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Am J Physiol Heart Circ Physiol 293: H2757-H2770, 2007. First published August 3, 2007; doi:10.1152/ajpheart.00477.2007
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Lentiviral vector-mediated expression of GFP or Kir2.1 alters the electrophysiology of neonatal rat ventricular myocytes without inducing cytotoxicity

Rajesh B. Sekar,1,* Eddy Kizana,2,* Rachel R. Smith,1,2 Andreas S. Barth,2 Yibing Zhang,1 Eduardo Marbán,2 and Leslie Tung1

1Department of Biomedical Engineering and 2Division of Cardiology, The Johns Hopkins University School of Medicine, Baltimore, Maryland

Submitted 20 April 2007 ; accepted in final form 31 July 2007


   ABSTRACT
TOP
 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Recombinant lentiviral vectors (LVs) are capable of transducing neonatal rat ventricular myocytes (NRVMs) and providing stable, long-term transgene expression. The goal of the present study was to comprehensively test whether transduction of NRVMs by LVs results in cytotoxicity and to examine the electrophysiological consequences of gene modification of NRVM monolayers by two vectors: one encoding a putatively inert enhanced green fluorescent protein (eGFP) and the other a major ion channel protein, inward rectifier K+ channel (Kir) 2.1. Freshly isolated NRVMs were transduced and cultured in monolayers. Immunohistochemistry, Trypan blue exclusion, annexin V binding followed by flow cytometry (FCM), and terminal transferase dUTP nick-end labeling assays were performed to assess for cytotoxicity. Optical mapping studies of action potential propagation in NRVM monolayers were performed to characterize the electrophysiological alterations following transduction. The cytotoxicity assays revealed that transduction had no adverse effects on NRVM cultures. However, eGFP-transduced monolayers exhibited a decrease in conduction velocity (CV) and action potential duration (APD) compared with monolayers transduced with LVs encoding LacZ or devoid of a transgene. In addition, small interfering RNA-mediated knockdown of eGFP expression corrected this phenotype. In contrast, Kir2.1 gene-modified monolayers showed an increase in CV and a predictable decrease in APD. This study demonstrates that LVs transduce NRVMs without cytotoxic effects. However, eGFP has a significant effect on APD and CV in this experimental system and calls into question the widely held belief that GFP is physiologically inert. In addition, LV-mediated overexpression of Kir2.1 opens up the prospect of studying the functional role of inward rectifier K+ current in cardiac arrhythmias.

lentiviral vectors; enhanced green fluorescent protein; inward rectifier K+ channel 2.1

GENE TRANSFER TO POSTNATAL cardiac myocytes has immense investigational benefits for understanding cardiac electrophysiology (27). Whereas the fetal heart grows by enlargement and proliferation of myocytes, the proliferative ability of myocytes is lost in the perinatal period and results in terminal differentiation (9). The loss of proliferative ability and the short life span of cardiac myocytes in vitro have previously been major hindrances to successful cardiac gene delivery (8). In addition, studies have shown that vector-associated cytotoxicity may disrupt cellular physiology (22). The emergence of lentiviral vectors (LVs), capable of transducing nondividing cells (17) and integrating into the host cell genome (2, 13, 25) to give long-term transgene expression (16), has placed postnatal genetic modification of cardiomyocytes within reach of gene transfer technology. Moreover, pseudotyped LVs cause little or no disruption of target cell physiology (3) and can potentially deliver up to 8 kb of gene sequence (27).

In the present study, we sought to characterize the level of cytotoxicity and the electrophysiological properties of neonatal rat ventricular myocytes (NRVMs) following LV transduction in an in vitro experimental model. Given recent reports that the expression of green fluorescent protein (GFP), a widely used reporter, can functionally impair striated muscle (1, 5, 18), we used optical mapping to study alterations in electrophysiological properties of NRVM monolayers following transduction with LVs encoding enhanced GFP (eGFP). Also, with the importance of the inward rectifier K+ current (IK1) in regulating cardiac action potential repolarization, maintenance of cardiac resting membrane potential, excitability, and arrhythmogenesis (19), we characterized changes in conduction velocity (CV) and action potential duration (APD) following transduction with LVs encoding inward rectifier K+ channel (Kir) 2.1, the molecular correlate of IK1. Our results revealed that LV-transduced and nontransduced NRVMs had comparable levels of apoptosis and that vector transduction did not induce excess cytotoxicity. Importantly, we found that eGFP expression lowers CV and shortens APD, raising questions about the appropriateness of this reporter as a control condition in muscle cells. In contrast, lentiviral transduction and overexpression of Kir2.1 resulted in increased CV and decreased APD over a period of 1 wk, demonstrating the utility of this approach for nontoxic, efficacious, and prolonged alteration of ion channel function.


   METHODS
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 ABSTRACT
 METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Molecular cloning. Vectors based on the human immunodeficiency virus type 1 (HIV-1) were used throughout this study (4, 26, 28–30). The third-generation constructs used include the self-inactivating (SIN) long-terminal repeat, the central polypurine tract (cPPT), and the woodchuck hepatitis virus posttranscriptional regulatory element (Wpre). The four plasmids required for vector production were kindly supplied by Professor Inder Verma from the Salk Institute, San Diego, CA. The four plasmids required for LV production (4) are as follows: 1) pRRL.SIN-18, the transfer construct containing HIV-1 cis-acting sequences and an expression cassette for the transgene; 2) pMDLg/pRRE, the conditional packaging construct expressing the gag and pol genes from the cytomegalovirus (CMV) promoter and intervening sequences and polyadenylation site of the human beta-globin gene; 3) pRSV-Rev, a non-overlapping construct expressing the rev cDNA; and 4) pMD2.VSV.G, the construct encoding for G protein of the vesicular stomatitis virus (VSV G), a heterologous envelope to pseudotype the vector. The cDNA for mouse Kir2.1-GFP fusion or LacZ was subcloned into the lentiviral plasmid, pRRLsin18.cPPT.CMV.eGFP.Wpre (designated as LV-eGFP) following the removal of eGFP. The resulting plasmids, pPPT.CMV.Kir2.1GFP and pPPT.CMV.LacZ, were designated as LV-Kir2.1 and LV-LacZ, respectively. For small interfering RNA (siRNA) experiments, an H1 promoter encoding short-hairpin RNAs targeting the GFP sequence or a nonsilencing sequence (24) was subcloned downstream of LacZ, resulting in plasmids PLZ.H1.shGFP and PLZ.H1.shNS, designated as LV-siGFP and LV-siNS, respectively. A control lentiviral plasmid devoid of a heterologous expression cassette was also constructed to produce an empty LV (LV-Empty).

Production of LVs. LV was produced by calcium-phosphate coprecipitation transfection of the four LV plasmids into human embryonic kidney (HEK) 293T cells (ATCC, Manassas, VA), as previously described (11). The supernatant from HEK cell flasks containing LVs was collected 48 and 72 h after transfection, filter sterilized using 0.2-µm cellulose acetate filter units (Corning, Cambridge, MA), and concentrated by ultrafiltration (100,000 MWCO, Centricon Plus-70, Millipore, Milford, MA). Transduction titer was assigned on concentrated viral stock by assessing transgene expression in HEK 293T cells using a limiting dilution assay in the presence of 8 µg/ml of Polybrene (Sigma-Aldrich, St. Louis, MO) 3 days after transduction. For LVs encoding eGFP (LV-eGFP and LV-Kir2.1GFP), expression was assessed by direct GFP fluorescence. For LVs encoding LacZ, expression was assessed by X-Gal staining. For LV-Empty, the titer was assigned by performing an ELISA for HIV-1 p24 core antigen (PerkinElmer Life Sciences, Boston, MA).

LV transduction of NRVMs. For all transduction experiments, the concentrated LV stock was applied at the indicated multiplicity of infection (MOI; i.e., the number of active vector particles per target cell) in the presence of 8 µg/ml of Polybrene. Vector stock stored at –80°C was thawed rapidly in a 37°C water bath before being added to cells for transduction. The target cells, NRVMs, which were freshly isolated, were suspended in culture medium containing 10% medium at a concentration of 106 cells/ml. Vector was added to this cell suspension, and cells were then plated on fibronectin-coated plastic coverslips. The time of plating is considered day 0 for the NRVM cultures. After 24 h of incubation at 37°C, the medium was aspirated from the transduced and adherent NRVM cultures, and fresh medium containing 10% serum was added. The medium for transduced cultures was changed every 2 days, similar to nontransduced cultures.

Quantitative reverse transcription polymerase chain reaction. To determine the gene-knockdown efficiency of the siRNA encoding LV construct, cells were cotransfected with PPT.CMV.eGFP and PLZ.H1.shGFP or PLZ.H1.shNS. Three days after transfection, total RNA was isolated from cells using RNeasy Plus Mini Kit (Qiagen, Valencia, CA), as per the manufacturer's instructions. For quantification of steady-state eGFP mRNA levels, the TaqMan gene expression assay (Applied Biosystems, Foster City, CA) was used. Primers and probe against the target mRNA were designed using the Primer-express software (Applied Biosystems). Single-step quantitative PCR was performed using a 7900HT Sequence Detection System (Applied Biosystems) under the following conditions. A total of 20 ng of RNA was used in each reaction in a 96-well optical plate with an initial RT step for 38 min at 48°C, followed by 10 min of RT inactivation before 40 cycles of PCR at 95°C/60°C. The 18S ribosomal subunit was used as an internal control, and each sample was run in quadruplicate. Results were analyzed according to the relative standard curve method using the SDS 2.1 software (Applied Biosystems).

Cell culture. HEK 293T cells used for vector production and titer assignments were maintained in DMEM culture medium (Gibco, Rockville, MD) supplemented with 10% heat-inactivated fetal bovine serum (Invitrogen, Carlsbad, CA) and 1% penicillin-streptomycin (Invitrogen). NRVMs were enzymatically dissociated from the ventricles of 2-day-old Sprague-Dawley rats (Harlan, Indianapolis, IN) with the use of trypsin (Amersham Biosciences, Piscataway, NJ) and collagenase (Worthington Biochemical, Freehold, NJ). Freshly isolated NRVMs were resuspended in M-199 culture medium (Gibco) supplemented with 10% FBS, glucose, 2 mM L-glutamine, penicillin, vitamin B12, HEPES buffer, and MEM nonessential amino acids (Gibco). Two 90-min preplating steps were performed to reduce fibroblasts and enrich cardiac myocyte content in the culture. The final cell suspension was collected, counted for NRVMs, and diluted at the desired plating concentration of 0.5 million cells in 1 ml of medium. For mapping experiments, 106 cells were plated on 21-mm plastic coverslips coated with fibronectin (Sigma-Aldrich) at a concentration of 25 µg/ml. After 24 h, the coverslips were washed with warm phosphate-buffered saline (PBS), and fresh medium with 10% serum was added. Starting from day 2 (2 days after plating), the cultures underwent media change on every second day and were maintained in medium containing 2% serum.

The protocol for tissue harvest from rat, RA07M247, was reviewed and approved by the Johns Hopkins University Animal Care and Use Committee (ACUC) on 06/25/2007. All animal experiments were performed in accordance with guidelines set by the Johns Hopkins Committee on Animal Care and Use and were in compliance with all federal and state laws and regulations. The animals, neonatal rat pups, were only used for harvesting cardiac tissue and were killed thereafter.

Immunostaining. Six-day-old control (nontransduced) and LV-transduced NRVM monolayers used for optical mapping studies were characterized thoroughly for their myocyte content using an immunostaining assay with antibodies against cardiac troponin I (cTnI). LV-Kir2.1-transduced NRVMs were characterized for their Kir2.1 expression using antibodies against Kir2.1. Hoechst (1:10,000, Invitrogen) was used as a nuclear counterstain. Cultured NRVMs were washed with warm Ca2+-free PBS and fixed in 4% paraformaldehyde for 10 min at room temperature. Cells were washed again with Ca2+-free PBS and permeabilized using 0.1% Triton X for 20 min. Cells were blocked using 10% goat serum in PBS plus 1% bovine serum albumin (BSA) for 1 h at room temperature. Primary antibodies against Kir2.1 (1:50, SC-28633, Santa Cruz, Santa Cruz, CA) and cTnI (1:50, SC-15368, Santa Cruz) were diluted in PBS plus 1% BSA. Cells were incubated with primary antibodies for 1 h at room temperature and washed again with Ca2+-free PBS. Cells were then incubated with Alexa-conjugated goat secondary antibodies (1:500, Invitrogen) for 1 h at room temperature and washed again with Ca2+-free PBS. Finally, cells were mounted in Vectashield (Vector Laboratories, Burlingame, CA). Kir2.1 levels were characterized by visual inspection of Kir2.1 immunostained images. The myocyte content in each culture was quantified by merging the transmitted light and cTnI-immunostained image for a given field of view and determining the percentage of cell-covered area of transmitted light image that was also positive for cTnI immunostaining. To obtain average myocyte content, each coverslip was studied over three randomly selected fields of view.

Cytotoxicity assays. Multiple complementary cytotoxicity assays, including Trypan blue, annexin V binding, and tranferase-mediated dUTP nick-end labeling (TUNEL) assays, were performed on 6-day-old cultures to determine the cytotoxicity of the LVs. The assays were performed on both nontransduced and transduced NRVMs, and the level of cell death in both of these cultures was quantified using microscopy or FCM.

For Trypan blue assay, culture medium was replaced with warm Ca2+-free PBS, and 0.5 ml of Trypan blue (Invitrogen) was added for every 1 x 106 NRVMs. The staining solution was replaced with Ca2+-free PBS after 10 min, and images were obtained. NRVMs permeabilized by exposure to 0.1% Triton X were used as positive control. To determine the long-term effects of transduction, the cultures were characterized by this assay throughout their maximal culture period of 10 days, and the percentage of dead cells in a given field of view was counted. Each culture system was studied using two coverslips from two different weeks of isolation and three randomly selected fields of view per coverslip.

For annexin V assay, NRVMs were washed with warm Ca2+-free PBS and dissociated using 0.25% trypsin-EDTA for ~10 min at room temperature. Cells were pelleted by centrifugation at 4°C and 750 rpm for 3 min. The single-cell suspension was washed again with Ca2+-free PBS and pelleted. Approximately 1 x 106 NRVMs were resuspended in 100-µl PBS containing 1% BSA and 1 mM Ca2+ for each test sample. Five-microliter annexin V APC (BD Pharmingen, San Diego, CA) was added to each test sample. Samples were incubated for 15 min at room temperature, and 400 µl of PBS containing 1% BSA and 1 mM Ca2+ were then added to each sample. Samples were stored on ice until analyzed by FCM using a FACSCalibur flow cytometer with CellQuest software (BD Biosciences). Gates were established by forward scatter and side scatter, and only cell debris was excluded from the gated population. Compensation percentages were set using unlabeled and single-dye labeled control samples. NRVMs maintained in a medium with no glucose and no serum and subjected to hypoxia (1% oxygen in a purged, closed chamber) for 48 h were used to test the integrity of the technique.

For TUNEL assay, NRVMs were fixed, permeabilized, and washed as for immunostaining. Cells were incubated with TUNEL TMR red reaction mix (Roche Applied Science, Indianapolis, IN) for 1 h at 37°C, washed again with Ca2+-free PBS, counterstained, and mounted as for immunostaining.

Fluorescence imaging. Wide-field fluorescence imaging of 6-day-old cultures was performed on a Nikon TE2000-U Eclipse inverted microscope using Image-Pro Plus software (Media Cybernetics, Silver Spring, MD).

Optical mapping. Optical mapping studies were performed on NRVM monolayers 6–7 days posttransduction. Cover slips supporting the monolayers were placed in a custom-designed chamber, stained with 10 µM di-4-ANEPPS (Molecular Probes, Eugene, OR) for 5 min, and continuously superfused with warm (37°C) oxygenated Tyrode solution, as previously described (15). Bipolar line stimulation via platinum electrodes was applied just above one edge of the monolayer. To determine the APD at 80% repolarization (APD80) and CV, cells were stimulated with monophasic, 10-ms pulses at 2 Hz delivered by the stimulus electrode at twice diastolic threshold. A 2-s recording was taken after a 10-beat drive train. Action potentials were recorded from 253 sites using a custom-built contact fluorescence imaging system (15). Using multisite optical recordings of transmembrane potential, sequential maps of the activation patterns of NRVM cultures were obtained, and normal and arrhythmic electrophysiological behavior were monitored.

Data analysis. Data were analyzed in MATLAB (The MathWorks, Natick, MA) with the use of custom-written scripts.

Statistics. Data are expressed as means ± SD. Paired Student's t-tests (GraphPad Prism, GraphPad Software, San Diego, CA) were used to compare APD80 and CV between nontransduced and transduced NRVM cultures. A P value of <0.05 was considered statistically significant.

The authors had full access to the data and take full responsibility for its integrity. All authors have read and agreed to the paper as written.


   RESULTS
TOP
 ABSTRACT
 METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Transduction efficiency of LVs. We have previously shown that the transduction efficiency of the vector increases with the MOI, and, at a MOI of 20, 95% of the cells are gene modified (10). We confirmed this previous finding for 6-day-old LacZ and eGFP gene-modified NRVMs. Compared with nontransduced NRVM monolayers (Fig. 1A), NRVM monolayers transduced with LV-Empty (Fig. 1B), LV-LacZ (Fig. 1, C and D), and LV-eGFP (Fig. 1, E and F) exhibited similar culture characteristics, such as confluency and morphology, as well as beating frequency. LV-eGFP-transduced NRVMs (Fig. 1G) that were immunostained for cTnI (Fig. 1H) showed that the gene-modified NRVM monolayer consisted almost entirely of myocytes (Fig. 1I). These findings suggested that the process of LV transduction does not compromise the culture quality of NRVMs. While nontransduced (Fig. 2, A and B), LV-Empty (Fig. 2, C and D), and LV-eGFP transduced (Fig. 2, E and F) NRVMs showed little or no significant Kir2.1 expression. LV-Kir2.1-transduced NRVMs (Fig. 2, G and H) showed increased Kir2.1 expression, as also seen from higher magnification images of Kir2.1 immunostain (Fig. 2I) and GFP fluorescence (Fig. 2J) of the Kir2.1-GFP fusion protein. Detailed inspection of LV-Kir2.1-transduced NRVM nuclei (Fig. 2K), and their merge with Kir2.1 immunostain and GFP fluorescence (Fig. 2L) showed a highly heterogenous expression of the Kir2.1-GFP transgene in individual NRVMs. Although some of the Kir2.1-GFP fusion protein could be seen in the membranes, most of it could be located in intracellular structures around the nuclei in the form of a ring.


Figure 1
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  		Fig. 1. Gene transfer potential and transduction efficiency of lentiviral vectors (LVs) studied in 6-day-old neonatal rat ventricular myocyte (NRVM) cultures. Transmitted light images of nontransduced (A), empty LV (LV-Empty; B), LV-LacZ (C), and LV-enhanced green fluorescent protein (eGFP; E) transduced monolayers show that, following vector transduction, cells still retained their culture characteristics, such as confluency and morphology. Color micrograph of beta-galactosidase staining of LV-LacZ transduced NRVMs (D) and eGFP fluorescent image of LV-eGFP transduced monolayer (F) show the gene transfer potential of the vector. LV-eGFP transduced NRVMs (G) that were immunostained for cardiac troponin I (cTnI; H) showed that the gene-modified NRVM monolayer consisted almost entirely of myocytes (I). Scale bar = 100 µm.

 

Figure 2
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  		Fig. 2. Levels of inward rectifier K+ channel (Kir) 2.1 expression studied in 6-day-old NRVM cultures. Transmitted light (left column) and fluorescent images of Kir2.1 immunostaining (middle column) show little or no significant Kir2.1 expression in nontransduced (A and B), LV-Empty (C and D), and LV-eGFP (E and F) transduced NRVMs. LV-Kir2.1 transduced monolayers (G and H) show increased expression of Kir2.1 as also shown in the higher magnification images of Kir2.1 immunostain (I) and green fluorescent protein (GFP) fluorescence from the Kir2.1-GFP fusion protein (J). Hoechst images (K) of LV-Kir2.1 transduced NRVM nuclei merged with the Kir2.1 immunostain, and GFP fluorescence signals from the Kir2.1-GFP fusion protein (L) show a heterogeneous expression profile of Kir2.1 in individual NRVMs with some of the protein on the membrane and most of it located in intracellular strucutures around the nuclei as part of increased protein trafficking in Golgi apparatus and endoplasmic reticulum. This increased trafficking and localization around the nuclei can be distinctively seen as rings (shown by arrows in I, J, and L). Scale bar = 100 µm.

 
LV transduction does not alter the survival rate of myocytes in culture. To determine the effect of LV transduction on culture composition, we began with a detailed characterization of our culture. Using transmitted light (Fig. 3, A–C), cTnI immunostain (Fig. 3, D–F), Hoechst (Fig. 3, G–I), merged transmitted light and cTnI (Fig. 3, J–L), and merged cTnI and Hoechst (Fig. 3, M–O) images of nontransduced (Fig. 3, left column), LV-Empty (Fig. 3, middle column), and LV-eGFP transduced (Fig. 3, right column) monolayers, we determined that the cultures consisted mostly of NRVMs, together with the presence of a small population of nonmyocytes, which were negative for cTnI immunostaining but were positive for Hoechst staining. Determination of cTnI-positive area out of the total cell-covered area on a transmitted light image (shown as percentage values in Fig. 3, J–L) revealed comparable levels of myocyte population in nontransduced (97.8 ± 0.9%), LV-Empty (96.7 ± 2.3%), and LV-eGFP (95.6 ± 1.7%) transduced monolayers. The data are representative of three fields of view per monolayer and total of n = 4 monolayers (2 replicates from 2 sets of cultures). The results confirmed that the vector transduction did not affect the survival rate of myocytes and noncardiac myocytes such as fibroblasts.


Figure 3
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  		Fig. 3. Characterization of myocyte composition in 6-day-old NRVM monolayers. Transmitted light (A–C), cTnI immunostain (D–F), Hoechst (G–I), merged transmitted light and cTnI (J–L), and merged cTnI and Hoechst (M–O) images of nontransduced (left column), LV-Empty (middle column), and LV-eGFP transduced (right column) monolayers reveal the presence of a large number of NRVMs and a small population of noncardiac myocytes. Fluorescent images of monolayers immunostained for cTnI reveal all NRVMs present in a given field of view, while Hoechst images reveal the total number of cells in the field of view. The merge of cTnI immunostain on transmitted light image of cultures revealed the percentage of cell-covered area on the latter that was cTnI positive. From the percentage values shown in J–L, it is clear that transduced monolayers are composed of NRVMs and noncardiac myocytes at levels comparable to those seen in nontransduced monolayers, and the morphology and confluency of transduced NRVMs are also comparable to those seen in nontransduced NRVMs. Scale bar = 100 µm.

 
Trypan blue assay confirms lack of vector-associated cytotoxicity. We performed a Trypan blue assay to assess the level of cytotoxicity that might be introduced by the vector transduction. As shown in Fig. 4, A, C, and E, both nontransduced and LV-empty and LV-eGFP transduced NRVM monolayers consisted almost entirely of live and healthy cells with intact membranes, as evidenced by the exclusion of the blue stain. A few Trypan blue positive cells (represented in terms of percentage of dead cells) could be detected in all cultures, as shown by the arrows in the high-magnification insets. The three culture systems studied had the same percentage of dead cells (~3%) for any field of view. To test the integrity of the technique, we then permeabilized both of these monolayers with 0.1% Triton X for 20 min and performed the same assay. The cultures now consisted almost entirely of dead cells, as seen from the dark blue stain in Fig. 4, B, D, and F. The data are representative of three fields of view per monolayer and total of n = 4 monolayers (2 replicates from 2 sets of cultures). Also, throughout the maximal culture period of 10 days, the percentage of total dead cells remained statistically similar in all of these cultures (Fig. 4G). The assay provided additional evidence that LV-vector transduction did not introduce any level of cytotoxicity to cultured NRVM monolayers.


Figure 4
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  		Fig. 4. Trypan blue assay of 6-day-old NRVM monolayers. A: nontransduced NRVM monolayers are devoid of apoptotic and necrotic cells. The few dead cells present in the monolayer are quantified and shown in the high-magnification inset. B: nontransduced NRVM monolayers permeabilized with 0.1% Triton X for 20 min now consist almost entirely of dead cells, as seen clearly from dark blue stain in the high-magnification inset. LV-Empty (C) and LV-eGFP (E) transduced monolayers are also devoid of dead cells and are comparable to nontransduced negative controls. Triton X-permeabilized transduced monolayers (D and F) consist entirely of dead cells. G: plot showing the percentage of dead cells in different culture systems (3 fields of view from n = 4 monolayers each) during their maximal culture period of 10 days. Scale bar = 100 µm.

 
Annexin V binding assay confirms lack of vector-related apoptosis. Using annexin V labeling, which would detect early apoptotic cells in addition to cells that had already undergone apoptosis or necrosis, we then quantified the level of total cell death in nontransduced and LV-transduced monolayers. The percentage of annexin-positive cells was comparable in NRVM cultures that were nontransduced or transduced with LV-Empty, LV-eGFP, or LV-LacZ. The total percentage of dead cells was similar for all four groups of NRVMs, as shown in Fig. 5A. To test the integrity of the technique, we performed the assay on NRVMs subjected to 48 h of hypoxia with no glucose or serum and compared it against nontransduced NRVM monolayers. As shown in Fig. 5B, the ischemic culture revealed a fourfold increase in the proportion of dead cells. The percentage of annexin-positive cells in different cultures is shown again as a bar graph in Fig. 5C. The data are representative of n = 4 monolayers (2 replicates from 2 sets of cultures). It should be noted that the relatively high percentage of dead or dying cells across all groups may be a consequence of the process of trypsinization during annexin V labeling of 6-day-old NRVMs that were well-attached to the coverslips.


Figure 5
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  		Fig. 5. Annexin V binding assay followed by flow cytometry (FCM) analysis to determine the total percentage of apoptotic and necrotic cells in NRVM cultures. A: FCM plots of nontransduced (gray), LV-Empty transduced (red), LV-eGFP transduced (green), and LV-LacZ transduced (blue) NRVMs are superimposed and show comparable levels of apoptotic and necrotic cells in these different cultures. The average percentage of cells positive for annexin V binding is ~17.4%. B: FCM plots of nontransduced and NRVMs subjected to 48 h of hypoxia with no glucose and no serum are superimposed and show a significantly larger percentage of cell death (~65%) in the latter. C: the percentage of annexin-positive cells is comparable in both nontransduced and LV-transduced NRVM monolayers. NS (nonsignificant) indicates P value >0.05 compared with nontransduced controls (n = 3 for each group).

 
TUNEL assay confirms lack of vector-associated cytotoxicity. We performed TUNEL assay in combination with Hoechst staining to determine the relative number of cells with DNA damage secondary to any apoptotic process induced by the vector. The number of TUNEL-positive cells was comparable in both nontransduced (Fig. 6, A–C) and LV-Empty-transduced (Fig. 6, D–F) monolayers and was low overall, consistent with our earlier findings that the LV vector was noncytotoxic. The data are representative of n = 6 monolayers (3 replicates from 2 sets of cultures). To test the integrity of the assay, we performed the assay on NRVMs subjected to ischemic conditions, which revealed a large population of TUNEL-positive nuclei (Fig. 6, G–I).


Figure 6
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  		Fig. 6. Transferase-mediated dUTP nick-end labeling (TUNEL) assay to characterize the level of DNA damage during apoptosis in NRVM cultures. Nontransduced (A, B, and C), LV-Empty transduced (D, E, and F), and ischemic (G, H, and I) NRVM monolayers reveal damaged nuclei, as shown by an arrow pointing to a representative TUNEL-positive nucleus in the TUNEL (A, D, and G), combined Hoechst and TUNEL (B, E, and H), and phase-contrast (C, F, and I) images. Transduced NRVM monolayers reveal very few damaged nuclei, similar to nontransduced controls. The monolayer subjected to ischemic conditions contains an abundance of TUNEL-positive nuclei in the TUNEL image and a large population of round dead cells in the phase-contrast image. Scale bar = 100 µm.

 
Does LV-eGFP transduction alter the electrophysiology of cardiac cells? Transduced NRVMs, like nontransduced NRVMs, survived a maximal culture period of 10 days, and their protein expression was maximal and maintained starting on day 3. Optical mapping studies of both nontransduced and LV-transduced NRVM monolayers were performed on day 6. With bipolar line stimulation at 2 Hz, planar waves were initiated that conducted across the monolayer. No wave breaks or arrhythmias resulted in any of the cultures, even with 4- to 6-Hz pacing, suggesting homogeneity of the cultures and uniform vector transduction. The planar waves propagated similarly in both nontransduced and LV-empty transduced NRVM monolayers. However, LV-eGFP-transduced NRVM monolayers propagated impulses with a decreased velocity compared with nontransduced monolayers. In contrast, LV-Kir2.1-transduced NRVM monolayers propagated impulses with a substantially increased velocity compared with nontransduced and LV-eGFP-transduced NRVM monolayers. Isochrone maps of action potential propagation for nontransduced (Fig. 7A), LV-Empty-transduced (Fig. 7B), and LV-eGFP-transduced (Fig. 7C) monolayers are shown. As seen from Fig. 7E, the average CV of nontransduced NRVMs was 19.1 ± 0.6 cm/s (n = 7), of LV-Empty-transduced NRVMs was 18.7 ± 1.0 cm/s (n = 7), and of LV-eGFP-transduced NRVMs was 12.8 ± 0.4 cm/s (n = 14). LV-eGFP-transduced NRVM monolayers had significantly decreased CV compared with nontransduced and LV-Empty-transduced NRVM monolayers. Analysis of the optical mapping data also revealed that eGFP expression shortened APD80 significantly compared with nontransduced and LV-Empty-transduced NRVM monolayers, as shown in the representative AP waveforms in Fig. 8, C, A, and B, respectively. The average APD80 of nontransduced NRVMs was 173 ± 14 ms (n = 7), of LV-empty-transduced NRVMs was 165 ± 10 ms (n = 7), and of LV-eGFP-transduced NRVMs was 114 ± 11 ms (n = 14) (Fig. 8E). To verify further that it was the eGFP transgene that altered the CV and APD80 values, we also mapped LV-LacZ-transduced NRVM monolayers. The average CV and APD80 were 18.1 ± 0.6 cm/s (n = 6, Fig. 7E) and 160 ± 10 ms (n = 6, Fig. 8E), respectively, and these values were comparable with those of nontransduced NRVMs. Thus it is clear from the mapping data that it was the eGFP transgene that decreased CV and APD80 of our monolayers, and that the vector by itself does not introduce any electrophysiological change to the monolayer.


Figure 7
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  		Fig. 7. Representative isochrone maps for action potential propagation in nontransduced and LV-transduced NRVM monolayers. Selected paths of wavefront propagation, along which conduction velocity (CV) values are calculated, are shown in black lines, and the corresponding values are also displayed. Nontransduced (A) and LV-Empty-transduced (B) display very similar CV characteristics. Monolayers transduced with LV-eGFP (C) show closely spaced isochronal lines (6-ms intervals), indicative of reduced CV. NRVM monolayers transduced with LV-Kir2.1 (D) show widely spaced isochronal lines, indicative of an increased CV. E: bar graph shows the average CV of the different culture systems tested [n = 7 each for nontransduced, LV-Empty, LV-short interfering GFP (siGFP), LV-short interfering nonsilencing sequence (siNS), LV-LacZ, and LV-Kir2.1, and n = 14 for LV-eGFP]. *P value <0.05 compared with nontransduced cultures. The graph shows restoration of CV values in LV-eGFP monolayers following siGFP-mediated knockdown of eGFP expression.

 

Figure 8
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  		Fig. 8. Representative action potential waveforms for nontransduced and LV-transduced NRVM monolayers. Nontransduced (A) and LV-Empty-transduced (B) monolayers display similar action potential characteristics. LV-eGFP-transduced monolayers (C) show significantly reduced action potential duration at 80% repolarization (APD80). LV-Kir2.1-transduced NRVM monolayers (D) show an even shorter APD80. E: bar graph shows the average APD80 of different culture systems (n = 7 each for LV-Empty, LV-siGFP, LV-siNS, LV-LacZ, and LV-Kir2.1, and n = 14 for LV-eGFP) that were studied. *P value <0.05 compared with nontransduced cultures (n = 7). The graph shows that the reduction of APD80 values in LV-eGFP monolayers is ameliorated by LV-siGFP vector. Horizontal grid spacing in A–D is 100 ms.

 
Altered electrophysiological values of LV-Kir2.1-transduced NRVM monolayers. As seen from the widely spaced isochrones of Fig. 7D, the average CV of LV-Kir2.1-transduced monolayers was significantly higher (27.4 ± 3.1 cm/s, n = 7) than that of nontransduced and LV-Empty-transduced monolayers (values stated earlier) (Fig. 7E). As expected from overexpression of Kir2.1 channels, LV-Kir2.1-transduced NRVMs had a much shortened APD80 (Fig. 8D) of 67 ± 9 ms (n = 7, Fig. 8E).

Altered electrophysiological properties due to eGFP are reversed by siRNA-mediated knockdown of eGFP expression. To confirm further that the altered electrophysiological properties of LV-eGFP transduced NRVM monolayers were due to eGFP transgene expression and that these effects could be reversed with removal of eGFP, LV-eGFP transduced NRVMs were subjected to additional transduction with LV-siGFP at a MOI of 50 (2.5 times that of LV-eGFP). As a negative control, we transduced eGFP-transduced NRVMs with LV-siNS at the same MOI. As expected, 6 days after transduction, the level of eGFP expression was diminished in LV-siGFP-transduced cultures (Fig. 9B). In contrast, eGFP expression was about the same in LV-eGFP- and LV-siNS-transduced cultures (Fig. 9, A and C). Notably, CV and APD80 of LV-eGFP + LV-siGFP-transduced NRVMs were restored to levels comparable to those of nontransduced or LV-empty-transduced NRVMs, as shown in Figs. 7E and 8E, respectively. However, LV-eGFP + LV-siNS-transduced NRVMs still exhibited the reduced CV and APD80 seen in NRVMs transduced with LV-eGFP alone. RT-PCR analysis of LV-siGFP and LV-siNS (n = 4 each) transfected cells showed that eGFP mRNA levels were decreased by 86% in the former compared with the latter (Fig. 9D). Thus these siRNA experiments confirmed that eGFP gene knockdown was able to rescue the abnormal electrophysiological phenotype conferred on NRVMs by eGFP expression.


Figure 9
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  		Fig. 9. Short interfering RNA (siRNA)-mediated knockdown of eGFP expression in NRVM cultures. A: NRVM cultures transduced with LV-eGFP at a multiplicity of infection (MOI) of 20. B: NRVMs transduced with both LV-eGFP (MOI = 20) and LV-siGFP (MOI = 50) reveal decrease in eGFP expression on day 5. C: NRVM cultures transduced with both LV-eGFP (MOI = 20) and LV-siNS (MOI = 50) show levels of eGFP expression comparable to that of NRVMs transduced only with LV-eGFP. D: bar graph showing the levels of eGFP mRNA in LV-siGFP- and LV-siNS-transfected (n = 4 each) cells. *P < 0.05. Scale bar = 100 µm.

 

   DISCUSSION
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With the goal in mind of gene delivery to cardiac tissue, we chose to study the LV system for many reasons. Prominent among these is the ease with which these vectors can be produced, permitting widespread application of this technology for use in basic cardiac research. While adenoviral vectors suffer from transient transgene expression and proven cytotoxicity of their viral proteins, adeno-associated viruses suffer from limited packaging capacity. LVs, on the other hand, are capable of stable, long-term expression of the transgene. In addition, the vector, unlike its parental derivative, has an excellent biosafety profile permitting its production and use at commonly used biosafety levels. Also, the use of VSV-G pseudotyped LVs broadens cell tropism beyond NRVMs, should transduction of additional target cell types be required (17). By comprehensively characterizing the cytotoxicity levels and the ability of LVs to alter NRVM physiology, we have established the utility of these vectors for in vitro gene modification studies. The main results of our study are as follows: 1) quantification of the lack of cytotoxicity associated with lentivirus-based gene delivery; 2) quantification of the adverse electrophysiological side-effects of eGFP expression; and 3) demonstration of changes in CV and APD with overexpression of the functionally active ion channel protein, Kir2.1.

It has been previously shown that LVs can efficiently transfer genes to neonatal and adult cardiac myocytes without necrotic damage over a culture period of 2 days, as assessed by creatine kinase assay (27). The present study characterizes and comprehensively quantifies the level of cytotoxicity associated with lentivirus-based gene delivery systems used with NRVMs over a prolonged culture period of 10 days. Transduction of cultured NRVMs did not alter the cellular composition of cultures, and there is no increase in apoptosis, necrosis, or cellular nuclear damage. Transduced NRVMs, like nontransduced cells, could be cultured for up to a period of 10 days, with functional electrophysiological activity on day 6. Following LV transduction of NRVMs, the protein expression was maximal on day 3 (72 h after transduction) and maintained through the remaining culture period.

GFP is currently widely used as a fluorescent reporter molecule. Although studies have previously reported adverse effects of GFP on the mechanical function of cardiac muscle (18) and on its ability to cause dilated cardiomyopathy (5) or impair actin-myosin interactions in skeletal muscle (1), no study has addressed the effects of GFP expression on cardiac electrophysiology, specifically on CV and cardiac APD. In the present study, we demonstrate for the first time that, in cultured NRVM monolayers, both CV and APD80 are negatively affected by eGFP transgene expression. The effect on CV is significant and a consistent observation. The adverse effects of eGFP expression on APD are equally prominent. We can speculate that GFP expression either reduces excitability by reduced sodium current availability or negatively impacts gap junctional coupling (12, 21, 23). Regarding the latter possibility, a recent study by Kizana et al. (10) confirmed a role for GFP in blocking the inhibitory effect of Cx43 internal loop mutants. Studies have also shown that GFP tagging at the extreme COOH-terminus of Cx43 can result in abnormal gap junctions by inducing conformational changes, which may interfere with the ability of the connexin to oligomerize with other connexin molecules (6, 7). Thus there is evidence for GFP effects on gap junctions, which may in turn alter cell-cell coupling and CV. Further studies are needed to fully elucidate the mechanisms of the depressive effects of eGFP expression observed in our study. Nevertheless, our findings suggest that GFP alone may not be an ideal control condition for cardiac research and that additional controls may be warranted for transgenic expression studies.

Finally, we demonstrated for the first time that, in cultured NRVM monolayers, CV of cardiac impulse propagation is increased significantly with Kir2.1 overexpression (Fig. 6E). Based on their simulation studies, Noujaim and coworkers (19) have already suggested increased sodium channel availability and greater excitability in transgenic mouse hearts with IK1 upregulation. Our findings of increased CV with Kir2.1 overexpression support their results. As expected, with increased expression of Kir channels, the final phase of AP repolarization is accelerated, and APD is significantly shortened (Fig. 7E). Recently, the short QT syndrome, SQTS3, has been identified as resulting from a de novo gain-of-function mutation in the KCNJ2 gene that encodes for Kir2.1 channels (20), and other studies have linked familial atrial fibrillation with enhanced Kir2.1 expression (14). Using an in vitro model of Kir2.1-overexpressed NRVMs, we can obtain useful insights into the role of IK1 in these arrhythmias, such as the maintenance and stabilization of initiated reentries (19).

In conclusion, the present study confirms the utility of, and enhances the prospect of, lentivirus-based vectors to become an important molecular tool for in vitro studies of gene-based cardiac arrhythmias.


   GRANTS
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
Funding for this work was provided by National Heart, Lung, and Blood Institute Grant R01-HL66239 to L. Tung. E. Marbán holds the Michel Mirowski, MD Professorship of Cardiology of the Johns Hopkins University. E. Kizana was funded by the Michel Mirowski, MD fellowship of the Heart Rhythm Society. A. S. Barth was supported by a grant from the German Research Foundation (DFG, Grant BA 3341/1-1).


   ACKNOWLEDGMENTS
 
We thank Peihong Dong for technical assistance with the molecular biology experiments.


   FOOTNOTES
 

Address for reprint requests and other correspondence: L. Tung, Dept. of Biomedical Engineering, The Johns Hopkins Univ. School of Medicine, 720 Rutland Ave., Traylor 703, Baltimore, MD 21205 (e-mail: ltung{at}jhu.edu)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

* R. B. Sekar and E. Kizana contributed equally to this work. Back


   REFERENCES
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