INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

CARDIAC INVOLVEMENT is a well-documented complication of human immunodeficiency virus (HIV) infection, although the incidence and pathogenesis have not been clearly established (1, 7). Myocardial dysfunction (DCM) has been documented by echocardiography with surprisingly high frequency (7). The pathophysiological mechanisms responsible for HIV DCM are not well understood. Potential mechanisms include indirect effects mediated through immune-stimulated cytokine production, direct effects of HIV on cardiac myocytes (CM), or a combination of both. Support for indirect effects of HIV infection on DCM has been provided by experimental evidence that cytokines and nitric oxide (NO) are endogenous myocardial depressants.

The potential for a direct effect of HIV on myocardial function must also be considered in view of the demonstration of HIV in endomyocardial biopsy specimens derived from HIV-infected patients with DCM (3). We attempted to clarify the potential direct contribution of HIV to DCM by studying the effects of recombinant proteins on isolated CM.

Several studies (9, 16, 19) have shown that interleukin-1 (IL-1) alone is sufficient to stimulate inducible NO synthase (iNOS) mRNA expression, iNOS protein synthesis, and NO production by neonatal rat CM in culture. The effects of recombinant HIV proteins on IL-1-stimulated NO production by CM have not been characterized. We now report that the HIV glycoprotein, glycoprotein120 (gp120), enhances IL-1-induced NO production by CM through a novel mechanism involving p38 mitogen-activated protein (MAP) kinase-mediated activation of NF-B. These findings support the hypothesis that interactions between cytokines and HIV proteins contribute to HIV-associated DCM.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Materials. All of the reagents were purchased from Sigma Chemical (St. Louis, MO) unless otherwise indicated. Cytokines were purchased from Genzyme (Boston, MA). The concentrations used were described in units per milliliters with the specific activity for IL-1 = 10⁸ U/mg protein. This value corresponds to 5 ng/ml of IL-1 for 500 U/ml as reported by others (9, 19). The recombinant HIV-1 proteins, gp120 and Tat, were obtained from the National Institutes of Health (NIH) AIDS Research and Reference Reagent Program.

Isolation of CM. Myocytes were prepared from the ventricles of 1- to 2-day-old rat pups as we have previously described (16). Briefly, the ventricles of 30-50 hearts obtained from three different litters were minced in Ca²⁺- and Mg²⁺-free Hank's balanced salt solution (HBSS) and digested for 15-min periods in 10 ml of a solution containing 0.1% trypsin (GIBCO-BRL, Grand Island, NY), 15 U/ml collagenase, and 0.1 mg/ml DNase (Worthington Biochemical, Freehold, NJ) in HBSS. Digestion was stopped by adding 10 ml of DMEM and Ham's F-12 solution (DMEM/F12; GIBCO-BRL) containing 5% calf serum. The cycles were repeated until all of the tissue was digested. The myocytes were cultured in DMEM/F12 culture medium supplemented with 5% calf serum, penicillin (50 U/ml), and streptomycin (50 mg/ml). Cells were seeded at a density of 1.25 × 10⁵ cells/cm² on various dishes (Falcon Plastics, Cockeysville, MD; Costar, Cambridge, MA) according to the experimental requirements. The culture medium was changed to fresh serum-free DMEM/F12 containing insulin, transferrin, selenium, and bovine serum albumin 48 h after plating. Myocytes formed confluent monolayers of spontaneously beating cells 24 h later. These cells were washed and fresh serum-free DMEM/F12 was added. IL-1 (Genzyme), N^G-methyl-L-arginine (L-NMNA), gp120, and Tat were added at this time and incubated as indicated.

Assay for NO₂ production. NO₂ assays on neonatal rat cardiac myocyte cell culture supernatants were performed as we described previously (18). Briefly, the stable metabolic end product of NO synthesis, NO₂, was used as a measure of NO production. Cell culture supernatants from 48-well plates were mixed with an equal volume of Greiss reagent for 1 h. The absorbance at 550 nm was measured with a microplate reader (Molecular Devices). We previously demonstrated that the ratio between NO₂ and total NO₂ + NO₃ did not significantly change throughout the various experiments. Thus the NO₂ levels accurately reflected the total amount of NO produced.

MAP kinase assay. p42/44 and p38 MAP kinase activities were determined by using phospho-p42/44 MAP kinase (Thr-202/Tyr-204) and phospho-p38 MAP kinase (Thr-180/Tyr-182) antibodies according to the manufacturer's recommendations as previously described by others and adopted by us (6). We lysed the cells by adding 100 µl of lyse buffer and then immediately scraped the 30-mm dish and transferred the extract to a microcentrifuge tube to keep on ice. This was followed by sonicating for 2 s and centrifuging at 10,000 g for 15 min at 4°C. The supernatant was transferred to a new centrifuge tube. A sample buffer was added to protein samples at a ratio of 2:1 and microcentrifuged for 30 s, followed by loading 20 µg of protein onto sodium dodecyl sulfate (SDS)-PAGE.

Electrophoretic mobility shift assay. Nuclear extracts were prepared as previously described (24) and stored at 80°C before use. The double-stranded oligonucleotide containing a consensus NF-B binding site, 5'-AGT TGA GGG GAC TTT CCC AGG C-3' (Santa Cruz Biotechnology, Santa Cruz, CA), was used to detect NF-B activity. Oligonucleotides were end-labeled with [-³²P]ATP (3,000 Ci/mmol; Amersham, Arlington Heights, IL) and T4 polynucleotide kinase (Promega, Madison, WI). ³²P-labeled oligonucleotides (~30,000 cpm) and 10 µg of nuclear protein were incubated for 20 min at room temperature in a total volume of 25 µl in the presence of (in mM) 2 Tris · HCl (pH 7.5), 8 NaCl, 0.2 EDTA, and 0.2 -mercaptoethanol, and 0.8% glycerol and 1 µg poly(dI-dC). Protein DNA complexes were resolved by electrophoresis on nondenaturing 5% polyacrylamide gels and visualized by autoradiography.

Northern blot analysis. Northern blots were prepared as previously described (10). After exposure of cells (2.5 × 10⁶ cells per 60-mm dish) to experimental conditions, total RNA was extracted using Tri Reagent (Molecular Research Center, Cincinnati, OH) according to the manufacturer's instructions. A 10-µg sample of total RNA per lane was subjected to electrophoresis on 1.2% agarose gels containing 2.2 M formaldehyde. RNAs were transferred onto Zeta-probe blotting membranes (Bio-Rad Laboratories, Hercules, CA) using Vacuum Blotter (model 785, Bio-Rad Laboratories) and ultraviolet autocross-linked (GS gene linker, Bio-Rad Laboratories). Membranes were hybridized 16 h at 62°C with HS-114 hybridization solution (Molecular Research Center) containing murine iNOS (Alexis, San Diego, CA) and human glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (Cayman Chemical, Ann Arbor, MI) cDNA probes labeled with [-³²P]dCTP (3,000 Ci/mM; Amersham) by random priming (Megaprime DNA labeling system, Amersham). The hybridized membranes were serially washed at 55°C with the use of 1× sodium citrate, sodium chloride, and 1% SDS solution and exposed to Kodak XAR-5 film overnight at 70°C with an intensifying screen.

Western blot analysis. Western blots were performed as previously described (10). CM were lysed directly in each plate (1.25 × 10⁶ cells in 30-mm plate) by application of a buffer containing (in mM) 10 Tris · HCl (pH 7.4), 150 NaCl, 2 EGTA, 2 1,4-dithiothreitol (DTT), 1 sodium orthovanadate and (in µg/ml) 100 phenylmethylsulfonyl fluoride, 10 leupeptin, and 10 aprotinin. Protein concentrations were determined by Bradford assay. The samples were treated with 2× Laemmli loading buffer and boiled for 5 min. Equal amounts (20 µg) of the denatured proteins per lane were subjected to 12% SDS-PAGE, transferred to a nitrocellulose membrane, and reversibly stained with Ponceau red to verify equal loading. The blots were probed with a 1:2,000 dilution of mouse monoclonal antibodies specific for iNOS (Alexis). The iNOS protein was detected by using the Amersham ECL system.

Statistical methods. Data represent the means ± SE of 9-15 different determinations derived from three individual wells from each of three to five completely separate myocyte preparations of 30-50 individual neonatal rat pup hearts/preparation from three litters/preparation. A total of 15 different litters of 150-250 rat pup hearts were used for a n = 5 (Fig. 1). A total of nine different litters of 90-150 rat pup hearts were used for a n = 3 (Figs. 2-4). ANOVA and the Student-Newman-Keuls test were used for multigroup comparisons. Values of P < 0.05 were considered statistically significant.

View larger version (10K): [in this window] [in a new window]   Fig. 1.   Bar graphs depicting the effects of interleukin-1 (IL-1, 500 U/ml) or glycoprotein120 (gp120, 1 µg/ml) alone or IL-1 + gp120 on nitric oxide (NO2) production and the effects of SB-203580 (SB) (p38 MAP kinase inhibitor) on IL-1 + gp120-stimulated NO2 production by neonatal rat cardiac myocytes (CM). Data are expressed as means ± SE of 15 different determinations derived from 3 wells each from 5 separate myocyte preparations from 15 different litters (n = 5). *P < 0.01 vs. vehicle. **P < 0.01 vs. IL-1.

View larger version (36K): [in this window] [in a new window]   Fig. 2.   A: representative Northern blot analysis of inducible NO synthase (iNOS) mRNA expression in CM exposed to IL-1, gp120, or IL-1 + gp120 and the effects of SB [p38 mitogen-activated protein (MAP) kinase inhibitor] on IL-1 + gp120-stimulated iNOS mRNA expression. The amount of iNOS mRNA expression was determined by densitometry and expressed as means ± SE in relative optical units derived from 3 separate experiments from 9 different litters (n = 3). From left to right: 4.2 ± 0.3, 26 ± 2.4, 3.9 ± 0.4, 70 ± 1.5, and 17 ± 1.2. B: representative Western blot analysis of iNOS protein in CM exposed to IL-1, gp120, or IL-1 + gp120 and the effect of SB (p38 MAP kinase inhibitor) on IL-1 + gp120-induced iNOS protein. The amount of iNOS protein was determined by densitometry and expressed as means ± SE in relative optical units derived from 3 separate experiments from 9 different litters (n = 3). From left to right: 5.2 ± 1.2, 18 ± 0.8, 6.8 ± 0.4, 42 ± 1.4, and 19 ± 1.2.

View larger version (25K): [in this window] [in a new window]   Fig. 3.   Representative MAP kinase activity assay illustrating MAP kinase activation by IL-1, gp120, and IL-1 + gp120 and the inhibition of this effect by the known p38 MAP kinase inhibitor SB (A) and PD-98059 (B), a p42/44 MAP kinase kinase inhibitor. MAP kinase activity was quantified by densitometry and expressed as means ± SE in relative optical units derived from 3 separate experiments from 9 different litters, n = 3. From left to right: A, 6.1 ± 1.4, 16.8 ± 1.2, 15.9 ± 0.8, 40 ± 0.8, and 20 ± 1.2; B, 3 ± 0.2, 14.7 ± 0.2, 10.5 ± 0.1, 12.4 ± 0.2, and 8.2 ± 0.2.

View larger version (55K): [in this window] [in a new window]   Fig. 4.   Representative NF-B activity reflected in electrophoretic mobility shift assay illustrating NF-B activation by IL-1, gp120, and IL-1 + gp120 and the inhibition of this effect by SB, a p38 MAP kinase inhibitor. NF-B activity was quantified by densitometry and expressed as means ± SE in relative optical units derived from 3 separate experiments from 9 different litters, n = 3. From left to right: 40 ± 4.6, 92 ± 10.7, 52 ± 6.8, 184 ± 12.7, and 96 ± 12.6.

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Exposure of CM to IL-1 alone (500 U/ml) resulted in a significant increase in NO₂ production at 48 h as we and others have previously reported (P < 0.01, n = 5) (10, 16, 17) (Fig. 1). Exposure of CM to recombinant HIV glycoprotein, gp120, alone (1 µg/ml) had no effect on NO₂ production over vehicle alone (P = not significant, n = 5). The addition of gp120 to IL-1 resulted in a statistically significant increase in NO₂ production compared with IL-1 alone (P < 0.01, n = 5) (Fig. 1). Another recombinant HIV protein, Tat, had no effect alone or in combination with IL-1 (data not shown). Potential mechanisms involved in gp120 enhancement of IL-1-stimulated NO production were explored. The gp120-mediated increase in IL-1-stimulated NO₂ production was totally abolished by the addition of 10 µM SB-203580 (SB), a selective p38 MAP kinase inhibitor (P < 0.01, n = 5) (Fig. 1).

The addition of gp120 to IL-1 considerably enhanced both iNOS mRNA expression by Northern blot analysis and iNOS protein synthesis by Western blot analysis compared with IL-1 alone (Fig. 2, A and B). The p38 MAP kinase inhibitor SB greatly reduced the gp120 enhancement of IL-1-stimulated iNOS mRNA expression and protein synthesis.

The essential role of MAP kinase activation in gp120 enhancement of IL-1-stimulated NO production was further confirmed by MAP kinase assay. gp120 significantly increased both p42/44 and p38 MAP kinase activities, which were reduced by the addition of the MAP kinase kinase inhibitors SB (Fig. 3A) and PD-98059 (PD) (Fig. 3B), respectively. However, the addition of gp120 further potentiated IL-1-stimulated p38 MAP kinase activity but not IL-1-stimulated p42/44 MAP kinase activity (Fig. 3, A and B).

We have previously shown by immunohistochemistry that nuclear translocation of NF-B is essential for IL-1-stimulated NO production by CM (17). We now studied the effect of gp120 and IL-1 on NF-B activation determined by electrophoretic mobility shift assay (Fig. 4). IL-1 and gp120 each increased NF-B activity at 2 h. The addition of both gp120 and IL-1 together greatly potentiated NF-B activation to a greater extent than either alone (Fig. 4). SB reduced NF-B activity that followed exposure to both IL-1 and gp120 (Fig. 4).

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Increased levels of iNOS have been reported in patients with HIV-associated DCM (4). Both IL-1 and gp120 have been shown in nonmyocyte cells to activate multiple second messenger signaling pathways, including activation of MAP kinase (14). We have recently implicated MAP kinase activation in the regulation of IL-1-induced NO production by CM (10, 11). Therefore, we examined the potential involvement of MAP kinase in gp120-mediated NO production in CM. SB, a specific inhibitor of p38 MAP kinase, completely abolished gp120 enhancement of IL-1-stimulated NO production (Fig. 1). This suggests that p38 is involved in gp120-regulated NO production by CM. Both Northern and Western blot analyses confirmed an inhibitory effect of SB (Fig. 2, A and B).

gp120 and IL-1 activation of MAP kinase was directly demonstrated by MAP kinase assay. gp120 greatly increased both p42/44 and p38 MAP kinase activities that were inhibited by SB and PD, respectively (Fig. 3, A and B). gp120 also potentiated IL-1-stimulated p38 but not p42/p44 MAP kinase activities (Fig. 3, A and B). Together, our data indicate that gp120 enhances IL-1-stimulated NO production through a p38 MAP kinase pathway. Activation of this MAP kinase alone by gp120 may be sufficient to induce transient expression of iNOS. However, it is clearly not sufficient to stimulate NO production in CM.

We previously reported (17) that nuclear translocation of NF-B is essential for IL-1-stimulated NO production in neonatal rat CM. Four NF-B enhancer elements were identified upstream in the human iNOS promoter that confer inducibility to cytokines (21). An effect of MAP kinase on the regulation of these NF-B enhancer elements has not been reported. Therefore, we investigated the potential role for gp120-activated MAP kinase cascades in NF-B activation. IL-1 and gp120 each increased NF-B activity. The effect of IL-1 on NF-B activity was greater than gp120 as indicated by electrophoretic mobility shift assay (Fig. 4). The addition of gp120 to IL-1 greatly potentiated the effect of IL-1 on NF-B activity. SB reduced gp120 plus IL-1-stimulated NF-B activity (Fig. 4). Our data indicate that both IL-1 and gp120 increase NF-B activity. However, gp120-induced NF-B stimulation was not associated with NO production (Fig. 1). Thus activation of NF-B is necessary but not sufficient for functional iNOS protein synthesis and NO production.

We recently also reported induction of iNOS mRNA expression without resulting in iNOS protein synthesis by neonatal rat CM after exposure to tumor necrosis factor- (TNF-) (11). The addition of TNF- to IL-1 also enhanced iNOS mRNA expression, protein synthesis, and nitrite production (11). These modulatory effects of gp120 and TNF- on IL-1-induced NO production may suggest potentially important mechanisms to control cardiac myocyte NO production under physiological and/or pathological conditions. It is interesting to note that elevated circulating levels of cytokines and iNOS have each been associated with patients with HIV DCM (4). The pathophysiological relevance of these cytokine and iNOS levels and/or their effect on HIV DCM remains to be determined (4).

The p38 MAP kinase inhibitor SB inhibited the gp120 enhancement of IL-1-stimulated NF-B activity (Fig. 4). This observation suggests that gp120 plus IL-1 stimulates NF-B activity mainly through a p38 MAP kinase signaling pathway. This is consistent with reports that IL-1 can activate three MAP kinase cascades, namely p46/54 c-jun NH₂-terminal kinase, p38 (MAP kinase), and extracellularly regulated kinase 1/2 (ERK-1/2), with maximal activation of 25-fold with p38 and only 3-fold with ERK-1/2 (13).

We report for the first time that the HIV coat protein gp120 directly regulates a cytokine effect on CM. The physiological consequences of the increase in iNOS expression and NO production were not explored in these spontaneously beating neonatal rat CM. Preliminary evidence for a direct inotropic effect of gp120 has been reported in adult CM (5). Chen et al. (5) reported in a recently published and presented abstract that gp120 directly depressed contractility in isolated rabbit ventricular myocytes in vitro by modulating transsarcolemmal Ca²⁺ influx through L-type Ca²⁺ channels. A negative inotropic effect mediated through L-type Ca²⁺ channels is consistent with previous reports indicating that both IL-1 and NO each mediate inotropic effects in CM through L-type Ca²⁺ channels (20, 22). Taken together, these findings support a novel hypothesis that interactions between cytokines and HIV proteins contribute to HIV-associated cardiomyopathy. Considerably more work needs to be done before definitive conclusions can be drawn about the relevance of our in vitro studies to HIV infection.

The direct effects of gp120 on neurons have been proposed as potential mechanisms contributing to HIV dementia (15). It appears that gp120 exerts its effect on the brain by binding to the N-methyl-D-aspartate (NMDA) receptor (23). However, NMDA receptors have not been demonstrated in the heart. However, the NMDA receptor antagonist (+)-MK801 has been shown to exert a positive inotropic effect on the rat heart (8). This may provide evidence for the existence of a homologous NMDA binding site in the heart. gp120 binding to this site would trigger the MAP kinase signaling pathway. Alternatively, gp120 could bind to a structure homologous to its primary binding site, the CD4 receptor (12). No such receptor has been reported in the heart. Thus identification of this gp120 binding site may represent a novel cardiac receptor.

Elevated circulating levels of cytokines and iNOS have been described in HIV patients with DCM (4). The pathophysiological relevance of cytokines, HIV coat protein gp120, and NO in clinical conditions such as DCM is unclear. These mediators may contribute to DCM via direct depression of contractility and/or induction of myocyte apoptosis (2). Exploring the mechanisms involved in cytokine and gp120-mediated NO production by CM may provide novel insights relevant to designing management strategies for HIV patients with DCM. Our proposed gp120 binding site and/or the P38 MAP kinase described in this study may represent potentially novel therapeutic targets.