HIV1 induces cardiomyopathy by cardiomyocyte invasion and gp120, tat, and cytokine apoptotic...

HIV-1 and Cardiomyocyte Apoptosis 97

Cardiovascular Toxicology Humana Press Volume 4, 200497

*Author to whom allcorrespondence and reprintrequests should beaddressed: Milan Fiala, MD,UCLA CHS 63-090,10833 Le Conte Avenue,Los Angeles, CA 90095-1760. E-mail: [email protected].

Received: 12/6/2003

Revised: 2/6/2004

Accepted: 2/6/2004

Cardiovascular Toxicology,vol. 4, no. 2, 97–107, 2004

HIV-1 Induces Cardiomyopathyby Cardiomyocyte Invasion and gp120,Tat, and Cytokine Apoptotic SignalingMilan Fiala,*,1,2 Waldemar Popik,3 Jian-Hua Qiao,4

Albert S. Lossinsky,5 Timothy Alce,3 Kenix Tran,1

Wendy Yang,1 Kenneth P. Roos,2 and James Arthos6

1Department of Medicine, Greater Los Angeles VA Medical Center;2Cardiovascular Research Laboratory; 3Oncology Center, The JohnsHopkins University School of Medicine, Baltimore, MD; 4Departmentof Pathology, David Geffen School of Medicine at UCLA, Los Angeles, CA;5Huntington Medical Research Institutes, Pasadena, CA; and 6Laboratoryof Immunoregulation, National Institute of Allergy and Infectious Diseases,National Institutes of Health, Bethesda, MD

Cardiovascular Toxicology (2004) 04 97–107 $25.00 (http://www.cardiotox.com)© Copyright 2004 by Humana Press Inc. All rights of any nature whatsoever reserved. 1530-7905/01Humana Press

AbstractWe examined heart tissues of AIDS patients with or without HIV cardiomy-

opathy (HIVCM) by immunohistochemistry, in situ polymerase chain reac-tion, in situ riboprobe hybridization, and the TUNEL technique for apoptosis.In HIVCM tissues, only inflammatory cells, but not endothelial cells or car-diomyocytes, displayed HIV-1 DNA and RNA. However, macrophages, lym-phocytes, and—in a patchy fashion—cardiomyocytes and endothelial cellsexhibited virus envelope protein gp120. Macrophages infiltrated the myocar-dium in a perivascular fashion and expressed tumor necrosis factor familyligands; adjacent cardiomyocytes suffered apoptosis. In vitro HIV-1 stronglyinvaded neonatal rat ventricular myocytes (NRVMs) and coronary arteryendothelial cells (CAECs) and induced microvilli but did not replicate. HIV-1,gp120, or Tat induced Erk 1/2 phosphorylation, activation of caspase-3, andapoptosis of NRVMs and CAECs; all of these were inhibited by a MAPK/ERK-kinase (MEK) inhibitor U0126. The pathogenesis of HIVCM involves HIV-1replication in inflammatory cells and induction of cardiomyocyte apoptosisby (1) the extrinsic pathway through apoptotic ligands and (2) the intrinsicpathway through direct virus entry and gp120- and Tat-proapoptotic signaling.Key Words: HIV cardiomyopathy; cardiomyocyte apoptosis; HIV-1 envelopeprotein gp120; HIV-1 protein Tat; macrophage.

Introduction

Cardiovascular complications of HIV-1 infection involve a range of conditions

from contractile dysfunction, arrhythmias, and pulmonary hypertension to end-

stage dilated HIV cardiomyopathy (HIVCM). Although HIV-1 infection is consid-

ered to be a necessary cause of HIVCM in severely immunosuppressed patients,

opportunistic viral and nonviral infections are contributory. HIV-1 invades the

Fiala (04-119).p65 8/10/2004, 3:26 PM97

98 Fiala et al.


heart by transcellular passage through coronary

artery endothelial cells (CAECs) (1) and by “Tro-

jan” transport in infiltrating monocyte/macrophages

(2). HIV-1 invades neonatal rat ventricular myocytes

(NRVMs) by macropinocytosis but does not repli-

cate in these cells (3). Previous studies of HIVCM

hearts identified HIV-1 RNA and DNA in heart tis-

sues but did not identify the infected cells (4–6). Some

hearts with HIVCM have shown a complete lack of

HIV-1 footprints. Enteroviruses are considered to

cause viral myocarditis by overt or undetected infec-

tion of cardiomyocytes (7), and their presence has

been detected in some AIDS hearts (8). Besides end-

stage damage, HIV-1 also exerts earlier pathophys-

iological effects on heart contractility (9), possibly

mediated by oxidative and nitration stress, as sug-

gested in rat cardiomyocytes (10) and a murine model

(11). Nucleoside reverse transcriptase inhibitors cause

mitochondrial toxicities (12), which could also lead

to heart failure (13). Consequently, the role of HIV-1

in HIVCM has not been completely delineated.

A critical mechanism of HIVCM is cardiomyocyte

apoptosis, which is found in over 50% of cardiomyo-

cytes in a patchy fashion adjacent to infiltrating HIV-

1-infected macrophages. These macrophages pro-

duce inflammatory cytokine tumor necrosis factor-�

(TNF-�) which induces cardiomyocyte apoptosis by

the extrinsic pathway and the viral protein gp120,

which causes intrinsic apoptosis. In a previous study,

gp120 expression was significantly associated with

HIVCM, whereas HIV-1 proteins Tat and Nef were

not significantly associated (3).

To elucidate which cells are the source and targets

of infectious HIV-1 and cytokines in the cardiovascu-

lar system, we examined virus nucleic acids and pro-

teins and cardiomyocyte apoptosis in the hearts of

infected patients. HIV-1 DNA was found only in in-

flammatory cells, whereas viral protein was detected

in inflammatory cells, endothelial cells, and cardio-

myocytes. We investigated the relationship between

cardiomyocyte apoptosis and infected macrophages

to detect a paracrine mechanism of apoptosis in heart

tissues. In HIVCM heart tissues, but not in other

AIDS heart tissues without HIVCM, cardiomyocytes

suffered an extremely high rate of apoptosis in a focal

fashion. Using cultured cardiomyocytes and endo-

thelial cells, we examined gp120- and Tat-induced

cell signaling and demonstrated the role of Erk phos-

phorylation in apoptosis.

Materials and Methods

Heart Tissues

This study was performed with formalin-fixed heart

tissues from the left ventricle of 21 patients with AIDS,

of which 10 were diagnosed as HIVCM and 11 had no

history or biochemical evidence of heart failure. The

tissues were provided by the Manhattan HIV Brain

Bank (R24MH59724) and Texas Repository for AIDS

Neuropathogenesis Research (R24MH 59656 and

R24NS 45491). The UCLA Office for Protection of

Research Subjects approved all work with human

tissues and blood cells. We previously described the

immunohistochemistry (IHC) of 17 hearts from this

group of patients (3).

HIV-1 and HIV-1 Proteins

HIV-1

HIV-1NL4-3, incorporating Vpr-GFP (named HIV-

1NL4-3 Vpr-GFP), was produced by cotransfection of

293 cells with pNL4-3 DNA and an expression vec-

tor coding for GFP-Vpr fusion protein (3).

gp120

Recombinant soluble gp120 from macrophage-

tropic strain JR-FL was produced in Chinese hamster

ovary (CHO) cells, essentially as described for simian

immunodeficiency virus (SIV) envelope (14). An HIV-

1 env was inserted into a mammalian expression vec-

tor and transfected into the CHO cell. A cell clone

expressing a high level of soluble gp120 was selected.

Concentrated culture supernatants of this clone were

purified on a lectin column, eluted with �-mannopyra-

noside, and then passed through an anti-�-macroglo-

bulin column.

Tat

HIV-1 Tat was expressed in Escherichia coli by

using wild-type pGEM2 Tat bacterial expression

vector and purified on an Aquapore RP-300 column

(Applied Biosystems, Foster City, CA) by reverse-

phase chromatography, as described (15).

Cell Culture

Primary CAECs were purchased from Clonetics/

BioWhittaker (Walkersville, MD) and cultured by

using an EGM®-2-MV Bullet Kit medium, as described

(16). NRVMs were prepared from neonatal rat heart

tissues with full approval of the UCLA Animal Re-

search Committee, as described (3). CD4- HeLa cells

Fiala (04-119).p65 8/10/2004, 3:26 PM98



were obtained from S. Chow, UCLA Department of

Pharmacology.

In Situ Riboprobe Hybridization (ISH)

The technique followed that of Bruggeman et al.

(17,18) with the following modifications relating to

the choice of probe derivation and labeling: Sense

and antisense nef mRNAs were transcribed from plas-

mid pGM93 (AIDS Research and Reference Reagent

Program, NIH). For fluorescein-labeled sense RNA,

pGM93 was digested with EcoRI and transcribed

with T7 RNA polymerase and fluorescein-dUTP by

using a kit from Roche (Mannheim, Germany). Fluo-

rescein antisense transcripts were synthesized from

HindIII-digested pGM93 by using SP6 RNA poly-

merase and fluorescein-dUTP (Roche). The transcrip-

tion products were then purified by using an RNeasy

kit (Qiagen, Valencia, CA). The 1.1-kb sense and anti-

sense transcripts were randomly fragmented by incu-

bation in 0.1M carbonate buffer (pH 10.2) at 60°C for

45 min and subsequent neutralization with 100 mM

sodium acetate (pH 6.0) and 0.5% (v/v) acetic acid.

Paraffin-embedded heart and lung sections were

de-paraffinized, subjected to Target Retrieval Solu-

tion (DAKO, Carpinteria, CA), treated with protein-

ase K (RTU), treated with 0.3% hydrogen peroxide in

methanol, prehybridized to a prehybridization solu-

tion (4XSSC, 0.45 formamide, sheared herring sperm

DNA [Sigma, St. Louis, MO] and heated transfer RNA

[Sigma]), and hybridized to the probe (30 ng RNA)

in situ hybridization solution (DAKO) overnight at

42°C in a humidified chamber. The sections were

washed in 2XSSC once at room temperature and once

at 42°C, and then in 0.2XSSC at 50°C. The brown

color of the probe was developed by using Genpoint

fluorescein-horseradish peroxidase (HRP) (DAKO),

as recommended by the manufacturer. In some expe-

riments, a blue color was developed by using an anti-

fluorescein isothiocyanate (FITC)-alkaline phospha-

tase (Sigma) and BCIP/nitroblue tetrazolium (NBT)

substrate (DAKO). The section was counterstained

by using Methyl Green.

In Situ Polymerase Chain Reaction

(IS-PCR) for HIV-1 DNA

The IS-PCR technique is based on incorporation

of FITC-labeled dUTP into PCR-amplified DNA by

using HIV-1 gag GE1 (5'GAAGGAGCCACCCCACA

AGATT) and GE2 (5'TAGGTGGATTATTTGTCAT

CCA) primers, as previously published (19), with some

modifications. Tissues were deparaffinized in xylene

and alcohol and digested with proteinase K (1:200

for 15 min at room temperature; Promega,Madison,

WI). The PCR reaction was performed in a volume of

50 µL by using a PCR Core Kit (Roche) with FITC-

dUTP (1:8), Ampli Taq-Gold polymerase (5 units;

Applied Biosystems) (20), and forward (100 pM) and

reverse (100 pM) Gag primers. Ampli Taq-Gold poly-

merase was added at 70°C. The PCR reaction was per-

formed in a PCR machine (PTC-100, MJ Research,

Waltham, MA) at 94°C for 2 min 30 s, at 55°C for

1 min 30 s, and then for 20 cycles of 94°C for 55 s

and 55°C for 45 s. The reaction mix was enclosed by

using a coverslip sealed with a nail polish and min-

eral oil overlay. After the reaction, the coverslip was

removed and the slide was washed once with xylene

for 10 min, once with 100% ethanol for 5 min, and

twice with 0.5XSSC for 3 min. The tissue was then

blocked with 0.6% hydrogen peroxide in methanol

for 5 min, rinsed with water, and stained by using

a DAKO GenPoint Fluorescein method (DAKO),

which detected incorporated FITC-dUTP by tyra-

mide signal amplification or an anti-FITC-alkaline

phosphatase/BCIP/NBT technique.

Immunohistochemistry

Formalin-fixed tissues were immunostained by

using an Envision Double Stain System (DAKO) after

antigen retrieval, as described (2). The cardiomyocyte

diameter was measured at a magnification of �40

after staining with wheat germ agglutinin (Sigma).

Apoptosis and Caspase-3 Assays

To induce apoptosis, CAECs or NRVMS were

treated with recombinant gp120JRFL (5–500 ng/mL)

or Tat (50 ng/mL) for 4 h or overnight. Apoptotic

cells were detected by (1) the terminal deoxynucleo-

tidyl transferase-mediated dUTP nick-end labeling

(TUNEL) technique (Apoptosis Detection Kit with

dUTP, Promega) by using the FITC-conjugated nucle-

otide mix and simultaneous staining with DAPI or

Hoechst 33342 (Molecular Probes, Eugene, OR), or

by (2) the FAM FLICA DEVD-FMK caspase-3/7

assay kit (Immunochemistry Technologies, Bloom-

ington, MN). Active caspase-3 was assayed by using

the Caspase-3 Colorimetric Protease kit (BioSource,

Camarillo, CA) and one 60-mm culture dish of CAECs

or one 100-mm dish of NRVMs per experimental

Fiala (04-119).p65 8/10/2004, 3:26 PM99

100 Fiala et al.


sample (50–200 µg of protein). The reaction was

developed by using a Reaction Buffer® and DEVD-

pNA substrate at 37°C for 2 h and read at 405 nm in

a spectrophotometer.

Western Blotting

After reaching confluence, approx 1 million cells

in a 60-mm culture dish were maintained overnight

in an EBM-2 basal medium (Clonetics/BioWhittaker)

without serum. The cells were stimulated by using

100 µL of prewarmed viral protein for 1–20 min and

harvested into 250 µL of ice-cold RIPA buffer (50

mM HEPES at pH 7.4, 10 mM EDTA, 150 mM NaCl,

1% NP-40, 0.1% SDS, 0.5% sodium deoxycholate,

complete protease inhibitor [Roche], and phospha-

tase inhibitor cocktail [Sigma]). The lysate was incu-

bated for 5 min on ice, centrifuged at maximum speed

in a microfuge, and frozen until the assay was per-

formed by Western blotting, as described (1).

Transmission Electron Microscopy (TEM)

and Scanning Electron Microscopy (SEM)

TEM and SEM were performed with cells grown

in Transwell inserts, as described (1). For TEM, the

inserts were gently washed with PBS and fixed with

3% glutaraldehyde buffered with 0.1M sodium phos-

phate or sodium cacodylate salts (pH 7.4) for 1 h at

25°C, stored in a 0.1M sodium cacodylate buffer con-

taining 0.2M sucrose, postfixed in 1% osmium tetrox-

ide in a 0.1M sodium cacodylate buffer for 1 h at

4°C, dehydrated in a graded series of ethanol, and

embedded in Epon. For SEM, after osmication and

dehydration, the inserts were dried with hexamethyl-

disalisane (21), attached to aluminum SEM stubs by

using double-sticky carbon tape, and then coated with

gold and palladium and scanned on an FEI ESEM

XL30 SEM.

Results

HIV Cardiomyopathy Is Hypertrophic and

Associated with Epi-, Peri-, and Endocarditis

In our material comprising the heart tissues of

21 patients with AIDS, 10 hearts showed HIVCM, 1

HIVCM heart also showed epi- and pericarditis and

vasculitis, and 2 hearts had endocarditis. The hearts

with HIVCM were hypertrophic (the average weight

of the hearts with HIVCM was 580 g, as compared

with the 337-g average weight of the hearts without

HIVCM). The average cardiomyocyte diameter in

the hearts with HIVCM was increased (41 µ vs 23 µ).

HIV-1 DNA and RNA

Are Restricted to Inflammatory Cells,

but Envelope Protein gp120 Is Detected

in Inflammatory Cells and Cardiomyocytes

We initially tested IS-PCR, ISH, and IHC by using

infected and uninfected CD4 HeLa cells. Positive

staining was detected only in infected cells using

HIV-1 primers, antisense RNA probe, or anti-gp120

antibody, respectively (Fig. 1). The results with un-

infected cells or when the correct primers, probe, or

antibody were omitted were negative with the excep-

tion of a weak staining of multinucleated giant cells

by both sense and antisense riboprobes. To detect

HIV-1 infection in heart tissues from patients with

and without HIVCM, we performed IS-PCR, ISH,

and IHC. These techniques detected virus footprints

only in HIVCM hearts. Virus DNA was detected by IS-

PCR in inflammatory cells, macrophages (some mul-

tinucleated), and T cells, whereas endothelial cells

and cardiomyocytes were negative (Fig. 1b,c). ISH

showed HIV-1 RNA in inflammatory cells with a halo

of positive reactivity surrounding these cells (Fig. 1i).

Endothelial cells adjacent to adherent mononuclear

cells were positive in a patchy fashion (Fig. 1h). HIV-

1 gp120 was detected in perivascular macrophages

(Fig. 1e), in a sarcomeric pattern in cardiomyocytes

(Fig. 1f), and in some endothelial cells. Six of the

10 hearts with HIVCM but none of the 4 without

HIVCM were positive for gp120 on infiltrating macro-

phages and lymphocytes. As noted previously, some

inflammatory cells expressed Nef and Tat, but these

viral proteins were not significantly associated with

HIVCM (3).

Apoptotic Cardiomyocytes

Are Found Adjacent to HIV-1-Infected

Perivascular Macrophages

Inflammatory cells infiltrated HIVCM hearts in a

patchy and perivascular distribution, and some were

diffusely scattered among cardiomyocytes. Apoptotic

cardiomyocytes were detected by TUNEL technique

adjacent to gp120-positive perivascular macrophages

(Fig. 2). We previously showed that TNF-� family

ligands Fas and TNF-��caspases-3 and -9, as shown in

Fig. 2 (3), and caspase-8 (22,3) were strongly expressed

in heart tissues with HIVCM.

Fiala (04-119).p65 8/10/2004, 3:26 PM100



Fig. 1. Heart tissues of patients with HIVCM show HIV-1 DNA and RNA in inflammatory cells and gp120 in inflam-

matory cells, cardiomyocytes, and endothelial cells. (A) IS-PCR with Gag primers (Genpoint-FITC-HRP). a, HIV-1-

infected HeLa-CD4 (inset shows uninfected cells). b and c, Heart tissue from a patient with HIVCM shows positive staining

of inflammatory perivascular cells but no staining of cardiomyocytes (inset shows negative staining in control heart

tissue). (B) IHC for gp120. a, HIV-1-infected HeLa-CD4 (inset shows uninfected HeLa-CD4; HRP-diaminobenzidine).

b, Perivascular CD68-positive macrophages (alkaline phosphatase-BCIP) are positive for gp120. c, Cardiomyocytes in

patchy distribution display positive staining for gp120 in sarcomeric pattern. (C) ISH with HIV-1 riboprobe (Genpoint-

FITC-HRP). a, HIV-1-infected HeLa-CD4 (inset shows uninfected HeLa-CD4). b, Positive staining of two mononuclear

cells and adjacent endothelial cells. c, Positive staining of inflammatory cells surrounded by a halo of positive staining

(inset shows negative staining of control heart tissue). Color image available for viewing at www.humanapress.com.

In Vitro Effects of HIV-1

Proteins on NRVMs and CAECs

To analyze the role of HIV-1 proteins in cardio-

myocyte apoptosis, we induced apoptosis with recom-

binant gp120 or Tat in cultured cells.

gp120 Activates Caspase-3in CAECs and NRVMsand Induces Apoptosis

gp120 induced apoptosis of NRVMs, as shown by

TUNEL assay (Fig. 3B), and CAECs, as shown by cas-

Fiala (04-119).p65 8/10/2004, 3:26 PM101

102 Fiala et al.


Fig. 2. Apoptotic cardiomyocytes are adjacent to infected perivascular macrophages. (A–C) Small-vessel vasculitisin the myocardium (hematoxylin-eosin). (D) The vasculitis involves infiltration by CD68-positive macrophages (alka-line phosphatase-BCIP). (E) The macrophages are gp120-positive (alkaline phosphatase-BCIP). (F) The cardiomyocytesadjacent to perivascular macrophages are TUNEL-positive (HRP-diaminobenzidine). Color image available for viewing

at www.humanapress.com.

Fig. 3. (Opposite page) gp120 and Tat activate caspase-3 and induce apoptosis of cardiomyocytes and CAECs. (A)gp120, Tat, or actinomycin D cleaves DEVD-p-nitroanilide (OD at 400 nm; colorimetric apoptosis assay [Biosource]in CAECs treated with gp120 [50 ng/mL] for 18 h). (B) gp120 induces NRVM apoptosis (fluorescent TUNEL assay incultured NRVMs treated with gp120 [50 ng/mL] for 48 h). a, Primary NRVM cultures comprise mostly cardiomyocytes(note most nuclei [DAPI-positive] colocalize with the cytoplasm stained red by antisarcomeric antibody MF-20/TR).b, Most cardiomyocytes colocalize with TUNEL staining and are apoptotic. (C) gp120 induces CAEC apoptosis (fluo-rescent caspase-3 assay in CAECs treated with gp120 [50 ng/mL] for 18 h). a, Hoechst 33342 staining of apoptotic nuclei(note shrunken [bright]) nuclei in the right lower corner). b, Fluorescent caspase-3 staining of cells with apoptotic nuclei(FLICA assay). Color image available for viewing at www.humanapress.com.

pase-3 colorimetric assay and fluorescent caspase-

3/Hoechst 33342 staining (Fig. 3C). A MEK inhib-

itor U0126 inhibited caspase-3 induction by viral

proteins. In CAECs and NRVMs, gp120 and Tat acti-

vated caspase-3 to a similar extent after 4 h or over-

night stimulation. The average ratio of caspase-3

optical density (OD) of CAECs treated with gp120

(50 ng/mL) compared with OD of CAECs treated

with medium was 2.18 (four experiments), and the

average ratio of OD of NRVMs treated with gp120

(50 ng/mL) compared with OD of NRVMs treated

with medium was 1.6 (two experiments). The stimu-

lation of caspase-3 by Tat (50 ng/mL) was 2.5-fold

in CAECs and 1.7-fold in NRVMs (Fig. 3A). U0126

decreased caspase-3 induction to baseline or below

the baseline and, as shown previously, reduced apop-

tosis by 80% (3).

gp120 Induces Phosphorylation of Erk1/2

To determine the initial virus interaction with car-

diomyocytes and CAECs, we examined mitogen-

activated protein kinase (MAPK) cell signaling by

gp120 and Tat. gp120 induced Erk 1/2 phosphoryla-

tion more rapidly (2 min) in NRVMs (Fig. 4A) than

in CAECs (10 min; Fig. 4B). Tat also induced signal-

ing in CAECs with slower kinetics (10 min; Fig. 4C).

The MEK inhibitor U0126 abolished Erk 1/2 phos-

phorylation in NRVMs by gp120 (Fig. 4A). No c-Jun

N-terminal kinase (JNK) signaling by gp120 was

found.

HIV-1 Enters and Remodels

CAECs and NRVMs

As previously shown by confocal microscopy,

within 3 h after exposure, HIV-1NL4-3 Vpr-GFP mas-

Fiala (04-119).p65 8/10/2004, 3:26 PM102



sively enters NRVMs (3) and CAECs (1) by the mech-

anism of macropinocytosis, which involves, as pre-

viously shown, induction of microvilli on the sur-

face of endothelial cells. To examine whether HIV-

1 also re-models cardiomyocytes, we performed SEM

of NRVMs. A 3-h HIV-1 exposure induced micro-

Fiala (04-119).p65 8/10/2004, 3:26 PM103

104 Fiala et al.


Fig. 4. gp120 and Tat induce Erk 1/2 phosphorylation

in cardiomyocytes and CAECs. (A) gp120 treatment of

NRVMs induced Erk1/2 phosphorylation with a maximum

at 2 min and a second maximum at 15 min postexposure.

U0126 blocked Erk 1/2 phosphorylation. (B) gp120 treat-

ment of CAECs induced Erk1/2 phosphorylation with a

maximum at 10 min and a second maximum at 30 min.

(C) Tat treatment of CAECs induced Erk1/2 phosphory-

lation with a maximum at 10 min.

villi, as shown by SEM (Fig. 5, cf. A,B). HIV-1NL4-3

Vpr-GFP strongly enters NRVMs, as observed by

confocal microscopy (3). TEM of NRVMs exposed

to HIV-1 for 3 h showed virion-sized (100-mµ diam-

eter) particles in vacuoles (Fig. 5D) and scattered in

the cytoplasm. Some of these particles had a typical

HIV nucleocapsid core (Fig. 5C).

Discussion

This study by nucleic acid hybridization tech-

niques complements previous studies by IHC (3) and

underlines the critical role of macrophage infiltra-

tion and production of viral and cytokine products

inducing cardiomyocyte apoptosis. The hearts with

HIVCM display multifaceted pathology with focal

myocarditis, complicated in some cases by epicardi-

tis, vasculitis in small intramyocardial vessels, myo-

cardial infarction, and endocarditis. The root of large

vessels is infiltrated by HIV-1-infected macrophages.

Coronary vessels were not available for examination

in this study. The hearts with HIVCM have cardio-

megaly (22,3) and hypertrophic cardiomyocytes. IHC

of HIVCM shows inflammatory pathology with infil-

tration by macrophages and T cells in a patchy peri-

vascular pattern. However, because of its focal nature,

unless many sections are examined, the inflamma-

tion may remain undetected.

As shown by nucleic acid hybridization, HIV-1

was found to productively infect inflammatory cells,

macrophages, and lymphocytes. We did not find evi-

dence by hybridization techniques of HIV-1 infec-

tion of cardiomyocytes, which had been previously

suspected (4,6,23). Although no viral DNA was found

in cardiomyocytes or endothelial cells, these cells

displayed gp120, indicating that the virus may invade

them without replication. HIV-1 proteins that are re-

leased when virions are degraded in lysosomes (as

observed in brain microvascular endothelial cells

[BMVECs; 1]), in particular gp120, could cause mito-

chondrion-controlled apoptosis by binding to cardio-

myocyte mitochondria. gp120 induced cytochrome

c release from NRVM mitochondria (3). Therefore,

cardiomyocyte apoptosis seems to be produced in a

paracrine fashion by death receptor family ligands

and viral products from infected macrophages.

The in vitro effects of HIV-1 observed in BMVECs

(1), CAECs (16), and NRVMs (this study) explain

the mechanisms of HIV-1 entry and apoptosis of tar-

get cells in the heart and probably the brain. First,

HIV-1 easily enters CAECs and BMVECs by macro-

pinocytosis (1) and traverses these cells into target

organs by transcellular and pericellular processes

(24,25). Second, in target organs, HIV-1 infects and

is replicated by perivascular macrophages, and the

extent of macrophage infiltration appears to deter-

mine the risk of HIVCM (2). Third, HIV-1 and gp120

signal in CAECs and NRVMs through the MAPK

pathway by Erk1/2 phosphorylation, and this signal-

ing is necessary for HIV-1- or gp120-induced apop-

tosis. Finally, HIV-1 remodels cardiomyocytes by

inducing microvilli on the cell surface. Using IEM

Fiala (04-119).p65 8/10/2004, 3:26 PM104



Fig. 5. HIV-1 induces microvilli and enters cardiomyocytes. (A) NRVMs were exposed to HIV-1NL4-3 (m = 104 RNA

copies per cardiomyocyte) for 3 h, and examined by SEM (�5000). Note surface microvilli. (B) NRVMs were not exposed

to HIV-1 and were examined as in a (�2500). Note smooth cell surface. (C) NRVMs were exposed to HIV-1NL4-3 (m = 104

RNA copies per cardiomyocyte) for 3 h. Some particles have a typical dense core (arrows; TEM, �18,000). (D) Virus

particles in cardiomyocyte vacuoles (arrows; TEM, �58,000).

and TEM (1), we previously demonstrated that HIV-

1 upregulates ICAM-1, displaying microvilli on

BMVECs. We speculate that the microvilli induced

by HIV-1 on endothelial cells and cardiomyocytes

might be displaying ICAM-1 and chemokine recep-

tors to attract and immobilize inflammatory cells in

the heart and to entangle virions and deliver them for

macropinocytosis (1). Further studies of ICAM-1 and

chemokine receptors on cardiomyocytes and endo-

thelial cells are in progress in our laboratory.

Several well-designed studies attempted to disst

the role in HIVCM of a limited repertoire of viral

proteins, such as gp120, Tat (26,27), or Nef (28), in

transgenic models. Unfortunately, such analyses did

not evaluate the effects of infectious HIV-1, in par-

ticular those effects bearing on macrophages, which

are critical cells replicating the virus in the heart.

The best model of HIVCM appears to be a primate

model using SIV infection. The pathogenesis of car-

diomyopathy induced by infectious SIV resembles

that of HIVCM: SIV myocarditis is lymphocytic (29)

and, therefore, resembles the inflammatory nature of

HIV-1 myocarditis (3). It is also intriguing that coro-

nary arteriopathy was frequent in chronically infected

monkeys because coronary arteritis has been observed

in our patients and in a previous study (30).

In conclusion, end-stage HIVCM correlates with

cardiomyocyte apoptosis (3), which is induced by

virions and viral proteins gp120 and Tat as well as by

TNF ligands shed from infected macrophages. In vitro,

cardiomyocytes are damaged both by the virions,

which enter cardiomyocytes with ease, and Erk 1/2 sig-

Fiala (04-119).p65 8/10/2004, 3:26 PM105

106 Fiala et al.


naling through viral proteins gp120 and Tat. Inhibition

of cell signaling by gp120 inhibits cardiomyocyte

apoptosis. Inhibitors of MAPK signaling might be

useful in the therapy of HIVCM.

Acknowledgments

We thank Sergei Nekhai for the supply of recom-

binant Tat; Sam Chow for CD4-HeLa; M. Ross, L.

Bruggeman, and P. Klotman for advice regarding

the IS-PCR and ISH techniques; and Mithulan Jega-

pragasan for cardiomyocyte measurements. The AIDS

Research and Reference Reagent Program, NIAID,

NIH provided the listed reagents.

References

1. Liu, N.Q., Lossinsky, A.S., Popik, W., Li, X., Gujuluva,

C., Kriederman, B., et al. (2002). Human immunodefi-

ciency virus type 1 enters brain microvascular endothelia

by macropinocytosis dependent on lipid rafts and the mito-

gen-activated protein kinase signaling pathway. J. Virol. 76:

6689–6700.

2. Liu, Q.N., Reddy, S., Sayre, J.W., Pop, V., Graves, M.C.,

and Fiala, M. (2001). Essential role of HIV type 1-infected

and cyclooxygenase 2-activated macrophages and T cells

in HIV type 1 myocarditis. AIDS Res. Hum. Retroviruses

17:1423–1433.

3. Twu, C., Liu, N.Q., Popik, W., Bukrinsky, M., Sayre, J.,

Roberts, J., et al. (2002). Cardiomyocytes undergo apop-

tosis in human immunodeficiency virus cardiomyopathy

through mitochondrion- and death receptor-controlled path-

ways. Proc. Natl. Acad. Sci. USA 99:14386–14391.

4. Grody, W.L.C. and Lewis, W. (1990). Infection of the

human heart by the human immunodeficiency virus. Am.

J. Cardiol. 66:203–206.

5. Lipshultz, S.E., Fox, C.H., Perez-Atayde, A.R., Sanders,

S.P., Colan, S.D., McIntosh, K., et al. (1990). Identifica-

tion of human immunodeficiency virus-1 RNA and DNA

in the heart of a child with cardiovascular abnormalities and

congenital acquired immune deficiency syndrome. Am. J.

Cardiol. 66:246–250.

6. Rodriguez, E.R., Nasim, S., Hsia, J., Sandin, R.L., Ferreira,

A., Hilliard, B.A., et al. (1991). Cardiac myocytes and den-

dritic cells harbor human immunodeficiency virus in infected

patients with and without cardiac dysfunction: detection

by multiplex, nested, polymerase chain reaction in individ-

ually microdissted cells from right ventricular endomyocar-

dial biopsy tissue. Am. J. Cardiol. 68:1511–1520.

7. Kandolf, R., Ameis, D., Kirschner, P., Canu, A., and Hof-

schneider, P.H. (1987). In situ detection of enteroviral

genomes in myocardial cells by nucleic acid hybridization:

an approach to the diagnosis of viral heart disease. Proc.

Natl. Acad. Sci. USA 84:6272–6276.

8. Cioc, A.M. and Nuovo, G.J. (2002). Histologic and in situ

viral findings in the myocardium in cases of sudden, unex-

pected death. Mod. Pathol. 15:914–922.

9. Chen, F., Shannon, K., Ding, S., Silva, M.E., Wetzel, G.T.,

Klitzner, T.S., et al. (2002). HIV type 1 glycoprotein 120

inhibits cardiac myocyte contraction. AIDS Res. Hum. Retro-

viruses 18:777–784.

10. Kan, H., Xie, Z., and Finkel, M.S. (2000). HIV gp120

enhances NO production by cardiac myocytes through p38

MAP kinase-mediated NF-kappaB activation. Am. J. Phys-

iol. Heart Circ. Physiol. 279:H3138–H3143.

11. Chaves A.A., Mihm M.J., Schanbacher B., Basuray, A.,

Liu, C.Y., Ayers, L.W., et al. (2003). Cardiomyopathy in

a murine model of AIDS: evidence of reactive nitrogen

species and corroboration in human HIV/AIDS cardiac tis-

sues. Cardiovasc. Res. 60:108–118.

12. Carr, A. and Cooper, D.A. (2000). Adverse effects of anti-

retroviral therapy. Lancet 356:1423–1430.

13. Frerichs, F.C., Dingemans, K.P., and Brinkman, K. (2002).

Cardiomyopathy with mitochondrial damage associated

with nucleoside reverse-transcriptase inhibitors. N. Engl.

J. Med. 347:1895–1896.

14. Mossman, S.P., Bex, F., Berglund, P., Arthos, J., O’Neil,

S.P., Riley, D., et al. (1996). Protection against lethal sim-

ian immunodeficiency virus SIVsmmPBj14 disease by a

recombinant Semliki Forest virus gp160 vaccine and by a

gp120 subunit vaccine. J. Virol. 70:1953–1960.

15. Deng, L., Ammosova, T., Pumfery, A., Kashanchi, F., and

Nekhai, S. (2002). HIV-1 tat interaction with RNA polymer-

ase II C-terminal domain (CTD) and a dynamic association

with CDK2 induce CTD phosphorylation and transcription

from HIV-1 promoter. J. Biol. Chem. 277:33922–33929.

16. Gujuluva, C., Burns, A.R., Pushkarsky, T., Popik, W.,

Berger, O., Bukrinsky, M., et al. (2001). HIV-1 penetrates

coronary artery endothelial cells by transcytosis. Mol. Med.

7:169–176.

17. Bruggeman, L.A., Dikman, S., Meng, C., Quaggin, S.E.,

Coffman, T.M., and Klotman, P.E. (1997). Nephropathy

in human immunodeficiency virus-1 transgenic mice is due

to renal transgene expression. J. Clin. Invest. 100:84–92.

18. Bruggeman, L.A., Ross, M.D., Tanji, N., Cara, A., Dikman,

S., Gordon, R.E., et al. (2000). Renal epithelium is a pre-

viously unrecognized site of HIV-1 infection. J. Am. Soc.

Nephrol. 11:2079–2087.

19. Strappe, P.M., Wang, T.H., McKenzie, C.A., Lowrie, S.,

Simmonds, P., and Bell, J.E. (1998). In situ polymerase

chain reaction amplification of HIV-1 DNA in brain tis-

sue. J. Virol. Methods 70:119–127.

20. Nuovo, G. (1997). PCR in Situ Hybridization: Protocols and

Amplifications, 3rd ed. New York, NY: Lippincott-Raven.

21. Lossinsky, A.S. and Shivers, R.R. (2003). Studies of cere-

bral endothelium by scanning and high-voltage electron

microscopy. In: Nag, S. (ed.). The Blood-Brain Barrier:

Biology and Research Protocols. Totowa, NJ: Humana

Press, pp 67–82.

22. October 2002. Available at: http://www.pnas.org. Accessed

February 27, 2004.

23. Barbaro, G. and Lipshultz, S.E. (2001). Pathogenesis of HIV-

associated cardiomyopathy. Ann. NY Acad. Sci. 946:57–81.

24. Fiala, M., Looney, D.J., Stins, M., Way, D.D., Zhang, L.,

Gan, X., et al. (1997). TNF-alpha opens a paracellular

route for HIV-1 invasion across the blood-brain barrier.

Mol. Med. 3:553–564.

Fiala (04-119).p65 8/10/2004, 3:26 PM106



25. Zhang, L., Looney, D., Taub, D., Chang, S.L., Way, D.,

Witte, et al. (1998). Cocaine opens the blood-brain barrier

to HIV-1 invasion. J. Neuro. Virol. 4:619–626.

26. Reid, W., Sadowska, M., Denaro, F., Rao, S., Foulke J. Jr.,

Hayes, N., et al. (2001). An HIV-1 transgenic rat that

develops HIV-related pathology and immunologic dysfunc-

tion. Proc. Natl. Acad. Sci. USA 98:9271–9276.

27. Bruggeman, L.A., Thomson, M.M., Nelson, P.J., Kopp,

J.B., Rappaport, J., Klotman, P.E., et al. (1994). Patterns

of HIV-1 mRNA expression in transgenic mice are tissue-

dependent. Virology 202:940–948.

28. Kay, D.G., Yue, P., Hanna, Z., Jothy, S., Tremblay, E.,

and Jolicoeur, P. (2002). Cardiac disease in transgenic

mice expressing human immunodeficiency virus-1 nef in

cells of the immune system. Am. J. Pathol. 161:321–335.

29. Shannon, R.P., Simon, M.A., Mathier, M.A., Geng, Y.J.,

Mankad, S., and Lackner, A.A. (2000). Dilated cardiomy-

opathy associated with simian AIDS in nonhuman pri-

mates. Circulation 101:185–193.

30. Barbaro, G., Barbarini, G., and Pellicelli, A.M. (2001).

HIV-associated coronary arteritis in a patient with fatal myo-

cardial infarction. N. Engl. J. Med. 344:1799–1800.

Fiala (04-119).p65 8/10/2004, 3:26 PM107

HIV1 induces cardiomyopathy by cardiomyocyte invasion and gp120, tat, and cytokine apoptotic...

Documents

Transcript of HIV1 induces cardiomyopathy by cardiomyocyte invasion and gp120, tat, and cytokine apoptotic...