Increased Expression of Integrin-Linked Kinase Attenuates Left Ventricular Remodeling and Improves Cardiac Function After Myocardial Infarction
Background— Left ventricular (LV) remodeling is associated with the development of heart failure after myocardial infarction. Here we investigated whether integrin-linked kinase (ILK) may regulate LV remodeling and function after myocardial infarction.
Methods and Results— Adenoviral vector expressing ILK (n=25) or empty adeno-null (n=25) was injected into rat peri-infarct myocardium after left anterior descending coronary artery ligation. ILK expression was confirmed by Western blotting and immunofluorescence. Echocardiographic and hemodynamic analyses demonstrated relatively preserved cardiac function in adeno-ILK animals. ILK treatment was associated with reduced infarct scar size, increased scar thinning ratio, and preserved LV diameter, wall thickness, cardiomyocyte size, and myofilament density. Enhanced angiogenesis and reduced fibrosis were observed in the adeno-ILK group, along with reduced apoptosis as demonstrated by terminal deoxynucleotidyl transferase–mediated dUTP nick-end labeling analysis. Moreover, increased cardiomyocyte proliferation was found in adeno-ILK animals, as measured by proliferating cell nuclear antigen, Ki-67, and phosphohistone-H3 staining. At long-term follow-up, most indices of cardiac function and hemodynamics showed no difference between adeno-ILK and control animals by 9 weeks, although LV end-systolic diameter and infarct scar size were reduced in the adeno-ILK group at this time point. Additionally, ILK overexpression was found to exert a rescue effect on remodeling when administered in a delayed fashion 1 week after coronary artery ligation.
Conclusions— ILK gene therapy improves cardiac remodeling and function in rats after myocardial infarction and is associated with increased angiogenesis, reduced apoptosis, and increased cardiomyocyte proliferation. This may represent a new approach to the treatment of postinfarct remodeling and subsequent heart failure.
Received November 5, 2008; accepted June 22, 2009.
Heart failure is an increasing public health problem worldwide, with the most common cause currently being cardiac remodeling after myocardial infarction (MI).1,2 Cardiomyocyte death in the context of MI in combination with increased preload and afterload triggers a cascade of biochemical intracellular signaling processes that modulate cardiac remodeling (which includes dilatation, hypertrophy, and fibrosis),3 and prevention or attenuation of these signaling processes is an important therapeutic goal.
Clinical Perspective on p 773
Integrin-linked kinase (ILK), a widely expressed serine/threonine protein kinase, is highly expressed in the heart.4 Previous studies have shown that ILK plays an important role in transducing cell–matrix interaction–induced biomechanical signals via binding to the cytoplasmic domain of β-integrins, which regulate cytoskeletal remodeling and angiogenesis, as well as cell growth, proliferation, survival, and differentiation.4,5 Recently, ILK has been reported to be an important factor regulating cardiac contractility,6 compensatory hypertrophy,7 survival, and repair.8 Targeted ILK deletion in the murine heart causes spontaneous dilated cardiomyopathy and heart failure.9 Additionally, ILK controls recruitment of endothelial progenitor cells to ischemic tissue.10 However, whether ILK has therapeutic potential after MI is not clear.
We hypothesized that ILK gene therapy would attenuate remodeling and improve cardiac function after MI. The purpose of this study was therefore to investigate the effect of ILK overexpression in the peri-infarct area on markers of cardiac ischemic damage, angiogenesis, and function.
Recombinant Adenovirus Construction
Wild-type ILK-encoding plasmid was a gift from Dr Hyo-Soo Kim (Seoul National University College of Medicine). Recombinant adenoviral vector (E1, E3 deleted) harboring human wild-type ILK cDNA and humanized recombinant green fluorescent protein (hrGFP) driven by the cytomegalovirus promoter were made by the GenScript Corporation (China Branch, Nanjing, China). Corresponding virus with null content was used as a control.
Animal Model of MI and Adenoviral Vector Delivery
Animal experiments were performed following the guidelines in the Guide for the Care and Use of Laboratory Animals published by the National Institutes of Health (National Institutes of Health publication No. 85-23, revised 1985). Adult, male Sprague-Dawley rats (n=50) weighing 180 to 200 g were anesthetized by intraperitoneal administration of a mixture of ketamine hydrochloride (50 mg/kg) and diazepam (5 mg/kg). Rats were endotracheally intubated and mechanically ventilated (Jiangxi Teli, China) with supplemental oxygen. The heart was exposed through a thoracotomy at the left fourth intercostal space, and the left anterior descending coronary artery (LAD) was ligated by a 6-0 silk suture 1 to 2 mm below the tip of the left atrial appendage. After ligation, lungs were fully inflated by positive end-expiratory pressure. During ligation of the LAD, rats were randomized to treatment with either adeno-ILK or adeno-null; immediately after LAD ligation, a total of 100 μL adeno-ILK (2×109 viral particles) or adeno-null (2×109 viral particles) was injected into 5 regions in the peri-infarct zone with the use of a syringe attached to a 29-gauge needle. The chest cavity, muscles, and skin were sutured in 3 layers.
Protein Expression After Adenoviral Vector Delivery
In vivo expression of exogenous protein after adenoviral delivery was detected by Western blotting and immunofluorescence analysis. hrGFP was used as a marker for exogenous gene expression. Full details are given in the expanded Methods section in the online-only Data Supplement.
ILK Kinase Assay
ILK kinase activity was determined by in vitro kinase assays as described in the online-only Data Supplement. The level of in vivo phosphorylated Akt1/PKBa (a downstream target of ILK) was further confirmed by Western blotting analysis.
Echocardiography and Hemodynamic Assessment
Four weeks after viral delivery, cardiac function was evaluated by transthoracic echocardiography. Thereafter, cardiac catheterization was performed in animals for hemodynamic study. All measurements were performed by an observer blinded to treatment. Full details are given in the expanded Methods section in the online-only Data Supplement.
Tissue Section Preparation
After hemodynamic evaluation, heart samples were collected and used for histological, immunohistochemical, and terminal deoxynucleotidyl transferase–mediated dUTP nick-end labeling (TUNEL) examination, as fully described in the online-only Data Supplement.
Paraffin-embedded sections were stained with hematoxylin-eosin for morphological examination or Masson’s trichrome for assessment of interstitial fibrosis. Infarct scar size, thinning ratio, left ventricular (LV) thickness, cardiomyocyte size, myofilament density, collagen volume fraction, and LV diameter were measured as described in the online-only Data Supplement.
Analysis of Microvessel Density
Microvessel density in the border area was evaluated by CD31 staining performed on frozen sections. Full details on the analysis of microvessel density can be found in the expanded Methods section in the online-only Data Supplement.
Cardiomyocyte apoptosis was measured by triple immunofluorescence staining of TUNEL, α-sarcomeric actin, and DAPI (see the online-only Data Supplement).
Immunohistochemical Analysis of Cardiomyocyte Proliferation
To detect whether ILK promotes cardiomyocyte proliferation, immunohistochemical analysis was performed for phospho-histone H3 (Ser10), proliferating cell nuclear antigen, and Ki-67. Full details of these procedures are in the online-only Data Supplement.
Longer-Term Follow-Up and Effect of Delayed ILK Administration
To investigate whether the improvement seen with ILK overexpression after MI is sustained or temporary, a subset of animals (n=6 per group) was intramyocardially injected with adeno-ILK or adeno-null vector in the border zone immediately after LAD ligation and examined by echocardiography 7 weeks and 9 weeks after adenoviral delivery. Hemodynamics and histology were evaluated 9 weeks after delivery.
To ascertain whether ILK overexpression can also have a rescue effect on the remodeling process, a separate cohort of rats (n=6 per group) was injected with adeno-ILK or adeno-null vector intramyocardially in the border zone 1 week after LAD ligation. Four weeks later, echocardiographic, hemodynamic, and histological analyses were performed.
All values were expressed as mean±SEM. All data analysis was performed with the use of SPSS 13.0 or STATA statistical software. Statistical significance was defined as P<0.05 (2-tailed). The normality or otherwise of distribution of the continuous variables was assessed with the Shapiro-Wilk test. Measurements in border or remote areas were compared within the adenovirus or control groups with the use of paired Student t test (when values were normally distributed) or Wilcoxon signed rank test (when distributions were not normal). Because some echocardiographic parameters were obtained 7 and 9 weeks after gene transfer, when repeated measures were made, the generalized estimating equations statistical method8 with an independent correlation structure was used to estimate differences between groups. When significant differences between the 2 groups (P<0.05) over time were found by the generalized estimating equations approach, a post hoc comparison between 2 groups at each time point was made by Student t test with Bonferroni correction. Changes from baseline in echocardiographic parameters before and after delayed adenoviral delivery (4-week value minus baseline value) were compared between 2 groups by use of an unpaired Student t test. Comparisons of other parameters between groups were performed by unpaired Student t test (when distributions were normal) or Mann-Whitney U test (when distributions were not normal).
The authors had full access to and take full responsibility for the integrity of the data. All authors have read and agree to the manuscript as written.
Fifty rats underwent LAD ligation and were injected with adeno-ILK or adeno-null, of which 42 survived (20 in the adeno-ILK group and 22 in the adeno-null group). Fifteen rats per group were randomly selected to undergo echocardiography, of which 1 rat in the adeno-null group died before echocardiographic examination could be performed as a result of anesthetic complications. After echocardiography, 10 rats per group were randomly selected to undergo hemodynamic testing, of which 1 rat in the adeno-null group died as a result of internal bleeding during the catheterization procedure. Forty rats (20 per group) were euthanized at the terminal time point. Forty hearts were used for frozen section (10 hearts per group ×2 groups) and paraffin section (10 hearts per group ×2 groups).
In Vivo Adenoviral Expression
After injection of adenovirus containing ILK and hrGFP or corresponding null vector in normal hearts, Western blotting analysis of ILK and hrGFP indicated higher ILK levels as well as expression of hrGFP levels in the adeno-ILK group compared with adeno-null controls between 3 and 28 days. In the adeno-ILK group, ILK and hrGFP protein levels rose progressively between 3 and 14 days after adenoviral delivery, after which ILK and hrGFP levels gradually declined and tapered off by 35 days (Figure 1A), indicating transient expression of this protein after treatment. Elevated expression of ILK as well as expression of hrGFP was also seen in infarcted hearts 3 days after adeno-ILK injection within the border area (Figure 1B), as defined by Evan’s blue dye staining (Figure I in the online-only Data Supplement).
Although Western blot analysis demonstrated successful expression of exogenous ILK, the morphological distribution of exogenous ILK was unknown. Therefore, heart slices from MI animals 5 days after transfection were stained for hrGFP. hrGFP-positive cells appeared in the border and noninfarcted areas around the injection sites in adeno-ILK animals (Figure 1C). High-magnification views showed that the majority of the hrGFP-positive cells were cardiomyocytes, as evidenced by simultaneous staining of α-sarcomeric actin (Figure 1D), although a small number of transgene-expressing noncardiomyocytes were also seen in the border and infarct area. Figure 1Dc shows hrGFP-positive (spindle-shaped) cells, which appear to be cardiac fibroblasts.
ILK Kinase Activity
We measured the in vitro kinase activity of ILK in infarcted hearts 3 days after adeno-ILK or adeno-null treatment (Figure 1E). The in vitro kinase assay revealed an increased ILK kinase activity in the adeno-ILK group compared with the adeno-null group, as evidenced by an increase in in vitro phosphorylation of Akt. Western blot analysis with the use of denatured myocardial tissue lysate similarly showed an upregulation of phospho-Akt in the adeno-ILK group, thereby confirming the in vitro findings.
Cardiac Function and Morphology
Macroscopically, the hearts of adeno-null rats were more spherical relative to those of adeno-ILK and untreated rats (Figure 2A), reflecting attenuation of LV global remodeling after adeno-ILK treatment.
Four weeks after adenoviral injection, echocardiography was used to evaluate cardiac function. The systolic function index percent fractional shortening demonstrated significant improvement in the adeno-ILK animals compared with adeno-null animals (Table). In parallel with this, LV end-diastolic diameter and LV end-systolic diameter were both lower in adeno-ILK animals, with no significant difference in interventricular septal thickness at diastole or LV posterior wall thickness. Hemodynamic analysis substantiated the beneficial effects of adeno-ILK on cardiac function. An improvement was seen in both LV systolic pressure and LV end-diastolic pressure in adeno-ILK animals compared with adeno-null animals, with an increase in the former and a decrease in the latter (Figure 2B and 2C). The adeno-ILK group also had a greater maximum +dP/dt and a lower maximum −dP/dt than the control group (Figure 2D). Heart rate was not different between the 2 groups (Figure 2E).
After removal of the heart, LV diameter was measured at the level of the papillary muscle, and other parameters of ventricular morphology were acquired from 3 blocks (base, midregion, and apex; 5 slides per block) of each heart and averaged (Figure 3A). Cardiomyocyte size and myofilament density were analyzed in border and remote zones (Figure 3B). Infarct scar size was reduced in adeno-ILK animals compared with the adeno-null group (Figure 3C). This translates into an 18% relative reduction in infarct size in adeno-ILK animals. Preservation of LV cavity size was noted in the adeno-ILK group compared with adeno-null animals, as manifested by a decrease in LV diameter (Figure 3D). Myofilament density was decreased in the border zone of adeno-null rats compared with adeno-ILK rats (Figure 3E). The thinning ratio was markedly increased in adeno-ILK compared with adeno-null animals (Figure 3F). Peri-infarct (border) zone thickness was lower in adeno-null animals than in adeno-ILK animals and was decreased to remote zone values in adeno-null rats (Figure 3G). Cardiomyocyte size was larger in adeno-null rats than in adeno-ILK rats within the border zone and was not different between the 2 groups within the remote zone (data not shown); in both adeno-null and adeno-ILK rats, cardiomyocyte size was considerably increased in the border region (Figure 3H).
Microvessel Density and Fibrosis
Masson’s trichrome staining for interstitial fibrosis in the border zone is shown in Figure 4A and 4B in the case of an adeno-null and an adeno-ILK rat, respectively. The border zone was confirmed by hematoxylin-eosin staining on serial sections (Figure 4C and 4D). Microvessel density was determined in the border zone by CD31 staining (Figure 4E and 4F). Collagen volume fraction was reduced in the adeno-ILK compared with the adeno-null group (Figure 4G), and microvessel density was found to be increased in adeno-ILK compared with adeno-null animals (Figure 4H).
Cardiomyocyte apoptosis was quantified by TUNEL assay. In the border zone, the percentage of TUNEL-positive cardiomyocytes was markedly reduced in the adeno-ILK relative to the adeno-null group (Figure 5A and 5B). Interestingly, although the percentage of TUNEL-positive cardiomyocytes was low in the remote region of adeno-null rats, it was reduced to still lower levels in adeno-ILK animals (Figure 5B), suggesting that injection of adenovirus containing ILK in the peri-infarct region causes a reduction in cardiomyocyte apoptosis even in areas remote from this.
Phosphohistone-H3 was found to localize to α-sarcomeric actin–stained cells, suggesting that this represented cardiomyocyte proliferation; in the border zone, phosphohistone-H3 was seen to a much greater level in adeno-ILK rats than in adeno-null animals (Figure 6A, 6B, and 6G), although in the remote zone it was present at a low level in both groups (Figure 6G).
Ki-67 expression (Figure 6C and 6D) as well as proliferating cell nuclear antigen expression (Figure 6E and 6F) localized to cardiomyocytes, and both were markedly upregulated in adeno-ILK animals compared with adeno-null rats (Figure 6H and 6I) in the border zone; interestingly, although proliferating cell nuclear antigen and Ki-67 expressions were low in the remote zone of both groups and comparable to levels seen in the border zone of adeno-null rats, expression of both was increased to a small but significant degree in the remote zone of adeno-ILK rats (Figure 6H and 6I). In the case of phosphohistone-H3, a trend was seen toward an increase in the remote zone of adeno-ILK animals, but this did not reach significance (Figure 6G). Collectively, these data confirm that injection of adenovirus containing ILK in the peri-infarct region gives rise to a marked increase in cardiomyocyte proliferation within that region and, additionally, a smaller increase in cardiomyocyte proliferation in more remote areas.
Longer-Term Follow-Up and Effect of Delayed ILK Administration
At 7 weeks after LAD ligation and adenoviral delivery, echocardiography revealed improved cardiac function (percent fractional shortening; Table I in the online-only Data Supplement) in the adeno-ILK compared with the adeno-null group; at 9 weeks, cardiac function (percent fractional shortening; Table I in the online-only Data Supplement) and hemodynamics (Figure IIC in the online-only Data Supplement) showed no difference between adeno-ILK and adeno-null animals, despite relative preservation of LV end-systolic diameter in the adeno-ILK group (Table I in the online-only Data Supplement). Morphological parameters were measured in paraffin-embedded heart sections with hematoxylin-eosin staining at 9 weeks (Figure IIB and IID to IIG in the online-only Data Supplement). The adeno-ILK animals showed a reduced infarct scar size compared with adeno-null animals (Figure IID in the online-only Data Supplement). There were no significant differences in LV diameter (Figure IIE in the online-only Data Supplement) and thinning ratio (Figure IIG in the online-only Data Supplement) between the 2 groups, although a trend to slight improvement in these parameters over adeno-null animals appeared to be present. In the adeno-ILK group, the wall thickness of the border area was increased, whereas that of the remote area was diminished compared with adeno-null animals (Figure IIF in the online-only Data Supplement).
In other animals, adenoviral injection was performed 1 week after LAD ligation to assess the potential of this therapy to rescue cardiac function and remodeling after MI. Four weeks after adenoviral injection, improved cardiac function was seen in the adeno-ILK group, with an enhanced percent fractional shortening (Table II in the online-only Data Supplement) and a greater maximum +dP/dt (Figure IIIC in the online-only Data Supplement) compared with adeno-null animals. Relative to adeno-null animals, preserved LV diameter and increased border zone wall thickness were observed in the adeno-ILK group, although there was no statistical difference in infarct scar size (Figure IIID in the online-only Data Supplement), remote-zone wall thickness (Figure IIIF in the online-only Data Supplement), or thinning ratio (Figure IIIG in the online-only Data Supplement) between the 2 groups.
LV remodeling after MI involves functional, geometric, cellular, and molecular changes,2 and these manifest as deterioration of cardiac function, LV chamber dilatation, infarct wall thinning, compensatory thickening in noninfarcted regions, cardiomyocyte hypertrophy, cardiomyocyte necrosis/apoptosis, and increased fibrillar collagen.11–14 In the present study, ILK therapy was found to attenuate LV remodeling and to improve cardiac function after MI. After 4 weeks of treatment, the ILK group exhibited improved cardiac function, reduced infarct scar size, relatively preserved LV diameter, increased border zone wall thickness and thinning ratio, decreased cardiomyocyte hypertrophy, and increased myofilament density, as well as attenuated interstitial fibrosis and myocyte apoptosis. Preserved scar size and thinning ratio may be associated with reduced infarct extension. Increased border zone wall thickness may relate to adaptive hypertrophy and increased myofilament density.
Postinfarction remodeling has been divided into an early (within 72 hours) and a late phase (beyond 72 hours).3 Although we detected ILK expression from 3 days after infarction in our study, it is known that recombinant adenoviruses can produce physiologically significant levels of transgene as early as 2 to 4 hours after infection,15 rendering it possible that the effect of adeno-ILK in this system commenced earlier than 3 days and within the early phase of remodeling. Our results demonstrate that expression of ILK itself and hrGFP disappeared by 35 days (5 weeks), whereas the effect of ILK treatment persisted for at least 7 weeks, as judged by hemodynamic alterations as well as structural and morphological changes in the heart. Besides the therapeutic effects of ILK injection at the time of coronary ligation, a rescue effect of ILK overexpression was also seen on the remodeling process when injected 1 week after coronary ligation.
ILK has been shown to be a critical component of the cardiac stretch sensor and plays an important role in integrin-mediated mechanosensing/mechanotransduction in the heart.6 It links membrane-bound β-integrins and the actin cytoskeleton to central signaling pathways, which have the effect of preserving cardiac structure and function, including cardiomyocyte contractility, postinfarct cell migration, and cell attachment.6,8,9 The results presented here suggest that ILK treatment may similarly give rise to an increase in signaling that beneficially influences remodeling in a variety of ways. Bendig et al6 showed that ILK regulates cardiomyocyte contractility, whereas mutation of ILK impairs cardiac contractility and reduces atrial natriuretic peptide and vascular endothelial growth factor expression. Therefore, ILK overexpression is likely to enhance cardiomyocyte contractility as well as atrial natriuretic peptide and vascular endothelial growth factor expression after infarct, and atrial natriuretic peptide could in turn reduce LV remodeling.16 Moreover, ILK has been reported to contribute to angiogenesis as well as cell growth, proliferation, survival, and differentiation in noncardiological studies,5 which could also contribute to the therapeutic effect of ILK in postinfarct remodeling. Our results bear out the cardioprotective, proangiogenic, proliferative, and antiapoptotic effects of ILK.
Apoptosis contributes to the progression of MI, and antiapoptotic treatment at an early stage reduces the infarct size.17 Myocardial apoptosis has been found to be a major determinant of unfavorable LV remodeling.18 Furthermore, increased apoptosis in areas remote from the infarct contributes to late LV remodeling after MI.19 Previous studies have indicated that ILK may reduce noncardiomyocyte cell apoptosis.20 In the present study, we have found that ILK reduces cardiomyocyte apoptosis, and this is true not just in the border zone (where the adenovirus was injected) but also more remotely. Overall, our results suggest that overexpression of ILK may attenuate remodeling by reducing MI size as well as progressive infarct extension and fibrous replacement secondary to apoptosis.
One important component of the remodeling process is neoangiogenesis. After MI, neoangiogenesis is normally unable to compensate for the decreased blood supply and to support the tissue growth required for contractile compensation and the greater demands of the hypertrophied but viable myocardium; this may contribute to the death of otherwise viable myocardium, leading to progressive infarct extension and fibrous replacement. In the present study, we found an increase in angiogenesis in adeno-ILK animals, and this may contribute importantly to the reduction in remodeling. Lee at al10 demonstrated that ILK as a hypoxia-responsive molecule may control the recruitment of endothelial progenitor cells to ischemic tissue. Moreover, Friedrich et al20 found that ILK is crucial for vascular development via integrin-matrix interactions and endothelial cell survival. Additionally, ILK can promote vascular endothelial growth factor expression.6,21 In our model, therefore, we would predict that overexpression of ILK after vector injection may enhance local vascular endothelial growth factor expression, protect endothelial cells, promote endothelial progenitor cell recruitment, and improve vascular development, resulting in enhanced angiogenesis.
In the border zone of MI, differentiated cardiomyocytes have been shown to reenter the cell cycle and recommence proliferation, as assessed by increased cardiomyocyte DNA synthesis.22 Sustained cardiomyocyte replacement may attenuate the cell loss after MI, resulting in improved ventricular remodeling. Our data show enhanced cardiomyocyte proliferation in the border zone after ILK treatment. Yamabi et al23 have shown that overexpressed ILK can promote stem cell amplification. It has also been reported that overexpression of ILK can induce the expression of cyclin D2,24 and cyclin D2 can promote regenerative growth in injured hearts.25 Furthermore, ILK may reduce apoptosis of viable cardiomyocytes after infarction, thereby contributing to cardiomyocyte proliferation. However, the precise mechanisms of ILK-induced cardiomyocyte proliferation need to be evaluated in further studies.
Although the majority of transfected cells were cardiomyocytes, some other transgene-expressing cells that look morphologically like fibroblasts were also seen. Previous studies have shown that cardiac fibroblasts can migrate from the border zone to the infarct zone after MI and thereby contribute to the postinfarct healing process by proliferation, collagen synthesis, and transformation into myofibroblasts, resulting in a firm scar.26 It has been reported that overexpression of wild-type ILK can protect fibroblasts from apoptosis,27 whereas ILK-deficient fibroblasts exhibit impaired cell migration and reduced proliferation rates.28 It is therefore possible that ILK overexpression may promote cardiac fibroblast viability, migration, and proliferation, which might in turn contribute to reduced infarct scar expansion. Further studies are needed to determine the precise role of ILK-overexpressing cardiac fibroblasts during remodeling.
In the present study, we found an increased kinase activity of ILK in the infarcted hearts after adeno-ILK delivery, which might contribute to its therapeutic effect. However, whether ILK itself or its kinase activity is essential to the effect remains unclear from the present study, and further investigation using vectors expressing a kinase-dead ILK is required.
In previous studies, evaluation of efficacy in adenovirus-mediated gene therapy has usually been performed within 4 to 6 weeks. In the present study, we performed a longer (up to 9 weeks) follow-up study to investigate the long-term efficacy of adeno-ILK treatment. The data demonstrate that the improvement in cardiac function after adeno-ILK transfection persists up to at least 7 weeks, and, although cardiac function and hemodynamics were not different between adeno-ILK and adeno-null animals at 9 weeks, nevertheless LV end-systolic diameter and infarct scar size were reduced in the adeno-ILK group at this time point. In future studies, it would be useful to examine the usefulness of alternative vectors that can express the target gene for much longer periods of time at a high level, such as recombinant adeno-associated virus.29
Considering the potential for detrimental angiogenesis and hypertrophy in response to prolonged ILK expression, we also examined both macroscopically and histologically the liver, kidneys, and spleen from ILK-treated animals 4 and 9 weeks after treatment, but no instances of neoplasia were observed. However, longer observation intervals with larger numbers of animals are required to exclude the possibility of neoplastic transformation. Cellular and humoral immune responses to adenovirus-mediated gene therapy should also be assessed in future studies. Nevertheless, our observations suggest that an ILK overexpression strategy may be of therapeutic benefit in preventing the evolution of post-MI LV remodeling and heart failure.
We thank Drs Di Xu and Qiang Zhou for expert technical assistance in performing the experiments outlined here.
Sources of Funding
This work was supported in part by grants from the National Natural Science Foundation of China (research grant 30170370) and from the Jiangsu Key Laboratory for Molecular Medicine, Nanjing University (research grant 2008). A.F. receives funding from the British Heart Foundation (Project Grant PG/06/068).
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Postinfarction ventricular remodeling, consisting of changes in cardiac geometry and function, is an important cause of heart failure after myocardial infarction. Current clinical approaches to preventing this include early recanalization of the infarct-related artery and pharmacological interventions (for example, angiotensin converting-enzyme inhibition, β-blockade), but despite such approaches, progressive remodeling still occurs with resultant ventricular dysfunction. Integrin-linked kinase (ILK), a multifunctional protein kinase, has been reported to play important roles in regulating angiogenesis, cell proliferation, and survival and has also been shown to be crucial in the maintenance of cardiac structure and function in mice with cardiac-specific ILK ablation. However, to date, its role, if any, in postinfarction ventricular remodeling has not been shown. The present study indicates that overexpression of ILK by adenoviral gene transfer after myocardial infarction in rats attenuates cardiac remodeling and improves cardiac function. This is accompanied by enhanced neovascularization, reduced myocyte apoptosis, decreased fibrosis, and elevated cardiomyocyte proliferation. ILK also exhibits a rescue effect on the remodeling process after delayed delivery (1 week after myocardial infarction). Our findings suggest that interventions including gene therapy designed to increase ILK expression may be a promising therapeutic approach to prevent postinfarction ventricular remodeling and consequent heart failure.
The online-only Data Supplement is available with this article at http://circ.ahajournals.org/cgi/content/full/CIRCULATIONAHA. 109.870725/DC1.