Download Infection of Human Fetal Cardiac Myocytes by a Human

Infection of Human Fetal Cardiac Myocytes by a Human
Immunodeficiency Virus-1–Derived Vector
Michael A. Rebolledo, Paul Krogstad, Fuhua Chen, Kevin M. Shannon, Thomas S. Klitzner
Abstract—Cardiomyopathy associated with HIV-1 infection is a well-recognized complication. However, it is unknown
whether direct cardiomyocyte infection is involved in the pathogenesis of the cardiomyopathy. An HIV-1– based
lentiviral vector and wild-type HIV-1 were used to infect human fetal cardiac myocytes in a primary culture.
Quantitative polymerase chain reaction, viral p24 antigen determination, and immunofluorescence were used to detect
the synthesis of HIV-1 DNA and proteins after the infection. High-efficiency infection occurred using the HIV-1– based
lentiviral vector, although no infection occurred with the wild-type HIV-1 strain. Dual-labeling immunofluorescence for
HIV-1 proteins and myosin confirmed that cardiomyocytes were infected. This in vitro analysis suggests that direct
myocyte infection with wild-type HIV-1 may not be involved in the pathogenesis of HIV-1 cardiomyopathy. However,
HIV-1– based vectors may prove useful for ex vivo cardiovascular gene therapy. (Circ Res. 1998;83:738-742.)
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Key Words: fetal heart n gene therapy n cardiomyopathy n human immunodeficiency virus n cell culture
C
ardiomyopathy is a well-recognized complication associated with HIV-1 infection.1– 4 The pathogenesis remains unknown. Possible mechanisms include direct cardiac
myocyte infection by HIV-1; toxicity from locally secreted
cytokines secondary to myocarditis5; coinfection with other
cardiotropic viruses, especially cytomegalovirus6; postviral
autoimmunity7; cardiotoxicity from nucleoside analogues or
illicit drugs8; and nutritional deficiencies seen in end-stage
HIV-1 infection.
Several studies provide evidence that HIV-1 may infect
cells in cardiac tissues. In one report, HIV-1 nucleic acid
sequences were detected by in situ hybridization in 6 of 22
postmortem samples of cardiac tissue from adults with
AIDS.9 In a case report, others used in situ hybridization and
polymerase chain reaction (PCR) to detect HIV-1 RNA and
DNA in postmortem tissue from an HIV-1–infected 13month-old child.10 Data from a recent study of cardiac tissue
from 3 infants with AIDS who died suddenly revealed
evidence of HIV-1–infected myocytes, vascular pericytes,
and macrophage infiltration in pericardial and myocardial
tissue.11 An extensive study of endomyocardial biopsy samples from 15 adults provided additional evidence of infection
of myocytes and dendritic cells in fixed cardiac tissue.12 In all
of these studies, the number of infected cells was low,
consistent with a model, as has been proposed for HIV-1
neuropathogenesis, in which infection of a small number of
parenchymal or accessory cells may lead to organ
dysfunction.13
It is presently unclear how HIV-1 infects endothelial cells
or cardiomyocytes, because neither cell type exhibits the cell
surface expression of the primary HIV-1 receptor, CD4.9
However, HIV-1 infection of CD4 negative cells such as
human lung fibroblasts and epithelial cells from embryonic
lung has been demonstrated.14 One possible explanation is
that infection of cells without surface expression of CD4 may
occur as a consequence of contact with infected leukocytes.15
Antibody enhancement of virus uptake also has been
suggested.16
We set out to determine whether freshly isolated human
fetal cardiac myocytes (HFCMs) in primary culture are
permissive for productive HIV-1 infection using wild-type
HIV-1,17 a virus isolated from a child with cardiomyopathy,
and with an HIV-1– based lentiviral vector pseudotyped with
vesicular stomatitis virus envelope glycoprotein G (VSV
envelope).18 We elected to use HIVNL4 –3 because it had been
previously shown to infect CD4 negative cells.19 Also,
HIVNL4 –3 has been shown to be syncytium inducing, although
the patient isolate (EF) is non–syncytium inducing. We
speculated that using two divergent phenotypic strains of
HIV-1 would improve the chances of demonstrating the
infection of HFCMs.
Materials and Methods
Approval (HSPC No. 94 – 09-327-B) from the UCLA Human Subjects Protection Committee was received in order to obtain human
fetal cardiac tissue from abortuses for this study. Human fetal hearts
were procured from abortuses of 14 to 18 weeks’ gestation. The
mother provided blood for HIV-1 antibody testing. The human fetal
heart was shipped overnight on wet ice in high-glucose 0.025-mol/L
DMEM (4500 mg/L) with penicillin (50 000 U/L) and streptomycin
(50 mg/L). Ventricular myocytes were isolated by enzymatic digestion, as previously described.20 The aorta was cannulated and
attached to a sterile Langendorff perfusion apparatus. The coronary
arteries were perfused for 3 minutes with warmed (37°C), oxygenated
Received December 22, 1997; accepted July 2, 1998.
From the Department of Pediatrics, University of California Los Angeles School of Medicine, Los Angeles, Calif.
Correspondence to Michael A. Rebolledo, MD, UCLA Medical Center, Department of Pediatrics, Division of Cardiology, Box 951743, Room B2-427
MDCC, Los Angeles, CA 90095-1743. E-mail [email protected]
© 1998 American Heart Association, Inc.
738
Rebolledo et al
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calcium-free Tyrode’s solution containing the following (mmol/L):
NaCl 136, KCl 5.4, NaH2PO4 0.33, MgCl2 1, Na-HEPES 10, D-mannitol
4, thiamine 0.6, glucose 10, and pyruvic acid 2 and titrated to a pH 7.3
with NaOH. A peristaltic pump controlled the rate of perfusion at 2.5
mL/min. All perfusion solutions were sterilized by filtering through a
0.2-mm filter. The heart was digested enzymatically by perfusion with
collagenase (900 mg/L, type 1, Sigma Chemical Co) and protease (76
mg/L, type XIV, Sigma) for 5 to 7 minutes. The pericardium and atrial
appendages were removed. The perfusate was switched to 0.1 mmol/L
calcium Tyrode’s solution for 3 minutes. The ventricles were removed,
and isolated ventricular myocytes were manually dispersed in
0.1 mmol/L calcium Tyrode’s solution. Other connective tissue was
removed by filtering through a sterile gauze (500-mm pore size, Fisher
Scientific). Ventricular myocytes were centrifuged at 400g for 1 minute.
Ventricular myocytes were resuspended in high-glucose DMEM containing 10% (vol/vol) heat inactivated FBS, 23 DMEM vitamins
(Sigma), 13 DMEM nonessential amino acids (Sigma), human apotransferrin 0.13 mmol/L (10 mg/mL), bovine insulin 1.7 mmol/L (10
mg/mL), penicillin (50 000 U/L), streptomycin (50 mg/L), and
2 mmol/L L-glutamine. Aliquots of myocardial cells were loaded into a
2-step discontinuous Percoll (Sigma) gradient diluted with Hanks’
balanced salt solution and centrifuged at 340g for 20 minutes. A
low-density fibroblast and a high-density myocyte layer were obtained.
The fibroblast layer was aspirated and discarded. Myocytes were
washed once with appropriate medium. The concentration of viable
HFCMs was determined using trypan blue dye exclusion. Viability was
typically $80%. After 4 to 10 days in culture, flow cytometric analysis
revealed that 60% to 90% of the cells excluded 7-aminoactinomycin D,
suggesting continued membrane integrity. Freshly isolated myocytes
demonstrated a normal current-voltage relationship and an increase in
inward calcium current in response to isoproterenol (data not shown).
HFCMs were incubated in high-glucose DMEM plus additives at a
density of 13 105 cells/ml. The cells were maintained at 37°C in a
humidified 95% air/5% CO2 atmosphere. The medium was changed
every other day.
HIV-1 Stock Production
Virus stocks of HIV-1NL4 –3 were prepared by collecting the supernatants of COS-7 cells transfected by electroporation with the plasmid
vector pNL4-3,17 as previously described.21 High-titer stocks of
pseudotyped-HIV–1 vectors were prepared by the electroporation of
COS cells with a mixture of the pNLthyDBgl, an HIV proviral clone
with an envelope gene deletion, and pHCMV-G, which encodes the
envelope protein of VSV.22 On the second day after transfection,
virus in these COS-7 cell supernatants was pelleted at 150 000g.
After removing the overlying medium, the virus pellets were
resuspended in 0.13 Hanks’ balanced salt solution, using a recently
described procedure.23 This pseudotyped virus will infect a broad
range of mammalian cells but is capable of only 1 round of
replication.24 Stocks of an HIV-1 isolate from an infant (EF) who
died of AIDS (encephalopathy, cytomegalovirus retinitis, and severe
cardiomyopathy) were prepared by a coculture with
phytohemagglutinin-stimulated peripheral blood lymphocytes
(PHA-stimulated PBMCs), then expanded by infection of PHAstimulated PBMCs. Nucleotide sequence analysis of the patient
isolate gp120 coding sequence demonstrated that it is similar to other
North American non–syncytium inducing isolates (data not shown).
Virus in these cultures (and in experimental infections) was measured using a commercially available ELISA (Coulter Corp). The
infectious titer of the HIV-1NL4 –3 and the patient virus stocks was
determined by calculating the 50% tissue-culture infectious dose per
million PHA-stimulated PBMCs using a streamlined end-point
dilution assay.25
Infections
To infect PHA-stimulated PBMCs, cells were incubated at 37°C for
1 to 2 hours with HIV-1NL4 –3, VSV-G–pseudotyped HIV-1, or patient
isolate (EF) virus stocks, then washed thoroughly with media before
being placed in culture. PHA-stimulated PBMCs were infected with
either HIV-1NL4 –3 or the patient isolate (EF) at a multiplicity of
infection of 0.04. The estimated multiplicity of infection for the
October 5, 1998
739
HFCMs was 0.1 to 0.4. For experiments analyzed by PCR, virus
stocks were filtered and treated with DNase I, as described earlier.26
An aliquot of these stocks was heat inactivated (60°C for 20 minutes)
for use as a noninfectious control to reveal the degree of residual
contamination of virus stocks by HIV-1 DNA. Cultures were
incubated at 37°C with 5% CO2 for 10 days. After the incubation,
HFCMs were washed 2 times with RPMI. Supernatant was harvested
at 3, 6, and 9 days and stored at 220°C for HIV-1 p24 antigen assay.
Fresh medium was added at each harvest to keep the culture volume
constant. Three days after the infection, HFCMs were lysed with
urea lysis buffer, and total cellular DNA was purified using phenol
extraction and ethanol precipitation. Subsequently, quantitative PCR
was used to detect HIV proviral DNA, as described below. Three
days after the infection, HFCMs infected with the VSV-G–
pseudotyped HIV-1 were subjected to dual-labeling immunofluorescence (as described below) using MF-20 and pooled antisera from
HIV-1–infected adults.
Detection of HIV-1 Proviral DNA by PCR
Quantitative PCR detection of human b-globin gene DNA and
HIV-1 DNA sequences was performed as previously described,26
except primers SK38 and SK3927 were used to detect HIV-1 gag
sequences. Products of amplification were separated electrophoretically through a 6% polyacrylamide gel. Dried gels were exposed to
film to produce autoradiographs. Incubation of cells with heat
inactivated virus was performed as a control to reveal any residual
DNA contamination in virus stocks.
Indirect Immunofluorescence Labeling
Isolated myocytes were fixed with 2% buffered formaldehyde for 15
minutes according to previously published methods.28 The fixed cells
were quenched in Na1 borohydrate (0.2%) for 15 minutes and then
treated with Triton X-100 (0.05% or 0.1%) for 5 minutes. All the
cells were kept in blocking solution (3% BSA and 5% goat serum)
for 45 minutes, followed by incubation for 1 hour with both a
polyclonal antibody against HIV-1 (1:200) and a monoclonal antibody against myosin (MF 20, 1:20), (Developmental Studies Hybridoma Bank, University of Iowa, Iowa City). The myocytes were
then incubated with FITC-labeled goat anti– human secondary antibody (1:50, for HIV-1) and Cy3-labeled goat anti–mouse secondary
antibody (1:100) for 45 minutes at room temperature. The combination of FITC and Cy3 produces minimal spectral overlap. Finally, the
myocytes were washed several times in Tyrode’s solution and
mounted on slides with mounting medium (90% glycerol plus 2%
DABCO [1, diazobicyclo-2,2,2-octane], a photobleaching inhibitor).
Control experiments were performed by exposing the myocytes to
the secondary antibodies alone. Immunofluorescence microscopic
pictures were taken using a Nikon fluorescence microscope. Pooled
antisera from HIV-1–infected adults were used to detect HIV-1
proteins. The secondary antibody FITC-conjugated goat anti-human
antibody was purchased from Cappel (Durham, NC). Cy3 conjugated
goat anti-mouse antibody was purchased from Jackson ImmunoResearch Laboratory (West Grove, Pa).
Results
Numerous reports have suggested that HIV-1 infection of
cardiac myocytes occurs in vivo in some patients with
HIV-1–related cardiomyopathy. We attempted to confirm this
by infecting HFCMs in vitro with HIV-1NL4 –3 and a virus
isolate from a child with cardiomyopathy. Figure 1A examines p24 antigen production from HIV-1–infected human
donor PHA-stimulated PBMCs and HFCMs. Both HIV-1
strains productively infected PHA-stimulated PBMCs, as
indicated by the marked increase in p24 antigen production at
6 and 9 days after the infection. However, neither strain
elicited p24 antigen production from the HFCMs. The p24
antigen measured in the supernatant from the HFCMs decreased as a result of media changes. Furthermore, quantita-
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HIV-1 Infection of Human Fetal Cardiac Myocytes
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Figure 2. PCR analysis of DNA from PHA-stimulated PBMCs
and HFCMs infected in vitro with HIV-1. Bottom, Autoradiographic bands that reflect PCR detection of b-globin gene
sequences using serial dilutions of DNA from uninfected PHAstimulated PBMCs as controls. Top, Serially diluted plasmid
DNA is used as a quantitative standard for similar detection of
HIV-1 DNA. No HIV-1 DNA is detected in HFCMs exposed to
either HIV-1NL4 –3 or HIV-1 isolated from a patient (EF) with
severe cardiomyopathy. HI, negative control lanes containing
DNA from cells incubated with heat-inactivated virus.
Figure 1. P24 antigen production from PHA-stimulated PBMCs
or HFCMs infected with HIV-1. A, Cells infected with the
replication-competent strains HIV-1NL4 –3 (black curve) or HIV-1
from a patient (EF) with severe cardiomyopathy (red curve). B,
Cells infected with VSV-G–pseudotyped HIV-1 vector. In both
panels, circles correspond to PHA-stimulated PBMCs, and
boxes correspond to HFCMs.
tive DNA PCR analysis failed to demonstrate HIV-1 DNA
within HFCMs lysed at 3 days after the infection (Figure 2),
suggesting that the virus was unable to infect the HFCMs and
complete its life cycle. Amplification of human b-globin gene
DNA sequences demonstrated that the samples contained no
inhibitors of amplification.21
To address the possibility that circulating HIV-1 antibodies
might bind to the virus and promote virus uptake by the
HFCMs, we preincubated the virus with serial dilutions
(1:5000 to 1:50 000) of pooled antisera from HIV-1–infected
adults. We found no evidence of antibody enhancement of
infection under these conditions; ie, no HIV-1 DNA sequences were detected in HFCMs after incubation with this
pretreated virus (data not shown).
We suspected that the lack of productive infection by
wild-type HIV-1 was most likely explained by the lack of
CD4 molecules on HFCMs and not an inability to support the
HIV-1 life cycle. Therefore, we chose a lentiviral-based
HIV-1 vector pseudotyped with VSV envelope.
Figure 1B examines p24 antigen production from VSV-G–
pseudotyped HIV-1 vector–infected HFCMs (performed in
duplicate) and PHA-stimulated PMBCs. By 3 days after the
infection, there was a 2-fold increase in p24 antigen in
infected HFCMs. Viral p24 antigen production continued to
increase up to 6 days after the infection. A lesser response
was noted from the PHA-stimulated–PBMCs infected with
the VSV-G–pseudotyped HIV-1 vector in agreement with
previous studies.23
Dual-labeling immunofluorescence was performed to determine the purity of the cultured HFCMs and to determine
the cellular origin of the viral particles detected in the
supernatant. Figure 3 shows a series of immunofluorescence
micrographs of cultured cardiac myocytes from an 18-week
human fetal heart. These HFCMs were cultured for 3 days
and then infected with VSV-G–pseudotyped HIV-1 vector
and prepared for immunofluorescence 4 days after the infection. The cells were dual-labeled with polyclonal antibodies
against HIV-1 (secondary antibody conjugated with FITC)
and a monoclonal antibody against light meromyosin (MF-20
[secondary antibody conjugated with Cy3]). The upper 2
panels represent uninfected control HFCMs, and the lower
panels represent infected cells. No HIV-1 proteins were
detected in the control culture HFCMs (upper right panel),
and immunolabeling against myosin alone is shown (upper
left panel). HIV proteins were detected in approximately
one-half of the HFCMs exposed to the VSV-G–pseudotyped
HIV-1 vector (lower right). Immunolabeling for light meromyosin indicates that these infected cells contained myosin
and were ventricular myocytes and not fibroblasts (lower
left). Similar results were obtained in 3 additional
experiments.
Discussion
The purpose of this study was to determine whether HFCMs
in primary culture will allow productive and efficient in vitro
replication of wild-type HIV-1 or a VSV-G–pseudotyped
HIV-1 vector. Based on the p24 antigen and quantitative PCR
analysis, it appears that wild-type HIV-1 does not readily
infect HFCMs in primary culture. Pretreatment with pooled
antisera from HIV-1–infected adults did not overcome the
intrinsic resistance to HIV-1 infection. In addition, incubation
with HIVNL4 –3, shown previously to infect CD4 negative fetal
Rebolledo et al
October 5, 1998
741
Figure 3. Dual-labeled immunofluorescence micrographs of HFCMs
infected with VSV-G–pseudotyped
HIV-1 vector. HIV-1–infected (lower
panels) and – uninfected HFCMs
(upper panels) were fixed 4 days after
the infection and examined by indirect immunofluorescence, using primary antisera against myosin (left
panels) and pooled sera from HIV-1–
infected adults (right panels). Note
that all of the HIV-1–infected cells are
cardiomyocytes, and approximately
half of the cardiomyocytes are
infected. Magnification 3400.
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brain cells also failed to establish infection.19 Pseudotyping
HIV-1 virions with the VSV envelope led to efficient and
productive infection, showing that HFCMs can support the
HIV-1 life cycle.
We recognize that the establishment of HFCMs in primary
culture may select for a subset of myocytes that are refractory
to HIV-1 infection. In addition, cultured HFCMs probably
represent a subpopulation of cells that may not be reflective
of all cardiomyocytes. However, our evaluation of these cells
revealed typical responses to b-adrenergic stimulation and
other features of normal cardiomyocytes.
The major finding of this study is that a lentiviral-based
vector can replicate efficiently in HFCMs in vitro. The
VSV-G–pseudotyped HIV-1 vector has a broad host range
because it only requires binding with the phospholipid component of the plasma membrane of a target cell.24 In contrast,
no in vitro replication of wild-type HIV-1 occurred, suggesting that direct myocyte infection may not play a direct role in
the pathogenesis of HIV-1 cardiomyopathy. However, we
have not excluded the existence of cardiotropic strains of
HIV-1, nor have we evaluated the potential influence of
cytokines that could promote or sustain HIV-1 infection in
cardiomyocytes. Although there are a host of immunological
mediators present in vivo that were not present in this in vitro
study, other studies have suggested that HIV-1 infection of
cardiomyocytes in vivo is a rare event. We speculate that
HIV-1–mediated cardiac injury is probably the result of
indirect processes. For example, direct HIV-1 infection of
cardiac myocytes may not be needed in order for viral
proteins to have deleterious effects. Several HIV proteins
found in a soluble form in plasma (eg, gp120, Tat or Vpr)
have been shown to have a wide variety of cellular effects,
including apoptosis, interference with b-adrenergic stimulation, and transcriptional activation of various cellular genes.16
Although in situ hybridization has demonstrated HIV-1
RNA within the myocytes of patients with HIV-1, the
mechanism of viral entry remains unclear. Recent research
has suggested that HIV-1 may bind to other receptors such as
galactosylceramide.16 It is possible that infiltrative macrophages or other antigen presenting cells may facilitate HIV-1
entry into CD4 negative host cells. This mechanism of
transmission was recently demonstrated in a report in which
electron micrographs demonstrate the transfer of HIV-1 from
infected T lymphocytes to a cervical carcinoma cell line at the
point of cell-to-cell contact.15 In addition, the local immunologic milieu may be altered by cytokines that induce alternative receptors facilitating viral entry.
This study suggests that pseudotype lentiviral vectors may
be useful to transduce genes into human cardiac myocytes.
Although adenovirus has been used for the transduction of
stationary somatic cells, only transient gene expression has
been recognized.29 This is caused partially by an immunologic clearance of the adenoviral-based vector. Our results
suggest that the VSV-G–pseudotype HIV-1 vector is capable
of infecting HFCMs with high efficiency and stable gene
expression. However, because HFCMs are a rapidly dividing
cell population, it is unclear whether the cardiac myocytes of
infants or adults would be susceptible to infection by
pseudotype lentiviral vectors. It is generally believed the
human cardiac myocytes in vivo achieve terminal differentiation during the first month of life.30 Nonetheless, lentiviral
vectors are capable of efficient expression in terminally
differentiated cells,31 in part because the interaction of the
HIV-1 protein Vpr with the nuclear pore protein that promotes nuclear entry of the viral genome.32 Because the
genome of lentivirus-based vectors integrate into the host
genome, repeated transduction should not be required. We
have demonstrated that lentiviral-based vectors are suitable
for ex vivo cardiovascular gene therapy.31,33 It remains to be
seen whether lentiviral-based vectors will be good vehicles
for in vivo gene therapy because these vectors would probably infect other cell types instead of a resting cardiomyocyte.
These and other issues related to cardiovascular gene therapy
have been reviewed elsewhere.34,35
742
HIV-1 Infection of Human Fetal Cardiac Myocytes
Acknowledgments
The authors thank E. Garratty for her expert technical advice and Drs
S. Kaplan and Y. Bryson for invaluable editorial advice. The
monoclonal antibody MF 20, developed by Dr D.A. Fischman, was
purchased from the Developmental Studies Hybridoma Bank maintained by the Department of Pharmacology and Molecular Sciences,
Johns Hopkins University School of Medicine, Baltimore, Md, and
the Department of Biological Sciences, University of Iowa, Iowa
City, Iowa, under contract N01-HD-2-3144 from the National
Institute of Child Health and Human Development.
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Michael A. Rebolledo, Paul Krogstad, Fuhua Chen, Kevin M. Shannon and Thomas S. Klitzner
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Circ Res. 1998;83:738-742
doi: 10.1161/01.RES.83.7.738
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