sign in sign up
<<
Home , Mitochondrion

Cardiomyocytes undergo in human immunodeficiency cardiomyopathy through - and death receptor-controlled pathways

Cheryl Twu*†, Nancy Q. Liu*†, Waldemar Popik‡, Michael Bukrinsky§, James Sayre¶, Jaclyn Roberts*†, Shammas Rania*†, Vishnu Bramhandam*†, Kenneth P. Roos†, W. Robb MacLellan†, and Milan Fiala*ʈ

*Department of Medicine, Greater Los Angeles Veterans Affairs Medical Center, Los Angeles, CA 90073; †Cardiovascular Research Laboratory, University of California, 3645 MRL, Los Angeles, CA 90095-1760; ‡Oncology Center, The Johns Hopkins University School of Medicine, Baltimore, MD 21231; §Department of Microbiology and Tropical Medicine, George Washington University, Washington, DC 20037; and ¶Department of Biostatistics, School of Public Health, University of California, Los Angeles, CA 90095-1760

Edited by Louis J. Ignarro, University of California School of Medicine, Los Angeles, CA, and approved August 30, 2002 (received for review May 31, 2002)

We investigated 18 AIDS (5 with and 13 without cardiomy- (SIV)mac 239 infection produced more severe immune suppres- ⌬ opathy) by using immunocytochemistry and computerized image sion and cardiac fibrosis compared with SIVmac 239 nef (12). analysis regarding the roles of HIV-1 and tumor necrosis In addition to HIV-1, opportunistic cardiotropic , such as factor ligands in HIV cardiomyopathy (HIVCM). HIVCM and cardi- adenoviruses and cytomegalovirus (13), may contribute to omyocyte apoptosis were significantly related to each other and to HIVCM. the expression by inflammatory cells of gp120 and tumor necrosis Cardiomyocyte apoptosis underlies many forms of dis- factor-␣. In HIVCM heart, active caspase 9, a component of the ease, including viral cardiomyopathy (14, 15). The inducers of mitochondrion-controlled apoptotic pathway, and the elements of cardiomyocyte apoptosis include myocardial stretch, pres- the death receptor-mediated pathway, tumor necrosis factor-␣ and sure overload, radicals, nitric oxide, cytokines such as Fas ligand, were expressed strongly on macrophages and weakly TNF-␣, and viruses (16). In failing human hearts, cardiomyocyte on cardiomyocytes. HIVCM showed significantly greater macro- apoptosis was found to be abundant, but the mechanism was not phage infiltration and cardiomyocyte apoptosis rate compared apparent (17). In experimental murine Coxsackievirus myocar- with non-HIVCM. HIV-1 entered cultured neonatal ventricular ditis, Fas ligand͞Fas pathway was noted to play a critical role myocytes by macropinocytosis but did not replicate. HIV-1- or (14). We hypothesized that apoptosis might underlie HIVCM, gp120-induced apoptosis of rat myocytes through a mitochondri- because HIV-1 can induce apoptosis of different cells both in on-controlled pathway, which was inhibited by heparin, AOP- vitro and in vivo (18). In this study, we examined the role of RANTES, or pertussis toxin, suggesting that cardiomyocyte apo- gp120, Nef, TNF-␣, Fas ligand, and caspases in virus-induced ptosis is induced by signaling through chemokine receptors. In apoptosis and in vivo. We showed that in vitro gp120 conclusion, in patients with HIVCM, cardiomyocytes die through induces cardiomyocyte apoptosis by a mitochondrion-controlled both mitochondrion- and death receptor-controlled apoptotic pathway, but in vivo death receptor ligands from macrophages pathways. are a major cause of apoptosis, and the apoptotic signaling may occur through chemokine receptors. IV cardiomyopathy (HIVCM) is a complication of HIV-1 Methods Hinfection with high cardiovascular morbidity and mortality in young and middle-aged adults. Its annual incidence was Heart Tissues. The Texas Repository for AIDS Neuropathogen- estimated by echocardiographic monitoring to be 15.9 cases per esis Research and the Manhattan HIV Brain Bank provided 1,000 asymptomatic Italian HIV-1-positive patients (1). Despite paraffin-embedded formalin-fixed heart tissues from the left its growing importance, the molecular mechanisms of HIVCM ventricle and plasma specimens of 22 patients with AIDS, of pathogenesis remain poorly understood. which nine were diagnosed as HIVCM (‘‘HIVCM-positive Immunopathological examination of AIDS heart tissues hearts’’), and 13 had no history or biochemical evidence of heart showed that HIVCM is strongly inflammatory and related to the failure (‘‘HIVCM-negative hearts’’). The University of Califor- density of the prostacyclin cyclooxygenase-2 nia, Los Angeles (UCLA), Resource Bank provided heart and the macrophage marker CD68 (2, 3). Infected hearts showed tissues from a normal patient (death from a motor vehicle a strong expression of the viral envelope gp120 and the accident) and four patients with non-HIVCM (three idiopathic regulatory protein Nef in infiltrating macrophages and T cells and one adriamycin-induced). The UCLA Office for Protection but only a weak or absent gp120 and Nef expression in cardio- of Research Subjects approved all work with human tissues and myocytes (3). This observation argues against a productive blood cells. infection of cardiomyocytes by HIV-1 as a primary mechanism of HIVCM, as previously proposed (4). Instead, it suggests that Primary Neonatal Rat Ventricular Myocyte (NRVM) Cultures. Primary indirect mechanisms initiated by inflammatory cells are plausi- NRVM cultures were prepared from neonatal 1- to 3-day-old ble, such as secretion of cytokines, in particular tumor necrosis Sprague–Dawley with full approval of the UCLA factor-␣ (TNF-␣) (5), nitric oxide (6, 7), and viral proteins, in Research Committee, essentially as described previously (19). particular gp120 (7, 8) and Tat (9). In agreement with an indirect

model, transgenic mice expressing the entire HIV-1 coding This paper was submitted directly (Track II) to the PNAS office. sequences in T cells and monocyte͞macrophages developed a Abbreviations: HIVCM, HIV cardiomyopathy; TNF-␣, tumor necrosis factor-␣; NRVM, neo- multitude of complications (10), including multifocal cardiomy- natal rat ventricular myocyte; BNP, brain natriuretic ; TUNEL, terminal deoxynu- opathy (P. Jolicoeur, personal communication), which were cleotidyl -mediated dUTP nick-end labeling; DAPI, 4Ј,6-diamidino-2-phenylin- found to be related to Nef expression (11). A simian study was dole; U test, Mann–Whitney U test. also suggestive of the role of Nef, because simian immunovirus ʈTo whom correspondence should be addressed. E-mail: fi[email protected].

14386–14391 ͉ PNAS ͉ October 29, 2002 ͉ vol. 99 ͉ no. 22 www.pnas.org͞cgi͞doi͞10.1073͞pnas.212327899 Downloaded by guest on October 1, 2021 Ventricles of Ϸ100 hearts were digested four times, 20 min each, ages of this area were acquired, and the images were scanned by in 30 ml of enzyme digestion solution containing 0.1% collage- using IMAGE-PRO PLUS 4.1 (Media Cybernetics, Silver Spring, nase (Worthington)͞12% pancreatin 4ϫ (GIBCO͞BRL)͞0.06% MD) (3). Ads buffer (116.4 mM NaCl͞5.4 mM KCl͞5.6 mM dextrose͞10.9 The antibodies included those to sarcomere (MF20, ͞ ␮ ͞ mM NaH2PO4 405.7 M MgSO4 20 mM Hepes, pH 7.3). Developmental Studies Hybridoma Bank, University of Iowa), NRVMs were purified by using a Percoll (Pharmacia) step gp120 (clones ID6 and 5F7), Nef (EH1 ascites) (AIDS Research gradient in Ads buffer (comprising layers with final densities of and Reference Reagent Program, National Institute of Allergy 1.130, 1.082, and 1.059 g͞ml) and washed in Ads buffer. Fibro- and Infectious Diseases), p10 fragment of caspase-9, Fas ligand blast cells were removed by aspiration. The myocytes (banding (BioSource International, Camarillo, CA), TNF-␣ (Sigma), and between the 1.082- and 1.059-g͞ml layers) were finally washed in CD68 and CD3 (DAKO). To study virus entry, cardiomyocytes plating media (4:1 DMEM͞M199͞10% horse serum͞5% neo- were pretreated with cholera toxin B–Alexa 594 (Molecular natal calf serum͞1% ) and plated at a density of 1 ϫ Probes), methyl-␤-cyclodextrin, heparin, 5-(N,N-dimethyl)- 106 cells͞60-mm dish or 104 cells in a four-well chamber slide amiloride (all from Sigma), AOP-RANTES (gift of A. Proud- coated with 1% gelatin. They were cultured in serum-free foot, Serono, Geneva), SDF-1 (PeproTech, Rocky Hill, NJ), medium, a 1:1 mixture of DMEM͞M199. The purity of cultured U0126 (Promega), or pertussis toxin (List Biological Laborato- NRVMs was 95–100% when the number of cell nuclei [4Ј,6- ries, Campbell, CA), as indicated. diamidino-2-phenylindole (DAPI)-positive] was compared with the number of myocytes (MF20-positive). Apoptosis Assays. Apoptosis of cardiomyocytes and NRVMs was detected by the terminal deoxynucleotidyl transferase-mediated Virus and Viral Proteins. HIV-1 protein gp120SF2 was obtained dUTP nick-end labeling (TUNEL) technique (Apoptosis De- from the AIDS Research and Reference Reagent Program, tection Kit with dUTP, Promega) by using the FITC-conjugated National Institute of Allergy and Infectious Diseases. HIV-1MN, mix, and simultaneous staining with DAPI or either active or aldrithiol-inactivated, and control vesicles from Hoechst 33342 (Molecular Probes). Apoptotic inhibitors in- uninfected cells were provided by J. Lifson (Science Applications cluded a general caspase inhibitor, Z-VAD-FMK, a preferential International, Frederick, MD). The inactivated virus has an caspase-8 inhibitor, Z-IETD-FMK, and a preferential caspase-9 intact envelope but inactivated nucleocapsid (20). HIV-1NL4-3 inhibitor, Z-LEHD-FMK (Enzyme Systems Products, Liver- was prepared by transfecting 293 cells with DNA of an infectious more, CA) (23). For detection of mitochondrial features of ␮ molecular clone (21). HIV-1NL4-3 incorporating Vpr-GFP (HIV- apoptosis, live cells were stained by using Hoechst 33342 (2 M) ЈЈ ЈЈ ЈЈ ЈЈ 1NL4-3 Vpr-GFP) was produced by cotransfection of 293 cells and 5,5 ,6,6 -tetrachloro-1,1 ,3,3 -tetraethylbenzimidazolyl- with pNL4-3 DNA (10 ␮g) and expression vector coding for carbocyanine iodide (JC-1, Molecular Probes) (2 ␮M) for 30 min GFP-Vpr fusion protein (20 ␮g) (22). A pseudotyped luciferase- at 37°C in complete medium. c release was deter- expressing NL HXB2 Env(ϩ)LUC(ϩ) virus was obtained by mined by confocal microscopy in cells fixed with 4% parafor- cotransfection of DNA-encoding envelope from a T- maldehyde and stained with monoclonal anticytochrome c tropic HIV-1 clone HXB2 and pNL4-3 Env(Ϫ)LUC(ϩ) into antibody. 293T cells (22). Western Blotting. An equal volume of tissue from each heart was Virus Assays. In the luciferase assay, 106 NRVMs were infected extracted by dounce homogenization in ice-cold cell lysis buffer. with 3 ϫ 107 RNA copies of NL HXB2 Env(ϩ)LUC(ϩ) and, at An equal amount of protein (40 ␮g) was run on 7.5% SDS– specified times postinfection, the cells were washed and lysed by acrylamide gel, transferred to polyvinylidene membranes, incu- using the Luciferase Cell Culture Lysis Reagent (Promega). The bated with primary and secondary antibodies, and developed by lysate was tested by using the Luciferase Assay Reagent (Pro- chemiluminescence, as described (24). Mouse anti-human mega) and scintillation counting (22). caspase-8 (p18) (Biosource) was used as a primary antibody. For the RT-PCR assay, 106 cardiomyocytes were exposed for ϫ 7 4hto5 10 copies of HIV-1LAI that had been pretreated for Statistical Analysis. The significance of data was determined by t 1 h at room temperature with 200 units͞ml of RNase-free DNase test analysis performed with the statistical software SPSS, Version I (Roche Molecular Biochemicals) in PBS containing 0.04 M 10.0 (SPSS, Chicago) and, when indicated, by nonparametric MgCl2, washed four times, and cultivated. At indicated times, the analysis using the Mann–Whitney U test (U test). The interaction cells were scraped, centrifuged, and the cell pellets were lysed in of gp120 and TNF-␣ in apoptosis was analyzed by median PCR buffer. After protein digestion with proteinase K (2 h at regression (25). 56°C) and its inactivation (10 min at 95°C), the lysate was subjected to amplification with the primers for LTR R͞U5 Results [(forward) 5Ј-GGC TAA CTA GGG AAC CCA CTG-3Ј, (re- Because the relative importance of gp120, Nef and TNF-␣, and verse) 5Ј-CTG CTA GAG ATT TTC CAC ACT GAC-3Ј], and apoptosis in HIVCM is unknown, we examined these parameters gag [(forward) 5Ј-CAG ATA TCC ACT GAC CTT TGG-3Ј, in the hearts of HIV-1-positive patients with and without (reverse) 5Ј-GCT TAA TAC TGA CGC TCT CGC A-3Ј], as HIVCM. Our previous study identified gp120 and Nef in mac- described (22). rophages and T cells but not cardiomyocytes within the infected heart (3), suggesting that these proteins might act in a paracrine Brain Natriuretic Peptide (BNP) Assay. BNP, a peptide rather than an autocrine fashion. whose plasma concentration is raised in congestive , was measured by a competitive enzyme immunoassay (American HIVCM Is Associated with gp120 Expression and Cardiomyocyte Apo- MEDICAL SCIENCES Laboratory Products, Windham, NH). ptosis, and the Apoptosis Is Related to both gp120 and TNF-␣. We examined apoptosis and HIV-1 proteins in the heart tissues of 18 Immunocytochemistry and Immunofluorescence. Formalin-fixed tis- HIV-1-positive patients, 5 HIVCM-positive patients (mean age sues were immunostained by DAKO LSAB 2 or EnVision 40 years), and 13 patients HIVCM-negative (mean age 44 years) Double Stain System kits (3). Cultured myocytes were stained by (see Table 1, which is published as supporting information on the indirect immunofluorescence. The stained sections were exam- PNAS web site, www.pnas.org). The hearts of HIVCM-positive ined at low power (ϫ10 objective) to locate the area of the patients generally showed increased mass [mean weight: highest staining density, three high-power (ϫ40 objective) im- HIVCM-positive ϭ 580 Ϯ 220 g; HIVCM-negative ϭ 346 Ϯ 57 g

Twu et al. PNAS ͉ October 29, 2002 ͉ vol. 99 ͉ no. 22 ͉ 14387 Downloaded by guest on October 1, 2021 Fig. 1. Relation of gp120, Nef, and TNF-␣ to HIV-1 cardiomyopathy and cardiomyocyte apoptosis. The hormone BNP was determined in the plasma of 12 patients by using a competitive enzyme immunoassay (A). The left ventricular sections from 18 AIDS hearts were stained by the trichrome technique to detect fibrosis (B), immunocytochemistry with anti-gp120, -Nef (C), and -TNF-␣ (E) , and the TUNEL and DAPI techniques (D) (note bright contracted nuclei corresponding to TUNEL-positive apoptotic nuclei). Maximum staining was identified at low power, and then three high-power fields were scanned in a predetermined order by using IMAGE PRO software. Zero value indicates that no positive staining was found anywhere. The mean Ϯ SD for each group (HIVCM-positive n ϭ 5 and HIVCM-negative, n ϭ 13) was plotted on the y axis and the data were analyzed by the t test (P values: BNP ϭ 0.004, fibrosis ϭ 0.091, gp120 ϭ 0.008, Nef ϭ 0.315, apoptosis ϭ 0.004, TNF-␣ ϭ 0.051), and the Mann–Whitney nonparametric test (P values: BNP ϭ 0.001, fibrosis ϭ 0.04, gp120 ϭ 0.08, Nef ϭ 0.3315, apoptosis ϭ 0.004, TNF-␣ ϭ 0.003).

ϭ ϭ Ϯ (P 0.002)], fibrosis [mean: HIVCM-positive 162,008 weak on rare myocytes (Fig. 2d2). In addition, p18 fragment of 290,568 pixels (px); HIVCM-negative ϭ 9,913 Ϯ 5,147 px (P ϭ caspase-8 was detected by Western blotting in left ventricular 0.09)], and elevated BNP serum levels [mean: HIVCM- heart tissue of four patients with HIVCM in relation to the BNP positive ϭ 1,100 Ϯ 703 units; HIVCM-negative ϭϽ342 units plasma titer in each patient (see Fig. 4, which is published as (P ϭ 0.004)] (Fig. 1). HIVCM-positive hearts had a significantly supporting information on the PNAS web site). higher expression of gp120 [mean: HIVCM-positive 153 Ϯ 146 To further analyze the molecular mechanisms by which HIV-1 ϭ Ϯ ϭ ϭ px, HIVCM-negative 5.2 8.1 px (U test, P 0.08; t test, P and gp120 induce cardiomyocyte apoptosis, we used NRVMs as ␣ ϭ Ϯ 0.008)], and TNF- [mean: HIVCM-positive 132 215 px, a model, because they survive and function in vitro for at least Ϯ ϭ ϭ HIVCM-negative 6.3 12.9 px, (U test, P 0.003; t test, P 72 h and express apoptotic death on TNF-␣ stimulation (26). Rat 0.051)]. Nef expression was not significantly different between cells are permissive for early virus products only after transfec- the two groups [mean: HIVCM-positive 65 Ϯ 106 px, HIVCM- ϭ Ϯ ϭ ϭ tion with human CD4, chemokine receptors, and cyclin T1 (27). negative 72 33 px (U test, P 0.315; t test P 0.916)]. However, HIV-1 enters NRVMs by macropinocytosis (see be- HIVCM was significantly associated with cardiomyocyte apo- Ϯ ϭ low), which bypasses CD4 and chemokine receptors, and thus ptosis [mean: HIVCM-positive 82 51 px, HIVCM-negative NRVMs may serve as a model of the nonproductive infection of 7.4 Ϯ 16.0 (U test, P ϭ 0.008; t test, P ϭ 0.004)]. When stepwise human cardiomyocytes. median regression was performed with apoptosis as the depen- dent variable and gp120 and TNF-␣ as independent variables, HIV-1 Binds to Ganglioside GM1 and Enters Cultured NRVMs by there was a significant relation with both (R ϭ 0.5, P Ͻ 0.001). Macropinocytosis, Which Is Inhibited by Cholesterol Extraction, Hep- To clarify which apoptotic pathways are involved in the hearts with HIVCM or non-HIVCM, we examined heart expression of arin, and AOP-RANTES. In brain and coronary endothelial cells, HIV-1 binds to ganglioside GM1 and enters by macropinocytosis the activated mitochondrial caspase, caspase-9, and the TNF ͞ superfamily ligand, Fas ligand. susceptible to inhibition by the Na K channel inhibitor dimeth- ylamiloride, cholesterol-extracting agent cyclodextrin, the polya- Components of both Extrinsic and Intrinsic Apoptotic Pathways Are nion heparin, and U0126 (24). In cultured NRVMs, HIV-1-Vpr- Present in HIVCM Hearts. The p10 cleavage fragment of both GFP also bound to the ganglioside GM1, as seen by confocal caspase-9 and Fas ligand was significantly overexpressed in microscopy 3 h postinfection (see Fig. 5, which is published as hearts with HIVCM (Fig. 2a) compared with hearts with non- supporting information on the PNAS web site), and the same HIVCM (Fig. 2b) or HIV-1 infection without HIVCM (Fig. 2c), compounds, except U0126, blocked virus entry, suggesting that and they were absent from a control heart. In HIVCM, their HIV-1 may use a similar entry mechanism in different cardio- expression was robust on round inflammatory cells (Fig. 2d1) and vascular cells. However, unlike in endothelial cells, the chemo-

14388 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.212327899 Twu et al. Downloaded by guest on October 1, 2021 Fig. 2. Cleaved caspase-9 and Fas ligand are overexpressed in HIVCM compared with non-HIVCM. Heart tissues from a control patient, three AIDS patients without cardiomyopathy, and four patients with non-HIV-1 cardiomyopathy were immunostained by using antiactive caspase-9 and anti-Fas ligand antibodies [ϫ40 (a, b, e, and f); ϫ100 (c, d, g, and h)].

kine analogue AOP-RANTES had a significant inhibitory effect of gp120 (100°C, 15 min) completely eliminated its apoptotic on entry into cultured NRVMs. activity. Finally, apoptosis was blocked by treatment with gp120 Virus entry into NRVMs did not lead to productive infection, monoclonal antibody (1:100, 30 min) (Fig. 3B). as demonstrated by using luciferase and RT-PCR assays. The To clarify the mechanisms of apoptosis induction, we inves- luciferase activity in NRVM lysates collected 3 and 4 days tigated the effects of several agents previously demonstrated to postinfection did not rise significantly above the background in inhibit gp120 interaction with target cells. any sample (negative data not shown). Reverse was only detected with R͞U5 primers, which amplify an early gp120-Induced Apoptosis Is Inhibited by AOP-RANTES. Interaction of product, but not with gag primers amplifying a late product (see gp120 with CCR5 or CXCR4 on target human cells initiates Fig. 6, which is published as supporting information on PNAS signaling cascades (28, 29), which might initiate apoptosis in web site), indicating that, as in brain endothelial cells (22), cardiovascular cells. Although the extent of between HIV-1 infection in rat myocytes is abortive. Taken together, rat and human CCR5 is unknown, the observation that a these results suggest that HIV-1 infection proceeds in a similar low-level infection of rat cells by HIV-1 can be achieved (27) fashion in different heart cells. suggests that HIV-1 might use rat CCR5 or CCR3. We therefore tested whether AOP-RANTES, a CCR5 antagonist and a potent HIV-1 and HIV-1 Envelope Protein gp120 Induce Apoptosis of Cultured competitive inhibitor of gp120-CCR5 interaction (30, 31), and Rat Myocytes Through a Mitochondrion-Controlled Pathway. Both pertussis toxin, which ADP ribosylates Gi␣2 and Gi␣3 and thus infectious HIV-1MN and noninfectious vaccine HIV-1MN with inhibits G protein-coupled chemokine receptor signaling (32, its nucleocapsid zinc finger inactivated by aldrithiol treatment 33), would inhibit gp120-induced apoptosis of NRVMs. AOP- but intact envelope (20) induced apoptosis of NRVMs (Fig. 3). RANTES strongly inhibited the induction of apoptosis by gp120 This result suggested that the HIV-1 envelope was sufficient (Fig. 3B), supporting the hypothesis that gp120 signaling from for apoptotic effect. This supposition was confirmed by ex- CCR5 or CCR3 leads to NRVM apoptosis, as previously shown periments with recombinant gp120 (0.01–1 ␮g͞ml), which in T cells (34). CCR3 is overexpressed on cardiomyocytes in induced NRVM apoptosis with rapid kinetics (49.6% after 5 AIDS hearts (N.Q.L. and M.F., unpublished data), creating an min, 76.2% after 30 min, and 100% after 4 h). All three opportunity for strong gp120 signaling. Consistent with the role preparations of recombinant gp120 from T-tropic strains of gp120- and chemokine-induced signaling, apoptosis was HIV-1MN, HIV-1SF2, and HIV-1IIIB/LAV had approximately strongly inhibited by the polyanion heparin, which inhibits equal apoptotic effects. NRVM apoptosis was also revealed by HIV-1 (35), Tat (36), and chemokine (37) binding to cells DAPI and Hoechst 33258 staining, which showed nuclear (Fig. 3B). condensation and fragmentation (Fig. 3A). To determine the apoptotic pathway induced by gp120, we Discussion relied on inhibitors of specific caspases and staining with the The presence of either gp120, Nef, or both in each of 18 AIDS dye JC-1 and antibody to . The caspase-9 inhib- hearts in this study showed that HIV-1 may invade the heart even itor z-LEHD-FMK (Fig. 3B) completely blocked induction of in patients without overt heart failure. Several etiological agents apoptosis by gp120, whereas the preferential caspase-8 inhib- of HIVCM have been proposed but not proven, including HIV-1

itor s-IETD-FMK was ineffective. A reduced mitochondrial itself (38), opportunistic cardiotropic viruses (13), abused drugs, MEDICAL SCIENCES , as shown by a low JC-1 red͞green ratio, and highly active antiretroviral therapy (5, 39–41). Although and release of cytochrome c in gp120-treated myocytes (Fig. previous studies investigated the roles of gp120 (42), Tat (43), 3C) supported a mitochondrion-dependent mechanism. and Nef (44) in neurological complications and the effects of Tat To exclude the possibility that apoptosis was due to the (45), gp120 (45), and Nef (11) in transgenic animal models, none presence of endotoxin in recombinant gp120 preparations, we of these studies addressed the role of these HIV-1 proteins in performed several control experiments. First, the endotoxin AIDS heart. content in gp120 preparations was found below the sensitivity of We show here that HIVCM is significantly related to the the Limulus lysate test (Ͻ0.003 ng͞ml). Second, heat inactivation viral protein gp120 but not Nef, and cardiomyocyte apoptosis

Twu et al. PNAS ͉ October 29, 2002 ͉ vol. 99 ͉ no. 22 ͉ 14389 Downloaded by guest on October 1, 2021 Fig. 3. Apoptosis of cultured rat myocytes is induced by HIV-1 or gp120 through the mitochondrial pathway. (A) NRVMs were sham-infected (a), infected with HIV-1MN (b), or treated with gp120 (1 ␮g͞ml) (c) and were stained with DAPI (Left), TUNEL (Center), antisarcomeric antibody MF-20 (Right, b), or anti-gp120 (Right, a and c). (B) NRVMs were treated as indicated with HIV-1 (active HIVMN or vaccine HIVMN) or gp120, intact, boiled or neutralized by antibody. gp120-treated cells were pretreated with the caspase-8 inhibitor Z-IETD-FMK (10 ␮M), caspase-9 inhibitor Z-LEHD-FMK (10 ␮M), the CCR5 antagonist AOP-RANTES (100 nM), pertussis toxin (Ptx) (1 ␮g͞ml), or heparin (20 ␮g͞ml), and stained by the TUNEL technique. The results were scanned and mean OD plotted. (C) NRVMs were either sham-infected or infected with HIV-1MN and were stained with Hoechst 33342 (a), JC-1 (b), and anticytochrome C (c). JC-1 staining (red and green) results were scanned. A high red͞green ratio indicates a high mitochondrial membrane potential.

is a critical mechanism in HIVCM. In the AIDS heart, ganglioside GM1 and entered these cells by macropinocytosis apoptosis was significantly related to gp120 and the extrinsic (22). AOP-RANTES inhibited cardiomyocyte apoptosis induced ␣ pathway ligand TNF- . As previously demonstrated, in by gp120MN, which is a CXCR4-using envelope protein. This HIVCM, gp120 is expressed abundantly in macrophages and T result suggests that a signal from AOP-RANTES through CCR3 cells but poorly or not at all in cardiomyocytes (see Fig. 7, or CCR5 interferes with the apoptosis-inducing signal from which is published as supporting information on the PNAS web gp120MN through CXCR4. Heparin inhibited both virus entry site) (3); furthermore, despite efficient entry, HIV-1 does not and apoptosis, suggesting that virus entry could be a significant replicate in rat cardiomyocytes in vitro. Therefore, the proapo- proaptotic stimulus. However, sulfated polysaccharides, includ- ptotic stimuli, gp120 and TNF ligands, appear to originate ing the heparan sulfate proteoglycans syndecans, bind both to the from infected macrophages and lymphocytes, and cardiomy- virus and the chemokines with basic residues, including RAN- ocytes may be affected secondarily. TES and SDF-1 (35, 37), suggesting that heparin could inhibit In previous studies in immune and neural cells, apoptosis induction of apoptosis by binding chemokines rather than the was induced by HIV-1 proteins, gp120 (46, 47), Tat (48, 49), virus. Regardless of the underlying mechanisms, robust effects of Nef (50, 51), and vpr (52). gp120 produced apoptosis in T cells several inhibitors on HIV-1-induced cardiomyocyte apoptosis by Fas signaling (53) but also by mitochondrial membrane suggest interesting opportunities for management of HIVCM. depolarization independently of Fas signaling (46). The latter In conclusion, gp120 alone initiates a mitochondrion- apoptosis was inhibited by the CXCR4 ligand SDF-1 (54). In controlled pathway of apoptosis in cultured myocytes, whereas another study, the mechanism of diverged between the apoptotic signaling in human cardiomyocytes in vivo R5 HIV env, being caspase-8-dependent, and X4 HIV-1 env involves the components of both mitochondrial and extrinsic (55), being caspase-8- and -9-independent. HIV-1 Tat (56) and pathways. Nef (50, 57) proteins protected infected cells but caused apoptosis of bystander cells. Tat induced apoptosis of endo- We thank J. Reed for advice concerning caspase inhibitors, M. thelial cells through caspase activation but not by TNF-␣ or Fas Schibler for assistance with confocal microscopic imaging, S. Antrobus pathways (58). Vpr induced apoptosis through caspase activa- and R. Ross for neonatal rat cardiomyocytes, V. Kermani (Immuno- tion (59). Thus, whereas several HIV-1 proteins have been biogene, Cardiovasular Research Laboratory, University of California shown to induce in vitro apoptosis through different pathways, at Los Angeles, West Hills, CA) for BNP assays, and M. Jegapragasan for skillful technical assistance. This work was supported by Grants the impact of these proteins on apoptosis in vivo has not been HL63639 and HL63065 from the National Institutes of Health resolved. (to M.F.), Grants AI42557 and AI50461 from the National Insti- In cultured rat myocytes, recombinant gp120 alone was suf- tutes of Health (to W.P.), and the Laubisch Endowment (to K.P.R. ficient to induce apoptosis by a mitochondrion-controlled path- and W.R.M.). The Manhattan HIV Brain Bank, New York way. The virus avidly bound to the myocytes through the (R24MH59724), and the Texas Repository for AIDS Neuropathogen-

14390 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.212327899 Twu et al. Downloaded by guest on October 1, 2021 esis Research, University of Texas, Galveston (R24MH59656), of the Program, National Institute of Allergy and Infectious Diseases, Na- National NeuroAIDS Tissue Consortium provided the heart tissues of tional Institutes of Health, provided the listed HIV-1 strains and HIV-1-positive patients. The AIDS Research and Reference Reagent antibodies.

1. Barbaro, G., Fisher, S. D. & Lipshultz, S. E. (2001) Lancet Infect. Dis. 1, 30. Simmons, G., Clapham, P. R., Picard, L., Offord, R. E., Rosenkilde, M. M., 115–124. Schwartz, T. W., Buser, R., Wells, T. N. C. & Proudfoot, A. E. (1997) Science 2. Fiala, M., Liu, N. Q., Reddy, S. & Graves, M. (2001) Alzheimer’s Rep. 4, 1–7. 276, 276–279. 3. Liu, Q. N., Reddy, S., Sayre, J. W., Pop, V., Graves, M. C. & Fiala, M. (2001) 31. Mack, M., Luckow, B., Nelson, P. J., Cihak, J., Simmons, G., Clapham, P. R., AIDS Res. Hum. 17, 1423–1433. Signoret, N., Marsh, M., Stangassinger, M., Borlat, F., et al. (1998) J. Exp. Med. 4. Grody, W., Cheng, L. & Lewis, W. (1990) Am. J. Cardiol. 66, 203–206. 187, 1215–1224. 5. Lewis, W. (2000) Prog. Cardiovasc. Dis. 43, 151–170. 32. Beals, C. R., Wilson, C. B. & Perlmutter, R. M. (1987) Proc. Natl. Acad. Sci. 6. Barbaro, G., Di Lorenzo, G., Soldini, M., Giancaspro, G., Grisorio, B., USA 84, 7886–7890. Pellicelli, A. & Barbarini, G. (1999) Circulation 100, 933–939. 33. Amatruda, T. T., 3rd, Gerard, N. P., Gerard, C. & Simon, M. I. (1993) J. Biol. 7. Kan, H., Xie, Z. & Finkel, M. S. (2000) Am. J. Physiol. 279, H3138–H3143. Chem. 268, 10139–10144. 8. Chen, F., Shannon, K., Ding, S., Silva, M. E., Wetzel, G. T., Klitzner, T. S. & 34. Cicala, C., Arthos, J., Rubbert, A., Selig, S., Wildt, K., Cohen, O. J. & Fauci, Krogstad, P. (2002) AIDS Res. Hum. Retroviruses 18, 777–784. A. S. (2000) Proc. Natl. Acad. Sci. USA 97, 1178–1183. 9. Raidel, S. M., Haase, C., Jansen, N. R., Russ, R. B., Sutliff, R. L., Velsor, L. W., 35. Saphire, A. C., Bobardt, M. D., Zhang, Z., David, G. & Gallay, P. A. (2001) Day, B. J., Hoit, B. D., Samarel, A. M. & Lewis, W. (2002) Am. J. Physiol. 282, J. Virol. 75, 9187–9200. H1672–H1678. 36. Rusnati, M., Tulipano, G., Spillmann, D., Tanghetti, E., Oreste, P., Zoppetti, 10. Hanna, Z., Kay, D. G., Cool, M., Jothy, S., Rebai, N. & Jolicoeur, P. (1998) G., Giacca, M. & Presta, M. (1999) J. Biol. Chem. 274, 28198–28205. J. Virol. 72, 121–132. 37. Sweeney, E. A., Lortat-Jacob, H., Priestley, G. V., Nakamoto, B. & Papayan- 11. Hanna, Z., Kay, D. G., Rebai, N., Guimond, A., Jothy, S. & Jolicoeur, P. (1998) nopoulou, T. (2002) Blood 99, 44–51. Cell 95, 163–175. 38. Lipshultz, S., Fox, C. & Perez-Atayde, A. (1990) Am. J. Cardiol. 66, 246–250. 12. Shannon, R. P., Simon, M. A., Mathier, M. A., Geng, Y. J., Mankad, S. & 39. Lipshultz, S. E. (1998) N. Engl. J. Med. 339, 1153–1155. Lackner, A. A. (2000) Circulation 101, 185–193. 40. Lewis, W., Grupp, I. L., Grupp, G., Hoit, B., Morris, R., Samarel, A. M., 13. Bowles, N. E., Kearney, D. L., Ni, J., Perez-Atayde, A. R., Kline, M. W., Bruggeman, L. & Klotman, P. (2000) Lab. Invest. 80, 187–197. Bricker, J. T., Ayres, N. A., Lipshultz, S. E., Shearer, W. T. & Towbin, J. A. 41. Lewis, W. (2001) Ann. N.Y. Acad. Sci. 946, 46–56. (1999) J. Am. Coll. Cardiol. 34, 857–865. 42. Jones, M. V., Bell, J. E. & Nath, A. (2000) AIDS 14, 2709–2713. 14. Seko, Y., Kayagaki, N., Seino, K., Yagita, H., Okumura, K. & Nagai, R. (2002) 43. Bonwetsch, R., Croul, S., Richardson, M. W., Lorenzana, C., Valle, L. D., J. Am. Coll. Cardiol. 39, 1399–1403. Sverstiuk, A. E., Amini, S., Morgello, S., Khalili, K. & Rappaport, J. (1999) 15. Alter, P., Jobmann, M., Meyer, E., Pankuweit, S. & Maisch, B. (2001) J. Neurovirol. 5, 685–694. Cardiovasc. Pathol. 10, 229–234. 44. Mankowski, J. L., Flaherty, M. T., Spelman, J. P., Hauer, D. A., Didier, P. J., 16. MacLellan, W. R. & Schneider, M. D. (1997) Circ. Res. 81, 137–144. Amedee, A. M., Murphey-Corb, M., Kirstein, L. M., Munoz, A., Clements, 17. Olivetti, G., Abbi, R., Quaini, F., Kajstura, J., Cheng, W., Nitahara, J. A., J. E., et al. (1997) J. Virol. 71, 6055–6060. Quaini, E., Di Loreto, C., Beltrami, C. A., Krajewski, S., et al.(1997) N. Engl. 45. Reid, W., Sadowska, M., Denaro, F., Rao, S., Foulke, J., Jr., Hayes, N., Jones, J. Med. 336, 1131–1141. O., Doodnauth, D., Davis, H., Sill, A., et al. (2001) Proc. Natl. Acad. Sci. USA 18. Yang, Y. & Ashwell, J. D. (2001) Apoptosis 6, 139–146. 98, 9271–9276. 19. Paradis, P., MacLellan, W. R., Belaguli, N. S., Schwartz, R. J. & Schneider, 46. Roggero, R., Robert-Hebmann, V., Harrington, S., Roland, J., Vergne, L., M. D. (1996) J. Biol. Chem. 271, 10827–10833. Jaleco, S., Devaux, C. & Biard-Piechaczyk, M. (2001) J. Virol. 75, 7637–7650. 20. Rossio, J. L., Esser, M. T., Suryanarayana, K., Schneider, D. K., Bess, J. W., 47. Scorziello, A., Florio, T., Bajetto, A. & Schettini, G. (1998) Cell. Signalling 10, Jr., Vasquez, G. M., Wiltrout, T. A., Chertova, E., Grimes, M. K., Sattentau, 75–84. Q., et al. (1998) J. Virol. 72, 7992–8001. 48. Li, C. J., Friedman, D. J., Wang, C., Metelev, V. & Pardee, A. B. (1995) Science 21. Popik, W. & Pitha, P. M. (2000) J. Virol. 74, 2558–2566. 268, 429–431. 22. Liu, N. Q., Lossinsky, A. S., Popik, W., Li, X., Gujuluva, C., Robertson, J., 49. Nath, A., Haughey, N. J., Jones, M., Anderson, C., Bell, J. E. & Geiger, J. D. Pushkarsky, T., Bukrinsky, M., Kriederman, B., Witte, M., et al. (2002) J. Virol. (2000) Ann. Neurol. 47, 186–194. 76, 6689–6700. 50. Ameisen, J. (2001) Nat. Med. 7, 1181–1182. 23. Li, P., Nijhawan, D., Budihardjo, I., Srinivasula, S. M., Ahmad, M., Alnemri, 51. Geleziunas, R., Xu, W., Takeda, K., Ichijo, H. & Greene, W. C. (2001) Nature E. S. & Wang, X. (1997) Cell 91, 479–489. 410, 834–838. 24. Liu, N. Q., Lossinsky, A. S., Popik, W., Li, X., Gujuluva, C., Kriederman, B., 52. Patel, C. A., Mukhtar, M. & Pomerantz, R. J. (2000) J. Virol. 74, 9717–9726. Roberts, J., Pushkarsky, T., Bukrinsky, M., Witte, M., et al. (2002) J. Virol. 76, 53. Bottarel, F., Feito, M. J., Bragardo, M., Bonissoni, S., Buonfiglio, D., De- 6689–6700. Franco, S., Malavasi, F., Bensi, T., Ramenghi, U. & Dianzani, U. (1999) AIDS 25. Harris, A. (1988) Am. Stat. 4, 14–15. Res. Hum. Retroviruses 15, 1255–1263. 26. Krown, K. A., Page, M. T., Nguyen, C., Zechner, D., Gutierrez, V., Comstock, 54. Berndt, C., Mopps, B., Angermuller, S., Gierschik, P. & Krammer, P. H. (1998) K. L., Glembotski, C. C., Quintana, P. J. & Sabbadini, R. A. (1996) J. Clin. Proc. Natl. Acad. Sci. USA 95, 12556–125561. Invest. 98, 2854–2865. 55. Vlahakis, S. R., Algeciras-Schimnich, A., Bou, G., Heppelmann, C. J., Villasis- 27. Keppler, O. T., Yonemoto, W., Welte, F. J., Patton, K. S., Iacovides, D., Keever, A., Collman, R. C. & Paya, C. V. (2001) J. Clin. Invest. 107, 207–215. Atchison, R. E., Ngo, T., Hirschberg, D. L., Speck, R. F. & Goldsmith, M. A. 56. McCloskey, T. W., Ott, M., Tribble, E., Khan, S. A., Teichberg, S., Paul, M. O., (2001) J. Virol. 75, 8063–8073. Pahwa, S., Verdin, E. & Chirmule, N. (1997) J. Immunol. 158, 1014–1019. 28. Davis, C. B., Dikic, I., Unutmaz, D., Hill, C. M., Arthos, J., Siani, M. A., 57. Wolf, D., Witte, V. & Lafferty, B. (2001) Nat. Med. 7, 1217–1224. Thompson, D. A., Schlessinger, J. & Littman, D. R. (1997) J. Exp. Med. 186, 58. Park, I. W., Ullrich, C. K., Schoenberger, E., Ganju, R. K. & Groopman, J. E. 1793–1798. (2001) J. Immunol. 167, 2766–2771. 29. Popik, W., Hesselgesser, J. E. & Pitha, P. M. (1998) J. Virol. 72, 6406–6413. 59. Stewart, S. A., Poon, B., Song, J. Y. & Chen, I. S. (2000) J. Virol. 74, 3105–3111. MEDICAL SCIENCES

Twu et al. PNAS ͉ October 29, 2002 ͉ vol. 99 ͉ no. 22 ͉ 14391 Downloaded by guest on October 1, 2021