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Toronto General Research Institute, Division of Cardiovascular Surgery, Department of Surgery, Toronto General Hospital, University of Toronto, Toronto, Canada
Submitted 23 August 2004 ; accepted in final form 27 January 2005
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ABSTRACT |
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 TOP ABSTRACT METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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myocardial infarction; ventricular modulation; gene therapy; cell transplantation
The myocardium has few elastic fibers. Physiologically, elasticity of the myocardium is due to cardiomyocyte muscle bundles. After a myocardial infarction, necrosed myocardium is replaced by fibroblasts and predominantly type III collagen. No elastic fibers are present in the scar tissue. The infarcted regional elasticity depends on the ratio of fibrous to muscular tissue and the density of the collagen cross-links (2, 12). Because matured myocardial scar tissue is mainly composed of nonelastic type III collagen fibers and few cells, increased left ventricular (LV) chamber stiffness results in diastolic dysfunction (4, 14, 16). Because the infarct region is weak and the intraventricular pressure stresses the infarcted area with each contraction, the scar expands and the LV chamber dilates over time (9, 15). Stiffness and enlargement of the scar and LV decrease cardiac function (18). Our hypothesis is that alteration of scar composition by the introduction of elastin should increase the compliance and elasticity of the infarcted region and preserve scar size, ventricular chamber size, and cardiac function. Because the life span of the elastic fibers is in terms of years, cardiac function could also be stabilized for years.
In the present study we transfected a fragment of the elastin gene into a cell line that was implanted into a myocardial infarct scar before scar expansion occurred. Although the implanted COS-7 cells alone did not affect scar size and cardiac function, the presence of elastin fragment expressed by genetically modified COS-7 cells implanted in the scar limited scar expansion, ventricular dilation, and onset of heart dysfunction.
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METHODS |
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Preparation and transfection of rat tropoelastin gene. With total RNA isolated from rat aorta, cDNAs of a fragment of rat tropoelastin gene were synthesized by the RT-PCR technique with primers (forward primer: AAA AAA CTC GAG ATC CCA GGT GTT GGG, reverse primer: AAA AAA TCT AGA TCC AAG ATC ACC AGG). The cDNA was confirmed by sequence, tagged with NH2-terminal chicken cartilage link protein 60 (LP60) protein, and then ligated with a plasmid vector (pcDNA3.0, Invitrogen). The plasmids were amplified with Escherichia coli, and purified plasmids were used for the studies.
COS-7 cells (American Type Culture Collection) were cultured for 24 h in culture medium [Iscove's modified Dulbecco's medium (IMDM) including 10% fetal bovine serum] before transfection. The cells were washed with phosphate-buffered saline, and 10 ml of serum-free IMDM was added to the flask. The plasmid containing elastin gene (50 µg) or plasmid vector and 20 µl of Lipofectamine 2000 (Invitrogen Life Technologies) were suspended in 1 ml of serum-free IMDM. The COS-7 cells were incubated with this mixture for 6 h. The cells were then washed and cultured for 48 h before cell transplantation.
Assessment of recombinant elastin expression. To assess the expression of the recombinant elastin, 2 x 106 cells were collected, lysed, and centrifuged. The supernatants were used for the assays. The culture medium was also collected and centrifuged, and the supernatants were used for the assays. Each sample was denatured in SDS solution, and 30 µg of protein was electrophoresed in stacking and separating gels containing 4 and 10% polyacrylamide, respectively. The gels were blotted onto nitrocellulose membranes. After the membranes were blocked, the membranes were immersed with the primary antibody against NH2-terminal LP60 protein (a gift from Dr. B. B. Yang, Sunnybrook Hospital, Toronto, ON, Canada) at 4°C overnight. After being washed, the membrane was incubated with the secondary antibody (goat anti-mouse IgG antibody) at room temperature for 1 h, followed by another 1-h wash. The membranes were then exposed to ECL reagents (Amersham) and X-ray films, followed by film development.
Stretch test. The physical characteristics of the recombinant elastin in a gelatin mesh (Gelfoam, Pharmacia & Upjohn, Kalamazoo, MI) were measured by stretch testing with the Instron 8501 Dynamic Biomechanical Testing System.
CELL SEEDING.
The gelatin Gelfoam mesh (30 x 30 x 2 mm) was incubated in culture medium for 2 days before cell seeding. The meshes were then attached to Bio-Stretch cell culture dishes, which were rectangular and allowed for fixation of the mesh by clipping to the inner aspect of the dish margin. The opposite free margin of each mesh was clipped to a cylindrical coated steel bar. COS-7 cells were transfected with the elastin gene (Elastin group) or the plasmid vector (Vector group) as described in Preparation and transfection of rat tropoelastin gene. The transfected cells in suspension were seeded onto a Gelfoam mesh (5 x 106 cells/mesh). After 2 days of incubation, the seeded meshes (n = 5) were subjected to cyclical mechanical stretch.
MECHANICAL STRETCH.
Mechanical stretch was applied to the cell-seeded Gelfoam mesh with the Bio-Stretch apparatus (1). The Bio-Stretch culture dish is placed in front of a magnet, and the movement of the steel bar is controlled by dynamically changing the magnetic field on the controller. The Bio-Stretch apparatus was programmed to deliver a 70% stretch length at a frequency of 90 cycles/min for 2 days, followed by a uniaxial tension test as described previously (1).
ANALYSIS OF TENSILE STRENGTH.
Uniaxial tension testing was performed with the Instron 8501 Dynamic Biomechanical Testing System (1). Gelfoam meshes were attached on opposite ends to the test apparatus. One arm of the test apparatus progressively stretched (0.5 mm/min) the mesh until failure (complete tear). The passive tensile strength of the patch was continuously recorded during the displacement. The data were plotted as the displacement in millimeters and the force in newtons. The tissue elasticity was determined as the area under the curve, the tissue resistance to stretch as the slope of the curve, and the maximum tensile strength as the peak force (1).
Histological study. After mechanical testing the Gelfoam meshes were rinsed three times in PBS and fixed in 10% phosphate-buffered formalin solution for 5 days. The meshes were embedded in paraffin and sectioned at a 10-µm thickness. The sections were stained with hematoxylin and eosin and trichrome staining as described by the manufacturers.
In Vivo Study
All experimental procedures were approved by the Animal Care Committee of the University Health Network and conformed to the guidelines in the Guide for Care and Use of Laboratory Animals published by the National Institutes of Health (NIH Publication No. 85-23, Revised 1985). The rats used in the experiment were male Lewis rats (Charles River, Canada).
Myocardial infarction. Under general anesthesia (20 mg/kg ketamine, 22 mg/kg pentobarbital, and inhalation of isoflurane 1–2%), rats were ventilated and the left anterior descending artery (LAD) was ligated via left thoracotomy. The rats received antibiotics (Duplocillin LA, 150,000 IU/kg; Intervet Canada) intramuscularly every 3 days for a week after surgery and buprenorphine (0.01–0.05 mg/kg) subcutaneously after the surgery and later as required. To minimize variation of infarct size and cardiac function, echocardiography was performed 5 days after LAD ligation, and the animals were selected according to the following criteria: infarct length >0.7 cm and <1.1 cm and percent fractional area shortening (%FAS) >20% and <40%.
Cell transplantation. Six days after LAD ligation, the heart was exposed via median sternotomy. To estimate the effects of COS-7 cells alone on cardiac function, 2 x 106 COS-7 cells (Cell group, n = 7) were injected into scar tissue with an insulin syringe and a 28-gauge needle. LAD ligation alone was performed as a control (Control group, n = 7). Cyclosporine (5 mg·kg–1·day–1) was injected subcutaneously into the rats in both groups daily during the study. Cardiac function was evaluated by echocardiography before and 8 wk after cell transplantation and by Langendorff study at 8 wk after cell transplantation as described in Measurement of cardiac function. After the functional study, the hearts were arrested with saline containing 20 meq/l potassium chloride and fixed with formalin at a pressure of 30 mmHg. The fixed hearts were cut into 3-mm-thick slices, and the scar size was measured by planimetry as described previously (17).
To estimate the in vivo function of the recombinant elastin, 30 rats were selected and randomly divided into two groups at 6 days after LAD ligation. COS-7 cells (2 x 106) transfected with the elastin gene (Elastin group) or the plasmid vector (Vector group) were transplanted into the infarcted area. Cyclosporine (5 mg·kg–1·day–1) was injected subcutaneously daily during the study. Two rats in each group were killed at 2 days and 2 and 5 wk after cell transplantation, and the scar was divided into two pieces. One half was fixed with 10% formalin for histology, and the other half was immediately frozen and stored at –80°C for assessment of elastin expression. The remaining nine rats in each group were used for functional assessment. Echocardiography was performed at 2 days and 2, 5, and 8 wk after cell transplantation, and the Langendorff study was performed at 8 wk. After the functional study, the scar was divided into two pieces for histology and assessment of the recombinant elastin. The frozen samples were homogenized, lysed, and centrifuged, and the supernatant was used for assessment of the gene expression as described in Assessment of recombinant elastin expression.
Measurement of cardiac function. Cardiac function was monitored with echocardiography (Sequoia Echocardiography System, ACUSON). The rats were anesthetized and maintained by 1.5–2.0% of inhaled isoflurane. The heart rate was kept higher than 300 beats/min. The short-axis views were recorded at the site where the maximal infarct area could be seen. The infarct length was determined as the maximum length of the akinetic wall at the end-diastolic phase. The area of the LV at the end-diastolic phase and the end-systolic phase was measured, and %FAS was calculated as [(LV end-diastolic area) – (LV end-systolic area)]/(LV end-diastolic area). The parameters were measured at 6 beats and averaged.
The cardiac function was also assessed 8 wk after cell transplantation with a Langendorff apparatus as we described previously (8). In brief, under general anesthesia, the heart was quickly excised and perfused in a Langendorff apparatus with filtered Krebs-Henseleit buffer equilibrated with 5% carbon dioxide and 95% oxygen. A latex balloon was inserted into the LV. After 20 min of stabilization, balloon volume was increased in 0.02-ml increments by addition of saline. LV systolic and end-diastolic pressures and maximum and minimum change of pressure with time (dP/dtmax, dP/dtmin) were recorded at each balloon volume. Developed pressure (DP) was calculated as the difference between the systolic and diastolic pressures.
Histological assessment. The fixed scar tissue were embedded in paraffin and cut into 10-µm-thick slices. The slices were stained with hematoxylin and eosin staining and elastica-van Gieson staining. To assess immunorejection, the tissues were also immunostained with antibody against CD4.
Data Analysis
All results are presented as means ± SE. Comparison in the stretch test was performed by Student's t-test. Comparisons of ventricular functional data over the 8 wk by echocardiography were performed by two-way repeated-measures ANOVA. The ventricular functional data from the Langendorff preparation were analyzed with SPSS system software. A value of P < 0.05 was considered statistically significant.
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RESULTS |
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The transfection efficiency of the cultured COS-7 cells was >90%. Newly synthesized recombinant elastin molecules were observed in both the cell lysate and the culture medium in the Elastin group (Fig. 1, A and B). No elastin gene expression was detected in the Vector group.
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As measured by echocardiography, transplantation of COS-7 cells (Cell group, n = 7) into the infarct scar did not affect %FAS and infarct length (P = 0.85, 0.78) compared with the non-cell-transplanted control infarct scar (n = 7). No difference was detected in the Langendorff study between the two groups (DP: P = 0.95; dP/dtmax: P = 0.43). Planimetry data also showed no difference in scar size between the two groups (P = 0.69).
When the elastin gene- or vector-transfected COS-7 cells were implanted into infarcted regions, differences in cardiac function and ventricular morphology were observed by echocardiography between the Elastin (n = 9) and Vector (n = 9) groups. Before cell transplantation there were no statistical differences in infarct length (0.83 ± 0.11 cm vs. 0.82 ± 0.08 cm) and %FAS (31.9 ± 4.7% vs. 30.5 ± 4.4%) between the two groups. Over the 8-wk study, the infarct length in the Vector group doubled, which was greater (P < 0.05) than the 58% increase in the Elastin group (Fig. 4A). Cardiac function in the Vector group deteriorated (%FAS: 30.5 ± 4.4% to 12.9 ± 2.8%) more (P < 0.05) than in the Elastin group (%FAS: 31.9 ± 4.7% to 22.6 ± 4.5%) (Fig. 4B)
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DISCUSSION |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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Recently, cardiomyocytes, skeletal muscle cells, smooth muscle cells, and bone marrow cells have been implanted into infarcted myocardium and have improved cardiac function after infarction (1, 3, 17, 20). The mechanism of action of transplanted cells is unknown. Accumulated evidence has indicated that implanted cell contractility is not essential to improve global function because no evidence showed contractility of implanted cells and transplantation of nonbeating cells, such as skeletal muscle cells and smooth muscle cells, also prevented heart failure (3, 20). In addition, the survival rate of implanted cells is only 20–30% and continues to decline with time (10). Therefore, it is unlikely that the remodeling benefits of cell transplantation can be attributed to the number, type, or contractile properties of implanted cells alone. We hypothesized that the implanted cells increase the elastic properties of the infarct scar and make it more resistant to the stresses of ventricular systole.
To confirm that newly synthesized elastin fragments had elastic properties and added tensile strength we seeded elastin fragment gene-transfected COS-7 cells (fibroblasts) into a gelatin Gelfoam mesh. The cells grew throughout the mesh and expressed elastin fragments. In addition, the elastin-transfected cells also secreted collagen in the mesh. Both elastin and collagen were structurally organized in the direction of the stretching force on the mesh. The physical properties of the Gelfoam mesh showed that the elastin fragments and collagen increased the mesh's elasticity and the tensile strength required to break down the mesh compared with the properties of the Gelfoam mesh seeded with vector-transfected COS-7 cells. The elastin fragments and collagen did not affect the biomaterial's stiffness.
To alter matrix composition and change the properties of the infarcted tissue, we introduced a fragment of the elastin gene by cell transplantation into a recent myocardial infarct scar of an adult rat. The gene-transfected COS-7 cells expressed a 600-base pair fragment of elastin that accumulated in the extracellular matrix at the implant site. The elastin and likely collagen were expressed as early as 2 days after cell transplantation. The accumulated elastic fragments and collagen fibers were laid down in the infarcted zone for at least 5 wk. In contrast, few elastin fibers and no significant increase in collagen content were observed in the Vector group. Significant differences of the infarct size and heart function were observed. The scar and ventricular chamber sizes in the Elastin group were smaller, and cardiac function was better preserved than in the vector-transfected COS-7 cell-transplanted hearts. The decrease of LV compliance in the elastin-transfected hearts was consistent with the infarct having recoil and tensile properties that contributed to cardiac output. The beneficial effect seen in the Elastin group was also likely contributed to by overexpression of collagen and collagen fiber formation as seen in the in vitro studies. We speculate that expression of the elastin fragments stimulated the synthesis and secretion of collagen in vivo and that these molecules formed an organized extracellular matrix structure that inhibited the negative structural changes of ventricular remodeling. Although the elastic fragment content in the scar was almost absent by week 8, scar size was still smaller, LV dysfunction was less, and compliance was better than those of the Vector control group. The loss of the elastin fragments and possibly collagen in the elastin-transplanted hearts would be expected to lead to the development of ventricular dysfunction similar to that found in the vector control hearts.
There are a number of possible mechanisms by which elastin fragments and likely synthesized collagen in a myocardial scar tissue could delay the onset of ventricular dysfunction. A more elastic, stronger scar could function as a shock absorber to cushion the stress and the tearing effect of the acute increase in intraventricular pressure on the scar during systole. The recoil of the elastic fragments in the scar could provide passive energy to return the scar size and ventricular chamber volume to precontractile dimensions. The matrix modulation caused by newly synthesized elastin and collagen should inhibit scar expansion and ventricular dilation. Alteration of myocardial matrix by overexpression of elastin and collagen fiber could affect apoptosis and decrease cardiomyocyte loss through an extracellular matrix modulation mechanism after myocardial infarction. However, more research is needed to define the beneficial changes in the infarcted heart.
Elastin fibers consist of a tropoelastin as a core and a cross-link with microfibrillin that provides elasticity to tissue (7, 11, 13, 19). Although it would have been better to have expressed the full length of the elastin molecule in the myocardial scar tissue, the rat elastin cDNA, 2595 base pairs long, was thought to be too challenging for us to clone and express. Because a fragment of elastin has been reported to have some properties of the elastin molecule in term of extensibility and resilience (5), we elected to clone a 600-base pair segment of rat tropoelastin. The fragment was located at the site between 1201 and 1800 base pairs. To identify the elastin fragment synthesized, the 600-base pair fragment was tagged with LP60. The protein synthesized by the plasmid was a fusion protein of an elastin fragment and a 60-amino acid chicken protein.
We used COS-7 cells as a carrier of elastin gene in the current study for the following reasons. 1) Transplantation of COS-7 cells alone into infarcted myocardial scar tissue did not limit scar expansion and did not improve cardiac function. Therefore, the COS-7 cell line provided an opportunity to investigate the functional effectiveness of an overexpressed elastin fragment in an infarct. 2) Nonviral transfection efficiency of COS-7 cells is >90%, and the cells actively synthesized and secreted the elastin fragment, which cannot be achieved with primary cultures of rat cells. The disadvantage of using COS-7 cells, which originated from the African green monkey kidney, is immunorejection of the xenogeneic cells. Immunorejection would kill the COS-7 cells and decrease the rate of synthesis of the elastin fragments. The fusion protein would have also been recognized as foreign and would probably cause immunorejection. The decrease of elastin synthesis from loss of the transplanted transfected COS-7 cells and the accelerated fusion protein degradation from the immunorejection could explain why the amounts of elastin fragments were decreased at 5 and 8 wk after transplantation. Future research is planned to evaluate an in vivo untagged elastin fragment dose-cardiac function response curve after genetically modified autologous cells are transplanted into myocardial scar tissue. In vitro measurements of the infarct scar's elastic and strength properties will be performed. The loss of elastin fragments from immunorejection will be minimal. In addition, the effect of expressed elastin on changes in collagen content, organization, and structure in the implanted area as well as in the noninfarcted myocardium will be evaluated.
In summary, early elastin fragment expression and likely collagen synthesis in an infarct scar altered the myocardial modulation process, prevented scar expansion, and delayed the onset of heart dysfunction. The results suggest that alteration of the extracellular matrix and an increase in the elastin and collagen content in a myocardial infarct can benefit myocardial contractile function.
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GRANTS |
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FOOTNOTES |
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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.
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