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Am J Physiol Heart Circ Physiol 276: H1968-H1976, 1999;
0363-6135/99 $5.00
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Vol. 276, Issue 6, H1968-H1976, June 1999

Angiotensin II and mechanical stretch induce production of tumor necrosis factor in cardiac fibroblasts

Tomoyuki Yokoyama1, Kenichi Sekiguchi1, Toru Tanaka1, Koichi Tomaru1, Masashi Arai1, Tadashi Suzuki2, and Ryozo Nagai1

1 Second Department of Internal Medicine and 2 Department of Laboratory Sciences, Gunma University School of Medicine, Maebashi 371, Japan


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

To determine whether ANG II as well as mechanical stress affect the production of tumor necrosis factor (TNF) in the heart, neonatal rat cardiac myocytes and fibroblasts were cultured separately and treated for 6 h with ANG II, lipopolysaccharide (LPS), or cyclic mechanical stretch. LPS induced the production of TNF in cardiac myocytes and fibroblasts. However, TNF synthesis in fibroblasts was 20- to 40-fold higher than in myocytes. ANG II (>= 10-8 M) and mechanical stretch stimulated the production of TNF in cardiac fibroblasts but not in myocytes. Furthermore, both ANG II and LPS increased the expression of TNF-alpha mRNA in cardiac fibroblasts. Isoproterenol inhibited both LPS- and ANG II-induced production of TNF in cardiac fibroblasts with increasing intracellular cAMP level. Moreover, both isoproterenol and dibutyryl cAMP inhibited LPS-induced TNF-alpha mRNA expression. Thus activation of the renin-angiotensin system, as well as mechanical stress, can stimulate production of TNF in cardiac fibroblasts. Furthermore, beta -adrenergic receptors may be responsible for the regulation of TNF synthesis at the transcriptional level by elevating intracellular cAMP.

myocyte; rat; isoproterenol; adenosine 3',5'-cyclic monophosphate


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

TUMOR NECROSIS FACTOR (TNF), which was originally described as a protein causing necrosis in certain transplantable mouse tumors, is now recognized as a cytokine that possesses multiple biological functions in different cell types (2, 32). Recent clinical observations indicate that the concentration of TNF is elevated in patients with advanced heart failure or hypertrophic cardiomyopathy (21-23). We previously demonstrated (36) that acute exposure to TNF causes a negative inotropic effect in the ventricle and in isolated adult cardiac myocytes, can increase cardiac protein synthesis, and causes cardiac hypertrophy (35). Furthermore, chronic infusion of TNF in rats produces left ventricular contractile dysfunction and dilation (3). However, the exact source and stimulus for TNF production in the setting of heart failure and cardiac hypertrophy are poorly understood. Recent experimental studies showed that the adult mammalian myocardium produces biologically active TNF in response to pressure overload (18). It has been shown in clinical studies that the circulating TNF concentrations increase in cachectic patients with chronic heart failure. This increase in TNF is associated with marked activation of the renin-angiotensin system in patients with end-stage cardiac disease (21). Furthermore, ANG II induces TNF production in isolated tubules from rat medullary thick ascending limb (8).

The role of TNF in the progression of heart failure and hypertrophic cardiomyopathy led us to hypothesize that ANG II, as well as mechanical stress, may affect the production of TNF in the myocardium. We therefore examined TNF production in isolated cardiac myocytes and cardiac fibroblasts after stimulation with ANG II and compared it with TNF production induced by lipopolysaccharide (LPS) or mechanical stretching. Because LPS-induced TNF release was shown to be modulated in macrophages and monocytes by alpha - and beta -adrenergic receptors (15, 25, 27), additional studies were performed to evaluate the effect of beta -adrenergic stimulation on TNF production induced by LPS or ANG II.


    METHODS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Reagents. Cell culture reagents and fetal calf serum were obtained from Life Technologies (Gaithersburg, MD). dl-Isoproterenol hydrochloride, dl-propranolol, and l-phenylephrine hydrochloride were obtained from Wako Pure Chemical Industries (Osaka, Japan). Dibutyryl cAMP was from Sigma (St. Louis, MO). Phentolamine was kindly provided by CIBA-GEIGY (Basel, Switzerland).

Preparation of neonatal rat cardiac myocytes. Primary neonatal rat cardiac ventricular myocyte cultures were prepared by the method of Simpson and Savion (26) with minor modifications. Briefly, ventricles were excised from 1- to 2-day-old Wistar rats anesthetized with ethyl ether and minced with scissors in Ca2+-free Krebs-Henseleit buffer (KHB; in mM: 118 NaCl, 4.0 KCl, 1.2 MgCl2, 1.1 KH2PO4, 25 NaHCO3, 5.0 glucose, and 20 HEPES, pH 7.4). The cells were dispersed with 0.05% trypsin and 0.05% collagenase type II in KHB. The cells were stirred with the use of a small magnetic stirrer bar for 10 min at 37°C, the supernatants were transferred to cold DMEM containing 10% fetal calf serum, and the digestion was repeated four times. The resulting cell suspension was centrifuged, and the pellet was resuspended in DMEM containing 10% fetal calf serum.

The cells were plated in culture flasks for 1.5 h to remove nonmyocytes. The unattached cells were removed and seeded (8 × 105 cells) in 35-mm gelatin-coated culture dishes in DMEM containing 10% fetal calf serum, 100 U/ml penicillin, and 100 µg/ml streptomycin. After incubation for 48 h at 37°C with 5% CO2, the medium was replaced with DMEM containing 10 µg/ml insulin, 5.5 µg/ml sodium transferrin, 6.7 ng/ml sodium selenite, 2.0 µg/ml ethanolamine, 0.1% BSA, and the above antibiotics, and the cells were incubated for another 24 h. Using this method, we routinely obtained cardiac myocyte-rich cultures with >95% of the cells being cardiac myocytes, as assessed by immunohistochemical staining with a monoclonal antibody against sarcomeric alpha -actinin (Sigma).

Preparation of cardiac fibroblasts. Neonatal rat cardiac fibroblasts were prepared using the method of Kinugawa et al. (19) with minor modifications. Adherent cells obtained during the preplating procedure described in Preparation of neonatal rat cardiac myocytes were cultured in the same culture media as the myocytes. After the second passage, cells were plated (2 × 105 cells) in 35-mm culture dishes and grown in medium containing 10% fetal calf serum. Two days later the medium was replaced with the same serum-free medium used for the myocyte culture, and the cells were incubated for another 24 h. In this nonmyocyte culture, <0.5% of the cells contained sarcomeric alpha -actinin. Immunostaining with an antibody against von Willebrand factor (DAKO, Glostrup, Denmark) revealed that <0.1% of cells were endothelial cells. The nonmyocyte cells did not stain with an antibody directed against smooth muscle alpha -actin (DAKO). These findings suggest that >99% of the cells in the nonmyocyte culture were cardiac fibroblasts.

Stimulation of cardiac myocytes and fibroblasts. The cardiac myocyte and fibroblast cultures were incubated in the presence or absence of isoproterenol and/or propranolol plus either LPS (Escherichia coli 0111:B4; Sigma) or ANG II (Sigma). After 6 h of incubation at 37°C, cell-free supernatants were collected and stored at -80°C until the time of analysis. The cells were solubilized in 500 µl of 0.5% SDS, 1 mM dithiothreitol, and 50 mM Tris · HCl, pH 7.5. Protein concentrations were measured using a commercially available kit [bicinchoninic acid (BCA), Pierce, Rockford, IL] using BSA as a standard.

Mechanical stretch of cardiac myocytes and fibroblasts. Cyclic mechanical stretch was performed in vitro using a Flexcell strain unit (Flexcell International, McKeesport, PA; Ref. 1). Cardiac myocytes or fibroblasts were cultured in six-well plates with flexible collagen-coated silicone rubber membranes at the bottom of each well. A vacuum (~28 kPa) was applied at a frequency of 6 cycles/min (4-s on time, 6-s off time) to the flexible membrane from the base of the plate. The maximal percent elongation of the culture surface was 18%. In a preliminary experiment, we measured the rate of protein synthesis in cardiac myocytes exposed to cyclic mechanical stretch for 6 h. [3H]phenylalanine incorporation in cardiac myocytes stimulated with mechanical stretch was 40.8 ± 10.8% (mean ± SE) higher than that in control cells. This effect was similar to the previous observation using neonatal rat cardiac myocytes stimulated with simple linear stretch (20). Thus the cyclic mechanical stretch using a Flexcell strain unit may be sufficient to cause stretched myocytes to produce relevant proteins.

TNF assay. Supernatants were tested for the presence of TNF using a colorimetric assay for L929 cell viability according to previously published protocols (11). Briefly, L929 cells were plated in 96-well flat-bottomed plates at a density of 1 × 104 cells/well in DMEM containing 10% fetal calf serum. The cells were incubated overnight and then were incubated for an additional 24 h in the presence of three different dilutions of supernatants containing actinomycin D (1 µg/ml, Boehringer Mannheim, Mannheim, Germany). At the end of the incubation period, the medium was removed and cells were stained for 10 min with 0.2% crystal violet in 12% Formalin and 10% ethanol. The cells were then washed with water and solubilized with ethanol in phosphate buffer. The absorbance of each well at 550 nm was determined using a Vmax Kinetic Microplate Reader (Molecular Devices, Menlo Park, CA). A TNF-alpha standard was kindly provided by Dainihon Pharmaceutical (Suita, Japan). The lowest value for the standard curve was 15 pg/ml. In a preliminary experiment, to determine whether the cytolytic effect on L929 cells was caused by TNF-alpha , samples from cardiac fibroblasts treated with 10 ng/ml LPS were incubated in both the presence and the absence of anti-rat TNF-alpha antibody (R&D Systems, Minneapolis, MN) capable of neutralizing 10 ng/ml of rat TNF-alpha . The addition of the anti-TNF-alpha antibody for 2 h at 37°C caused >88% inhibition of the observed cytolytic effect in the samples. However, this bioassay did not differentiate between the various forms of TNF (i.e., TNF-alpha , TNF-beta ). Furthermore, to facilitate the comparison between cell isolations, the concentration of TNF in samples was normalized by the total protein concentration of cells. Therefore, all results were expressed as picograms of TNF per milligram of protein.

cAMP assay. Cyclic nucleotides were extracted using cold 6% trichloroacetic acid, and cAMP was measured using a commercially available kit (Amersham, Amersham, UK).

Northern blot analysis of TNF mRNA. Cardiac fibroblast cultures (1 × 106 cells/100-mm culture dish) were incubated with LPS, ANG II, or diluent for 4 h at 37°C in DMEM supplemented with 0.1% BSA. After incubation, the cells were harvested and total cellular RNA was isolated using the acid guanidinium thiocyanate-phenol-chloroform extraction method (5). RNA (20 µg) was fractionated on 1.2% formaldehyde-agarose gels and capillary transferred onto nylon membranes. The blots were prehybridized for 4 h at 42°C in 40% formamide, 0.1% SDS, 5× SSC (1× SSC is 0.15 M NaCl and 0.015 M sodium citrate, pH 7.0), 5× Denhardt's solution (0.1% Ficoll, 0.1% BSA, and 0.1% polyvinylpyrrolidone), and 0.01 mg/ml denatured salmon sperm DNA. Hybridization was carried out at 42°C for 12 h using a restriction fragment labeled via nick translation using a random primer DNA labeling kit obtained from Boehringer Mannheim. The restriction fragment used as the probe included mouse TNF-alpha DNA (4). The nylon membranes were stained with methylene blue after transfer and photographed. After hybridization, the blots were washed two or three times with 0.1% SDS-2× SCC at 42°C and exposed to Kodak X-OMAT AR film at -80°C.

Statistical analysis. Values are expressed as means ± SE. One-way analysis of variance was used to evaluate differences between groups. Where appropriate, post hoc multiple-comparison tests were performed to evaluate differences between the control and experimental groups. A P value <0.05 was accepted as statistically significant.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

TNF production in cardiac fibroblasts after stimulation with LPS, ANG II, or mechanical stretch. The production of TNF from LPS-stimulated cardiac fibroblasts was found to be concentration dependent for LPS concentrations between 0.01 and 10 ng/ml (Fig. 1). To determine whether ANG II could stimulate the production of TNF in cardiac fibroblasts, cells were incubated for 6 h with ANG II (10-9-10-5 M). As shown in Fig. 2, ANG II also stimulated the production of TNF in a concentration-dependent manner. The production of TNF by 10-8 M ANG II was significantly higher than in control cells. However, the production of TNF induced by 10-5 M ANG II was similar to that induced by a concentration of LPS between 0.001 and 0.01 ng/ml.


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  		Fig. 1.   Tumor necrosis (TNF) production in cardiac fibroblasts after stimulation with lipopolysaccharide (LPS). Values represent means ± SE for 6 cultures/group. In comparison with control fibroblasts (Cont; open bar), treatment with LPS (filled bars) resulted in concentration-dependent increase in TNF production. * P < 0.05 in comparison with control value.



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  		Fig. 2.   TNF production in cardiac fibroblasts after stimulation with ANG II. Values represent means ± SE for 7 cultures/group. In comparison with control fibroblasts (open bar), treatment with ANG II (filled bars) resulted in concentration-dependent increase in TNF production. * P < 0.05 in comparison with control value.

To determine whether mechanical stretch stimulates the production of TNF in cardiac fibroblasts, we cultured cells on collagen-coated flexible silicone rubber supports and then applied cyclic strain for 6 h. As shown in Fig. 3, cyclic mechanical strain caused a threefold increase in TNF production in cardiac fibroblasts compared with quiescent control cells. The amount of TNF produced by mechanical stretch was similar to that induced by a concentration of LPS between 0.001 and 0.01 ng/ml.


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  		Fig. 3.   TNF production in cardiac fibroblasts and myocytes after stimulation with in vitro cyclic mechanical stretch. Values represent means ± SE for 8 cultures/group. In comparison with control fibroblasts, treatment with mechanical stretch resulted in significant increase in TNF production (* P < 0.0001). In contrast, treatment with mechanical stretch did not induce production of TNF in cardiac myocytes.

In this study, TNF production was measured using a bioassay. Thus we could not exclude the possibility that LPS, ANG II, or mechanical stretch increased the levels of immunoreactive TNF that was bound by soluble TNF receptors and hence biologically inactive. This was a potential limitation of this study.

TNF production in cardiac myocytes after stimulation with LPS, ANG II, or mechanical stretch. The production of TNF by LPS-stimulated cardiac myocytes was found to be concentration dependent for LPS concentrations between 10 and 100 ng/ml (Fig. 4). However, our cardiac myocyte preparation included ~5% nonmyocytes, and the same concentration of LPS induced a 20- to 40-fold greater production of TNF in cardiac fibroblasts than in myocytes (Fig. 1). Therefore, we could not exclude the possibility that the production of TNF in cardiac myocyte cultures resulted from contamination by nonmyocytes. We examined the production of TNF in cardiac myocytes stimulated with either ANG II (10-7-10-5 M) or mechanical stretch for 6 h. Neither ANG II nor mechanical stretch induced TNF production in cardiac myocyte cultures (Table 1 and Fig. 3).


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  		Fig. 4.   TNF production in cardiac myocytes after stimulation with LPS. Values represent means ± SE for 6 cultures/group. In comparison with control myocytes (open bar), treatment with LPS (filled bars) resulted in concentration-dependent increase in TNF production. * P < 0.05 in comparison with control value.


                              
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  		Table 1.   TNF production in cardiac myocytes after stimulation with angiotensin II

Effects of isoproterenol and phenylephrine on TNF production in cardiac fibroblasts after stimulation with LPS or ANG II. On the basis of the above observations, we concluded that TNF production is higher in cardiac fibroblasts than in cardiac myocytes. Therefore, additional experiments were performed using cardiac fibroblast cultures to determine the role of alpha - and beta -adrenergic-receptor stimulation in TNF production induced by LPS or ANG II.

To address whether alpha 1- and beta -adrenergic receptors have the capacity to regulate production of TNF induced by LPS in cardiac fibroblasts, cells were incubated with the beta -adrenergic-receptor agonist isoproterenol (10-8-10-5 M) or the alpha 1-adrenergic-receptor agonist phenylephrine (10-8-10-4 M) in addition to LPS (10 ng/ml) for 6 h. Neither isoproterenol nor phenylephrine induced TNF production in the absence of LPS (data not shown). In contrast, isoproterenol caused a concentration-dependent inhibition of LPS-induced TNF production (Fig. 5), which was statistically significant at an isoproterenol concentration >= 10-6 M. Phenylephrine also inhibited LPS-induced TNF production (Fig. 6). However, this inhibition was observed only at high concentrations (10-5 and 10-4 M) of phenylephrine (Fig. 6).


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  		Fig. 5.   Effect of isoproterenol on TNF production in cardiac fibroblasts after stimulation with LPS. Values represent means ± SE for 8 cultures/group. In comparison with LPS + 0 isoproterenol group, in which cells were treated with 10 ng/ml LPS but not isoproterenol (open bar), treatment with isoproterenol (filled bars) resulted in concentration-dependent decrease in LPS-induced production of TNF. * P < 0.05 in comparison with value of LPS + 0 isoproterenol group.



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  		Fig. 6.   Effect of phenylephrine on TNF production in cardiac fibroblasts after stimulation with LPS. Values represent means ± SE for 4 cultures/group. In comparison with LPS + 0 phenylephrine group, in which cells were treated with 10 ng/ml LPS but not phenylephrine (open bar), treatment with high concentrations of phenylephrine (filled bars) resulted in significant decrease in LPS-induced production of TNF. * P < 0.05 in comparison with value of LPS + 0 phenylephrine group.

To determine whether the inhibition of TNF production by isoproterenol and phenylephrine was mediated by alpha 1- or beta -adrenergic receptors, cardiac fibroblasts were incubated for 6 h with LPS (10 ng/ml) and phenylephrine (10-5 M) or isoproterenol (10-6 M) in the presence or absence of the alpha -adrenergic-receptor antagonist phentolamine (10-6 M) or the beta -adrenergic-receptor antagonist propranolol (10-6 M). In the absence of isoproterenol and phenylephrine, neither phentolamine nor propranolol affected LPS-induced TNF production. Propranolol completely prevented the inhibitory effect of isoproterenol, and the concentration of TNF after incubation with LPS in the presence of isoproterenol and propranolol was similar to that after incubation with LPS alone (Fig. 7). In contrast, phentolamine did not affect the inhibitory effect of phenylephrine, whereas propranolol completely prevented the effect of phenylephrine (Fig. 8). These results suggest that relatively high concentrations of phenylephrine inhibit LPS-induced TNF production through beta -adrenergic-receptor activation.


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  		Fig. 7.   Propranolol attenuated inhibitory effect of isoproterenol on LPS-induced production of TNF in cardiac fibroblasts. Values represent means ± SE for 4 cultures/group. In comparison with LPS + 0 isoproterenol group, in which cells were treated with 10 ng/ml LPS but not isoproterenol (open bar), 10-6 M isoproterenol inhibited LPS-induced production of TNF (* P < 0.0001). Propranolol (10-6 M) completely prevented this inhibitory effect of isoproterenol. Propranolol itself did not have any effect on LPS-induced production of TNF.



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  		Fig. 8.   Propranolol attenuated inhibitory effect of phenylephrine on LPS-induced production of TNF in cardiac fibroblasts. Values represent means ± SE for 4 cultures/group. In comparison with LPS + 0 phenylephrine group, in which cells were treated with 10 ng/ml LPS but not phenylephrine (open bar), 10-5 M phenylephrine inhibited LPS-induced production of TNF (* P < 0.0001). Phentolamine (10-6 M) did not have any effect on this inhibitory effect of phenylephrine (* P < 0.0001 in comparison with LPS + 0 phenylephrine group). In contrast, 10-6 M propranolol completely prevented this inhibitory effect of phenylephrine.

To determine whether isoproterenol inhibits the production of TNF induced by ANG II, cardiac fibroblasts were incubated with isoproterenol (10-8-10-5 M) plus ANG II (10-6 M) for 6 h. Isoproterenol inhibited ANG II-induced TNF production in a concentration-dependent manner, which was statistically significant at an isoproterenol concentration >= 10-8 M (Fig. 9).


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  		Fig. 9.   Effect of isoproterenol on TNF production in cardiac fibroblasts after stimulation with ANG II. Values represent means ± SE for 4 cultures/group. In comparison with ANG II + 0 isoproterenol group, in which cells were treated with 10-6 M ANG II but not isoproterenol (open bar), treatment with isoproterenol (filled bars) resulted in concentration-dependent decrease in ANG II-induced production of TNF. * P < 0.05 in comparison with value of ANG II + 0 isoproterenol group.

TNF mRNA accumulation in cardiac fibroblasts stimulated with LPS or ANG II. To determine whether LPS or ANG II increased TNF mRNA expression, cardiac fibroblasts were incubated with LPS (10 ng/ml) or ANG II (5 × 10-6 M) for 4 h. Northern blot analysis was then performed on RNA extracts. Both LPS and ANG II increased TNF mRNA expression (Fig. 10). To determine whether isoproterenol mediated LPS-induced TNF mRNA synthesis, cardiac fibroblasts were incubated for 4 h with LPS (10 ng/ml) in the presence of isoproterenol (10-6 M). Isoproterenol inhibited the TNF mRNA accumulation induced by LPS (Fig. 11A). As shown in Fig. 11B, isoproterenol inhibited 68% of the TNF mRNA accumulation induced by LPS. This inhibitory effect of isoproterenol was similar to the above-mentioned observation in which isoproterenol inhibited 77% of TNF protein production induced by LPS.


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  		Fig. 10.   Northern blot analysis showing effect of ANG II (5 × 10-6 M, 4 h) and LPS (10 ng/ml, 4 h) on expression of TNF-alpha mRNA in cardiac fibroblasts. Expression of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA was used as internal control. cDNA for TNF-alpha hybridized to bands at 1.7 kb. Both ANG II (lane 2) and LPS (lane 3) increased TNF-alpha mRNA in comparison with control cells (lane 1). Similar results were obtained in 3 additional experiments.




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  		Fig. 11.   Northern blot analysis showing effect of isoproterenol (10-6 M, 4 h) on LPS-induced expression of TNF-alpha mRNA in cardiac fibroblasts. Expression of GAPDH mRNA was used as internal control. cDNA for TNF-alpha hybridized to bands at 1.7 kb. A: lane 1, control cells; lane 2, LPS-stimulated cells; lane 3, isoproterenol + LPS-treated cells. B: group data results, in which relative optical density of hybridization signal for TNF-alpha is normalized to hybridization signal for GAPDH. Values represent means ± SE for 4 experiments/group. Isoproterenol attenuated LPS-induced expression of TNF-alpha mRNA.

Effects of isoproterenol and phenylephrine on cAMP content in cardiac fibroblasts. To confirm that both isoproterenol and higher concentrations of phenylephrine inhibit LPS-induced TNF production through beta -adrenergic-receptor activation, we assayed cAMP levels in cardiac fibroblasts after addition of 10-6 M isoproterenol or 10-5 M phenylephrine. Treatment with isoproterenol resulted in a significant increase in the cAMP level compared with control cells. A higher concentration of phenylephrine also significantly increased the cAMP level (Fig. 12).


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  		Fig. 12.   Effect of isoproterenol and phenylephrine on cAMP levels in cardiac fibroblasts. Values represent means ± SE for 6 cultures/group. In comparison with control cells, treatment with isoproterenol resulted in significant increase in cAMP level. A higher concentration of phenylephrine also significantly increased cAMP level. * P < 0.05 in comparison with control value.

Effect of dibutyryl cAMP in TNF production and TNF mRNA accumulation in cardiac fibroblasts stimulated with LPS. On the basis of the observation that both isoproterenol and a higher concentration of phenylephrine increased intracellular cAMP levels in cardiac fibroblasts, we hypothesized that beta -adrenergic-receptor activation inhibits TNF production in cardiac fibroblasts via increased intracellular cAMP levels. Thus we examined whether exogenous administration of dibutyryl cAMP suppresses TNF production and/or TNF-alpha mRNA expression induced by LPS. Cardiac fibroblasts were incubated with dibutyryl cAMP (0.1 mM) for 15 min before the addition of LPS (10 ng/ml) stimulation. A total of 96% of LPS-induced TNF production was inhibited by dibutyryl cAMP (Fig. 13). Dibutyryl cAMP also markedly inhibited the TNF-alpha mRNA expression induced by LPS (Fig. 14).


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  		Fig. 13.   Effect of dibutyryl cAMP on TNF production in cardiac fibroblasts after stimulation with LPS. Cells were treated with dibutyryl cAMP for 15 min before addition of LPS. Values represent means ± SE for 5 cultures/group. In comparison with LPS + 0 dibutyryl cAMP group, in which cells were treated with LPS but not dibutyryl cAMP (open bar), treatment with dibutyryl cAMP (filled bar) resulted in significant decrease in LPS-induced production of TNF. * P = 0.013 in comparison with value of LPS + 0 dibutyryl cAMP group.



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  		Fig. 14.   Northern blot analysis showing effect of dibutyryl cAMP (0.1 mM) on LPS (10 ng/ml, 4 h)-induced expression of TNF-alpha mRNA in cardiac fibroblasts. Cells were treated with dibutyryl cAMP for 15 min before addition of LPS. Expression of GAPDH mRNA was used as internal control. cDNA for TNF-alpha hybridized to bands at 1.7 kb. Lane 1, control cells; lane 2, LPS-stimulated cells; lane 3, dibutyryl cAMP on LPS-treated cells. Dibutyryl cAMP markedly attenuated LPS-induced expression of TNF-alpha mRNA. Similar results were obtained in 2 additional experiments.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Clinical reports showed TNF mRNA and protein expression in the ventricles of patients with dilated or ischemic cardiomyopathy (12, 30). In contrast, explanted hearts from normal organ donors do not express TNF mRNA or protein (30). In experimental studies, myocardial TNF mRNA expression and protein production were increased in response to LPS stimulation or pressure overloading (17, 18, 34). These observations suggest that TNF expression is limited in the normal heart but increases in response to certain humoral factors or mechanical stress.

Activation of the immune system is generally responsible for increased TNF production. However, heart failure and cardiac hypertrophy are not associated with immune activation. Only one animal study has demonstrated that the adult mammalian heart produces biologically active TNF in response to pressure overloading (18).

It is now well established that activation of the circulating and tissue renin-angiotensin systems is involved in the pathophysiology of heart failure. In clinical studies, the plasma ANG II concentration increased in patients with asymptomatic heart failure and increased further in patients with overt heart failure (9). In hearts from animals with myocardial hypertrophy or heart failure, increased expression of angiotensin-converting enzyme (ACE) was noted (14, 24). In this study, we observed that cardiac fibroblasts produced biologically active TNF after stimulation with either ANG II or mechanical stretch. It is therefore conceivable that activation of the circulating or tissue renin-angiotensin system during heart failure can stimulate the production of TNF in the heart. Interestingly, ACE inhibitors suppress TNF production both in vitro and in vivo (10).

In previous in vitro studies, both adult cardiac myocytes and nonmyocytes were shown to synthesize TNF mRNA and protein after stimulation with LPS or pressure overloading (17, 18). The TNF protein content in nonmyocytes after stimulation was ~1.3-fold higher than in cardiac myocytes in those studies. However, the present study demonstrated that TNF protein synthesis in cardiac fibroblasts is 20- to 40-fold higher than in cardiac myocytes. Furthermore, both ANG II and mechanical stretch stimulated the release of TNF from cardiac fibroblasts but not from myocytes. Although the precise reason(s) for the discrepant findings of the present study and previous studies of adult cells is not apparent, it is likely that the differences may relate, at least in part, to developmental differences between neonatal and adult cell preparations (6). Furthermore, a previous study showed that neonatal and adult rat cardiac fibroblasts in culture express AT1 receptors for ANG II (33). ANG II stimulation of AT1 receptors results in increased gene expression for extracellular matrix proteins (33). However, no information regarding the difference of ANG II receptor numbers between cardiac myocytes and fibroblasts was available. Thus we could not exclude the possibility that ANG II induced the production of TNF in fibroblasts because of increased receptors in these cells compared with cardiac myocytes. Although we could not exclude the possibility that cardiac myocytes produce TNF, we believe that the cardiac fibroblasts play a much greater role in TNF synthesis in the setting of pathological conditions, e.g., heart failure or cardiac hypertrophy. Our hypothesis is supported by previous observations that the number of nonmyocytes exceeds the number of myocytes in the normal heart (37) and, further, that nonmyocytes increase in number in pathological conditions including pressure-overload hypertrophy and infarction (16).

In the present study, we showed that ANG II stimulates TNF mRNA expression and the production of biologically active TNF in cardiac fibroblasts in a concentration-dependent manner. However, the amount of TNF produced by stimulation with ANG II was less than that induced by LPS. The serum TNF concentration in the setting of various human cardiac diseases is usually less than several hundred picograms per milliliter (21, 23). In addition, the TNF protein content of right atrial specimens obtained from patients with severe heart failure was <5 pg/mg protein (7). The TNF level induced by stimulation with ANG II or mechanical stretch is similar to the TNF concentrations reported in the above-mentioned clinical studies.

In contrast to their well-documented physiological effects on the cardiovascular system, the effects of alpha - and beta -adrenergic-receptor agonists on myocardial cytokine production are not known. We have shown for the first time that the nonspecific beta -adrenergic-receptor agonist isoproterenol inhibits the LPS-induced production of TNF in cardiac fibroblasts. This is in keeping with previous observations in macrophage cultures (15) and whole blood cell preparations (25). Furthermore, ANG II-induced production of TNF is also inhibited by isoproterenol. Although high concentrations of the selective alpha 1-adrenergic-receptor agonist phenylephrine inhibited the LPS-induced production of TNF in cardiac fibroblasts, this effect was blocked by the beta -adrenergic-receptor antagonist propranolol but not by the alpha -adrenergic-receptor antagonist phentolamine. Furthermore, both isoproterenol and a higher concentration of phenylephrine increased intracellular cAMP levels in cardiac fibroblasts. Exogenous administration of dibutyryl cAMP also inhibited the LPS-induced production of TNF. Therefore, beta - but not alpha 1-adrenergic receptors may be responsible for the regulation of TNF synthesis in cardiac fibroblasts.

The biosynthesis of TNF is believed to be regulated primarily at the translational level (13), because TNF mRNA is constitutively expressed by a variety of tissues (31). However, it remains unclear whether the inhibitory effect of beta -adrenergic-receptor activation on TNF production is controlled at the transcriptional level or the translational level (25, 28). In this study, we demonstrated that both isoproterenol and dibutyryl cAMP inhibit not only the release of TNF but also TNF mRNA synthesis in cardiac fibroblasts after stimulation with LPS. Furthermore, previous work using macrophage cultures showed that treatment with dibutyryl cAMP also strongly suppresses LPS-induced TNF mRNA expression in a concentration-dependent manner (29). Thus beta -adrenergic-receptor agonists may regulate TNF synthesis in cardiac fibroblasts at the transcriptional level by elevating intracellular cAMP.

In conclusion, recent reports demonstrated that the cardiac myocyte may be an important source of TNF production in response to LPS stimulation or pressure overloading (17, 18, 34). However, we found that cardiac fibroblasts synthesize a greater amount of biologically active TNF than cardiac myocytes after stimulation with LPS, ANG II, or mechanical stretch. We therefore hypothesize that TNF production in cardiac fibroblasts may act on cardiac myocytes in a paracrine fashion in the setting of congestive heart failure or cardiac hypertrophy. We also found that the production of TNF in cardiac fibroblasts is inhibited by beta -adrenergic receptor stimulation. Thus cardiac myocyte and fibroblast interactions via angiotensin, catecholamines, and cytokines are likely to be important in the progression of heart failure and cardiac hypertrophy.


    FOOTNOTES

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Address for reprint requests and other correspondence: T. Yokoyama, Second Dept. of Internal Medicine, Gunma Univ. School of Medicine, 3-39-22, Showa-machi, Maebashi 371, Japan (E-mail: yokoyamt{at}news.sb.gunma-u.ac.jp).

Received 17 August 1998; accepted in final form 8 February 1999.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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Am J Physiol Heart Circ Physiol 276(6):H1968-H1976
0002-9513/99 $5.00 Copyright © 1999 the American Physiological Society


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