1Department of Physiology and Biophysics and 2Department of Chemistry, University of Illinois at Chicago, Chicago, Illinois
Submitted 26 April 2004 ; accepted in final form 9 September 2004
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ABSTRACT |
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cell adhesion; connexin43; focal adhesion kinase; surface chemistry
There are many ECM proteins, including the collagens, fibronectin, laminin, and elastin, to name but a few. Fibronectin molecules consist of three homologies that, in combination, have different cellular and ECM protein binding capabilities (15). These homologies, in addition to collagen and glycosaminoglycans, bind to cells through the RGD amino acid sequence. The laminins, on the other hand, are important components of the basement membrane and are composed of -,
-, and
-chains. Laminin can bind collagen IV, perlecan, and entactin and is thought to play a role in initiating the synthesis of the basement membrane (19). It also contains an RGD sequence on the
-chain and YIGSR on the
-chain, which facilitate cellular attachment. Although the ligands for cellular attachment are only a few peptides long, the complete size of these various ECM proteins is usually several thousand amino acids in length. The exact role of the remaining regions of these large molecules is largely unknown.
The ECM is often seen as a component of the tissue synthesized and regulated by the cells within it, but many cellular activities can also be regulated by the ECM by a process known as "outside-in signaling" through the cell surface integrins (26). For example, in endothelial cells, synthesis of endothelin-1 can be influenced by the type of collagen the cells are cultured on (9). In the myocardium, the ECM plays an important role in ventricular remodeling following mechanical overload and hypertrophy (20).
In vitro, neonatal myocyte cultures are frequently used to study the influence of mechanical stimulation on cell signaling and myocyte remodeling. However, a significant number of cells become detached in response to mechanical stimulation when ECM proteins are noncovalently adsorbed onto the culture surface, limiting the scope of such studies. It would be advantageous to firmly adhere the ECM to the culturing surfaces so that it would better withstand the effects of mechanical stimulation.
In this study, we have successfully adhered RGD and YIGSR synthetic peptides to the surface of silicone membranes for the purpose of cell culture. Cells cultured on these peptides were compared with their respective native peptides, fibronectin and laminin. This was done to determine the importance of the binding ligands on myocyte cellular adhesion, cell morphology, and function in the absence of the large noncellular binding domains of these proteins. We sought to determine whether covalently adhered peptides could better withstand mechanical stimulation compared with their noncovalently adsorbed native proteins. We also determined the effect of these synthetic peptides on cell behavior and whether they could evoke functional downstream signaling in response to mechanical stimulation.
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MATERIALS AND METHODS |
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Myocyte and fibroblast cell cultures. Primary heart cultures were obtained from neonatal rats according to Institutional Animal Care and Use Committee and National Institutes of Health (NIH) Guide for the Care and Use of Laboratory Animals (NIH Publication 85-23, Rev. 1985). Hearts were removed from 1- to 2-day-old neonatal Sprague-Dawley rats and placed in Moscona's saline (136.8 mM NaCl, 28.6 mM KCl, 11.9 mM NaHCO3, 9.4 mM glucose, and 0.08 mM NaH2PO4, pH 7.4) on ice. The atria were removed, and the ventricles were washed several times with cold Moscona's buffer and then with cold Krebs-Ringer buffer (KRB; 118.4 mM NaCl, 2.4 mM MgSO4, 4.7 mM KCl, 23.8 mM NaHCO3, 1.5 mM KH2PO4, and 11.1 mM glucose) with 1 mg/ml BSA fraction V and stock antibiotic/antimycotic solution diluted 1:100 (containing penicillin G/streptomycin/amphotericin; Sigma). Phenol red was used as a pH indicator, and the solution was gassed with 5% CO2, pH 7.4. The tissue was minced with dissecting scissors in this modified KRB solution.
The cells were dissociated at 37°C in a shaking water bath for 10-min periods at 50 oscillations/min with a collagenase type 2 at 0.42 mg/ml (Worthington Biochemical) in the modified KRB solution, except that we used a higher concentration of BSA fraction V (20 mg/ml). During the digestion, triturating the tissue through a cannula/syringe mechanically disrupted the tissue. The cells released after the first digestion were discarded (fibroblast- and debris-rich mixture), whereas the cells from subsequent digestions were added to 25 ml of KRB with the higher BSA concentration and kept on ice. After final collection, the cells were pelleted by centrifugation (5,000 rpm for 6 min at room temperature) and the supernatant was discarded. The cells were resuspended, filtered through a metal sieve to remove large material, and preplated in different media as described below.
For fibroblast-enriched cultures, we compared two media. One group of cells was resuspended, plated, and maintained in PC1 medium (Biowhittaker/Cambrex) containing L-glutamine, antibiotic/antimycotic solution (Sigma), and gentamicin (50 mg/l). The other group was placed in DMEM, which contained Nutrient Mixture F-12 Ham without L-glutamine (Sigma), standard amino acid concentrations plus palmitic (2.56 mg/l) and linoleic (0.84 mg/l) fatty acids, antibiotic/antimycotic solution, gentamicin (50 mg/l), and 5% serum. The resulting cell mixtures were preplated in either media for 1 h in a 37°C CO2 incubator. The adhered fibroblasts were cultured for another 2 days before passaging in either PC1 serum-free medium or DMEM complete medium with 5% serum. Fibroblast cell number was assessed by cytometric counting 1 and 5 days after trypsinization. The counts from four dishes were averaged to assess cell number on each surface.
For myocyte-enriched cultures, we used the PC1 medium for resuspension and preplating. The cells remaining in suspension were plated at high density (1,000 cells/mm2) and kept for 1 day in PC1 medium (with the additions as noted above), after which time only a small percentage of fibroblasts remained, yielding a myocyte-enriched culture. PC1 prevented further proliferation of any remaining fibroblasts during this time. After 1 day in PC1, the myocyte-enriched cells were kept in DMEM-M199 (4:1) for the remainder of their time in culture.
Mechanical cyclic stain of cultured cells. Myocytes were mechanically stretched after 48 h of culture with the use of the Flexcell strain unit (model FX-4000; Flexcell International) as described previously (17). Cells were cyclically strained at 20% maximum strain at 1 Hz for 48 h in PC1 serum-free medium to limit nonmyocytic proliferation. After this period, cells were rinsed with Moscona's buffer before undergoing further analysis.
Protein analysis by Western blotting.
Protein preparations, Western blots, and their analysis were performed as described previously (4). We measured the expression of 1-integrin (Research Diagnostics), connexin43 (Transduction Laboratories), focal adhesion kinase (FAK; Upstate Biotechnology), type 1 collagen antibody [Developmental Studies Hybridoma Bank (DSHB)], and myosin heavy chain (DSHB) in primary neonatal myocyte cultures. Peroxidase-conjugated secondary antibodies of donkey anti-goat, donkey anti-mouse, and donkey anti-rabbit (Research Diagnostics) were used. Proteins were then visualized using the ECL chemiluminescence system (Amersham).
Immunocytochemical staining. Immunostaining was performed as previously described (3) using anti-FAK antibody (Upstate Biotechnology), phalloidin (Molecular Probes), anti-connexin43 (Transduction Laboratories), anti-myosin (Iowa Hybridoma Bank), and appropriate fluorescence-labeled secondary antibodies. Counterstaining for actin was done using rhodamine-phalloidin (Molecular Probes). The rhodamine-conjugated phalloidin was kept relatively low (1 in 1,000) so that nonstriated actin staining in nonmyocytes was minimal. Membranes were then mounted on glass slides with the addition of Vectashield with 4',6'-diamidino-2-phenylindole (DAPI; Vector Laboratories) as a nuclear stain and an antifade reagent. Stains were visualized using a fluorescence microscope (Nikon Microphot-FXA), and images were digitally captured with a Spot RT color camera (Diagnostic Instruments).
Statistical analysis. Data are presented as means ± SE. Sample number (N) was defined as the number of separate cultures performed at a different time from different animals. Data groups were compared using one-way ANOVA followed by a Student-Newman-Keuls multiple-comparison test. Significance was taken at P < 0.05.
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RESULTS |
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Figure 1B shows that untreated silicone is very hydrophobic and had the lowest cellular attachment as measured by total protein per dish. Cells plated on the intermediate reaction steps of APTES and maleimide showed significantly higher levels of total protein compared with a normal commercial polystyrene tissue petri dish. These results were then compared with synthetic RGD peptide at two reaction concentrations. The results show that increasing the RGD peptide reaction concentration significantly increases the degree of cellular attachment. To determine whether the cellular attachment was due to the RGD sequence, we also plated cells on 100 µM RGE as a negative control in which aspartic acid had been replaced by glutamic acid. With the RGE peptide, the cellular attachment was very low and similar to that of untreated silicone. This also shows that the increased protein measurement was derived from the attached cells and was not due to indirectly measuring the attached peptide from the silicone surface.
Cells were subsequently plated on RGD and YIGSR peptides at varying concentrations and compared with the native proteins fibronectin and laminin, respectively. These results are shown in Fig. 2A. The density of cells on fibronectin was significantly higher than on laminin, as reflected in the total protein per dish. There was a steady increase in the cellular attachment as peptide concentration was increased for both RGD and YIGSR. At 100 µM, cellular attachment was identical to that of their respective native proteins. At this concentration, cellular attachment on the RGD peptide was also significantly higher than on YIGSR.
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To assess the contribution of nonmyocytes in the culture to our experimental findings, we plated cardiac fibroblasts in serum and PC1 serum-free medium. Figure 3A shows that after 5 days in culture, cardiac fibroblasts did not proliferate in PC1 medium. However, in serum medium, there was a fourfold increase in cell number from 1 day to 5 days of culture. To assess the possible contribution of nonmyocytic ECM production to myocyte cultures, we measured procollagen I by Western blotting, as shown in Fig. 3B. In cardiac cells cultured in PC1 medium, there was no detectable collagen I, unlike cells cultured in serum medium, which produced significant amounts of the protein. Subsequently, myocytes were cultured in PC1 medium to reduce the effects of fibroblast contamination. Therefore, in myocytes cultured in PC1, cell number after 2 days was a reflection of cell adhesion and not cell proliferation.
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DISCUSSION |
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The RGD peptide sequence is thought to be one of the most abundant cell integrin recognition sequences in adhesive ECM proteins (31). The size of these recognition sequences is small compared with many of the ECM proteins, but the exact role of the remaining regions and their contribution to cell homeostasis is largely unknown. In HeLa cells, cellular attachment to the RGD sequence requires the recruitment of proteins on the cytoplasmic domain of the integrin and an intact cytoskeleton (29). Our data show that myocyte attachment to the peptides was strong enough to resist 48 h of significant mechanical stimulation.
These data suggest that the strength of the integrin attachment is not compromised by the absence of the remaining portions of the laminin or fibronectin proteins. The detachment of cells from laminin was not due to detachment of the underlying matrix, because cyclic stretch did not alter this. This finding suggests that the cellular attachment is different between laminin and its YIGSR peptide. This can be seen from the different distributions of 1-integrin in cells from the two groups. Investigators at our laboratory previously showed (17) that cardiac fibroblasts cultured on these synthetic peptides were also more resistant to trypsin compared with those on native proteins.
In 3T3-derived fibroblasts, the degree of receptor aggregation at the points of cellular attachment appears to be important for determining the strength of the adhesion to the RGD sequence (12). In one study, myocytes cultured on fibronectin were three times larger than those plated on noncoated dishes (26). This and other fibronectin-mediated effects could be inhibited in a dose-dependent manner with soluble GRGDSP. This would suggest that although the synthetic peptides can bind the cell surface integrins, they do not mediate the same signaling as the native proteins. It also has been suggested that the unfolding of hidden coiled domains within ECM proteins during mechanical stimulation may play an important physiological role (8). Clearly, such complex dynamic changes within the matrix are not possible on the peptide surfaces. This may explain some of the differences in response to mechanical stimulation.
FAK localizes to sites of focal adhesions (33) and is important for maintaining cytoskeletal integrity, motility, and survival. Recruitment of FAK to the focal adhesions is part of the many signaling events that are thought to occur in response to integrin binding with the ECM. However, we show here that myocyte adhesion to YIGSR peptide does not induce the same degree of FAK expression as laminin. This results in formation of fewer striated myofibrils. Our data also show that the decreased FAK expression in myocytes cultured on YIGSR peptide is associated with an abnormal response to mechanical stimulation. FAK has been shown to be an important component in the early response to stretch in cardiac myocytes (28, 35). Work in our laboratory showed recently (21) that FAK is required for muscle sarcomere addition and myocyte remodeling in response to static stretch. Thus decreased FAK expression would be considered detrimental to myocyte homeostasis because it reduces the amount of phosphorylatable protein available to the cell. This in turn might affect potential signaling events required for an adequate response to mechanical stimulation.
In the myocardium, connexin43 plays an important role in cell-cell communication; however, the exact mechanism of this regulation is poorly understood (16). Our data show that connexin43 is more phosphorylated in myocytes cultured on synthetic peptides compared with the native proteins. Western blots also have been used to determine the phosphorylation of other phosphoproteins (2).
Atrial and ventricular myocytes show a different pattern of connexin expression in vitro (14). Our data show that there is also a different pattern of phosphorylation between the atrium and ventricle in the adult heart, with the atrium having more dephosphorylated connexin43. Physiologically, this is associated with higher gap junction communication; however, in vitro, the effects of connexin43 phosphorylation are less clear (1, 7, 16). In vivo, connexin43 can be phosphorylated by protein kinase A and protein kinase C through a number of signaling pathways (1, 7, 16). As yet, the role of the ECM in connexin regulation in the heart is unknown. In the hypertrophied and failing heart, there are often significant changes within the ECM (10, 27), including changes to matrix protein isoforms (6, 36). This is associated with significant and frequently detrimental changes to connexin43 expression and function (13, 32, 34). Our data presented here show that synthetic peptides RGD and YIGSR increase the phosphorylation of connexin43 in myocytes, an event that would decrease gap junction communication. In alveolar epithelial cells, fibronectin has been shown to increase connexin43 expression and intercellular communication, compared with laminin-rich Matrigel (22). Numerous other studies also have shown that the ECM can directly and indirectly alter gap junctional communication in various cell types (5, 24, 25). These results, in combination with our own, strongly suggest that the ECM can influence both the expression and function of gap junctions. Our data present the first such findings in myocytes and may have some important implications in the failing heart, where significant changes to the ECM occur as part of the remodeling process.
In this study, we have been able to decouple the effects of adhesion to the ECM and subsequent downstream signaling required for FAK recruitment to the focal adhesion and cytoskeletal integrity. We show that RGD and YIGSR peptides are all that is required to achieve the same degree of myocyte adhesion as their respective native proteins, fibronectin and laminin. Moreover, these covalently adhered peptides provide better resistance to mechanical strain in cultured neonatal myocytes. However, in the absence of a large portion of the native protein, adhesion was unable to induce the same degree of FAK expression even though 1-integrin levels were unchanged. This was associated with abnormal sarcomere and myofilament formation in these cultured myocytes. Therefore, although these covalently adhered peptides provide improved cellular adhesion in response to mechanical stimulation, they promote aberrant myocytic gene expression and cell morphology.
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GRANTS |
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ACKNOWLEDGMENTS |
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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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