Infection of Human Fetal Cardiac Myocytes by a Human Immunodeficiency Virus-1–Derived Vector

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 mrebolle{at}ucla.edu

	Abstract

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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.

Key Words: fetal heart • gene therapy • cardiomyopathy • human immunodeficiency virus • cell culture

	Introduction

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Cardiomyopathy is a well-recognized complication associated with HIV-1 infection.^{1 2 3 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 13-month-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 HIV_NL4–3 because it had been previously shown to infect CD4 negative cells.¹⁹ Also, HIV_NL4–3 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

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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 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-µm 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-µm 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, 2x DMEM vitamins (Sigma), 1x DMEM nonessential amino acids (Sigma), human apotransferrin 0.13 µmol/L (10 µg/mL), bovine insulin 1.7 µmol/L (10 µg/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 1x 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-1_NL4–3 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 pNLthyBgl, 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 0.1x 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 PHA-stimulated 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-1_NL4–3 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-1_NL4–3, 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-1_NL4–3 or the patient isolate (EF) at a multiplicity of infection of 0.04. The estimated multiplicity of infection for the 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 -20°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 ß-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 Na⁺ 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

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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-1_NL4–3 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, quantitative 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 ß-globin gene DNA sequences demonstrated that the samples contained no inhibitors of amplification.²¹

View larger version (14K): [in this window] [in a new window]   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.

View larger version (83K): [in this window] [in a new window]   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 ß-globin gene sequences using serial dilutions of DNA from uninfected PHA-stimulated 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.

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.

View larger version (122K): [in this window] [in a new window]   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 x400.

	Discussion

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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 HIV_NL4–3, shown previously to infect CD4 negative fetal 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 ß-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 ß-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}

	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.

Received December 22, 1997; accepted July 2, 1998.

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