Evidence that Myocardial Na/I Symporter Gene Imaging Does Not Perturb Cardiac Function

Recombinant Adenoviral Vectors

Ad.EGFP.NIS is a replication-deficient vector based on human adenoviral serotype 5 that contains cytomegalovirus promoter-driven expression cassettes for the EGFP gene and the full-length human NIS complementary DNA (cDNA) substituted for E1 through homologous recombination. This vector has been shown to produce parallel levels of separate NIS and EGFP proteins in rat myocardial tissue (11). Ad.EGFP, which carries a cytomegalovirus promoter-driven EGFP gene, was used as the control vector. The viruses were amplified in 293 cells, and viral titer was determined by plaque assay.

Cultured Cardiomyocyte Adenoviral Gene Transfer and ¹²³I Treatment

The cellular effects of adenovirus-mediated NIS gene expression and ¹²³I incorporation were evaluated in H9C2 cells that were differentiated into cardiomyocytes. H9C2 cells, myoblasts derived from rat embryonic cardiocytes, were obtained from the American Type Culture Collection and maintained in Dulbecco's modified Eagle medium supplemented with 10% fetal calf serum (FCS) under standard 37°C, 5% CO₂ in air conditions. Cells split at a low density in 12- or 24-well dishes were cultured in 10% FCS culture medium for 1 d and then differentiated into cardiomyocytes by methods described by Ménard et al. (20). Briefly, cells were cultured in medium containing 1% FCS with replacement of culture medium every 2 d. Stimulation was performed daily for 7 d with 10 nmol/L all-trans-retinoic acid (Sigma), using aliquots that were used only once after storage at −20°C and were freshly diluted at the time of experiments.

Cardiomyocyte differentiation was verified by immunofluorescent staining with a polyclonal antibody that specifically recognizes the cardiac marker myosin light chain 2v (MLC-2v) (C-17; Santa Cruz Biotechnology, Inc.). Cells rinsed with phosphate-buffered saline (PBS) were fixed with 4% paraformaldehyde for 30 min, permeabilized with 0.03% Triton X-100 (Sigma) for 5 min, and then washed with PBS 3 times. Blocking of nonspecific sites was performed by incubating cells with 3% bovine serum albumin at 37°C for 30 min. Cells were incubated for 1 h at 37°C with the primary antibody (1:100 dilution) in 3% bovine serum albumin solution. Cells were then washed with PBS and incubated for 1 h at 37°C with fluorescein isothiocyanate–conjugated donkey antigoat secondary antibody (1:400 dilution; Santa Cruz Biotechnology, Inc.), and finally inspected under a fluorescent microscope.

Cardiomyocyte differentiated cells were infected with 10 multiplicities of infection (MOIs) of Ad.EGFP or Ad.EGFP.NIS, whereas control cells were not infected. At 4 and 9 d after adenoviral infection, cells were treated with 0 or 185 kBq (0 or 0.5 μCi) of ¹²³I for 6 h. Cells then underwent evaluation for viability and proliferation using direct cell counting in conjunction with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays. Counting the number of cells was achieved by mixing equal volumes of trypan blue (Sigma) to trypsin-ethylenediaminetetraacetic acid–harvested cells followed by direct cell counting using a hemacytometer. For MTT assays, 50 μL of 2 mg/mL MTT stock solution (Sigma) were added to the culture medium. After incubation at 37°C for 4 h, the culture medium was aspirated and 200 μL of dimethyl sulfoxide (Sigma) were added to dissolve MTT-formazan crystals. One hundred microliters of solution from each well were transferred to a 96-well microplate and measured for absorbance at 570 nm.

Rat Intramyocardial Adenovirus Injection and Ischemia–Reperfusion Injury

Sprague–Dawley rats (300–350 g) were studied under protocols approved by the Institutional Guidelines on the Use and Care of Animals. Rats anesthetized with ketamine (80 mg/kg) and xylazine (10 mg/kg) were mechanically ventilated with room air, and sternotomy was performed to expose the beating hearts. The anterolateral wall of the left ventricle (LV) was injected with 3 × 10⁸ plaque-forming units (pfu) of either Ad.EGFP.NIS (n = 9) or Ad.EGFP (n = 8) in 100-μL volumes using a 30-gauge needle. All 17 rats underwent serial echocardiography after intramyocardial adenovirus administration, and all 9 rats of the Ad.EGFP.NIS group were also subjected to intravenous administration of 74 MBq of ¹²³I on days 2, 4, and 9. Separate rats underwent measurements of serum creatine kinase (CK) levels serially after Ad.EGFP.NIS (n = 3) or Ad.EGFP (n = 3) injection. After these animals were sacrificed, myocardial tissue was subjected to myocardial myeloperoxidase (MPO) assays and histologic assessment of inflammatory response.

Four rats underwent myocardial ischemia–reperfusion injury without adenoviral gene transfer as positive controls of myocardial injury. Briefly, rats anesthetized with xylaine and ketamine were mechanically ventilated with room air, and sternotomy was performed to expose the beating hearts. Myocardial ischemia was induced by ligation of the left anterior descending coronary artery for 40 min with a 6.0 silk suture approximately 3–4 mm from its origin. The chest wall was then closed, and the rats were sacrificed 3 d later.

Serial Scintigraphic Imaging with ¹²³I After Gene Transfer

Five rats of the Ad.EGFP.NIS group underwent scintigraphic imaging at 2, 4, and 9 d after myocardial gene transfer. Anesthetized animals were injected in a tail vein with 74 MBq of Na¹²³I (Korean Atomic Energy Research Center, Seoul, Korea), and a 20-min image was acquired 40 min later using a conventional γ-camera (Monad XLT; Trionix Research Laboratory) with a 20% energy window centered at the energy peak and a 256 × 256 pixel matrix. A lead pinhole collimator with a 5-mm aperture and a focal length of 18 cm was used, and the distance from the pinhole to the rats was approximately 6 cm.

To semiquantitate the level of myocardial ¹²³I uptake from the images, 5 × 5 pixel-sized rectangular regions of interest were placed on the myocardial region showing increased uptake and on the upper mediastinal region (as background activity), from which ratios of cardiac to mediastinal counts (C/M ratios) were calculated.

Echocardiographic Assessment of Cardiac Dimensions and Function

Animals of Ad.EGFP.NIS (n = 9) and Ad.EGFP (n = 8) groups underwent serial echocardiography at baseline (day –1) and on day 4 and day 9 after myocardial gene transfer. After animals were anesthesized, anterior chest hair was removed with clippers, and the rats were secured to a table. Transthoracic echocardiography was performed with a 13-MHz linear transducer (model 15L8 probe; Acuson), and parameters were obtained using current ultrasound technology following the American Society of Echocardiography guidelines (21). Briefly, M-mode and 2-dimensional echocardiographic images were recorded in the parasternal long- and short-axis views, and thickness of the intraventricular septum and the posterior wall and LV dimensions were determined at the tips of the papillary muscle. LV end-systolic and end-diastolic areas were traced in single-plane apical 4-chamber view, and the ejection fraction was calculated following Simpson's rule using the modified single-plane method. LV mass was calculated with a regression formula using wall thickness and LV dimension values as used previously for adult rats (22).

Total Serum CK Assays

Separate Ad.EGFP.NIS-injected (n = 3) and Ad.EGFP-injected (n = 3) rats were evaluated for evidence of myocardial injury as assessed by CK and MPO activity and histologic findings. These animals underwent serial assays for serum CK activity at 3, 7, and 14 d after myocardial gene transfer. Rats that did not undergo any intervention (n = 3) were negative controls, and rats at 3 d after myocardial ischemia–reperfusion injury (n = 4) were positive controls. Serum was obtained from 100 μL of blood sampled from the tail vein, and quantitative determination of total CK activity was performed with the N-acetyl cysteine–activated assay method on an automated clinical chemistry analyzer (Roche Diagnostics).

MPO Assays and Histologic Assessment of Inflammatory Response

Rat myocardium extracted 14 d after Ad.EGFP.NIS or Ad.EGFP injection was snap-frozen (n = 3 for each group). Cryosections of 10-μm thickness were obtained, fixed in 0.5% glutaraldehyde, and then washed with PBS. The sections were stained with hematoxylin, and a single pathologist who was unaware of the study groups inspected the sections under a light microscope to assess the magnitude of inflammatory reaction. Sections were visually graded for the extent of inflammatory reaction as localized or diffuse and for the level of inflammatory cell infiltration as mild, moderate, or severe.

The remaining myocardial tissue was homogenized and assayed for MPO activity as an index of inflammatory injury following a previously described method (23). Briefly, weighed frozen myocardial tissue was mixed with 5 mL of ice-cold 5 mmol/L phosphate buffer (pH 6), freeze/thawed 3 times, sonicated, and then homogenized in 0.5% hexadecyltrimethyl ammonium bromide (Sigma) in 50 mmol/L phosphate buffer (pH 6). After centrifugation at 12,500g at 4°C for 15 min, the supernatants were collected and reacted with 0.167 mg/mL of o-dianisidine dihydrochloride (Sigma) and 0.0005% H₂O₂ (Sigma) in 50 mmol/L potassium phosphate buffer (pH 6). The change in absorbance at 460 nm at 25°C was monitored with a spectrophotometer for several minutes. Results are expressed in units of MPO per gram of wet weight, which is defined as the enzymatic activity that degrades 1 μmol of H₂O₂ per minute at 25°C. MPO activities in myocardial tissue of negative control rats and rats 3 d after myocardial ischemia–reperfusion were also assayed for comparison.

Data Analysis

The significance of difference in cell number, MTT assay results, cardiac radiouptake ratio, echocadiographic measurements, serum CK level, and MPO activity was analyzed by Student t tests. Data are given as mean ± SD or mean ± SE as indicated, and P values < 0.05 were considered significant.

Effect of Adenoviral Gene Transfer and ¹²³I Incorporation on Cultured Cardiomyocytes

H9C2 cells stimulated with retinoic acid for 7 d exhibited positive staining for MLC-2v, consistent with cardiomyocyte differentiation of the cells. Infection of these cells with 10 MOIs of either Ad.EGFP or Ad.EGFP.NIS led to high transgene expression levels, as assessed by fluorescence intensities observed on day 2 (data not shown). There was no difference in viable cell numbers at 4 and 9 d between Ad.EGFP-infected, Ad.EGFP.NIS-infected, and uninfected control cells (with or without ¹²³I treatment) (Fig. 1A). Similarly, there was no difference in MTT assay results at days 4 and 9 after Ad.EGFP or Ad.EGFP.NIS application, either with or without ¹²³I treatment, compared with controls (Fig. 1B). These results indicate that, under our experiment conditions, neither NIS overexpression nor incorporation of ¹²³I causes significant effects on cultured cardiac cell viability or proliferation.

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FIGURE 1. 

Effect of adenoviral gene transfer and ¹²³I treatment on cultured cardiac cells. (A) Number of viable cells at 4 and 9 d after infection with 10 MOIs of Ad.EGFP or Ad.EGFP.NIS and in uninfected controls. Cells were incubated with 0 or 185 kBq (0 or 0.5 μCi) ¹²³I for 6 h at days 4 and 9. (B) MTT assay results of cells treated as in A. Cells are H9C2 myoblasts differentiated into cardiomyocytes by stimulation with retinoic acid. All results are mean ± SD of 4 samples obtained from a single experiment (representative of 2 experiments).

Serial ¹²³I Scintigraphy and Time Course of Cardiac Radiouptake

At 2 d after gene transfer, all 5 rats among the Ad.EGFP.NIS group that underwent ¹²³I scintigraphy demonstrated intense focal radiouptake localized to the virus-injected region of the heart. Scintigraphy repeated at 4 d also showed high cardiac radioactivity of comparable intensity. In contrast, the same animals imaged at 9 d revealed a complete loss of cardiac radiouptake (Fig. 2A). Semiquantitative measurements of image-based C/M ratios closely corresponded to the visual findings: The ratio was high on days 2 and 4 (5.04% ± 0.62% vs. 5.09% ± 0.99%, respectively; P = not significant) but was dramatically reduced to blood-pool levels by day 9 (1.38% ± 0.05%; P < 0.0001 compared with 2- and 4-d levels; Fig. 2B). These findings indicate that NIS protein is present in high levels in the heart between 2 and 4 d after direct NIS adenovirus injection but declines to levels below the threshold for detection with ¹²³I imaging by day 9.

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FIGURE 2. 

Time course of cardiac ¹²³I uptake. (A) Serial scintigraphic rat images at 2, 4, and 9 d after myocardial injection with 3 × 10⁸ pfu of Ad.EGFP.NIS (n = 5). Arrow denotes focal cardiac ¹²³I uptake consistent with site of adenovirus injection. (B) Semiquantitative C/M ratios expressed as mean ± SE of values obtained from 5 animals.

Serial Echocardiographic Measurements of Cardiac Function and Dimension

The results of serial transthoracic echocardiography measurements before and after myocardial adenoviral gene transfer are summarized in Table 1. At baseline, Ad.EGFP and Ad.EGFP.NIS group rats had similar heart rates, LV dimensions, and cardiac functional parameters, which are also consistent with previously reported values observed for normal Sprague–Dawley rats (21). Follow-up echocardiography demonstrated that, even after adenovirus injection, the Ad.EGFP and Ad.EGFP.NIS groups did not differ in heart rate, LV dimensions, and functional parameters at days 4 and 9. When postinjection parameters were compared with baseline levels, LV dimensions were increased slightly, and LVEF and LV fractional shortening (LVFS) were reduced slightly at day 9 for both Ad.EGFP and Ad.EGFP.NIS groups, but to similar magnitudes between the 2 groups. No arrhythmia or any other significant ECG abnormalities were observed during serial echocardiography in either AdV.EGFP.NIS or AdV.EGFP group.

Serum CK Activity and Myocardial MPO Levels and Inflammatory Cell Infiltration

Neither Ad.EGFP group nor Ad.EGFP.NIS group demonstrated a significant increase in serum total CK activity at 3, 7, and 14 d after myocardial transfer compared with negative controls. In contrast, positive controls that had undergone myocardial ischemia–reperfusion injury had serum CK levels elevated to 7-fold of negative controls (Fig. 3A). Similarly, myocardial tissue MPO activity 14 d after injection of Ad.EGFP and Ad.EGFP.NIS was not elevated compared with negative controls, whereas it increased significantly 3 d after myocardial ischemia–reperfusion injury (Fig. 3B).

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FIGURE 3. 

Serum CK and myocardial MPO activity. (A) Total serum CK activity (mean ± SE; IU/L) at 3 (D3), 7 (D7), and 14 (D14) d after myocardial injection of Ad.EGFP.NIS (n = 3) or Ad.EGFP (n = 3), in negative controls (n = 3), and at 3 d of ischemia–reperfusion (n = 4). (B) Myocardial MPO activity (mean ± SE; U/g) in rats as in A (14 d after adenovirus injection). NS = not significant.

Microscopic inspection of myocardial microsections revealed comparable mild inflammatory cell infiltration restricted to relatively small areas, consistent with the injection site for both Ad.EGFP and Ad.EGFP.NIS groups (Figs. 4A–4D).

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FIGURE 4. 

Hematoxylin-stained myocardial microsections obtained 14 d after intramyocardial injection of Ad.EGFP.NIS (A and B) or Ad.EGFP (C and D). Arrows indicate areas of localized inflammatory infiltration, consistent with injection site. Length of bar indicates 400 μm for A and C and 100 μm for B and D.