Departments of 1Medicine and 2Physiology and Pharmacology, West Virginia University School of Medicine, Robert C. Byrd Health Sciences Center, Morgantown 26506; and 3Louis A. Johnson Department of Veterans Affairs Medical Center, Clarksburg, West Virginia 26301
Submitted 11 February 2003 ; accepted in final form 19 August 2003
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ABSTRACT |
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cardiomyopathy; cell signaling
Evidence is accumulating that HIV gp120 may also play an important pathogenic role in HIV dementia and cardiomyopathy. Direct effects of HIV gp120 on rat neurons have been reported (24, 25). These effects appear to involve changes in intracellular ionized free calcium and p38 MAP kinase activation (13, 19, 24, 34). Altered calcium homeostasis and p38 MAP kinase activation have also been implicated in myocardial dysfunction (8, 21, 27, 31, 32, 35, 36, 40, 46). We previously reported (23) that HIV gp120 stimulates p38 MAP kinase activation in neonatal rat cardiac myocytes. The physiological consequences of these findings were not explored. We now report direct inotropic effects of recombinant HIV gp120 on adult rat ventricular myocytes (ARVM) mediated by a signaling pathway involving p38 MAP kinase activation. HIV gp120 may elicit a common stress response signaling pathway in ARVM that contributes to HIV and other cardiomyopathies.
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MATERIALS AND METHODS |
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Statistical methods. Data represent the means ± SE of 1215 different determinations derived from 1215 individual myocytes from 79 separate myocyte preparations from 79 different rats. Analysis of variance (ANOVA) was used for multigroup comparisons. Values of P < 0.05 were considered statistically significant.
Adult rat ventricular myocytes. Cells were isolated from the hearts of adult male Sprague-Dawley rats (250300 g) as previously reported by Kan et al. (22). Rats were anesthetized with pentobarbital sodium, and the hearts were removed rapidly and perfused with Krebs-Henseleit bicarbonate buffer (KHB) containing (in mM) 118.1 NaCl, 3.0 KCl, 1.8 CaCl2, 1.2 MgSO4, 1.0 KH2PO4, 27.3 NaHCO3, 10.0 glucose, and 2.5 pyruvic acid, pH 7.4, according to the method of Langendorff at a constant rate of 8 ml/min with a peristaltic pump. All buffer and enzyme solutions used during cell isolation were maintained at 37°C and preequilibrated with 95% O2-5% CO2. Hearts were perfused with KHB for 15 min, followed by changing to low-Ca2+ KHB containing (in mM) 105.1 NaCl, 3.0 KCl, 0.01 CaCl2, 1.2 MgSO4, 1.0 KH2PO4, 20.0 NaHCO3, 10.0 glucose, 5.0 pyruvic acid, 10.0 taurine, and 5.0 mannitol, pH 7.4, for an additional 10 min. The hearts were then immersed in recirculating KHB with low Ca2+ containing collagenase B (1.25 mg/ml; Boehringer Mannheim Biochemicals, Indianapolis, IN) for 40 min. The ventricles were minced and placed into a 50-ml centrifuge tube, adjusted to 25 ml with low-Ca2+ KHB, and centrifuged at 50 g for 2 min. The supernatant was aspirated, and the concentration of Ca2+ in KHB was increased in four increments (0.08, 0.6, 1.2, and 1.8 mM). Finally, the mixture was passed through 225-µm nylon mesh and centrifuged at 50 g for 2 min. The centrifuge procedure was repeated until the preparation was composed of at least 80% viable left ventricular myocytes. The myocytes exhibited typical striated and rod-shaped appearance when viewed by light microscope. Only those myocytes that were rod shaped, with striations and no blebs, and not spontaneously contracting were included for analyses (12). Physiological experiments were conducted with continuous superfusion of 95% O2-5% CO2 KHB. Myocytes typically retained their baseline fractional shortening (FS) for at least 4 h, and only freshly isolated cells were used for physiological experiments.
Measurement of myocyte contractile function. Measurements of the amplitude and velocity of unloaded single ARVM shortening and relengthening were made on the stage of an inverted phase-contrast microscope (Olympus, IX70-S1F2, Olympus Optical) with the Myocyte Calcium Imaging/Cell Length System, in which the analog motion signal was digitized and analyzed by EDGACQ edge detection software (Ionoptix, Milton, MA; Refs. 38 and 44). Electrical field stimulation was applied at 0.5 Hz, 3-ms duration, and 20 V to achieve threshold depolarization. Experiments were performed at 20% above threshold. Each cell served as its own control by continuous superfusion of buffer and drugs. The initial inotropic effects of HIV gp120 were confirmed with three different sources of recombinant gp120 (HIV-1 Bal and Cheng Mai from NIH and MN from Protein Sciences, Meriden, CT). The data presented reflect the results obtained from HIV-1 gp120 (Cheng Mai).
Intracellular ionized free Ca2+ concentration. Intracellular ionized free Ca2+ concentrations ([Ca2+]i) of the same ventricular myocytes were measured simultaneously by the same system (38, 44). Cells were loaded with fura 2-AM (2 µM). Calcium concentrations were analyzed by WIZARD fluorescence analysis software (Ionoptix). Coverslips with attached cells were placed in a temperature-controlled (37°C) chamber (series 20 Recording/Perfusion Chambers, Warner Instrument, Hamden, CT) and continuously superfused at 0.2 ml/min with oxygenated KHB. One cell per coverslip was used.
Phosphoprotein assays. Western analyses were used to determine phosphorylation of MKK3/MKK6, p44/42, JNK, p38 MAP kinase, and transcription factor ATF-2 by using phospho-MKK3/MKK6, phospho-p44/42, phospho-JNK, phospho-p38 MAP kinase (Thr180/Tyr182), and phospho-ATF-2 (Thr71) antibodies (Cell Signaling Technology, Beverly, MA) according to the manufacturer's recommendations as we previously reported (23). The quantity of each sample loaded was determined by using non-phospho-protein antibodies. Blots were detected by using the Amersham ECL system. Blots were also left in the stripping buffer (7 M guanidine-HCl, 2.5 M glycine, 0.05 mM EDTA, 0.1 M KCl, 20 mM mercaptoethanol) for 15 min, washed with distilled water, and reblotted with different antibodies. Lysis buffer (100 µl) was added, cells were scraped off the 30-mm dish, and the extract was transferred to a microfuge tube to keep on ice. This was followed by sonication for 2 s and centrifugation at 10,000 g for 15 min at 4°C. The supernatant was transferred to a new centrifuge tube. 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 SDS-PAGE.
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RESULTS |
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The initial positive inotropic effect of gp120 was associated with a corresponding increase in [Ca2+]i (Figs. 2 and 4). This is consistent with the most common mechanism of enhancing contractility by increasing [Ca2+]i (4). However, the delayed negative inotropic effect was not associated with a decrease in [Ca2+]i compared with controls (Figs. 2 and 4). A plausible mechanism by which binding of gp120 to the surface of a cardiac myocyte could lead to changes in contractile proteins remained to be determined.
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HIV gp120 has been reported to activate p38 MAP kinase in rat neurons (24). We also previously reported (23) that HIV gp120 activates p38 MAP kinase in isolated neonatal rat cardiac myocytes. Therefore, we sought to determine whether the inotropic effect(s) of gp120 could be altered by the p38 MAP kinase inhibitor SB-203580. Pretreatment of myocytes with 10 µM SB-203580 completely blocked the negative inotropic effect of gp120 while having no effect on the initial positive inotropic effect (Fig. 5). SB-203580 alone had no inotropic effect. Interestingly, SB-203580 also had no effect on the initial gp120-mediated increase in [Ca2+]i (Fig. 6).
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More direct evidence of a role for p38 MAP kinase in the negative inotropic effect of gp120 was provided by measuring phosphorylation of proteins involved in the p38 MAP kinase signaling pathway. Exposure of ARVM to gp120 (1 µg/ml) resulted in a time-dependent phosphorylation of p38 MAP kinase and its downstream effector, ATF-2 (Fig. 7). This pattern was selective for p38 MAP kinase and was not shared by p44/42 and JNK kinases (Fig. 7). HIV gp120 exposure had no apparent effect on p44/42 phosphorylation. JNK appears to be slightly and only transiently activated within a few minutes after exposure to gp120.
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The contribution of the components of the p38 MAP kinase pathway to the negative inotropic effect of HIV gp120 on ARVM was further explored. Exposure of ARVM to gp120 (1 µg/ml) also resulted in phosphorylation of MKK3/6, an upstream activator of p38 (Fig. 8). A lower concentration of SB-203580 was used to ensure greater specificity of inhibition of p38 MAP kinase activity. Five micromolar SB-203580 selectively blocked the phosphorylation of ATF-2 by p38 MAP kinase without blocking the phosphorylation of either MKK3/6 or p38 MAP kinase (Fig. 7).
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This lower concentration of SB-203580 (5 µM) also blocked the negative inotropic effect of gp120 as noted above for 10 µM SB-203580 (Figs. 5 and 8). In addition, the administration of 5 µM SB-203580 for 20 min before or 20 min after gp120 treatment blocked the gp120-induced negative inotropic effect (Fig. 9).
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DISCUSSION |
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The development of a cardiomyopathy is a well-recognized complication of HIV infection that confers a worse prognosis for those afflicted with AIDS (3, 26, 29). An autopsy study found myocarditis in 52% and dilated cardiomyopathy in 10% of AIDS patients (1). Echocardiographic evidence of decreased FS (myocardial dysfunction) has been reported to predict an increase in mortality among HIV-infected children (2, 3, 8, 28).
A pathogenic role for gp120 in HIV cardiomyopathy is very similar to the role already proposed for gp120 in HIV dementia (5, 10). HIV gp120 has been reported to have CD4-independent effects on rat neurons that may be mediated through the N-methyl-D-aspartate (NMDA) receptor (10, 41). Drugs that either deplete (caffeine, thapsigargin) or block release from (dantrolene) intracellular Ca2+ stores were effective in blocking the rise in intracellular Ca2+ and cell death in rat neural tissue caused by gp120 (34). In addition, gp120 has been reported to regulate p38 MAP kinase activity in neurons (24, 25). Our results provide evidence for both Ca2+-dependent and Ca2+-independent effects of gp120 in cardiac myocytes. Ca2+-independent effects of gp120 have not been reported in neurons. This suggests differences between cardiac myocyte and neuronal receptors or signaling pathways and/or differences in the techniques used to study neurons and myocytes. These observations in rat brain and heart support the view that clinically relevant insights into manifestations of HIV infection can be derived from studies in animal models that do not develop immunosuppression and death from HIV exposure. This may be true for HIV cardiomyopathy as well as dementia.
As noted above, this gp120 signaling pathway involves p38 MAP kinase activation. p38 MAP kinase is a member of a class of intracellular enzymes that phosphorylate proteins in response to inflammatory mediators (e.g., cytokines) and stress (e.g., ischemia) (40, 42). Evidence is rapidly accumulating in support of a pathogenic role for p38 MAP kinase in myocardial dysfunction in animal models and humans (8, 28). p38 MAP kinase activation has been implicated in ischemia, hypertrophy, apoptosis, and adrenergic signaling in cardiac myocytes (31, 32, 35, 36, 45, 46). We now provide compelling evidence implicating p38 MAP kinase activation in the negative inotropic effect of gp120 in ARVM. A relatively brief and transient p38 MAP kinase response may confer early adaptive benefits by uncoupling Ca2+ release from myocyte contraction. This could conserve energy and diminish oxidative stress. However, the more prolonged activation of neurons by HIV gp120 has been shown to result in apoptosis (10, 24). Similarly, prolonged and repeated activation of p38 MAP kinase in cardiac myocytes by gp120 could result in HIV cardiomyopathy in vulnerable individuals.
Adrenergic signaling is characteristically blunted in humans as well as animal models with myocardial depression, cardiomyopathy, and chronic heart failure from a variety of causes (14, 16, 21). A physiological role for p38 MAP kinase in adrenergic signaling in cardiac myocytes was recently suggested by an elegant study in a transgenic mouse model lacking 1-adrenergic receptors (46). Inhibition of p38 MAP kinase activation with SB-203580 was found to enhance the positive inotropic effect of the
-adrenergic agonist isoproterenol through the
2-adrenergic receptor. A physiologically relevant role for p38 MAP kinase in adrenergic signaling in cardiac myocytes was further supported by studies in avian cardiac myocytes (33). Magne et al. (33) provided strong evidence for the regulation of
2-adrenergic signaling through a MAP kinase/cPLA2 pathway. Human heart failure is associated with a relative decrease in
1- and an increase in
2-adrenergic receptors (6, 20). Thus activation of p38 MAP kinase by HIV gp120 would lead to blunted autonomic responses typical of human cardiomyopathies and chronic heart failure.
A negative inotropic effect of p38 MAP kinase was reported recently by Liao et al. (28). Activation of p38 MAP kinase was achieved by adenoviral gene transfer of an activated mutant of its upstream kinase, MKK3bE. The authors reported a [Ca2+]i-independent negative inotropic effect of the activated MKK3bE mutant. This is remarkably similar to the effect we report as a result of HIV gp120. Liao et al. (28) concluded that troponin I phosphorylation was not responsible for the negative inotropic effect of p38 MAP kinase. Their conclusion was based on the results of their experiments that did not reveal direct phosphorylation of purified troponin I protein by p38 MAP kinase in vitro. These observations would suggest that p38 MAP kinase does not directly phosphorylate troponin I. However, p38 MAP kinase could still participate in an upstream process that results in troponin I phosphorylation. Future studies are warranted to further elucidate the details of the steps involved in the physiological consequences of p38 MAP kinase activation in cardiac myocytes.
The activation of p38 MAP kinase by phosphorylation through a MAPKK-dependent pathway is well established (40, 42). Ge et al. (15) recently discovered an alternative MAPKK-independent mechanism of activation of p38 MAP kinase by autophosphorylation. The results of our studies in ARVM could not definitively distinguish between the MAPKK-dependent and MAPKK-independent pathways. We identified phosphorylation of both MKK3/6 and p38 MAP kinase by gp120 (Fig. 7). The blockade of the inotropic effect of gp120 by SB-203580 (5 µM) was associated with minimal changes in MKK3/6 and p38 MAP kinase phosphorylation along with a decrease in ATF-2 phosphorylation (Fig. 7). These observations are consistent with either MKK3/6- or p38 MAP kinase-mediated phosphorylation and activation of p38 MAP kinase signaling by gp120 in cardiac myocytes.
Our data are also consistent with a recent report by Chen et al. (7) that HIV gp120 depresses rabbit ventricular myocyte contractility. However, the activation of intracellular signaling pathways by gp120 was not explored. Our data and those of Chen et al. (7) indicate the presence of a high-affinity (nM) HIV binding site on cardiac myocytes. The previously identified binding sites for HIV gp120 include the CD4, CXCR4, CCR5, and NMDA receptors (10, 30, 37). Our data suggest the presence of structures homologous to CD4, CXCR4, CCR5, or NMDA receptors on ARVM. Alternatively, ARVM possess a completely novel receptor for HIV gp120. A single report of immunolocalization of CXCR4 to human cardiac myocytes has been published (11). The immunohistochemical data from human heart failure patients raise the interesting possibility that CXCR4 is the receptor responsible for the physiological effects of HIV gp120 on ARVM. This recently identified binding site has no previously known function in the heart. Unfortunately, the currently available antibodies to human CXCR4 do not cross-react with the rat. This signaling pathway is presumably part of the repertoire that cardiac myocytes (and other cells) have evolved to respond to infectious and/or ischemic injury. Elucidation of this receptor signaling pathway in ARVM may provide fundamental insights into cardiac myocyte adaptation to stressful stimuli.
Controlling the activation and termination of this gp120 signaling pathway in myocytes has considerable potential clinical relevance. These studies are likely to provide new insights into the mechanisms contributing to myocardial depression seen in HIV infection as well as other cardiomyopathies. The basic mechanisms involved in p38 MAP kinase activation are relevant to chronic heart failure, ischemia, ischemic preconditioning, and adrenergic signaling in cardiac myocytes as well (35, 36, 46). Management of the HIV epidemic has brought many challenges and new insights into basic biology and medicine. This gp120 signaling pathway in cardiac myocytes may provide a novel therapeutic target for HIV as well as other cardiomyopathies.
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ACKNOWLEDGMENTS |
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GRANTS
This research was supported by National Heart, Lung, and Blood Institute Grant R01-HL-70565, a Department of Veterans Affairs Veterans Integrated Service Network-4 Competitive Pilot Project Fund, and The West Virginia University Research Corporation.
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FOOTNOTES |
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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. Section 1734 solely to indicate this fact.
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