins in cardiac myocytes. We studied whether new synthesis of secondary proteins that were secreted from cardiac myocytes by mechanical strain suppressed iNOS mRNA expression in cytokine-stimulated NRVM. Addition of cycloheximide (10 M), a protein synthesis inhibitor, for 8 h slightly suppressed iNOS mRNA in cytokine-stimulated NRVM (Fig. 6). However, cycloheximide modestly inhibited (46%) the effect of 9% cyclic mechanical strain. This experiment supported the premise that cyclic mechanical strain directly suppresses iNOS mRNA expression without new protein synthesis.

Effects of TGF-␤ and anti-TGF-␤ Antibody on Suppression of iNOS mRNA Expression by Mechanical
Strain-TGF-␤, released by cardiac myocytes and acting in an autocrine and/or paracrine manner, may participate in myocardial remodeling by hypertrophic stimuli (35). Induction of iNOS is inhibited in cardiac myocytes by TGF-␤ (34,36,37). We investigated whether TGF-␤ is involved in the suppressive effect of mechanical strain in cytokine-stimulated NRVM using TGF-␤ and anti-TGF-␤ antibody. As shown in Fig. 7, addition of TGF-␤ (1 ng/ml) for 8 h suppressed iNOS mRNA expression in cytokinestimulated NRVM. In addition, anti-TGF-␤ antibody (30 g/ml) slightly augmented iNOS mRNA expression and did not abolish the suppressive effect of 9% cyclic mechanical strain (1 Hz) on iNOS mRNA expression in cytokine-stimulated NRVM (5% inhibition). Neither TGF-␤ nor anti-TGF-␤ antibody by themselves induced iNOS mRNA expression in NRVM. This experiment suggests that TGF-␤ secreted from cardiac myocytes does not play an important role in the suppressive effect of mechanical strain on iNOS mRNA expression.
Protein Kinase C and Tyrosine Kinase Inhibition-To examine the role of protein kinase C and tyrosine kinases in the suppression of iNOS mRNA expression by mechanical strain, we tested the effect of protein kinase C and tyrosine kinase inhibitors (15)(16)(17)(18)(19). As shown in Fig. 8, addition of staurospo-FIG. 2. Effects of mechanical strain on iNOS protein expression. NRVM were plated on 2 g/ml fibronectin in DMEM containing 7% FCS for 48 h. After serum deprivation for 24 h, myocytes were exposed to 0 or 9% cyclic mechanical strain (1 Hz) with or without IL-1␤ (4 ng/ml) ϩ IFN-␥ (100 units/ml) for 24 h. Cell extracts were subjected to SDS-polyacrylamide gel electrophoresis, followed by immunoblot analysis using mouse anti-iNOS antibody. The molecular mass of iNOS protein is 130 kDa. Data are representative of two independent experiments with nearly identical results.  6. Effects of cycloheximide on suppression of iNOS mRNA expression by mechanical strain. NRVM were plated on 2 g/ml fibronectin in DMEM containing 7% FCS for 48 h. After serum deprivation for 24 h, myocytes were exposed for 8 h to 0 or 9% cyclic mechanical strain (1 Hz) with or without IL-1␤ (4 ng/ml) ϩ IFN-␥ (100 units/ml) in the presence or absence of cycloheximide (10 M). Cycloheximide was applied to the myocytes 30 min before mechanical strain. Total RNA was isolated and analyzed by Northern blotting with 32 Plabeled iNOS (upper panel) and GAPDH (G3PDH; lower panel) cDNA probes. Data are representative of two experiments with nearly identical results. rine (100 nM; 100% inhibition), a nonselective protein kinase inhibitor, and calphostin C (1 M; 98% inhibition), a selective protein kinase C inhibitor, for 8 h completely abolished the suppressive effect of 9% cyclic mechanical strain on iNOS mRNA in cytokine-stimulated NRVM. Genistein (100 M), a tyrosine kinase inhibitor, partially inhibited (30% inhibition) the effect of strain. These inhibitors by themselves did not induce iNOS mRNA in NRVM. In addition, protein kinase C depletion by pretreatment with PMA (1 M) for 24 h inhibited the suppressive effect of strain by 92% (Fig. 9). Thus, the suppressive effect of cyclic mechanical strain is mediated at least partially via activation of protein kinase C. DISCUSSION Shear stress and cyclic mechanical strain represent important components of the mechanical environment that regulate gene expression in endothelium, vascular smooth muscle, and myocardium. In bovine aortic endothelial cells, cyclic mechanical strain augments endothelial NOS expression and NO production (28). In human aortic endothelial cells, pulsatile stretch increases both endothelial NOS protein and mRNA expression (29). Wagner et al. (38) reported that shear stress and cyclic strain induce the expression of heme oxygenase-1, the enzyme that forms carbon monoxide, but not of iNOS mRNA in cultured vascular smooth muscle cells. In the present study, mechanical strain did not induce NO production and iNOS expression in cardiac myocytes, whereas mechanical strain suppressed NO production and iNOS expression in cytokine-stimulated myocytes. These findings suggest that me-chanical strain may play an important role in iNOS expression in immunological and inflammatory conditions, including postcardiac transplantation, cardiomyopathy, myocarditis, and ischemia-reperfusion injury.
Although cardiac mechanical overload is known to alter the expression of several cardiac-specific genes, it has not yet been established whether deformation acts directly or via local paracrine and autocrine factors liberated in response to hemodynamic load. In particular, the local renin-angiotensin system may play an important role in the adaptation of the heart to pressure and volume overload (39,40). Angiotensin II secreted from cardiac myocytes is an important mediator of the straininduced hypertrophic response, but factors other than angiotensin II may be involved in cardiac hypertrophy induced by mechanical strain (23,(41)(42)(43). In the present study, losartan weakly inhibited the suppressive effect of mechanical strain on iNOS mRNA expression in cytokine-stimulated cardiac myocytes. Thus, it is likely that factors other than angiotensin II are also involved in the response to mechanical strain.
TGF-␤ is secreted from cardiac myocytes by hypertrophic stimuli (35). TGF-␤, which stimulates iNOS expression in 3T3 fibroblasts (44), diminishes iNOS expression in response to cytokines in cardiac myocytes (34,36,37) as well as microvascular endothelial cells (45) and vascular smooth muscle cells (46). Perrella et al. (47) demonstrated that the ability of TGF-␤ to suppress iNOS promoter/enhancer activity occurs through a site(s) other than the NF-B motif in vascular smooth muscle cells. In the present study, anti-TGF-␤ antibody did not alter the suppressive effect of mechanical strain on iNOS mRNA expression in cytokine-stimulated cardiac myocytes. It is therefore unlikely that TGF-␤ is the initial mediator for the suppressive effects of mechanical strain on iNOS mRNA expression. Furthermore, cycloheximide partially inhibited the effect of mechanical strain. These findings suggest that mechanical strain directly acts on iNOS gene expression in cardiac myocytes without new paracrine protein synthesis.
Protein kinase C pathways activated by mechanical strain play important roles in hypertrophy and gene expression in cardiac myocytes (19). Stretch-induced c-fos expression is inhibited by both protein kinase C inhibitors and down-regulation of protein kinase C (15,19). Recently, Obadia et al. (48) reported that protection against myocardial ischemia in rats could be induced by stretch, likely through activation of protein kinase C. On the other hand, many factors appear to be involved in the complex regulation of iNOS induction in response to cytokines (49 -53). In the present study, protein kinase C inhibitors and pretreatment with PMA abolished the suppres- FIG. 7. Effects of TGF-␤ and anti-TGF-␤ antibody on suppression of iNOS mRNA expression by mechanical strain. NRVM were plated on 2 g/ml fibronectin in DMEM containing 7% FCS for 48 h. After serum deprivation for 24 h, myocytes were exposed for 8 h to 0 or 9% cyclic mechanical strain (1 Hz) with or without IL-1␤ (4 ng/ml) ϩ IFN-␥ (100 units/ml) in the presence or absence of TGF-␤ (1 ng/ml), anti-TGF-␤ antibody (Ab; 30 g/ml), or TGF-␤ ϩ anti-TGF-␤ antibody. Total RNA was isolated and analyzed by Northern blotting with 32 Plabeled iNOS (upper panel) and GAPDH (G3PDH; lower panel) cDNA probes. Data are representative of two independent experiments with nearly identical results.
FIG. 8. Effects of protein kinase C and tyrosine kinase inhibitors on suppression of iNOS mRNA expression by mechanical strain. NRVM were plated on 2 g/ml fibronectin in DMEM containing 7% FCS for 48 h. After serum deprivation for 24 h, myocytes were exposed for 8 h to 0 or 9% cyclic mechanical strain (1 Hz) with or without IL-1␤ (4 ng/ml) ϩ IFN-␥ (100 units/ml) in the presence or absence of staurosporine (100 nM), calphostin C (1 M), or genistein (100 M). These inhibitors were applied to the myocytes 30 min before mechanical strain. Total RNA was isolated and analyzed by Northern blotting with 32 P-labeled iNOS (upper panel) and GAPDH (G3PDH; lower panel) cDNA probes. Data are representative of two independent experiments with nearly identical results.
FIG. 9. Effects of pretreatment with PMA on suppression of iNOS mRNA expression by mechanical strain. NRVM were plated on 2 g/ml fibronectin in DMEM containing 7% FCS for 48 h. After serum deprivation and pretreatment with or without PMA (1 M) for 24 h, myocytes were exposed for 8 h to 0 or 9% cyclic mechanical strain (1 Hz) with or without IL-1␤ (4 ng/ml) ϩ IFN-␥ (100 units/ml). Total RNA was isolated and analyzed by Northern blotting with 32 P-labeled iNOS (upper panel) and GAPDH (G3PDH; lower panel) cDNA probes. Data are representative of two independent experiments with nearly identical results. sive effect of mechanical strain on iNOS expression in cytokinestimulated cardiac myocytes, suggesting that this effect is mediated at least partially via activation of protein kinase C.
In conclusion, cyclic mechanical strain suppresses cytokineinduced iNOS expression in cardiac myocytes, at least partially via activation of protein kinase C. This mechanism may play a role in the pathophysiology of myocardial infarction, post-cardiac transplantation, cardiomyopathy, myocarditis, and congestive heart failure. In addition, these data demonstrate that mechanical stimuli are potent regulators of the molecular response of myocytes to cytokines.