INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

CARDIAC FIBROBLASTS (CFbs) play a crucial role in the regulation of extracellular matrix (ECM) metabolism in the heart by production of ECM components such as collagen as well as by producing enzymes that can either degrade or inhibit the degradation of ECM (34, 39). In the normal state, CFbs are generally quiescent and long lived so that the CFb population remains relatively stable. However, in pathological circumstances associated with cardiac remodeling, such as myocardial infarction or heart failure, CFbs can enter the cell cycle and proliferate, thereby altering the balance of cell populations in the heart (10). Proliferation of CFbs and production of ECM by CFbs play an important role in infarct healing following acute coronary occlusion (38). However, in noninfarcted myocardium, proliferation of CFbs and/or excess production of ECM can increase the stiffness of the ventricular wall, thereby leading to diastolic dysfunction (39). Conversely, insufficient fibroblast proliferation and matrix deposition after injury results in an abnormally thin ventricular wall at risk of rupture. Therefore, identifying factors that regulate the size of the CFb population is important for understanding the mechanisms involved in physiological repairs and during pathological remodeling of the left ventricle. One such factor that may be involved in the regulation of cell viability and proliferation of cardiac fibroblasts is nitric oxide (NO) produced by inducible NO synthase (iNOS), because NO has been demonstrated to induce cell death in several other cell types, including myocytes and smooth muscle cells (15, 25). iNOS expression is usually undetectable in the normal heart.

Myocardial infarction, allograft rejection, endotoxemia, or heart failure have been associated with increased levels of inflammatory cytokines, a number of which has been shown to induce iNOS expression in inflammatory and noninflammatory cells in the heart (4, 5, 23, 27). In the heart, neonatal CFbs have been shown to express iNOS in response to cytokines (11), although it is uncertain whether adult CFbs retain this ability. Furthermore, whether and how iNOS expression in adult CFbs might influence cell viability is not completely defined. Therefore, the aim of this study was to determine whether adult cardiac fibroblasts are able to express iNOS in response to cytokine stimulation and examine the impact of iNOS expression and exogenous NO on CFb viability. In several cell types, considerable data indicate that the proapoptotic effect of NO largely results from the formation of peroxynitrite (ONOO) (4, 13, 21). Thus this study also examined whether peroxynitrite formation is required for NO to act as a toxic effector in CFbs. To study these issues, primary cultures of CFbs isolated from adult rat hearts were treated with inflammatory cytokines or directly exposed to NO donors. We show here that IL-1 induces iNOS expression and NO production in adult CFbs, and this is associated with an increase in p53 and Bax expression, an increase in caspase-3 activity, and apoptotic cell death.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Reagents. Mouse recombinant IL-1, IFN-, TNF-, caspase-3 colorimetric assay kit, and caspase-3 inhibitor [Z-Asp-Glu-Val-Asp-fluoromethyl ketone (Z-DEVD-FMK)] were purchased from R&D Systems (Minneapolis, MN). The anti-iNOS antibody was from Transduction Laboratories (Lexington, KY). The antibodies against Bcl-2, Bax, Bcl-x_L, p53, and caspase-3 were from Santa Cruz Biotechnology (Santa Cruz, CA). The transferase-mediated dUTP nick-end labeling (TUNEL)-based staining kit was from Boehringer Mannheim (Indianapolis, IN). The NO donors diethylenetriaminepentaacetic acid (DPTA-NONOate) and Z-1[N-(2-aminoethyl)-N-(2-ammonoethyl)amino]diazen-1-ium-1,2-dioate (DETA-NONOate), peroxynitrite, and Mn(III) tetrakis(4-benzoic acid) porphyrin chloride (MnTBAP) were from Cayman Chemical (Ann Arbor, MI). FCS was from HyClone Laboratories (Logan, UT). All other reagents were from Sigma Chemical (St. Louis, MO).

Primary culture of cardiac fibroblasts. All animal procedures were conducted in accordance with guidelines published in the Guide for the Care of Laboratory Animals (National Institutes of Health) and were approved by the University of Minnesota Animal Care Committee. CFbs were isolated from adult male Sprague-Dawley rats (180-200 g) as previously described with minor modifications (9). Rats were anesthetized with pentobarbital sodium (25 mg/kg), and the heart from each rat was removed. The left ventricles were minced and washed in Hank's balanced salt solution. Cells were released by digesting the tissue with a mixture of 0.1% trypsin (GIBCO-RBL Life Technologies; Grand Island, NY) and 100 U/ml of collagenase (type IV) for 10 min per cycle at 37°C. Cells from the second to the fifth digestion cycle were cultured in flasks containing DMEM plus 20% FCS at 37°C, 10% CO₂-90% air for 2 h. The attached cells (>95% are CFbs) were allowed to grow in DMEM containing 10% FCS at 37°C and 5% CO₂-95% air until confluent. Cells were subcultured one or two more times before use. Fibroblasts were distinguished from other cell types by the presence of the fibroblast marker vimentin (V-5255, Sigma Chemical) and the absence of the endothelial cell marker von Willebrand factor (F-3520, Sigma Chemical) or the muscle cell marker desmin (D-1033, Sigma Chemical) when examined by immunofluorescent staining as previously described (32).

Induction of iNOS expression. CFbs were subcultured in dishes containing 10% FCS (at 37°C, 5% CO₂-95% air) for 16 h and then switched to DMEM with low serum (0.1% FCS) and incubated for 24 h. All subsequent experiments were performed in low-serum DMEM. Cells were treated with inflammatory cytokines (IL-1, IFN-, or TNF) or NO donors in the presence or absence of other chemical compounds for the indicated time intervals. The concentrations of chemical compounds used in this study were chosen based on results from pilot studies to optimize the effects of the interventions (data not shown). Medium from treated and nontreated cells was collected and stored at 70°C. Cells were lysed, and lysate protein was subjected to Western analysis for iNOS protein expression. A parallel set of cells was fixed with precooled methanol for immunostaining for morphological analysis.

NO measurement. Assessment of NO production in the culture medium was performed by using the Griess reagent that measures nitrite (NO₂), the major NO metabolite in the cell culture system (40). One hundred microliters of Griess reagent (a mixture of one part of Griess reagent A containing 0.1 g of N-[-1-naphythyl]-ethylenediamine hydrochloride in 100 ml of water and one part of Griess reagent B containing 1 g of sulfanilamide in 100 ml of 3 N HCl) were added to 100 µl of sample or standard (sodium nitrite served as the standard) in each well of a 96-well plate. After incubation at room temperature for 15 min, samples were read in a spectrophotometer at 550 nm, and the amount of nitrite was calculated from a standard curve constructed using NaNO₂ at concentrations of 0.1-80 µM.

Assessment of apoptosis. Apoptotic cells were identified by direct staining of the condensed nuclei or fragmented DNA with bis-benzimide (Hoechst 33258) or TUNEL-based staining. For Hoechst 33258 staining, the bis-benzimide stock solution was added directly into the culture medium (at a final concentration of 0.02%) and incubated with the cells for 20 min at 37°C. Methanol-fixed CFbs were subjected to microscopic analysis by using an inverted phase-contrast fluorescence microscope. Processing of images was performed using NIH Image, Adobe Photoshop, and a Fuji Pictography 3000 color printer; 500 cells per sample were counted, and TUNEL-positive cells were expressed as percentage of total cells.

Assessment of caspase-3 activity. Caspase-3 activity was detected by using the specific caspase-3 colorimetric substrate [Asp-Glu-Val-Asp-pNatural alanine (DEVD-pNA)]. Cells were collected by centrifugation and lysed by the addition of cell lysis buffer. The cell lysate was incubated on ice for 10 min and clarified by centrifugation at 10,000 g for 1 min. The measurement was carried out in a 96-well plate by adding 100 µg of total protein from each sample to the well followed by the addition of 50 µl of 2× reaction buffer and 5 µl of caspase-3 colorimetric substrate (DEVD-pNA). The reaction was allowed to proceed for 2 h at 37°C. Signal was detected by using a microplate reader at 405-nm wavelength, and the results are expressed as arbitrary optical density (OD) units.

Preparation of nuclei. CFb nuclei were prepared as described by Martin et al. (20). CFbs were harvested by centrifugation at 200 g for 10 min and washed three times in PBS (pH 7.2) and one time in 15 ml of nuclei isolation buffer (10 mM HEPES, pH 7.4, 10 mM KCl, 2 mM MgCl₂, 1 mM DTT, 1 mM cytochalasin B, and 1 mM PMSF). The cells were resuspended in 10 vol of nuclei buffer and incubated on ice for 20 min followed by gentle homogenization several times on ice with a Dounce homogenizer. The liberated nuclei were layered over 30% sucrose in nuclei buffer, centrifuged at 800 g for 10 min at 4°C, washed once with nuclei buffer, and resuspended in storage buffer (10 mM HEPES, pH 7.4, 80 mM KCl, 20 mM NaCl 1M, 250 mM sucrose, 5 mM EGTA, 1 mM DTT, 0.5 mM spermidine, 0.1 mM spermine, 1 mM PMSF, and 50% glycerol) at 2 × 10⁶ nuclei/ml and stored at 80°C in 20-µl aliquots until use.

Western blotting. Western blot analysis for iNOS, Bax, Bcl-x_L, p53, caspase-3, and -tubulin expression was performed as previously described with minor modification (33). Cells were harvested and lysed with lysis buffer (50 mM Tris · HCl, pH 7.5, 250 mM NaCl, 5 mM EDTA, 50 mM NaF, and 0.5% Nonidet P-40) containing a protease inhibitor cocktail (Boehringer Mannhem). The lysate was clarified by centrifugation at 16,000 g for 15 min at 4°C. Equal amounts of total protein were subjected to 8% SDS-PAGE and electrophoretically transferred to a High-Bond nitrocellulose membrane (Amersham Life Science; Arlington Heights, IL). After being blocked with Tween 20-Tris-buffered saline (TTBS; 20 mM Tris · HCl, pH 7.6, 137 mM NaCl, and 0.05% Tween 20) containing 5% nonfat milk for 1 h at room temperature, the membrane was incubated for 1 h at room temperature with the primary antibodies at 1:500 dilution in blotting buffer (TTBS with 5% nonfat milk). After being washed three times for 10 min each in TTBS, the membrane was incubated with an appropriately diluted horseradish peroxidase-labeled secondary antibody (1:2,000) in blotting buffer for 1 h at room temperature. The membrane was washed three times, reacted with ECL reagent (Amersham Life Science), and subjected to autoradiography. The strength of the signal was analyzed by using densitometry, and the results were expressed as arbitrary units. Protein levels were standardized by comparison with anti--tubulin antibody.

Statistical analysis. Each experiment was repeated at least three times. Data are presented as means ± SE. Comparison between groups was performed by two-way ANOVA. Significance was considered as P < 0.05.

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Induction of iNOS expression in CFbs by cytokines. To determine whether adult cardiac fibroblasts are able to express iNOS in response to stimulation with inflammatory cytokines, cells were exposed to IL-1 (5 ng/ml), IFN- (10 ng/ml), or TNF (10 ng/ml) individually or in combination for 16 h, and iNOS protein levels were detected by Western blot analysis (-tubulin was used as a loading control). Among these cytokines, only IL-1 was able induce iNOS expression. IFN- or TNF alone or combined had no effect on iNOS expression (Fig. 1A). NO production by these cells was consistent with the observed changes in steady-state iNOS protein levels (Fig. 1B); only cells exposed to IL-1 produced detectable NO. These results indicate that adult CFbs are capable of expressing iNOS on stimulation with IL-1, but not with IFN- or TNF, and this is associated with a marked increase in NO production. The induction of iNOS expression was associated with a significant increase in cell death (Fig. 1C); after stimulation with IL-1 for 16 to 24 h ~35% to 40% of CFbs showed evidence of cell death.

View larger version (56K): [in this window] [in a new window]   Fig. 1.   Effect of cytokines on inducible nitric oxide (NO) synthase (iNOS) expression and NO production. A: cardiac fibroblasts (CFbs) were treated with cytokines for 16 h. Cells were lysed, and equal amounts of total protein (100 µg) from the lysate were subjected to Western blot analysis for iNOS protein (130 kDa) using an anti-iNOS polyclonal antibody. Lane 1 is lysate from nontreated CFbs (control); lanes 2, 5, and 7 are lysates from cells treated with IL-1 (5 ng/ml), IL-1 (5 ng/ml) + IFN- (10 ng/ml), or IL-1 (5 ng/ml) + TNF (10 ng/ml), respectively. Lanes 3, 4, and 6 are from cells treated with IFN- (10 ng/ml), TNF (10 ng/ml), or IFN- + TNF (both at 10 ng/ml). -Tubulin was used as a loading control. OD, optical density. B: nitrite (NO2) concentration (µM) in culture medium collected from CFbs treated with cytokines for 24 h (n = 7). * P < 0.05 vs. control. # P < 0.05 vs. IL-1 + IFN-. C: phase-contrast microscopy of control CFbs (panel A), CFbs treated with IL-1 (5 ng/ml, panel B), IFN- (10 ng/ml, panel C), TNF- (10 ng/ml, panel D), IL-1 + IFN- (panel E), IFN- + TNF- (panel F), or IL-1 + TNF- (panel G). (Bar = 40 µm.)

View larger version (14K): [in this window] [in a new window]   Fig. 2.   Dose response and time course of IL-1-induced iNOS expression and NO production. A: CFbs were treated with IL-1 (0.3-25 ng/ml) for 16 h and subjected to Western blotting for iNOS protein expression. B: nitrite concentration (µM) in medium collected from cells treated with IL-1 for 24 h. C: Western blotting for iNOS protein expression in CFbs treated with IL-1 (5 ng/ml) for various time periods (0, 2, 4, 6, 8, 16, and 24). D: nitrite concentration (µM) in medium collected from cells treated with IL-1 for the various times as indicated.

iNOS expression induces CFb apoptosis. Studies were performed to determine whether exposure to IL-1 resulted in CFb apoptosis. Evaluation of apoptosis was carried out by using the TUNEL assay (Fig. 3A) or staining the cells with Hoescht 33258 (data not shown). Apoptosis was evidenced by cell shrinkage and nuclear condensation and fragmentation that occurred in cells treated with IL-1 (5 ng/nl) (Fig. 3A, panel B). The apoptotic cell number was significantly reduced by the selective iNOS inhibitor S-methylisothiourea (SMT) (Fig. 3A, panel C). Quantitative assay showed that the number of TUNEL-positive cells was increased from 1 ± 0.15% of control CFbs to 39.2 ± 1.47% of IL-1-treated cells (P < 0.05) (Fig. 3B). IL-1-induced CFb apoptosis was reduced from 39.2 ± 1.47% in CFbs treated with IL-1 alone to 5.6 ± 0.59% in cells treated with both IL-1+SMT (Fig. 3B). These findings demonstrate that the proapoptotic effect of IL-1 requires an intact iNOS-NO axis.

View larger version (103K): [in this window] [in a new window]   Fig. 3.   IL-1-induced iNOS expression and NO production is associated with apoptosis. A: inverted phase-contrast microscopic analysis for nuclear condensation of CFbs after labeling with transferase-mediated dUTP nick-end labeling (TUNEL)-based staining. Shown are control CFbs (panel A) and CFbs treated (for 24 h) with IL-1 (panel B) or IL-1 combined with S-methylisothiourea (SMT, 3 × 105 M, panel C), 1H-[1,2,4]-oxadiazolo[4,3-a]quinoxalin-1-one (ODQ, 2 µM, panel D), the caspase-3 inhibitor Asp-Glu-Val-Asp-fluoromethyl ketone (DEVD-FMK, 100 µM, panel E), or 8-bromocGMP (8-BrcGMP, 10 µM, panel F.) (Bar = 40 µm). B: percentage apoptosis induced by IL-1. * P < 0.05 vs. control, IL+SMT, IL+CSP3-I, or 8-BrcGMP.

Mechanism of IL-1-induced apoptosis. NO has been shown to induce vascular smooth muscle cell apoptosis through a cGMP-dependent pathway. To determine whether the same is true in CFbs, cells were treated with the guanylyl cyclase inhibitor 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ, 2 µM). As shown in Fig. 3A (panel D), ODQ did not alter IL-1-induced CFb apoptosis. Furthermore, no change in cell viability was observed when CFbs were exposed to the cGMP analog 8-bromo-cGMP (10 µM) (Fig. 3A, panel F, and Fig. 3B). These data demonstrate that IL-1-induced CFb apoptosis can occur in a cGMP-independent manner.

View larger version (31K): [in this window] [in a new window]   Fig. 4.   Effect of IL-1 on the expression of p53, Bcl-xL, and Bax. A: Western blot analysis of p53 protein levels in control CFbs (C), CFbs treated (for 16 h) with IL-1 (5 ng/ml) alone (IL), or combined with SMT (3 × 105 M). B: Western analysis of p53 protein levels in control CFbs (lane 1), CFbs treated (for 16 h) with IL-1 (5 ng/ml, lane 2), IL-1 + Mn(III)tetrakis(4-benzoic acid) porphyrin chloride [Mn(III)TBAP] (100 µM, lane 3), or IL-1 + Mn(III)TBAP + SMT (3 × 105 M, lane 4). C: Western analysis of p53 protein levels in control CFbs (lane 1), CFbs treated (for 4 h) with DETA-NONOate (3 × 105 M, lane 2), DETA-NONOate + Mn(III)TBAP (100 µM, lane 3), ONOO (100 µM, lane 4), or ONOO + Mn(III)TBAP (100 µM, lane 5). D: Western analysis of Bcl-xL protein levels in control CFbs (lane 1), CFbs treated (for 16 h) with IL-1 (5 ng/ml, lane 2) alone or combined with either SMT (3 × 105 M, lane 3) or ODQ (2 µM, lane 4). E: Western analysis of Bax protein levels in control CFbs (lane 1), CFbs treated (for 16 h) with IL-1 (5 ng/ml, lane 2) alone or combined with either SMT (3 × 105 M, lane 3) or ODQ (2 µM, lane 4). -Tubulin was used as a loading control.

View larger version (28K): [in this window] [in a new window]   Fig. 5.   Effect of IL-1 on caspase-3 expression and activity in CFbs. A: control CFbs (lane 1), CFbs treated with IL-1 (5 ng/ml for 16 h, lane 2) alone or combined with SMT (3 × 10 5 M, lane 3) or ODQ (2 µM, lane 4), and CFbs treated with 8-BrcGMP alone (2 µM, lane 5). -Tubulin was used as loading control. B: caspase-3 activity of control and treated CFbs. * P < 0.05 vs. control, IL+SMT, or 8-Br-cGMP.

NO-triggered apoptosis does not require conversion to ONOO. To determine whether the proapoptotic effect of NO was dependent on the formation of ONOO, CFbs were exposed directly to the NO donor DETA-NONOate (at concentrations from 3 × 10⁷ M to 3 × 10³ M) or ONOO (100 µM) for 4 h followed by Hoescht staining. When CFbs were exposed to DETA-NONOate at concentrations lower than 10⁶ M, no significant apoptosis was observed (data not shown). However, when CFbs were exposed to DETA-NONOate at 3 × 10⁵ M, apoptotic cells were increased to 50 ± 1.4%; this effect was only partially inhibited by the selective ONOO scavenger Mn(III)TBAP (100 µM) (Fig. 6). In contrast, ONOO (100 µM)-induced CFb apoptosis was essentially completely blocked by Mn(III)TBAP (100 µM) (Fig. 6). Mn(III)TBAP alone had no effect on cell viability. Furthermore, Mn(III)TBAP only partially inhibited IL-1-induced CFb apoptosis (Fig. 6C). These data demonstrate that NO is capable of inducing CFb apoptosis independent of conversion to ONOO.

View larger version (66K): [in this window] [in a new window]   Fig. 6.   NO donor DETA-NONOate induces CFb apoptosis in cultured CFbs. A: laser confocal microscopic analysis for the cytoskeletal protein vimentin (green) and nuclei with propidium iodide (red). Shown are control CFbs (panel A), CFbs treated with DETA-NONOate (3 × 105 M, panel B), DETA-+ Mn(III)TBAP (100 µM, panel C), ONOO (100 µM, panel D), and ONOO + Mn(III)TBAP (100 µM, panel E). Bar = 20 µm. B: apoptotic frequency in control or treated CFbs. * P < 0.05 vs. control or ONOO + Mn(III)TBAP. # P < 0.05 vs. DETA-NONOate or ONOO. C: apoptotic frequency in control, IL-1 (5 ng/ml), or IL-1 ± Mn(III)TBAP (100 µM)-treated CFbs. * P  0.05 vs. control. # P  0.05 vs. IL-1.

NO causes apoptotic changes in isolated nuclei. To determine whether NO or ONOO can directly cause apoptotic nuclear changes, isolated nuclei were exposed to DETA-NONOate or ONOO for 4 h followed by Hoescht staining. When isolated CFb nuclei were incubated with the NO donor DETA-NONOate or with ONOO, nuclear condensation and fragmentation was observed (Fig. 7A, panels B and D). As expected, the apoptotic changes produced by ONOO were largely blocked by Mn(III)TBAP (Fig. 7A, panel E). Quantitative assay indicated that more than 50% of the isolated nuclei underwent apoptotic morphological changes in response to DETA-NONOate (Fig. 7A, panel B), but the apoptotic nuclei number was not affected by Mn(III)TBAP (100 µM) (Fig. 7A, panel C). No nuclear condensation was observed in control nuclei (Fig. 7A, panel A). These results demonstrate that NO was capable of producing nuclear morphologic changes characteristic of apoptosis and suggest that conversion of NO to ONOO was not required for the proapoptotic effect of NO in CFb nuclei.

View larger version (19K): [in this window] [in a new window]   Fig. 7.   NO or ONOO induced apoptotic change in isolated nuclei. A: microscopic analysis of isolated CFb nuclei after exposure to DETA-NONOate and ONOO for 2 h followed by staining with Hoechst 33258. Shown are control nuclei (panel A), nuclei treated with the NO donor DETA-NONOate (3 × 105 M, panel B) alone or combined with the ONOO scavenger Mn(III)TBAP (100 µM, panel C), and nuclei treated with either ONOO alone (100 µM, panel D) or combined with Mn(III)TBAP (100 µM, panel E) (Bar = 40 µm). B: apoptotic frequency in control or treated nuclei. * P < 0.05 vs. control. # P < 0.05 vs. DETA-NONOate, DETA-NONOate + Mn(III)TBAP, or ONOO.

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

The present data demonstrate that exposure of adult rat CFbs to either IL-1 or an NO donor can induce apoptotic cell death. The ability of IL-1 to induce apoptosis was blocked by a selective inhibitor of iNOS, indicating that NO plays an essential role in IL-1-induced CFb apoptosis. IL-1-induced CFb apoptosis involved a p53-dependent mechanism that included upregulation of Bax expression and activation of caspase 3. Our findings suggest that apoptosis was at least partly due to direct NO-induced nuclear events and that NO is capable of directly triggering apoptosis in cardiac fibroblasts without conversion to ONOO.

Expression of iNOS in response to cytokine stimulation was originally observed in macrophages where it is involved in NO-mediated cell damage and apoptosis (18). Subsequent data have shown that other cell types, including smooth muscle cells and cardiac myocytes, are capable of expressing iNOS on stimulation with cytokines or microbial products even though iNOS is not expressed in these cells under basal conditions. In smooth muscle cells and cardiac myocytes (6, 15, 25), the proapoptotic effect of cytokines is usually mediated by the induction of iNOS protein, which only occurs in pathological conditions and is associated with high levels of NO production (18). Interestingly, NO can function as either a proapoptotic or an antiapoptotic effector, depending on the levels of NO generated locally as well as the antiapoptotic response of each cell type to NO (18). Thus NO has been found to inhibit apoptosis in hepatocytes and endothelial cells (18, 31), presumably in its signaling role. Low levels of NO generated by endothelial NOS may have a protective effect, whereas the higher levels produced by iNOS may exert a proapoptotic effect.

Apoptosis is an important mechanism for maintaining the balance of cell populations within tissues. In the heart, apoptosis may be important for restoring the cell population balance in the context of fibroblast proliferation that can occur with hypertension or following myocardial infarction (39). Conversely, excessive apoptosis might also lead to impairment of function (19, 24). Several investigators have reported increased levels of circulating cytokines in the setting of heart failure (4, 5, 23). Expression of iNOS protein in the myocardium has been observed in heart failure, and cytokine-induced NO production has been linked to evidence that apoptosis occurs in the failing heart (19, 23). With the use of immunohistochemical techniques, in hearts from patients with dilated cardiomyopathy or ischemic heart disease, several investigators have demonstrated iNOS in cardiac myocytes (12, 14, 28), as well as in endothelial cells and smooth muscle cells (36). From these considerations, it is tempting to speculate that the increased circulating cytokine levels found in the setting of heart failure might result in induction of iNOS and apoptotic loss of CFbs. This could be of importance, because loss of the integrity of the myocardial ECM appears to be a critical factor in the left ventricular dilatation that occurs in the failing heart (24). Anand et al. (2) found that contractile function of isolated cardiac myocytes obtained from the myocardial region remote from an infarct produced by coronary artery ligation in rats was normal. Because this remote region demonstrated systolic dysfunction in vivo, the normal in vitro myocyte function implied that nonmyocyte factors contributed to the abnormal function of the noninfarcted left ventricular wall. Such nonmyocyte alterations could include abnormalities of the extracellular matrix with inefficient force transmission due to impaired coupling between individual myocytes. This is of interest because the rat model of myocardial infarction is associated with expression of iNOS in the remote noninfarcted myocardium (2). It is well known that NO produced by iNOS can depress myocyte contractility (16); it is also possible that NO-induced alterations of CFb viability or function could impair ventricular chamber function by affecting the coupling between individual myocytes within the wall. Future studies are needed to determine whether iNOS expression can be demonstrated in cardiac fibroblasts in vivo in the diseased heart.

NO has been shown to cause apoptosis in vascular smooth muscle cells by stimulating the soluble guanylyl cyclase pathway (8). Similarly, a cGMP-dependent pathway has been reported to be involved in NO-induced cardiac myocyte apoptosis (7). However, in other cell types, including neutrophils and endothelial cells, the proapoptotic effect of NO is independent of cGMP accumulation (29, 37). In the present study, blockade of guanylyl cyclase with ODQ did not alter the apoptotic frequency either in cells in which iNOS was induced with IL-1 or in cells directly treated with the NO donor. Furthermore, administration of the cGMP analog 8-bromo-cGMP did not cause CFb apoptosis. These data indicate that the proapoptotic effect of NO in adult CFbs can occur independently of the guanylyl cyclase pathway.

The mitochondria act as central integrator of the apoptotic response to cellular stress. Whereas the precise steps transducing pathological stress into mitochondrial release of cytochrome c and activation of the apoptosome remains to be elucidated, p53 and Bcl-2 family of proteins play a prominent role (26). As a product of a tumor suppressor gene, p53 has been implicated in the apoptotic response to numerous stimuli including NO-induced macrophage apoptosis and from stressors that produce DNA damage (21). We found that p53 protein levels were increased in IL-1-treated CFbs and that this change occurred before the onset of CFb apoptosis. Induction of p53 was mitigated by inhibition of iNOS activity with SMT. In contrast to IL-1-induced apoptosis, the NO donor DETA-NONOate or ONOO caused apoptotic nuclear changes without accumulation of p53 protein. Interestingly, Kibbe et al. (17) found that vascular smooth muscle cells from p53 null mice were more sensitive to the propapoptotic effects of NO than were p53 competent cells, indicating that the mechanism of NO-induced apoptosis is complex and does not predictably require the participation of p53. Of note, our data revealed that the NO donor DETA-NONOate and ONOO caused apoptotic changes in isolated nuclei. Thus our data can be interpreted to suggest that IL-1-dependent apoptosis may involve direct DNA damage by NO, thus triggering a p53-dependent death mechanism, as well as by activating downstream pathways operating through NO as a signaling intermediate. Definitive resolution awaits further studies using p53 null CFbs.

In several cell types, NO-mediated apoptosis has been shown to result from the reaction of NO with superoxide to form the potent oxidant ONOO (3, 18, 22). Thus Arstall et al. (3) demonstrated that the ONOO scavenger Mn(III)TBAP was able to fully protect cultured neonatal rat ventricular myocytes from NO-induced apoptosis. In the present study, Mn(III)TBAP provided only partial protection against NO-mediated apoptosis in CFbs, although it fully protected the cells from apoptosis induced by exogenous ONOO. To determine whether NO could exert a direct proapoptotic effect, isolated CFb nuclei were exposed to the NO donor DETA-NONOate. DETA-NONOate was used as an NO donor because it avoids the nitrosation and thiolation that can occur when nitrosothiols are used as a source of NO (7). DETA-NONOate has an added advantage of a relatively long half-life (~56 h at pH 7.4), so that it can produce a physiologically relevant steady-state NO concentration (8); the concentration of DETA-NONOate used was at the low range of concentrations used by previous investigators (20-1,000 µmol/l) (7, 22, 25). DETA-NONOate caused condensation and fragmentation of the isolated CFb nuclei that were not blocked by the ONOO scavenger Mn(III)TBAP. These findings suggest that in CFbs conversion to ONOO is not required for NO to induce cell death. Whether ONOO and NO-induced apoptosis are mediated through the same mechanisms remains to be defined.