In Vivo Gene Transfection of Human Endothelial Cell Nitric Oxide Synthase in Cardiomyocytes Causes Apoptosis-Like Cell Death
Identification Using Sendai Virus–Coated Liposomes
Background Nitric oxide (NO) has various actions on the cardiovascular system, although its pathophysiological significance in myocardial cells remains obscure. The aim of the present study was to identify direct NO actions on cardiomyocytes by gene transfection in vivo using a newly developed vector under physiological conditions.
Methods and Results Liposomes containing the β-galactosidase (β-gal) gene alone or with the human endothelial cell nitric oxide synthase (ecNOS) gene were coated with UV-inactivated Sendai virus and injected into the left ventricular wall of rat heart in vivo. Histological examination confirmed that the transfection efficiency was comparable to adenovirus-mediated transfection and that the new vector per se caused no inflammation. β-Gal expression was confined to cardiomyocytes between two intercalated discs, suggesting that the transfected gene did not permeate the discs. An immunohistochemical study showed that cotransfection of the ecNOS gene induced massive myocardial cell shrinkage in both transfected cells and the adjacent myocytes in a time- and dose-dependent manner. Histochemical findings in shrunk cells coincided with apoptosis as identified by terminal deoxynucleotidyl transferase–mediated dUTP-biotin nick-end labeling. Electron microscopy of the lesion revealed myofibrillar degradation and accumulation of mitochondria but no apoptotic bodies. Pretreatment with the NOS inhibitor Nω-nitro-l-arginine methyl ester abolished these morphological alterations.
Conclusions The efficient expression of the human ecNOS gene in vivo suggests that NO or its toxic metabolite caused myocardial degradation, a part of which was compatible with apoptosis of the transfected cardiomyocytes themselves and the adjacent cells as a paracrine effect. These morphological features mimicked acute myocarditis or ischemic injury.
Nitric oxide has a variety of actions on the cardiovascular system: vessel relaxation, inhibition of the proliferation of smooth muscle cells and endothelial cells, and suppression of platelet adhesion.1 2 The recent report of Balligand et al3 that ecNOS protein is constitutively expressed not only in coronary endothelial cells but in myocardial cells suggests autocrine and paracrine effects on cardiomyocytes, as is the case in vascular cells.4 5 Compared with NO released from activated macrophages, which might contribute to the pathogenesis of viral or bacterial myocarditis or septic shock,1 2 the pathophysiological significance of NO produced from myocardial cells is still obscure.
In general, overexpression or knockout of a specific gene in myocardial tissue is of great use for the accurate elucidation of the actual function of its corresponding product. In a physiological setting, transmural pressure, passive stretching during diastole, and local coronary flow in the presence of several cytokines, which might modify gene expression, are preserved. Accordingly, local modulation of gene expression would be very significant for evaluation of the gene product.
In this study, the human ecNOS gene was successfully transfected by using Sendai virus–coated liposomes. This vector caused less inflammation than the adenovirus-mediated methods previously reported.6 7 Massive necrosis of myocardial cells, a part of which satisfied the criteria of apoptosis, is also described.
Plasmid Construction and Design of Vectors
The β-gal gene with a CMV promoter at an Xba I site and SV40 poly A signal at a BamHI site was inserted into a pBluescript II KS+ plasmid (Stratagene). A 4.1-kb Xba I fragment of human ecNOS cDNA8 was inserted into a plasmid containing the CMV promoter with SV40 poly A signal (pcDNA 3, InVitrogen) at an EcoRI site.
Sendai virus (hemagglutinating virus of Japan)–coated liposomes were prepared.9 10 Nuclear protein (high-mobility group 1, 65 μg) and histochemical marker (β-gal) gene alone (200 μg) or with the ecNOS gene (200 μg) were mixed and incubated for 1 hour at room temperature. The liposomes were then vortexed and sonicated with a lipid mixture (phosphatidylcholine, cholesterol, and phosphatidylserine) and incubated with UV-inactivated Sendai virus for 1 hour at 37°C.
Gene Transfer In Vivo
After anesthetizing 300-g male Wistar rats (n=28) with pentobarbital sodium (25 mg/kg IP), the Sendai virus–coated liposomes were percutaneously injected into the left ventricular wall under the guidance of two-dimensional echocardiography. To examine the dose dependency of ecNOS gene transfection on histology, a high (50 μg) or low (5 μg) dose of ecNOS gene was administered.
NOS Inhibition by L-NAME and Its Verification
L-NAME was administered to rats in drinking water (12 mg·kg body wt−1·d−1, 100 mg/L) for 7 days before the transfection11 to clarify the effects of L-NAME on NOS inhibition. After transfection, L-NAME treatment was continued in both the control (β-gal transfection alone) and NOS gene (NOS plus β-gal transfection) groups for an additional 7 days. NOS inhibition was physiologically verified by measuring systemic pressure in the tail artery by sphygmomanometry and histologically examined by inspecting the medial thickening of coronary arteries.12
Light and Electron Microscopy
Rats were killed on days 3, 7, 10, and 14 (n=4 in each group) after the in vivo transfection. The hearts were excised, fixed in 2% paraformaldehyde in PBS for 4 hours at 4°C and sliced (2-mm thickness). After X-gal staining,13 the extent of β-gal expression was measured by obtaining the ratio of the X-gal–stained area to the cross-sectional area of the left ventricular wall. Paraffin-embedded 4-μm-thick sections were then prepared for light microscopic assessment.
Other tissues fixed in PBS containing 2.5% glutaraldehyde and 2% paraformaldehyde for 12 hours at 4°C were postfixed in 2% osmium tetroxide for 2 hours at 4°C, embedded in epoxy resin 812, sectioned to 60-nm thickness, and stained with uranyl acetate for 15 minutes and lead citrate for 20 seconds at room temperature. Electron microscopy was performed with a Hitachi H 7000 electron microscope.
Immunohistochemical Assessment of Gene Expression and Macrophage Infiltration
Cryostat sections (6 μm thick) of the hearts removed on day 7 after the transfection were fixed with 2% paraformaldehyde in PBS for 10 minutes. Endogenous peroxidase activity was quenched with 2% hydrogen peroxide in 60% methyl alcohol for 30 minutes at room temperature. The specimen was then permeabilized with 0.1% Triton X-100 in PBS for 20 minutes and incubated with either monoclonal antibody specific to ecNOS protein (5 μg/mL, Transduction Laboratories) or monoclonal antibody specific to rat macrophage (2 μg/mL, Serotec) overnight at 4°C. These sections were incubated with biotinylated rabbit anti-mouse IgG for 30 minutes at room temperature, and immunoproducts were visualized by using the avidin-biotin complex method (ABC kit, Vectastain). After rinsing, the slides were counterstained with Mayer’s hematoxylin solution and mounted for light microscopy.
Assessment of Apoptosis
DNA was extracted from the transfected heart, and electrophoresis was performed in agarose gel to detect the ladder.14 For the in situ detection of apoptosis, cryostat sections fixed in 10% paraformaldehyde solution for 10 minutes at room temperature were treated according to the instructions for the apoptosis detection kit (ApopTag Plus, Oncor), which is a modification of the TUNEL method.15
Briefly, after endogenous peroxidase was quenched with 2% hydrogen peroxide in PBS for 5 minutes at room temperature, specimens were incubated with terminal deoxynucleotidyl transferase enzyme in a humidified chamber for 1 hour at 37°C and then anti–digoxigenin peroxidase for 30 minutes at room temperature. They were then stained with diaminobenzidine substrate for 3 minutes at room temperature and counterstained in Mayer’s hematoxylin solution for 1 minute at room temperature.
For morphometry, pictures of five or six vision fields were taken at ×400 magnification; observation fields were restricted to the area where the β-gal expression was evident in X-gal staining. Values are expressed as mean±SE. The results were considered significant if P<.05. Statistical significance was estimated among the various groups by using one-way ANOVA. Group-to-group comparisons were conducted by using Student’s t test.
Efficiency of Gene Transfection by Sendai Virus–Coated Liposomes
The expression of β-gal was clearly detected in the cross section of the transfected hearts (Fig 1⇓). Enzyme activity was visualized as blue color in the left ventricular wall in all hearts transfected with the β-gal gene alone or cotransfected with ecNOS genes. β-Gal expression was detected 3 through 14 days after transfection (Fig 2⇓). It peaked on day 7, when the extent of β-gal expression reached 11.6±1.1% (n=4) in cross section (Fig 2⇓).
In contrast, expression after the administration of plasmid DNA alone without Sendai virus–coated liposomes attained only 1.1±0.1% (n=4, P=.0003 versus virus-coated liposome vector; Fig 2⇑). Cotransfection of the ecNOS gene did not affect the extent of β-gal expression (11.6±1.7%, n=4; Figs 1⇑ and 2⇑). However, when animals were treated with L-NAME to examine the effect of NOS inhibition, β-gal expression decreased even when Sendai virus–coated liposome vectors were used in the cotransfection with ecNOS (3.4±0.4%, n=4, P=.0022 versus without treatment; Fig 2⇑) or β-gal transfection alone (3.6±0.2%, n=4). It is unclear why β-gal expression decreased with L-NAME treatment. Changes in systemic or coronary pressure and circulation with L-NAME treatment may influence β-gal gene expression.
Histological and Physiological Findings of β-Gal Expression and ecNOS Transfection With or Without L-NAME Treatment
Microscopic examination revealed characteristic features of gene expression after transfection (Fig 3⇓). β-Gal activity was detected throughout the entire myoplasm of the transfected cells and was clearly distinguished by an intercalated disc from adjacent cells where β-gal was not expressed. Infiltration of white blood cells in the transfected portion was slight, and the inflammatory process due to vector administration was much less than for the adenovirus-mediated method (Fig 3A⇓).6 7
Furthermore, ecNOS gene transfection together with the β-gal gene demonstrated markedly different findings from the marker gene transfection alone (Fig 3B⇑ and 3C⇑). At the low dose (5 μg) of the ecNOS gene no or minimal lesions were documented (Fig 3B⇑), but at the high dose (50 μg) a clear border was delineated between the intact cardiac tissue and the injured area (Fig 3C⇑). The lesion corresponded to the transfected area of the ecNOS gene because β-gal staining was restricted to that lesion (Fig 3B⇑ and 3C⇑). The sizes of most cells in the transfected area were greatly reduced, and the myoplasm of the degenerated cells was much decreased (Fig 3C⇑).
Small cells in the lesion could be classified into four types according to the shape (elongated or round) and stain (blue or not stained). Elongated cells stained blue were scattered throughout the necrotic lesion (arrows in Fig 3⇑), while elongated, unstained cells were detected in the periphery (arrowheads in Fig 3B⇑ and 3C⇑). These elongated cells may have originated from denatured myocardial cells, while the round cells might be infiltrating macrophages, as identified later. For ecNOS gene transfection alone without the marker (β-gal) gene, the injured area had the same changes as observed for cotransfection (Fig 3D⇑).
The pathological alterations in the NOS gene group were markedly reduced with L-NAME treatment (Fig 3E⇑) because the number of shrunk cells decreased to the same level as the control. The cardiomyocytes transfected with β-gal preserved their original histological structure. These findings suggest that the pathological degeneration after ecNOS gene transfection was due to NO or its metabolites. The medial thickening of coronary arteries was not histologically obvious in the L-NAME–treated group, probably because the dose and period were smaller than in the previous report.12 Accordingly, these drastic alterations in the transfected lesion would be due to the ecNOS gene and not the β-gal gene.
L-NAME treatment confirmed the NOS inhibitory effect since the systolic pressures of rats increased from 117±3 to 158±3 mm Hg (n=4, P<.01) in the control group (β-gal transfection alone) and from 120±2 to 160±2 mm Hg (n=4, P<.01) in the NOS gene group (NOS plus β-gal transfection).
Characterization of the Transfected Lesion
Immunohistochemical staining with antibody specific to ecNOS demonstrated endogenous NOS protein on myocardial cells3 as well as endothelial cells in coronary arteries (Fig 4A⇓). In overtransfected myocardial cells, additional staining was visualized as thick staining. Cotransfection of the ecNOS and β-gal genes resulted in the mixed color of brown (immune complex with ecNOS protein) and blue (β-gal activity; arrows in Fig 4A⇓). Small elongated cells without blue staining (arrowheads in Fig 4A⇓) were also documented at the border between the degraded lesion and normal myocardial cells. Occasionally, either β-gal activity or ecNOS protein was clearly detected on normal-sized myocardial cells, and sarcomeres were clearly visible in these cells. This might be due to the selective transfection of one of the two genes in each myocardial cell.
The infiltration of a large number of macrophages was detected with specific antibody in the injured area, indicating that a portion of the round small cells found in the transfected lesion (Fig 4B⇑) represent invading leukocytes and macrophages.
Recent studies have indicated that apoptosis occurs with cell necrosis.16 End labeling of fragmented DNA with the TUNEL method demonstrated that 0.5% of the nuclei in small cells in the degenerated lesion were positive for the apoptotic reaction in situ, whereas none of the control myocardial cells were positive (Fig 4C⇑). All cells that were positively stained shrank in volume.
Azan staining revealed deposition of collagen fibers in the necrotic lesion (Fig 4D⇑), suggesting that the degenerative process after the ecNOS gene transfection was accompanied by fibrosis. The pathological findings of leukocyte invasion and fibrosis were similar to those of the inflammatory lesion in myocardial infarction at the subacute stage or myocarditis of viral or bacterial origin.
Electron Microscopic Assessment
Electron microscopy of the untransfected area showed no pathological deterioration, and myofibrillar array and mitochondrial structure were both well preserved (Fig 5A⇓). However, in the transfected area, mitochondrial accumulation and swelling were found between thin myofibrils (Fig 5B⇓). The mean numbers of these mitochondria in the transfected and untransfected areas, respectively, were 95±7 and 82±5 per 100 μm2 (P<.05). The mean diameter of these mitochondria in the transfected area was 1.16±0.04 μm, compared with 0.91±0.04 μm (P<.01) in the untransfected area. The mitochondria were round or elliptical in the untransfected area; some of the mitochondria between the sparse myofibrils in the transfected area were deformed and shaped like confetti.
In addition to these mitochondrial abnormalities, various changes within the myocytes were observed, including intracellular edema, depletion of glycogen granules, and sparse myofilament arrays (Fig 5C⇑). However, the integrity of the cytoplasmic membrane was preserved, with both apoptotic bodies and chromatin condensation being absent (Fig 5C⇑). As described in the light microscopic study (Fig 3A⇑), a clear contrast bordered by an intercalated disc was observed in two myocytes (Fig 5D⇑). In other words, most of the mitochondria were destroyed on one side of the intercalated disc.
We have made five major observations. First, in contrast to adenovirus vector,6 7 the newly developed Sendai virus–coated liposomes were very useful for the efficient transfection of gene in vivo with little associated inflammation. Second, both the reporter gene β-gal and the physiologically significant gene ecNOS were transfected to cardiomyocytes in living animals. Third, expression of the β-gal gene was limited to the cardiomyocytes between two intercalated discs, providing a clear contrast with adjacent cells. Fourth, overexpression of the ecNOS gene caused unique cell degeneration, including a reduction in myoplasm volume, mitochondrial accumulation, collagen deposition, and macrophage infiltration. Finally, these morphological alterations were ameliorated with L-NAME pretreatment.
Biological Characteristics of Sendai Virus–Coated Liposome Vector
To elucidate the specific function of an expressed gene, gene transfer is superior to transgenic animals for the following reasons. When the gene product is systemically expressed, it may obscure the local function. This is especially true when the gene product is essential for maintaining cell viability or the overexpressed product is lethal to the animal. Local expression or knockout of a specific gene is of great significance for identifying the physiological function of the gene product in situ. In this setting, the vector should not cause a secondary effect.
Adenovirus vector is not appropriate for this purpose because the vector itself causes local inflammation.6 7 Sendai virus–coated liposomes were vastly superior to adenovirus-mediated transfection because Sendai virus has no pathogenicity in either humans or rodents. UV-irradiation of Sendai virus is beneficial for preventing infection in other species, and the process does not weaken the transfection efficiency of the liposomes. Local administration of the newly developed vector in vivo induced no harmful action other than the needle puncture (Fig 3A⇑). Furthermore, expression of the β-gal gene was confined to myocytes between two intercalated discs, suggesting that the transfected gene did not permeate the discs. Comparison of transfected cardiomyocytes and nontransfected adjacent cells with electron microscopy (Fig 5D⇑) was particularly useful.
Morphological Alterations After ecNOS Gene Transfection and Relation to Apoptosis
The classic criteria for apoptosis17 18 19 include a reduction of cytoplasm volume, which was shown in most cells in the degraded lesion (Fig 3B⇑ and 3C⇑), detection of apoptotic reaction as identified by end labeling of fragmented DNA (Fig 4C⇑), and no disruption of the cytoplasmic or nuclear membranes of the degraded cells as assessed by electron microscopy (Fig 5C⇑). Other characteristics of apoptosis not observed in the present study include the presence of apoptotic bodies, chromatin condensation, and a DNA ladder after gel electrophoresis. Thus, the present data satisfied some but not all of the features of typical apoptosis.
Pathological examination also demonstrated an inflammatory process in the transfected myocardium. Double staining of β-gal activity and ecNOS protein (Fig 4A⇑) or macrophages (Fig 4B⇑) using specific antibodies, TUNEL analysis of apoptosis (Fig 4C⇑), and azan staining (Fig 4D⇑) revealed coexisting myocardial cell necrosis, apoptosis, and lymphoplasmacytic infiltration in the transfected myocardium. Pinsky et al20 have indicated that NO production in vitro from activated macrophages causes cytotoxic action on myocardial cells in tissue culture. Their results, together with the present findings, suggest that an inflammatory process does not exclude the possibility of apoptosis.
Pathological Significance of Injured Cells
Occasional myocytes located at the periphery of the transfected lesion showed degradation without expression of ecNOS protein (Fig 4A⇑). This finding may indicate that the cells transfected by the ecNOS gene and adjacent nontransfected cells were injured by NO or its secondary metabolites, such as peroxynitrite (ONOO−), in an autocrine or paracrine manner, respectively. This scenario is likely since NO is a diffusible gas and is actually excreted from NO-producing cells.5 20
A very small number of atrophic myocytes stained positively for the apoptotic reaction in cryostat sections in situ, whereas none of the control myocardial cells were positive (Fig 4C⇑). All cells that stained positively by the TUNEL method had decreased cell volumes. It should be emphasized that nucleus fragmentation, which is characteristic of apoptosis, was not detected at all. The heterogeneity of the apoptotic reaction among the atrophic cells suggests that a stage in the degradation process is required for apoptotic reaction; hence, the other denatured cells could not react to TUNEL staining.
The electron microscopy results revealed mitochondrial accumulation and swelling of the injured cells (Fig 5B⇑) compared with control cells in the untransfected portion of the same myocardial tissue (Fig 5A⇑). This indicates that mitochondria in a limited area were affected by ecNOS gene transfection and that the overexpression of ecNOS did not cause any morphological changes in myocytes distant from the transfected lesion. Impairment of oxidative phosphorylation and energy metabolism might lead to failure of the mitochondrial cation pump, and subsequently progressive swelling before mitochondrial rupture.21 The accumulation and swelling of mitochondria in degenerated lesions may be a defense mechanism against free radicals, including ONOO−, because mitochondria are one of the main sources of toxic free radicals.22 Hibbs et al23 report that activated macrophages cause inhibition of mitochondrial respiration.
In addition to the mitochondrial abnormalities, other changes within the myocytes were observed, including intracellular vacuole formation, depletion of glycogen granules, and sparse myofilament arrays. These findings suggest deterioration of the myofibrils and their subsequent absorption (Fig 5C⇑). Most damaged cells with mitochondrial abnormalities did not exhibit typical apoptotic bodies or dense chromatin, which are characteristic of apoptosis.19 Although these changes satisfied some of the oncosis criteria proposed by Majno and Joris,24 the present findings lacked blebbing and increased permeability of the cytoplasm membrane.
The findings of the present study suggest that overexpression of ecNOS by in vivo gene transfection probably produced NO or its toxic metabolites, which caused unique myocardial cell death. These cell injuries fulfilled some but not all of the criteria for apoptosis in the transfected cardiomyocytes themselves and in the adjacent cells as a paracrine effect. It would be attractive to assume that similar cell death may be involved in the progression of acute myocardial infarction, viral myocarditis, the development of cardiomyopathy, or the regression of cardiac hypertrophy.
Selected Abbreviations and Acronyms
|ecNOS||=||endothelial cell nitric oxide synthase|
|L-NAME||=||Nω-nitro-l-arginine methyl ester|
|NOS||=||nitric oxide synthase|
|TUNEL||=||terminal deoxynucleotidyl transferase–mediated dUTP-biotin nick-end labeling|
A portion of the present study was financially supported by the Ministry of Education, Science and Culture and the Ministry of Health and Welfare, Japan, the Research Foundation for Clinical Pharmacology and Molecular Cardiology, the Fugaku Trust Foundation for Medical Research, and the Mochida Research Foundation. The authors thank Dr James K. Liao, Division of Vascular Medicine, Brigham and Women’s Hospital, Boston, Mass, for kindly donating human ecNOS gene. The assistance of Dr Satoru Fukuda, Chief Director, Laboratory of Electron Microscopy, University of Tokyo, Mari Kataoka, and Yoshitaka Nakagawa is gratefully acknowledged.
Drs Kawaguchi and Shin contributed equally to the present study.
- Received September 25, 1996.
- Revision received December 11, 1996.
- Accepted December 11, 1996.
- Copyright © 1997 by American Heart Association
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