Temporal Response and Localization of Integrins β1 and β3 in the Heart After Myocardial Infarction
Regulation by Cytokines
Background— Integrins are involved in structural remodeling and tissue repair. This study aimed to elucidate the role of the β-integrins in cardiac remodeling after myocardial infarction (MI).
Methods and Results— The MI model was created by ligation of the left anterior descending coronary artery in rats. We detected cardiac integrins β1 and β3 gene expression (quantitative in situ hybridization) and protein production (Western blot and immunohistochemistry) and potential regulation by tumor necrosis factor (TNF) using neonatal ventricular myocytes and TNF−/− knockout mice. Integrins β1 and β3 gene expression and protein production were low in sham-operated hearts. After MI, the β1 and β3 mRNA and proteins were significantly increased at the site of MI at day 3, reached a peak at day 7, and gradually declined thereafter. Integrin β1A localized primarily in fibroblasts and inflammatory cells, β1D localized in myocytes, and integrin β3 was associated primarily with endothelial and smooth muscle cells in peri-infarct vessels. In cultured myocytes, there was isoform transition from the adult β1D to the fetal β1A on exposure to TNF-α. This was confirmed in vivo in the peri-infarct myocytes, but the transition was voided in TNF−/−-knockout mice.
Conclusions— Integrins β1 and β3 are significantly activated in the infarcted myocardium. Integrin β1 is active particularly at sites of inflammation and fibrosis, whereas integrin β3 localizes to vessels in the peri-infarct zone in a temporally coordinated manner. Integrin β1D to β1A isoform transition in myocytes is regulated by TNF-α.
Received August 9, 2002; revision received November 7, 2002; accepted November 8, 2002.
Integrins are a family of transmembrane heterodimeric receptors that are widely expressed on the cell surface and provide a physical and biochemical bridge between components of the extracellular matrix and the intracellular physiological environment. They are composed of α and β subunits that individually consist of a large extracellular domain, a transmembrane region, and a relatively short cytoplasmic domain for signaling.1–4
Integrins mediate cell–cell and cell–matrix interactions in response to stress that leads to intracellular signal transduction, cytoskeletal rearrangements, wound healing, and cell proliferation, differentiation, and death, processes important in tissue repair.5 Integrin expression is also associated with changes in extracellular matrix, coordinating synthesis of collagen, fibronectin, and angiogenesis.6,7
After myocardial injury such as infarction (MI), extensive remodeling takes place both in myocytes and in the extracellular matrix. Adverse remodeling can set the stage for ventricular dysfunction and heart failure. During remodeling, the extracellular domains of existing integrins can be shed into the extracellular space, and new isoforms can be reexpressed on the cell surface to rapidly generate de novo cell–matrix connections.8 Previous work has demonstrated significant changes in several α-integrin subunits (α1, 2, and 5) after MI.9 β1 integrins may play an important coordinating role in extracellular matrix synthesis and remodeling, as demonstrated in skin and lung after injury.5 Another potentially important role of integrins in the heart is their ability to serve as mechanotransducers during development and in response to physiological and pathological signals.10–13 β1 integrin comes in 2 isoforms in the heart: β1A, the fetal isoform, responsible for cardiac morphogenesis and plasticity, and β1D, the adult isoform, important for facilitation of contractility.14 Integrin β3 is essential for smooth muscle and endothelial cell migration15,16 and blood vessel formation in granulation tissue.17 Currently, the potential role of β1 and β3 in post-MI remodeling remains uncertain, and whether β1 isoforms can be regulated by inflammation and cytokines in the myocytes is unknown.
In the present study, we sought to determine (1) the temporal expression and spatial localization of integrins β1 and β3 in the infarcted heart and (2) whether the functional isoforms of β1 integrins can be regulated by cytokines such as tumor necrosis factor (TNF)-α.
Left ventricular MI was created in 12-week-old male Sprague-Dawley rats (Charles River Canada, Quebec, Canada) by ligation of the left anterior descending coronary artery as previously described by our laboratory.18 Animals were randomized into sham-operated controls (n=7) and infarcted animals that were randomly euthanized on postoperative days 3, 7, 14, and 28 (n=7 per time point). Hearts were immediately harvested, frozen, and stored at −80°C.
To examine the role of TNF on integrin β1 isoform expression in vivo, TNF-knockout mice (TNF−/−) and wild-type controls (WT) (C57BL/6-TNFtm1Gk1, Jackson Laboratory, Bar Harbor, Me) were also subjected to ligation of the left anterior descending coronary artery similar to the protocol outlined above.
For details of methodology on cell culture and in situ hybridization, please refer to the online-only Data Supplement.
Generation of Integrin β1D, β1A Polyclonal Antibody
Rabbit polyclonal anti-peptide antibodies against the 17-mer CPINNFKNPNYGRKAGL of β1D integrin, which contains a unique amino acid sequence present only in the β1D but not the β1A isoform, were generated. In addition, rabbit polyclonal anti-peptide antibodies against the 12-amino-acid sequence (CTTVVNPKYEGK) of β1A integrin, which are isoform specific and do not cross-react with β1D and other known β subunit variants, as previously documented, were generated.19 Rabbits were immunized subcutaneously with 1 mg of the respective conjugate and boosted 5 weeks later with 0.5 mg of the same conjugate. Pooled antiserum was affinity-purified and stored at 4°C in the presence of 2% BSA. Antibody specificity was confirmed by Western blot.
Western Blot Analysis
Protein was extracted from the freshly prepared tissues/cells with lysis buffer after homogenization. It was diluted 1:1 with 2× SDS sample buffer (Invitrogen Novex). Equal amounts of protein (40 μg) were loaded in each lane of 8% to 16% Tris-glycin gel (Helixx). Proteins were separated by electrophoresis and transferred from the gel to a nitrocellulose membrane with an electroblotting apparatus. Membranes were first incubated with 5% BSA for 1 hour and then incubated with anti-integrin β1, β3 (PharMingen), β1A, and β1D overnight at 4°C. After incubation, samples were washed and subsequently incubated with peroxidase-conjugated secondary antibody and detected by use of the ECL detection Kit (Amersham).
Cryostat sections (5 μm) were prepared, air-dried, and fixed in cold acetone (−20°C) for 10 minutes. The endogenous peroxidase activity was blocked by 0.3% hydrogen peroxide and incubated with 10% normal mouse/goat serum. Reaction with primary antibody was performed overnight at 4°C. After a washing in PBS, secondary antibodies, IgG-peroxidase–conjugated goat anti-mouse polyclonal antibody (PharMingen) for β3 and goat anti-rabbit biotinylated antibody (Vector) for β1D and βA were used. Bound antibodies were detected with streptavidin-peroxidase complex with DAB. Negative control sections were incubated with secondary antibody alone.
Reverse Transcription–Polymerase Chain Reaction
Total RNA was extracted from cultured myocytes. RT-PCR was performed with primers for β1, β3, α1, αv, α7, and GAPDH (Table). With PCR buffers and Taq1 polymerase, the cycle parameters were as follows: denaturation at 95°C for 2 minutes, annealing at 60°C for 1 minute, and extension at 72°C for 2 minutes for 33 cycles with 5 minutes of final elongation at 75°C. Reaction mixture without template cDNA was used as a negative control.
Statistical analyses of in situ hybridization and Western blot findings were performed by ANOVA (SAS). Values are expressed as mean±SEM, with P<0.05 considered significant.
After coronary ligation, myocardial necrosis in the ligated zone was evident on day 3, with sarcolemmal disruption and nucleus dropout. By day 7, inflammatory cells were observed in large numbers in the peri-infarct region. New blood vessel formation and fibroblast proliferation were also evident at the site of infarction. On day 14, inflammatory infiltration subsided, and necrotic tissue was partially replaced by fibroblast-like cells. By day 28, necrotic tissue was completely replaced with fibrotic tissue.
Myocardial Gene Expression of Integrin β1
With in situ hybridization (Figure 1), we observed low levels of expression of integrin β1 mRNA in both ventricles of the sham-operated hearts. After MI, integrin β1 was markedly increased at the site of MI at day 3, with particularly robust expression in the peri-infarct border zone. Integrin β1 mRNA levels in the infarcted myocardium reached a peak at day 7. At days 14 and 28, integrin β1 mRNA gradually declined at the site of infarction but remained significantly higher than in the contralateral wall. (Quantitative analysis of β1 and β3 is available online.)
Localization of Integrin β1 in the Normal and Infarcted Myocardium
To determine whether change in integrin β1 mRNA transcripts results in actual changes in integrin protein, immunostaining with specific integrin β1A and β1D antibodies was performed (Figure 2). In sham-operated rat heart, interstitial fibroblasts, vascular smooth muscle cells, and endothelial cells were labeled with anti-integrin isoform β1A antibody (A), whereas myocytes were labeled only by antibodies specific to integrin isoform β1D (B). After MI, within the infarct zone, integrin β1A was localized primarily to the inflammatory cells, blood vessels, and fibroblast-like cells at days 3 and 7. At days 14 and 28, integrin β1A was localized primarily in fibroblasts (C, E, G, I). Integrin β1D was not found in nonmyocyte tissue at any time (D, F, H, J).
Changes in protein production of the total integrin β1 were also quantified by Western blot. The integrin β1 isoforms do not display the standard mature and immature forms as distinct bands. At all time points, tissue of the infarcted model showed an increase in total integrin β1 levels similar to the pattern that was observed for gene expression (Figure 3). The highest level of integrin β1 protein was found in the infarct as well as on day 3: 7 peri-infarct zones. Contralateral zones β1 levels exhibited no significant change.
Myocardial Integrin β3 Gene Expression
Expression of the integrin β3 mRNA is shown in Figure 4. By in situ hybridization, low levels of integrin β3 mRNA were observed uniformly in both ventricles of sham-operated heart. After MI, integrin β3 followed a pattern similar to integrin β1, in which the mRNA was largely increased at the site of MI at day 3, with particular concentration at the border zone between the infarcted and noninfarcted regions. Integrin β3 mRNA levels in the infarcted myocardium reached a peak at day 7. At days 14 and 28, integrin β3 mRNA gradually declined. Compared with sham, integrin β3 mRNA levels were unchanged in noninfarcted myocardium.
Localization of Integrin β3 in the Normal and Infarcted Myocardium
As shown in Figure 5, the immunohistochemistry localized β3 primarily to blood vessels and interstitial cells (A). At early stages after MI, new vessels within the peri-infarct and infarct zones were strongly stained for β3, whereas inflammatory cells and fibroblast-like cells at the site of MI were, by contrast, only minimally stained (B, C). At days 14 and 28, interstitial cells expressed very low levels of integrin β3, whereas blood vessels still expressed high levels of integrin β3 (D).
Regional production of integrin β3 was evaluated quantitatively by Western blot (Figure 3). Integrin β3 protein showed low levels in the normal myocardium after MI. Its content in the infarct and peri-infarct myocardium was significantly increased at day 3 and remained elevated over the course of the 4-week observation. Compared with controls, integrin β3 levels in noninfarcted myocardium remained unchanged at all time points. (Quantitative data available online.)
Effect of TNF on Integrin β1 Gene Expression
Our previous studies showed that TNF is increased within hours after MI, with upregulation particularly in the infarct and peri-infarct zones,18 following a pattern very similar to that observed for integrin β1. To determine whether TNF may regulate integrin β1 expression in myocytes, we isolated and cultured neonatal myocytes and exposed them to TNF-α over different periods of time. Integrin mRNA expression over the time course is illustrated in Figure 5. Most interestingly, when myocytes in culture were exposed to TNF 12 hours later, there was a time-dependent decrease in integrin β1D expression but no change in integrin β1A. To determine whether this is unique to β1, we also assessed β3, α1, α3, αv, and α7 mRNA expression. But in all cases, TNF did not have an effect on any other integrin isoforms. The transcription levels of integrins β1A and β1D protein production were also demonstrated in Western blot (Figure 6).
To confirm that the mechanisms observed above are also relevant in vivo, we performed MI in mice homozygous for TNF−/− or their WT littermates as controls. We have found that in the WT animals, in which there is a significant increase in local TNF production in the peri-infarct zone, integrin β1D was detected at relatively low levels in the myocytes of the peri-infarct zone (Figure 7A, d and e). In the TNF−/− animals, however, the level of β1D was high in the corresponding region (Figure 7A, f). This suggested that there is also downregulation of β1D by TNF in the peri-infarct zone. No integrin β1A was expressed in myocytes (Figure 7A, a–c). Western blot analysis showed that the integrin β1D expression increased in TNF−/− at the site of the peri-infarct zone after MI day 7 but was decreased in WT mice. In contrast, the integrin β1A increased in both WT and TNF−/− mice (Figure 7, B and C).
Cell–cell and cell–extracellular matrix interactions provide a structural, chemical, and mechanical substrate that is essential for regulation of cellular phenotype in specific environments. Integrins provide the critical interface between the cell and the matrix and may serve as a candidate mechanotransducer during cardiac development or in response to injury during pathophysiological stress.12,13,20 In the present study, we observed that both integrins β1 and β3 gene expression were temporally upregulated after MI. In particular, β1D is associated with myocytes, β1A with interstitial and inflammatory cells, and β3 with new vascular structures. β1D is downregulated rapidly by cytokines such as TNF.
We demonstrated that upregulation of integrin β1A expression is synchronous with other changes in the extracellular matrix, including collagen, fibronectin, osteopontin, tenacin, and others.21 The changes in integrins are probably important to allow new connections to be made between the remaining viable cells and the new matrix in the peri-infarct zone, similar to those seen in models of hypertrophy.22–24
Recently, changes of α-integrins after MI have been documented. By day 7 after MI, the α1 integrin expression was elevated both in the remaining normal myocytes in the peri-infarct zone and in the remodeled tissue in the infarct zone.9
In our study, the early rise of integrin β1 production in the repairing myocardium suggests its potential participatory role in the local inflammatory process. Our immunohistochemical study reveals that inflammatory cells contribute to integrin β1A production in the infarcted myocardium. Cells expressing integrin β1A at the site of MI are primarily fibroblasts and inflammatory cells, whereas those positive in integrin β1D are primarily myocytes. These findings suggest that integrin β1 plays a role in inflammation and subsequent fibrosis, similar to the processes of tissue repair observed in liver,25 lung,26,27 and kidney.28
Studies have demonstrated that a variety of growth factors upregulate the expression of several integrins. These growth factors, including angiotensin II and transforming growth factor-β appeared to modulate expression of integrins in a paracrine or autocrine fashion. Our study demonstrated that inflammatory cytokine TNF also regulates integrin expression. After MI, there is an immediate and rapid upregulation of TNF-α in the myocardium as part of the immune injury response system.18 Our in vitro study demonstrated that integrin expression can be altered by TNF-α. When myocytes were exposed to TNF-α, there was a time-dependent downregulation of expression of integrin β1D (the adult isoform, Figure 6). Integrin β1 is present in 2 isoforms in the rat heart, β1A (fetal isoform) and β1D (adult isoform).27 They are coexpressed in embryo heart until embryonic day 17. From this stage on, β1A progressively decreased and β1D increased to become the dominant β-isoform in the adult cardiomyocytes.10 They share significant sequence homology in their alternatively spliced regions. Knockin of β1 integrin with only the β1D isoform was embryonically lethal because of the lack of proper heart formation, accompanied by multiple other defects,14 suggesting an essential function of β1A to confer cell motility and plasticity during embryogenesis.14 β1D, the dominant adult form of β1 in the myocardium, is found in the intercalated disks at cell–cell contact points of the cardiomyocytes.19 β1D interacts with structural proteins of the myocyte to form a stable cytoskeletal framework, permitting a stronger contractile force in the adult myocyte.24 Adult β1D knockout mice display abnormalities of cardiac function. Our study suggests that transition of the integrin from the β1D to β1A isoform in the myocardium after infarction may be regulated by cytokines such as TNF and may contribute to the decreased function and increased mobility of the myocytes to facilitate the remodeling process.
Another important development of the wound-healing process and inflammation is reestablishment of the capillary network, or angiogenesis. Angiogenesis is characterized by the invasion, migration, and proliferation of smooth muscle and endothelial cells. Several members of the integrin family of adhesion receptors are expressed on the surface of cultured smooth muscle cells and endothelial cells.29–31 We found integrin β3 mRNA and protein expressed primarily at the edge of infarcts, associated with vascular structures (Figure 5). Recent studies demonstrated that integrin αvβ3 is strongly expressed in endothelial cells of new blood vessels in tumors7 and oxygen-induced retinal neovascularization.32 Endothelial cells migrate on extracellular matrix components in vitro and very likely also in vivo.33 Cultured endothelial cells are dependent on αvβ3 for survival. If αvβ3 is blocked with arginine-glycine–aspartic acid motif (RGD)–containing peptides or antibodies selective for this integrin, endothelial cells grown in vitro and angiogenic endothelial cells in vivo will undergo apoptosis7,16 and inhibit the migration of smooth muscle cells.15 These observations suggest that integrin β3 upregulation may contribute to angiogenesis in the peri-infarct zone as part of the remodeling process.
In summary, we studied temporal and spatial responses of integrin β1 and β3 in the rat heart after MI. Integrin β1 and β3 expression is upregulated at the site of MI in both inflammatory and fibrogenic stages of healing. Integrin β subunits may play an important role in tissue repair and angiogenesis. The expressions of integrins in cardiac cells are also regulated by TNF-α, which can uniquely induce isoform transition from β1D to β1A dominance. This may account for some of the important effects of TNF on cardiac remodeling after MI.
This study was supported in part by grants from the Heart and Stroke Foundation of Ontario and the Canadian Institutes of Health Research. Dr Liu is the Heart and Stroke/Polo Chair Professor of Medicine at the University of Toronto.
A supplemental Methods section, including additional figures, is available in the online-only Data Supplement at http://www.circulationaha.org.
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