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Circulation Research. 2000;87:508-515

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(Circulation Research. 2000;87:508.)
© 2000 American Heart Association, Inc.

Molecular Medicine

Synthesis of Extracellular Matrix and Adhesion Through ß1 Integrins Are Critical for Fetal Ventricular Myocyte Proliferation

Lisa K. Hornberger, Sandra Singhroy, Tiscar Cavalle-Garrido, Wendy Tsang, Fred Keeley, Marlene Rabinovitch

From the Divisions of Cardiovascular Research (L.K.H., S.S., T.C.-G., W.T., F.K., M.R.), Cardiology (L.K.H., M.R.), Department of Paediatrics, The Hospital for Sick Children, University of Toronto, Canada.

Correspondence to Dr Lisa K. Hornberger, Division of Cardiology, The Hospital for Sick Children, 555 University Ave, Toronto, Ontario M5G1X8. E-mail hornberg{at}sickkids.on.ca


*    Abstract
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 *Abstract
down arrowIntroduction
 down arrowMaterials and Methods
 down arrowResults
 down arrowDiscussion
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Abstract—Extracellular matrix (ECM) regulates vascular smooth muscle cell proliferation. The role of ECM in myocardial growth is unexplored. We sought to determine whether human fetal ventricular myocytes (HFVMs) produce ECM and whether synthesis and attachment to ECM are necessary for their epidermal growth factor (EGF)–dependent and –independent proliferation. Cultured HFVMs proliferate in the presence but not absence of serum and EGF, as determined by increase in cell number and [3H]thymidine and [14C]leucine incorporation (measures of DNA and protein synthesis, respectively). Using a cyanogen bromide digestion technique to measure collagen and elastin and using affinity chromatography for fibronectin, we found that HFVMs synthesized collagen and fibronectin but not elastin. HFVMs grown on exogenous ECM (including fibronectin and type I collagen and laminin) demonstrated no change in proliferation or DNA and protein synthesis with or without EGF. However, inhibition of collagen synthesis using cis-4-hydroxyproline resulted in a decrease in EGF-related HFVM proliferation and DNA and protein synthesis, which was reversed by exposure to L-proline but not by growth on type I collagen. Use of ß1 but not ß3 integrin antibody to inhibit cell interaction with ECM resulted in a decrease in HFVM proliferation and DNA and protein synthesis in response to EGF. Furthermore, EGF-dependent proliferation was enhanced by {alpha}1ß1 and {alpha}5ß1 antibodies that act as functional ligands, but not {alpha}3ß1, the only ß1 subtype expressed in adult myocytes. In conclusion, proliferating HFVMs synthesize collagen and fibronectin. The proliferative response of HFVMs to EGF requires the synthesis of collagen as well as attachment to specific {alpha}/ß1 integrin heterodimers.


Key Words: ventricular myocyte • proliferation • extracellular matrix • ß1 integrins


*    Introduction
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 up arrowAbstract
 *Introduction
down arrowMaterials and Methods
 down arrowResults
 down arrowDiscussion
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Myocardial growth in the fetus occurs primarily as a result of cardiac myocyte proliferation.1 2 Shortly after birth, however, cardiac myocytes lose their ability to proliferate,2 3 4 and subsequent myocardial growth occurs secondary to cardiac myocyte hypertrophy and nonmyocyte proliferation.2 3 Although the mechanisms responsible for myocyte hypertrophy have been the focus of numerous investigations, the cellular and molecular mechanisms that regulate cardiac myocyte proliferation remain largely unknown.

The extracellular matrix (ECM) plays a critical role in the growth, division, and differentiation of a diversity of cells.5 6 Adhesion and interaction with the ECM is mediated in part by integrin receptors, a group of heterodimeric transmembrane glycoproteins that consist of an {alpha} and ß subunit. Integrins modulate changes in cell shape and signal-transduction events. They mediate cell signaling in the absence of growth factors7 8 9 but play an even more important role in enhancing the response of the cell to growth factors either directly10 11 12 13 or indirectly through modulation of focal adhesions.14 15

In the heart, the ECM is important for both myocardial structure and function16 17 and is involved in several important developmental processes during cardiac embryogenesis.18 19 20 21 The extensive change in ECM components within the perinatal period22 and the developmental changes in cardiac myocyte attachment to various components of the ECM that correspond to changes in integrin receptor expression suggest a potential role in regulating myocyte proliferation. For example, ß1 integrin expression is highest in fetal and lowest in adult cardiac myocytes.23 Of the {alpha}/ß1 heterodimers, fetal and, to a lesser extent, neonatal myocytes express {alpha}1ß1, {alpha}3ß1, and {alpha}5ß1 integrins, through which they attach to collagen types I, III, and IV; laminin; and fibronectin.23 Adult myocytes, however, only express {alpha}3ß1, through which they are only able to adhere to laminin and type IV collagen and poorly to other collagens and fibronectin.23 24

Although developmental changes in ß1 integrin expression suggest a potential role in cardiac myocyte proliferation, there is currently no direct evidence of this role. To investigate this, we used a unique human fetal ventricular myocyte (HFVM) culture system. These cells proliferate in response to growth factors, synthesize both collagen and fibronectin, and require synthesis of collagen to proliferate in the presence of epidermal growth factor (EGF). Growth on exogenous matrix, including proteolyzed and nonproteolyzed type I collagen, fibronectin, and laminin, does not alter their proliferative capacity. Furthermore, binding to matrix does not require Arg-Gly-Asp (RGD) sites. We further demonstrate that HFVMs not only express an abundance of ß1 integrins but need ligation of ß1 and not ß3 integrins, including specifically ß1 heterodimers {alpha}1ß1 and {alpha}5ß1, but not {alpha}3ß1, for EGF-induced proliferation.


*    Materials and Methods
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 up arrowIntroduction
 *Materials and Methods
down arrowResults
 down arrowDiscussion
 down arrowReferences
 
Cell Culture System
Hearts were harvested from 10- to 21-week gestational age human fetuses within 15 minutes of elective pregnancy termination and maintained on ice in PBS with penicillin, streptomycin, and fungizone (Gibco). Consent was obtained for use of fetal tissue as currently recommended by the Medical Research Council of Canada.

The ventricles were removed, minced, and enzymatically digested with 0.5 mg/mL collagenase form II (Worthington Biochemical Corp), 50 µg/mL trypsin (Gibco), and 24 µg/mL deoxyribonuclease (Worthington Biochemical Corp) for 10 minutes. Isolated HFVMs were removed, and the trypsin was neutralized with a standard medium of DMEM (Gibco) containing 12.5 mmol/L L-glutamine (Gibco), 10% FBS (Gibco), and antibiotics. HFVMs were then centrifuged at 4°C and 130g for 5 minutes and subsequently stored on ice. Isolated HFVMs were resuspended in half-strength DMEM, centrifuged, placed in standard medium, and plated in culture dishes. All experiments were performed after first passage of just-subconfluent HFVMs without ECM (unless specified) and at least in triplicate.

Assessment of Proliferation
After first passage in 24-well plates at a density of 5x104 cells/well (just subconfluent), HFVMs were cultured in standard medium for 24 hours and then for 24 hours in serum-free DMEM with insulin, transferrin, and selenium A (Gibco). Cells were treated with 0%, 2%, 5%, and 10% FBS or in the absence or presence of 50 ng/mL EGF (Gibco) for 48 hours in 24-well plates either with [3H]thymidine (1 µCi/mL, Amersham) and [14C]leucine (2 µCi/mL, Amersham) or without isotopes (cell counts). For measurements of DNA and protein synthesis, medium was removed and HFVMs were washed with cold PBS and treated for 1 hour with 5% trichloroacetic acid (TCA) and then washed with cold H2O and lysed in 0.4 mol/L NaOH for 30 to 60 minutes. Cell lysates were then placed in vials with scintillation fluid (Amersham), and counts were obtained using a dual-labeling program from a Winspectral scintillation counter. All experiments were performed in triplicate.

To assess the effect of inhibiting collagen synthesis, cis-4-hydroxyproline (cis-OH, Sigma), an analogue of proline that results in an inability to form a triple helix, was added at a concentration of 200 µg/mL.25 To determine whether HFVMs could be "rescued" from the effect of cis-OH, L-proline (400 µg/mL, Sigma) was added after 48 hours of treatment with cis-OH for an additional 48 hours. HFVMs were also exposed to 50 µg/mL ß-aminoproprionitrile fumarate (Sigma) to prevent cross-linking of secreted procollagen to form mature collagen fibrils or to 3.25BTC units/mL collagenase for 48 hours.

Assessing the effects of exogenous ECM, first-passage cells were plated on fibronectin 2.5 µg/mL, laminin 10 µg/mL, and type I collagen and proteolyzed collagen as previously described.26 HFVMs grown in the absence or presence of exogenous ECM (fibronectin and type I collagen) were also exposed to RGD peptides, using RAD peptides as controls.

To assess which of the integrin receptors might be relevant to HFVM proliferation, the cells were exposed either to inhibiting ß1 and ß3 integrin receptor antibodies (Chemicon) at the time of passage or to EGF treatment. Antibodies that act as functional ligands for {alpha}1ß1 (Chemicon), {alpha}3ß1 (clone P1E5, Gibco), and {alpha}5ß1 (clone P1D6, Gibco) were used to determine which of the ß1 heterodimers modulate HFVM proliferation.27

Immunohistochemistry
After growth on glass coverslips, primary and first-passage HFVMs were fixed with 3% paraformaldehyde at 4°C for 10 minutes and subsequently washed with PBS. Fixed cells were incubated for 1 hour at 37°C in 3% BSA. Cells were incubated with anti-human ß-myosin heavy chain (Sigma) at a 1:100 dilution for 1 hour. For integrin receptor detection, mouse antihuman ß1 (diluted 1:50, Sigma) and mouse antihuman ß3 (diluted 1:50, Sigma) antibodies were used. Collagen was probed with mouse antihuman collagen type I (clone col-1, 1:50 dilution, Sigma). IgG controls in washed buffer were used for all appropriate antibodies. The coverslips were incubated for 1 hour at 37°C with fluorescein-conjugated goat antimouse antibody (diluted 1:100, Sigma) or, for collagen, Texas Red–conjugated goat antimouse antibody (diluted 1:100, Sigma). After washing, all coverslips were mounted onto glass slides using Elvanol (Sigma). Observations and photomicrographs were obtained with a fluorescent microscope (Olympus Corp) using epifluorescence.

Collagen and Elastin Production
Synthesis of collagen and elastin was determined using a cyanogen bromide digestion technique as previously described for whole tissue.28

To assess collagen synthesis in the presence of cis-OH, after 24 hours of exposure to cis-OH 25 µCi of 2-[3H]glycine (Amersham) was added to the control wells and cis-OH wells for an additional 6 hours. The samples were precipitated overnight with 10% TCA at 4°C, and subsequent processing for scintillation spectrometry was as described by Fisher and Periasamy.25 Another set of cis-OH–treated cells were exposed to L-proline for an additional 48 hours. At 42 hours, 25 µCi of 2-[3H]glycine was added to the cis-OH/L-proline wells for 6 hours and processed. The fraction of collagenase-sensitive counts was determined by subtracting counts in the presence of collagenase from counts in the absence of collagenase and dividing the result by the counts in the absence of collagenase (total protein incorporating glycine). This fraction was multiplied by the total counts in the original sample to determine the amount of collagen and noncollagenous protein synthesized during 6 hours of labeling.

Immunoprecipitation
After 24 hours in serum-free medium, HFVMs were placed in DMEM free of glutamine, methionine, and cystine for 48 hours in the presence of 10 µCi/mL of [35S]methionine (Amersham) with and without 5% FBS. Using the conditioned medium, total cpm was normalized by TCA precipitation, and equal cpm values (5x105 cpm/sample) were immunoprecipitated with 75 µL of washed gelatin 4B-Sepharose (Pharmacia Biotech, Inc) overnight at 4°C. The fibronectin retained on the beads after 4 washes with 1 mL of Tris-buffered saline containing 0.5% Tween-20 was eluted in 75 µL of 2x SDS sample buffer after boiling for 5 minutes. Fibronectin was separated on 6% SDS-PAGE gels, fixed in 5% acetic acid/10% methanol for 30 minutes, dried, and exposed to film (X-Omat, Kodak). Using the autoradiograph as a template, the corresponding bands were cut from the gel and the radioactivity was determined by liquid scintillation spectometry.

Assessment of Apoptosis
Apoptosis was assessed using an Apoptag kit (s-7160, Oncor) that uses the terminal deoxynucleotidyltransferase–mediated dUTP nick-end labeling (TUNEL) assay.11 Both FITC signal and the propidium iodide counterstain were viewed with a fluorescein microscope.


*    Results
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 *Results
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HFVM Proliferation
We verified that primary culture and first-passage HFVMs consisted of >90% cardiac myocytes as determined by immunostaining for ß-myosin heavy chain (Figure 1Down). HFVMs demonstrated evidence of proliferation in the presence but not in the absence of FBS and demonstrated an increased response to increasing concentrations of FBS (Figure 2ADown). The increase in cell number correlated with [3H]thymidine and [14C]leucine incorporation; however, the amount of [3H]thymidine and [14C]leucine per cell remained unchanged with increasing concentrations of FBS, which suggests only a hyperplastic and not a hypertrophic response to growth factors (Figure 2BDown). No difference was observed in the proliferative capacity of HFVMs from 10 to 13 weeks compared with HFVMs from 15 to 21 weeks of gestation. Although exposure of HFVMs to 5% FBS resulted in a near doubling in cell number, a lesser but still statistically significant proliferative response was observed with exposure to 50 ng/mL of EGF (Figure 2CDown). Thus, HFVMs in culture at first passage are capable of proliferating in response to mitogens, which is consistent with the in vivo phenotype.



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  		Figure 1. Immunostaining of HFVMs after first passage for ß-myosin heavy chain. A, Negative control using propidium iodide and IgG. B, More than 90% of the cells stained positively for ß-myosin heavy chain.



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  		Figure 2. A, Proliferation in response to increasing concentrations of FBS for HFVMs from 8- to 13-week and 15- to 18-week gestational age fetuses. There was no significant difference between gestational age groups (data are mean±SE, n=6; *P<0.05 vs 0% FBS). B, [3H]Thymidine and [14C]leucine cpm per cell. There was no significant change in radioactivity cpm per cell, which suggests a primary hyperplastic response to FBS. C, Proliferation in response to EGF. HFVMs exposed to 50 ng EGF/mL demonstrated proliferation, but the response was significantly smaller than the response to 5% FBS (*P<0.05 vs 5% FBS and vs controls).

To determine the effect of attachment to different exogenous ECM matrices on EGF-induced proliferation, HFVMs were grown on type I collagen, fibronectin, laminin, and proteolyzed collagen (to expose ß3 binding sites26 ). We observed no significant difference in the attachment efficiency at 24 hours of HFVMs and no change in the proliferative response of HFVMs grown on any of the matrices with or without EGF. There was an obvious morphological change in HFVMs grown on type I collagen with a more stellate appearance, but not with either fibronectin or laminin compared with control cells grown on plastic.

Collagen and Fibronectin Synthesis by HFVMs
In the adult myocardium, the proliferating fibroblasts are believed to be the source of the ECM.29 30 In the neonatal myocardium, fibroblasts are responsible for production of fibronectin17 and types I and III collagen,22 and ventricular myocytes produce only type IV collagen as determined by in situ hybridization.22 In this study, we documented that HFVMs synthesize collagen in culture, with a significant increase in production in response to FBS (Figure 3Down). HFVMs did not, however, synthesize any measurable elastin using this technique. Type I collagen was identified in HFVMs by immunohistochemical staining (data not shown). Immunoprecipitation demonstrated synthesis of fibronectin with a significant increase in fibronectin expression by HFVMs in response to FBS (Figure 4Down). These data suggest that in the immature myocardium, proliferating HFVMs produce both collagen and fibronectin.



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  		Figure 3. Collagen synthesis by HFVMs. There was a significant difference in synthesis (both total per plate and newly synthesized based on [14C]proline incorporation) with exposure to FBS (data are mean±SE, P<0.05).



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  		Figure 4. Fibronectin (FN) production by HFVMs. A, Autoradiograph demonstrating fibronectin expression (at 240 kDa) by HFVMs from 4 different hearts of ages 16, 10, 16, and 17 weeks with and without 5% FBS (c indicates control; s, FBS). B, Assessment of [35S]methionine incorporation suggested a significant increase in fibronectin production by HFVMs in the presence of 5% FBS compared with controls (absence of FBS) (data are mean±SE; n=6, P=0.02).

To delineate the role of the ECM in HFVM proliferation, we treated the cells with the proline analogue cis-OH. Use of cis-OH has been shown in cardiac myocytes from chick embryos to significantly decrease collagen production and alter their morphology without altering the viability of the cells.25 Exposure of HFVMs to cis-OH resulted in a significant decrease in collagen production both in the absence and in the presence of EGF (TableDown). There was also a significant reduction in the HFVM proliferative response to EGF when treated with cis-OH (Figure 5Down), but there was no significant change in the absence of EGF and no change in HFVM morphology (data not shown). Growth on exogenous type I collagen did not significantly enhance the proliferative response to EGF. In addition, HFVMs exposed to ß-aminopropionitrile fumarate and collagenase did not demonstrate altered proliferation, which further suggests that collagen synthesis by the cell is necessary for mitogen-dependent HFVM proliferation. L-Proline exposure after 48 hours of cis-OH resulted in improved EGF-induced proliferation, which suggests that the effect of cis-OH was reversible.


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  		Table 1. cis-OH Effect on Stimulation of Collagen Production by EGF



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  		Figure 5. HFVM response to cis-OH (COH). Shown are changes in cell number and radioactivity counts relative to controls with exposure to EGF expressed as a percentage of no-EGF control. Decreased collagen production by cis-OH resulted in a significant reduction in EGF-induced proliferation (P<0.001) but did not affect baseline counts (not shown). This reduction was not significantly changed by growth on exogenous type I collagen but was reversed by the addition of L-proline (n=6, *P<0.01 relative to controls). COH indicates cis-OH; col I, type I collagen.

Integrin Receptors and HFVM Proliferation
ß1 Integrin expression has been demonstrated in both immature and mature rat cardiac myocytes; however, to date, ß1 integrin expression in human cardiac myocytes and ß3 integrin expression by cardiac myocytes has not been demonstrated. Furthermore, although both ß1 and ß3 integrins play a role in modifying the attachment of other cell types to the surrounding matrix and in their proliferative response to growth factors, no information exists concerning the role of these integrins in cardiac myocyte attachment and proliferation. Immunohistochemical staining revealed that HFVMs express an abundance of ß1 integrin receptors (Figure 6Down). ß3 was also observed but with less intense staining (data not shown). Use of a ß1 integrin–inhibiting antibody resulted in a significant decrease in the proliferative response of HFVMs to EGF (Figure 7Down) but did not affect the attachment of cells. The proliferative response was completely abolished without evidence of apoptosis when concentrations of the ß1-inhibiting antibody were doubled (data not shown). Attachment of HFVMs, however, was reduced when they were exposed to the ß3-inhibiting antibody at the time of plating, but exposure to the ß3-inhibiting antibody did not affect proliferation (P>0.05). We concluded that ß1 integrin receptors are critical for the proliferative response of HFVMs to EGF. Although not involved in HFVM proliferation, ß3 integrins may be important for HFVM attachment.



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  		Figure 6. ß1 integrin expression. A, Negative control with IgG as primary antibody. B, Immunostaining of first-passage HFVMs revealed abundant expression of ß1 integrins.



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  		Figure 7. Effect of ß1 integrin inhibition on HFVM proliferation. A, Use of an inhibiting ß1 integrin antibody (Ab) at the time of first passage had no significant effect on attachment of the cells as reflected by baseline cell numbers compared with controls, whereas ß3 inhibition did reduce attachment and baseline cell counts. B, Use of the ß1 inhibiting antibody at time of treatment with EGF significantly decreased the proliferative response of HFVMs to EGF (*P=0.03 vs controls) as demonstrated by percentage change in cell number and radioactivity counts compared with controls. Values are expressed as a percentage of no-EGF controls. cpm indicates counts per 106 cells; Thy, thymidine; and Leu, leucine. C, Effect of {alpha}/ß1 heterodimer ligation on HFVM proliferation. Ligation of {alpha}1ß1 and {alpha}5ß1 induced a significant increase in both cell number and [3H]thymidine and [14C]leucine incorporation in response to EGF compared with controls, whereas {alpha}3ß1 had no significant effect. *P<0.05% change compared with controls (data are mean±SE; n=6).

To determine whether interaction with fibronectin and collagen might require binding through the RGD-dependent receptors {alpha}5ß1 and potentially {alpha}3ß1,31 we exposed HFVMs grown in the presence and absence of fibronectin and type I collagen to RGD peptides using RAD peptides as controls. We found no significant difference in the proliferative response of HFVMs to EGF when exposed to RGD peptides with or without exogenous ECM (data not shown). We hypothesized that either integrins involved in RGD binding do not play a role in HFVM proliferation or the availability of other receptors for signaling and cell cycle progression resulted in RGD peptides having no effect. Finally, ligation of {alpha}1ß1 and {alpha}5ß1, the {alpha}/ß1 heterodimers expressed in fetal but not normal adult myocytes,23 resulted in an enhanced proliferative response to EGF, whereas ligation of {alpha}3ß1 did not (Figure 7CUp).


*    Discussion
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 up arrowIntroduction
 up arrowMaterials and Methods
 up arrowResults
 *Discussion
down arrowReferences
 
The majority of previous investigations into mechanisms that regulate proliferation of immature cardiac myocytes have been focused primarily on the role of growth factors and their receptors. To date, acidic and basic fibroblast growth factor,32 33 the insulin-like growth factors (IGFs),33 34 35 and EGF36 37 have all been shown to be potent mitogens of the immature cardiac myocyte. All 3 are produced by immature cardiac myocytes,32 38 39 40 41 and their receptors are expressed by immature and, to a significantly lesser extent, by mature myocytes.32 38 40 41

Although our work is not the first to demonstrate the capacity of human fetal cardiac myocytes to proliferate in culture in response to growth factors,36 42 we have begun to elucidate the critical role of the ECM and integrin receptors in mitogen-induced HFVM proliferation. Our data suggest that proliferating HFVMs synthesize the ECM components fibronectin and collagen, the latter of which is necessary for cell cycle progression in response to a mitogenic stimulus. Use of cis-OH has been shown to inhibit proliferation of other cell types.43 44 That it is the synthesis of collagen and not a toxic effect of cis-OH is supported by our ability to reverse the effect of cis-OH by exposure to L-proline. Furthermore, Ikeda et al43 showed a similar effect on hepatocyte proliferation by use of factors that impair translational modification of procollagen. As observed in our work, they also found that ß-aminoproprionitrile fumarate and collagenase do not alter hepatocyte proliferation, which suggests that collagen synthesis and not just the presence of extracellular collagen is necessary for cell proliferation. Although growth of the cells on type I collagen failed to rescue them from the effect of cis-OH, we have not excluded the possibility that another type of collagen, such as type III or IV, might have rescued the cells.

The exact mechanism whereby reduced intracellular production of collagen results in decreased EGF-dependent HFVM proliferation is unclear. We anticipated that cis-OH would induce a change in HFVM shape with rounding up, as previously shown in chick embryo myocytes.25 Prevention of cell spreading and altered cell shape has been previously shown in other cell types to alter proliferation.8 11 However, despite the reduced proliferative response of HFVMs to EGF, we did not observe an obvious morphological change in the majority of cells in response to cis-OH. HFVMs grown on exogenous collagen developed a more stellate shape, which suggests that collagen does influence cell shape. Perhaps reduced intracellular synthesis of collagen has a more subtle effect on the cytoskeleton or plays another role within the cell.

Our work further suggests that ß1 integrin receptors are expressed in abundance by HFVMs from 10 to 21 weeks in gestation and are necessary for the proliferative response of HFVMs to growth factors. Inhibition of cell adhesion through ß1 integrins resulted in a significant reduction in the proliferative response of HFVMs to EGF, which suggests a key role for integrin ligation in EGF-related signaling. We have demonstrated a role for the specific {alpha}/ß1 heterodimers. Ligation of {alpha}1ß1, a collagen and laminin receptor in the fetal and neonatal myocyte,23 and {alpha}5ß1, a fibronectin receptor, enhanced the hyperplastic response to EGF. In contrast, {alpha}3ß1, which is expressed in the adult myocyte, did not have any effect on the proliferative response of HFVMs. That RGD peptides did not affect HFVM proliferation was not unexpected, as {alpha}1ß1 integrins do not bind to RGD peptide sites, and therefore RGD peptides would only have affected {alpha}5ß1– and perhaps {alpha}3ß1–mediated proliferation.31 Both {alpha}1ß1 and {alpha}5ß1 have been shown to signal through Shc–mitogen-activated protein (MAP) kinase,27 which suggests duplicated pathways that may preserve proliferation when one integrin is blocked or not available. In support of this, {alpha}1-null mice have a normal cardiac phenotype both antenatally and postnatally.45 Furthermore, although absence of {alpha}5 as shown in {alpha}5-null mice is lethal by 9 days of gestation, cardiovascular development to this stage is preserved.46 Whether cardiac myocytes compensate for the lack of one ß1 subtype necessary for proliferation by increased expression of the other to maintain a normal proliferative response is unknown.

Although our work investigated only the relationship between EGF and the ECM, adhesion through ß1 integrins may be equally important for the response of HFVMs to other growth factors. For instance, Shc-MAP kinase is an important signaling pathway for the mitogenic response resulting from activation of both the EGF and IGF-1 receptors.47 As ligation of ß1 integrins enhances EGF receptor phosphorylation and clustering9 11 and induction of Shc-MAP kinase9 by EGF, leading to a stronger mitogenic response, one would anticipate a similar relationship for the IGF/IGF-1 receptor system and ß1 integrins. Furthermore, this relationship may occur solely with {alpha}1ß1 and {alpha}5ß1, the only {alpha}/ß1 heterodimers known to activate Shc-MAP kinase.27

With the temporal expression of {alpha}/ß1 heterodimers by cardiac myocytes and their involvement in regulating myocyte proliferation, it is tempting to consider a potential regulatory role for ß1 integrin expression in the transition from hyperplastic to hypertrophic growth. As is true for many fetal proteins in response to hemodynamic load, however, adult myocytes appear to re-express {alpha}1ß1 and {alpha}5ß1.23 Despite the re-expression of these {alpha}/ß1 heterodimers in the cardiac myocytes of hypertensive adult rats, Terracio et al23 observed a reduced attachment of hypertrophied adult cells to the different collagens and fibronectin compared with fetal and neonatal cells. This could suggest either more limited expression of the receptors or, as occurs in differentiating keratinocytes,4 altered function of these receptors. Either change could modulate signaling and result in an inability of adult myocytes to proliferate in response to mitogens. Changes in ECM adhesion through integrins have been shown to induce a differentiated state in other cell types48 49 50 51 ; however, as has been shown for primary mammary epithelial cells,52 complete functional differentiation may require the integration of signaling pathways activated by both ECM-integrin interaction and circulating differentiation factors.

In summary, proliferating HFVMs synthesize collagen and fibronectin. HFVMs must synthesize collagen for EGF-dependent proliferation. Finally, HFVM EGF-dependent proliferation appears to require ECM attachment to ß1 integrins, particularly {alpha}1ß1 and {alpha}5ß1, the heterodimers expressed by immature cardiac myocytes. Further investigation into the relationship between growth factors and their receptors and ß1 integrins in cell signaling, and the forces responsible for their reduced expression in the perinatal period, will likely provide insight into the mechanisms responsible for the transition between hyperplastic and hypertrophic growth of the cardiac myocyte.


*    Acknowledgments
 
This work was funded in part by a Physicians’ Services Incorporation Foundation Grant (to L.K.H.).


*    Footnotes
 
This manuscript was sent to Elizabeth G. Nabel, Consulting Editor, for review by expert referees, editorial decision, and final disposition.

Received June 2, 2000; revision received July 25, 2000; accepted August 9, 2000.


*    References
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 up arrowAbstract
 up arrowIntroduction
 up arrowMaterials and Methods
 up arrowResults
 up arrowDiscussion
 *References
 

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