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Cardiovascular Research 2000 47(4):715-725; doi:10.1016/S0008-6363(00)00140-1
© 2000 by European Society of Cardiology
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Copyright © 2000, European Society of Cardiology

Expression of {alpha} and β integrins during terminal differentiation of cardiomyocytes

Niranjan Maitraa, Irwin L. Flinka,*, Joseph J. Bahla and Eugene Morkina,b

aUniversity of Arizona Sarver Heart Center and Department of Medicine, 1501 N. Campbell Avenue, University of Arizona, Tucson, AZ 85724, USA
bDepartment of Physiology and Pharmacology, 1501 N. Campbell Avenue, University of Arizona, Tucson, AZ 85724, USA

* Corresponding author. Tel.: +1-520-626-6639; fax: +1-520-626-8408 iflink{at}u.arizona.edu

Received 14 December 1999; accepted 9 May 2000


   Abstract
Top
 Abstract
1 Introduction
 2 Methods
 3 Results
 4 Discussion
 References
 
Background: In the myocardium, myocyte cell division is irreversibly blocked shortly after birth. The signal that initiates cell cycle withdrawal is unknown. The purpose of this study was to relate changes in expression of β1 integrin and its associated {alpha} subunits to cardiomyocyte cell cycle progression during the fetal-to-neonatal developmental transition in rat. Methods and results: The developmental expression pattern and function of β1 integrin and several of its associated {alpha} subunits were examined using reverse transcription (RT) polymerase chain reaction (PCR) and β1 blocking antibodies. During the fetal to neonatal transition, a dramatic shift occurred in the levels of β1 and {alpha} isoforms. At the 17-day fetal stage only β1A was present, which remained relatively constant until immediately after birth then decreased by 30% at the adult stage. By contrast, β1D appeared at fetal day 18, increased at neonatal day 2, and afterwards remained constant. This resulted in a ratio of β1A to β1D of about 1:1 in the adult heart. The integrin β1-associated subunits, {alpha}3, {alpha}6, and {alpha}7, were expressed at extremely low levels in 17-day fetal cardiomyocytes. After birth {alpha}3 and {alpha}6 transiently increased at the 2-day neonatal stage, while {alpha}7 isoforms B, C, and X2 progressively increased to the adult stage. Unlike skeletal muscle cells, fluorescence-activated cell sorting analysis (FACS) showed no down regulation of the {alpha}5β1 fibronectin receptor during cell cycle withdrawal. Treatment of cultured cardiomyocytes with β1 blocking antibody inhibited the cell cycle in fetal but not in neonatal cells. Conclusion: These results suggest that progression through the cardiomyocyte cell cycle may be dependent upon cell attachment via integrin β1 and correlate with changes that occur in β1 spliced variants and their respective {alpha} isoforms.

KEYWORDS Cell culture/isolation; Developmental biology; Extracellular matrix; Myocytes

This article is referred to in the Editorial by J. Hescheler and B.K. Fleischmann (pages 645–647) in this issue.


   1 Introduction
Top
 Abstract
 1 Introduction
2 Methods
 3 Results
 4 Discussion
 References
 
Integrins are members of a large family of heterodimeric membrane glycoproteins containing various combinations of {alpha} and β subunits, which act as cell surface adhesion receptors linking the extracellular matrix (ECM) to cytoskeletal components. More than 20 different integrin heterodimeric receptors have been identified. Each {alpha} and β protein contains a large extracellular domain, a single membrane-spanning region, and a short, highly conserved cytoplasmic domain. The β subunit targets integrins to sites of ECM-cell adhesion, whereas the {alpha} subunit determines the specificity of ligand binding to various ECM proteins such as, fibronectin, laminin, and collagen [1]. A further level of complexity contributing to the diversity of integrin function results from alternative processing of specific {alpha} and β cytoplasmic domains. Cytoplasmic domain variants include members of the β1 [2–5], β3 [6–8], β4 [9], {alpha}3, {alpha}6 [10,11], and {alpha}7 [12–14] subtypes. Integrin receptors generate bidirectional signals in an ‘outside-in’ and an ‘inside-out’ manner that are initiated from the extracellular and cytoplasmic domains, respectively. These signaling cascades influence a variety of processes including cell proliferation, spreading, migration, tissue repair and remodeling, differentiation, and programmed cell death [15,16].

Changes in integrin isoforms appear to be critically important in cell cycle control in many tissues. For example, adhesion of β1 integrin to the ECM appears to be necessary for the induction of cyclin D1 and activation of the cyclin E-cdk2 complex [17–19]. In cardiac muscle, little information is available on the expression pattern of integrins during the transition from active cell proliferation to terminal differentiation. Brancaccio et al. [20] studied changes in the cytoplasmic spliced variants of β1 integrin by immunostaining. Both β1A and β1D were present in fetal mouse cardiomyocytes, but in adult cardiomyocytes only the β1D isoform could be demonstrated. Inactivation of integrin β1A and β1D subunits in knockin/knockout studies has been shown to mildly affect cardiac morphology but provided no information related to effects on the cell cycle.

The objective of the present study was to define the pattern of β1 and {alpha} integrin isoforms in developing rat primary cardiomyocytes and correlate changes in their expression with cell cycle exit. This study focuses primarily on the fetal-to-neonatal transition when cardiomyocytes are preparing for permanent withdrawal from the cell cycle. We find that β1A is expressed in 17-day fetal cells while β1D appears at fetal day 18 but then falls to a low level until after birth, when its expression increases to the adult stage. The present study also examined developmental changes in several {alpha} isoforms, which have not been previously studied during the proliferative/differentiation switch in cardiac muscle. The effect on the cell cycle of inhibiting attachment of the extracellular domain of β1 integrins with blocking antibody was examined. Treatment with blocking antibody was associated with cell cycle exit in fetal cells but not in neonatal cells.

The switch in integrin β1 isoforms and its {alpha} partners during the fetal-to-neonatal transition may be an important event during the developmental period when cardiac myocytes are undergoing the process of commitment, that is, the decision of permanent withdrawal from the cell cycle. At 17 days of fetal development a switch in partners of E2F/DP1 with Rb-family members from p107 to p130 occurs [21]. The complex of p130 with E2F/DP1 is thought to inhibit S-phase entry and block cells in G1 phase. Formation of the E2F/DP1 p130 complex commonly occurs during serum starvation of cultured cells, such as mouse embryo fibroblasts, and also is observed during the myoblast to myotube transition in skeletal muscle [22]. Changes in expression of β1 and its {alpha} partners in heart cells may contribute to cytoskeletal alterations and intracellular signaling that underlie withdrawal from the cell cycle and terminal differentiation.


   2 Methods
Top
 Abstract
 1 Introduction
 2 Methods
3 Results
 4 Discussion
 References
 
2.1 Preparation of cardiomyocytes
This investigation conforms with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85-23, revised, 1996). Primary cardiomyocytes were prepared from Sprague–Dawley rats at 17 days of fetal development and at 2 and 7 days postnatally as previously described [21]. Cardiomyocytes were differentially plated for 1 h to remove fibroblasts and other contaminating cell types and cultured overnight at 37°C in 5% CO2 at 1–2x106 cells/78-cm2 plate in Dulbecco's modified essential medium (DMEM) and 10% fetal bovine serum (Hyclone Lab., Logan, UT).

2.2 Measurement of integrin isoforms by reverse transcription (RT) polymerase chain reaction (PCR)
A semiquantitative RT-PCR assay was used to determine the relative amounts of integrin cytoplasmic and extracellular domain variants from 17-day fetal to the adult stage. The RT-PCR reaction was carried out according to the method of Zhao et al. [23], except that the level of each integrin isoform was determined by liquid scintillation counting and normalized to a glycerol 3-phosphate dehydrogenase (G3PDH) control reaction.

RNA was prepared from the 17-day fetal to 7-day neonatal stage using freshly isolated enriched cardiomyocytes according to Flink et al. [21]. Total cellular RNA was prepared from freshly isolated adult cardiac tissue using TRIsol (Gibco-BRL, Grand Island, NY) according to the manufacturer's instructions. First strand synthesis was carried out using 200 units of mouse moloney leukemia reverse transcriptase (Clonetech, Palo Alto, CA), 1.0 pmol oligo(dT)18 primer, and 50 ng of total RNA. The following oligonucleotide primer sets were designed to sequences that flank alternative splice sites of various integrin isoforms:

β1NZ1 (sense) 5'-TTGTGGAGACTCCAGACTGTCCTACT-3'
β1PE6 (antisense) 5'-TCATTTTCCCTCATACTTCGGATT-3' [3]
{alpha}3 (2032) (sense) 5'-AAGCCAAATCTGAGACTGTG-3'
{alpha}3 (2033) (antisense) 5'-GTAGTATCGGTCCCGAATCT-3' [10]
{alpha}6 (1157) (sense) 5'-GACTCTTAACTGTAGCGTGA-3'
{alpha}6 (1156) (antisense) 5'-ATCTCTCGCTCTTCTTTCCG-3'
{alpha}6 nested primer (sense) 5'-GAACTGTGTGAACATCAGA-3'
{alpha}6 nested primer (antisense) 5'-ATCCTTACAGCATGGTATCG-3' [10]
{alpha}7abc (sense) 5'-AGCCGTGCTTCATGTCT-3'
{alpha}7AB (antisense) 5'-GGCTGGTACAGCACAGTAAG-3'
{alpha}7C (antisense) 5'-TGGTACAGCACACTTGAAGAA-3' [13]
{alpha}7X1 (sense) 5'-GCCAGGGTGGAGCTCTG-3'
{alpha}7X1 (antisense) 5'-CTATCCTTGCGCAGAATGAC-3'
{alpha}7X2 (sense) 5'-GTGACCAACATTGATAGCTC-3' [14].

For each integrin, except {alpha}6, one-twentieth of the synthesized cDNA yielded a product that was within a linear range in a PCR reaction for 40 cycles at a primer concentration of 0.75 µM and 2.5 units of Taq polymerase in a DNA thermal cycler (Perkin-Elmer/Cetus, Wellesley, MA). For integrin {alpha}6, one-fiftieth of the first PCR reaction was used in a second round of PCR for 25 cycles using a set of nested primers. PCR products were separated by agarose gel electrophoresis, transferred to nylon membranes, and hybridized with a 32P-labeled gene-specific probe. Amplified fragments of the predicted size were excised, quantitated by liquid scintillation counting, and the CPM normalized to G3PDH PCR product as a control. To exclude the possibility that contaminating genomic DNA was the source of the obtained products, the PCR was performed in the absence of the RT reaction. Each PCR reaction was performed 2 to 4 times.

2.3 Fluorescence activated cell sorting
Cell surface expression of rat fibronectin receptor, {alpha}5β1, was analyzed by indirect immunofluorescent staining and flow cytometry as previously described [24]. After plating cells overnight, fetal and neonatal primary cardiomyocytes (5x105 cells) were harvested in trypsin–EDTA, rinsed in PBS, and incubated in PBA buffer containing PBS, 1% BSA, and 0.1% sodium azide. Cells were incubated on ice for 1 h with either anti-human fibronectin receptor primary antibody (#12118-014, Gibco-BRL) or isotype-matched rabbit IgG (10 µg/ml) and then rinsed 3 times with PBA. Secondary antibody, conjugated goat anti-mouse IgG phycoerythrin F(ab')2 fragment (Jackson Immunoresearch, West Grove, PA), was added for 1 h on ice in the dark. Cells were then fixed in 1% paraformaldehyde for 2 h on ice, resuspended in PBA, and analyzed on a FACScan (Becton-Dickinson) flow cytometer. FACS was carried out at the University of Arizona Cancer Center Flow Cytometry Facility. FACS scattergrams were analyzed using the WinMDI 2.8 software package (Scripps Institute, La Jolla, CA). For data evaluation, a gated cluster of cells was established that excluded low forward and side scatter light intensity. Two windows were set to include the distribution of fluorescent positive cells immunostaining with the fibronectin receptor antibody (M2) and autofluorescence obtained with control IgG antibody (M1). The relative amount of fibronectin receptor of fetal and neonatal cardiomyocytes was determined by subtracting the mean immunofluorescence value of control IgG in the fibronectin receptor window (≤5%) from the mean fibronectin receptor immunofluorescence. Statistical analysis was performed using Student's t-test. Data are presented as means±S.E.M.

2.4 Immunostaining
Paxillin (P13520 [GenBank] ) (Transduction Lab., Lexington, KY), rhodamine phalloidin, P1951 (Sigma, St. Louis, MO), and MF20 (University of Iowa, Developmental Studies Hybridoma Bank) antibodies were used for immunostaining. After preparation, primary cardiomyocytes were allowed to attach overnight onto glass coverslips in DMEM medium containing 10% fetal calf serum (FCS). Cells were rinsed in PBS solution, and immersed in 3.7% paraformaldehyde solution for 10 min. After fixation, cells were rinsed in PBS and incubated in blocking buffer for 20 min. Cells were incubated with primary antibody at room temperature for 45 min, washed, and incubated with secondary antibody for 1 h. Microscopic analysis was performed with a Zeiss Axioskop fluorescent microscope equipped with an automatic camera.

2.5 Treatment of cardiomyocytes with β1 blocking antibody
Immediately after preparation, primary cardiomyocytes (2.5x105 cells) were incubated for 30 min at room temperature with β1 blocking antibody (50 µg/ml), CD29 (Pharmingen, San Diego, CA), in the presence of DMEM containing 10% FCS, and plated overnight onto 35-mm glass coverslips in six-well microtiter plates. The next day, after cell attachment, colcemid (0.4 µg/ml) was added, and incubation was continued overnight. Cells were then prepared for FACS or immunostaining. Alternatively, after attachment overnight, cardiomyocytes were treated with β1 blocking antibody.


   3 Results
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
4 Discussion
 References
 
3.1 Developmental expression of integrin β1 and its associated {alpha} subunits during terminal differentiation of cardiomyocytes
In skeletal muscle, integrin β1 [3] and several of its associated {alpha} subunits [2,13,25] have been shown to display a regulated pattern of expression during terminal differentiation [26], suggesting they may play a role in cell proliferation and myogenesis. To determine whether integrin expression patterns are altered during the permanent withdrawal of cardiac muscle cells from the cell cycle, primary cultures of 17-day fetal and 2-day neonatal cardiomyocytes were chosen to represent the period when cell cycle exit occurs. Results in fetal and neonatal cells were compared with integrin expression in adult ventricular tissue. RT-PCR was performed with primer sets that span portions of the {alpha} and β subunit mRNAs encoding the transmembrane and cytoplasmic domains. The results demonstrate changes in the expression pattern of several alternatively spliced {alpha} and β isoforms during the fetal to neonatal transition, which begin as early as fetal day 18.

Of the four known β1 subunits that have been identified (β1A–β1D), only the mRNA encoding the β1A isoform was present in ventricular cardiomyocytes at the 17-day fetal stage (Fig. 1A). The level of β1A remained relatively constant to neonatal day 7, then decreased by about 30% at the adult stage. By contrast, the amount of β1D increased at neonatal day 2 and remained at a constant level through the adult stage. The ratio of β1A to β1D was {approx}3:1 at neonatal day 2, and decreased to {approx}1:1 at the adult stage because of the lower level of β1A. In each case, quantification of mRNA was determined by normalization to a G3PDH control (Fig. 1B).


Figure 1
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  		Fig. 1 Developmental changes of the integrin β1 subunit in primary cardiomyocytes demonstrated by RT-PCR. (A) β1A mRNA levels remain relatively constant until the adult stage, when it was decreased by about 30%. The onset of β1D expression was observed at neonatal day 2 and continued at a steady level to adulthood. (B) Glycerol 3-phosphate dehydrogenase was used as an internal control to normalize for differences in mRNA concentration. Autoradiograms are shown after hybridization of the RT-PCR product to their respective oligonucleotide probes designed to bind within the flanking primer sequences. Bands representing integrin isoforms were excised and quantitated by liquid scintillation counting. The molecular weight of each PCR product is indicated next to the arrow.

 
Among the {alpha} subunits examined, low amounts of {alpha}3B, {alpha}6A, and {alpha}6B were present at the 17-day fetal stage (Fig. 2A,B). Each of these isoforms increased at neonatal day 2 and declined by neonatal day 7. At the adult stage the level of {alpha}3B remained constant, {alpha}6A decreased, and {alpha}6B increased. At neonatal day 7, a decrease of {approx}50% occurred in the levels of {alpha}3B and the {alpha}6 isoforms. A similar decrease was observed by fluorescence-activated cell sorting (FACS) for {alpha}3B (results not shown). At the adult stage, integrin {alpha}6A was found to decrease to a barely detectable level. The {alpha}3A integrin subunit was not expressed in cardiomyocytes (results not shown).


Figure 2
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  		Fig. 2 Changes in integrin {alpha}3B, {alpha}6A, and {alpha}6B isoforms during cardiomyocyte development. Extremely low levels of (A) {alpha}3B and (B) {alpha}6A and B expression were detected by RT-PCR at day 17 of fetal development. Maximal expression of each isoform was observed at neonatal day 2. After this time their levels diminished, except {alpha}3B and {alpha}6A, which were present at the adult stage. Each isoform was normalized to G3PDH as described in Fig. 1.

 
In contrast to the complex expression patterns of integrin β1 and the {alpha}3 and {alpha}6 cytoplasmic domain isoforms during the fetal to neonatal transition, the {alpha}7 isoforms showed a uniform increase in expression from fetal day 17 to the adult stage. Barely detectable levels of {alpha}7 isoforms were observed at the fetal stage (Fig. 3A–C). At the adult stage, {alpha}7B and {alpha}7C were increased 21- and 13-fold, respectively. The time-course of expression of an {alpha}7 extracellular ligand binding domain variant, {alpha}7(X2) (Fig. 3C), showed a 5-fold increase at the adult stage, similar to {alpha}7B and {alpha}7C. The integrin {alpha}7A subunit does not appear to be expressed in cardiomyocytes. Each {alpha} isoform was normalized to G3PDH as shown in Fig. 1B.


Figure 3
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  		Fig. 3 Expression of integrin {alpha}7 isoforms during cardiomyocyte development. Message levels of the cytoplasmic domain variants, {alpha}7B and {alpha}7C, and the ligand binding domain variant, {alpha}7X2, were barely detectable at the 17-day fetal stage. In the adult, {alpha}7B and {alpha}7C increased {approx}21- and {approx}13-fold, respectively, and {alpha}7X2 increased 5-fold. The methods and use of G3PDH template cDNA as an internal control are the same as in Fig. 1.

 
3.2 The onset of integrin β1 and {alpha}7B expression begins at fetal day 18
A detailed time-course of β1 and one of its major partners, {alpha}7B, was carried out from the 17-day fetal to the 2-day neonatal stage (Fig. 4A–C). The results show that the mRNA levels of β1A, β1D, and {alpha}7B were increased from 3- to 20-fold at 18 days of fetal development. After this time, their levels varied until neonatal day 2, when the amount of β1A remained about the same, and β1D and {alpha}7B increased about 4- and 20-fold, respectively. At fetal day 19, the expression of each integrin diminished, with the most dramatic drop occurring in β1A. At fetal days 20 and 21, expression of β1A and β1D remained relatively constant, whereas {alpha}7B increased sharply. Interestingly, immediately before birth (fetal day 22), the level of each isoform diminished. Overall, the fold-increase in β1D and {alpha}7B, comparing Figs. 1 and 3Go with Fig. 4, is about the same during the fetal to neonatal transition. Differences in CPM are due to the age of [32P]oligonucleotide probe used in the hybridization. Each integrin isoform at each time point was normalized to a G3PDH control (Fig. 4D). To ensure that the RT-PCR reaction was within a linear range, synthesis of G3PDH was measured as a function of increasing amounts of mRNA. The results showed a linear amount of G3PDH was synthesized in the presence of increasing quantities of mRNA (results not shown).


Figure 4
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  		Fig. 4 Time-course of integrin β1 and {alpha}7B expression during the fetal to neonatal transition. Integrin expression was measured by RT-PCR from fetal day 17 through neonatal day 2. The methods are the same as described in Fig. 1. The mRNA levels of β1A (A), β1D (B), and {alpha}7B (C) increased at day 18 of fetal development. At day 19 each isoform decreased. By day 21, β1A and {alpha}7B increased and β1D decreased. Immediately before birth, at fetal day 22, the level of each isoform diminished. At day 2 of neonatal development, β1D and {alpha}7B increased above the level at fetal day 17, whereas β1A remained about the same. G3PDH was used to normalize for differences in mRNA concentration. The amount of G3PDH synthesized at each time point is shown in (D, top) with their respective values (D, bottom). Autoradiograms for each integrin are shown after hybridization to a [32P]oligonucleotide probe.

 
3.3 Fibronectin receptor level remains unchanged during cardiomyocyte withdrawal from the cell cycle
FACS analyses were performed on cardiomyocytes indirectly immunostained with {alpha}5β1 (fibronectin receptor) antibodies to determine whether a change occurs in the number of receptors during the fetal-to-neonatal transition. The data were analyzed using a gated population of events excluding particles consisting of low side scatter and forward scatter light intensity. Quantification of the results are shown in Table 1. The mean background immunofluorescence of fetal and neonatal cardiomyocytes stained with control IgG was 15±6 and 13±1, respectively. After subtracting the nonspecific IgG immunofluorescence, the mean specific immunofluorescence of fetal and neonatal cardiomyocytes stained with {alpha}5β1 was 1228±317 and 1448±277, respectively. No statistical difference was observed between the fetal and neonatal immunofluorescence values suggesting that the number of fibronectin receptors remains the same in these two cell types during the fetal-to-neonatal transition.


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  		Table 1 Fibronectin receptor expression in fetal and neonatal cardiomyocytesa

 
3.4 Inhibition of β1 function prevents sarcomeric organization, cell spreading, and attachment of cardiomyocytes
Since integrin β1 isoforms have been shown to change during terminal differentiation of skeletal muscle cells [2,3,5], it was of interest to study whether blockage of integrin β1 function would have an effect on cell proliferation. First, the effect of treatment with β1-integrin blocking antibody on the morphology of fetal cardiomyocytes was examined. In the absence of blocking antibody, cardiomyocytes stained with MF20 antibody [27] showed a regular pattern of sarcomeric myosin (Fig. 5A). After overnight treatment with β1 integrin blocking antibody, cells became rounded with disorganized myofibrils (Fig. 5B). Additionally, the regular pattern of sarcomeric thin filaments in cardiomyocytes stained with rhodamine–phalloidin (top of Fig. 5C) was lost after antibody treatment (Fig. 5D). Cardiomyocytes in Fig. 5C,D also were stained with paxillin antibody. Paxillin is widely distributed in most animal and human tissue [28] and colocalizes with focal adhesion kinase at focal adhesion sites [28]. Before treatment, a characteristic distribution of paxillin on the surface of spreading cardiomyocytes was present. After treatment with β1-blocking antibody, paxillin was arranged in a regular spoke-like pattern around the inner surface of rounded cells (Fig. 5D). In cells that appeared to be more spread after β1 blocking antibody treatment, other patterns of paxillin staining were observed.


Figure 5
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  		Fig. 5 The effect of integrin β1 blocking antibody on fetal cardiomyocyte morphology and cell shape. Fetal cardiomyocytes were incubated with β1 blocking antibody and immunostained with rhodamine phalloidin and anti-paxillin antibody or MF20 as described in Section 2. (A) IgG-treated control cardiomyocytes immunostained with MF20, which specifically interacts with sarcomeric myosin. The arrow indicates myofilament organization in control cardiomyocytes. (B) Cardiomyocytes treated with β1 blocking antibody and immunostained with MF20. The cells are more round and less well spread. (C) IgG-treated control cardiomyocytes immunostained with rhodamine phalloidin (red) and paxillin (FITC, green). Rhodamine phalloidin interacts with sarcomeric and cytoskeletal actins. The sarcomeric pattern is more observable in the two adjacent cells at the top of 5C. Paxillin (arrow) is located at focal adhesion plaques where integrins link the cell membrane to cytoskeletal components. (D) Cardiomyocytes treated with β1 blocking antibody and immunostained with rhodamine phalloidin and paxillin antibody. Sarcomeric and cytoskeletal actins are more diffusely stained with rhodamine phalloidin compared to cells treated with IgG. Immunostaining of paxillin (arrow) is arranged in a spoke-like pattern around the inner surface of the rounded cells. Other patterns of paxillin staining are also observable.

 
The percentage of cardiomyocytes attached to coverslips in the presence of β1 blocking antibody was determined by counting the number of MF20-positive cells in 15 high-power fields and analyzed by the Student's t-test. The average number of MF20 positive cells per high-power field in the control was 15. In the presence of blocking antibody, 68% (P<0.001) of the cells remained attached, and 38% (P<0.001) of these cells were more round in appearance. Cells treated with blocking antibody were examined for cell viability and apoptosis by acridine orange/ethidium bromide uptake and exclusion of trypan blue. Fourteen percent of the round cells appeared apoptotic or necrotic by these methods. These results suggest that β1 blocking antibody caused a general weakening of cells at adhesion sites with disruption of focal contacts, resulting in a lower number of cells attached and an increased number with altered morphology. Thus, integrin β1 appears to have a role in cardiomyocyte attachment and cell spreading. Cardiomyocyte attachment to ECM appears necessary, but not sufficient, for cell proliferation.

3.5 Integrin β1 blocking antibody inhibits cell cycle progression of fetal cardiomyocytes
Antibodies that block integrin β1 function were used to understand the function of the integrin β1 subunit in cardiomyocyte cell proliferation. Initial experiments demonstrated that β1 blocking antibodies influenced cell spreading and attachment. The β1 blocking experiments were performed five times for fetal cells and three times for neonatal cells. In preliminary experiments, the amount of β1 blocking antibody per plate and cell density were varied. Two sets of experiments were performed with fetal cells. In the first series of experiments, β1 blocking antibody was added at the time of cell plating. An equal amount of IgG in PBS, and an amount of PBS equivalent to the volume of antibody, were used as negative controls. Cell cycle kinetics were analyzed by FACS. In comparison with IgG-treated cells and the PBS control, antibody-treated cells showed a 43% and 39% decrease in the percentage of fetal cells entering S and G2/M phases, respectively (Table 2). These results demonstrate that interference with β1 function by blocking antibody is not the result of nonspecific interactions.


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  		Table 2 The effect of β1 blocking antibodies on fetal and neonatal cardiomyocyte cell cycle progressiona

 
Because addition of blocking antibody at the time of plating may have prevented some cells from attaching, a second series of experiments were carried out in which antibody was added to fetal cardiomyocytes that had been plated overnight. Under these conditions, the percentage of cells blocked in G2/M (40%) was similar to the first experiment, except for an increase in the percentage of cells entering S-phase. Thus, in fetal cells cardiomyocyte attachment to ECM appears necessary, but not sufficient, for transit through the cell cycle.

To determine whether cells could recover from β1 blocking antibody treatment, antibody-containing medium was replaced after 2 days with fresh growth medium. The results demonstrated a 13% increase in the percentage of cells entering G2/M compared to cells treated without antibody replacement (Table 3). The percentage of cells entering G2/M was close to the normal level (78%). Although these results do not rule out the possibility that the antibody ultimately exerts an adverse effect on cell survival, they indicate that the potential for cell proliferation was preserved during the time course of these experiments.


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  		Table 3 The effect of removal of β1 blocking antibodies on fetal cardiomyocyte cell cycle progressiona

 
Although the percentage of cell cycle inhibition varied in fetal cells ({approx}22–40% inhibition), results consistently showed a block in G2/M compared to neonatal cells (Table 2). These data suggest that the requirement of integrin β1A interaction with ECM components is more critical during the fetal period when this isoform is the only β1 subunit present in the ventricular myocardium. The fact that β1 blocking antibody did not stimulate or inhibit cell cycle progression of neonatal cells may indicate that the β1A and D isoforms are not functionally equivalent.


   4 Discussion
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
 4 Discussion
References
 
There is considerable evidence that the pattern of integrins expressed on the surface of the cell is closely related to the proliferative state of the cell. Attachment to the ECM through integrins is thought to stimulate progression through G1 phase of the cell cycle by inducing expression of cyclin D1 and activation of the cyclin E-cdk2 complex [17–19]. During terminal differentiation in permanent skeletal muscle lines, changes in integrin isoforms are associated with cell cycle withdrawal [25]. The role of integrin expression in cardiomyocyte cell cycle control has not been well characterized. In this study, the expression patterns of β1 integrin and several of its associated {alpha} subunits have been examined in cardiomyocytes during the fetal-to-neonatal transition when these cells are actively withdrawing from the cell cycle. The results show that during this period there is a change from the β1A to the β1D isoform, an increase in {alpha}3, {alpha}6A, {alpha}6B, and three isoforms of {alpha}7. In fetal cardiomyocytes, addition of a monoclonal β1 blocking antibody inhibits cell cycle progression, but this effect is essentially lost in neonatal cells. The changes in expression of β1 integrin isoforms and their {alpha} integrin partners during cell cycle withdrawal in cardiomyocytes are similar to those observed during the myoblast to myotube transition in skeletal muscle, with the exception that the {alpha}5β1 fibronectin receptor is not down regulated in cardiomyocytes.

Support for the importance of β1 integrin expression in cardiac cell cycle withdrawal has been demonstrated using a monoclonal β1-blocking antibody (Table 2), which inhibits cell cycle progression in fetal but not neonatal cells. This effect is similar to observations reported on treatment of cultured skeletal muscle myoblasts with the monoclonal antibody, CSAT, which interferes with integrin attachment to fibronectin and laminin [29], and irreversibly prevents the differentiation of myoblasts into myotubes [30]. In cardiomyocytes, the effects on the cell cycle may be related to the change in the β1 isoform from A to D during the fetal to neonatal transition. The switch in integrin isoforms results from alternative processing of the cytoplasmic domain, and has been correlated with terminal differentiation in skeletal muscle cells [3]. Another β1 cytoplasmic domain variant, β1C, also has been shown to inhibit DNA synthesis and block cell cycle progression near the G1/S boundary, but it is not present in heart [31].

The functional importance of integrin β1A and β1D subunits has been studied by inactivation in knockin/knockout studies [32] and in embryonic stem cells [33]. Although cellular morphology, adhesion, migration, and mild cardiac functional abnormalities were found, no attempt was made to obtain data about possible effects on proliferation of cardiomyocytes. The results of knockin/knockout studies suggested that β1 integrins may act as mechanotransducers and are important in cellular migration during morphogenesis. In β1-null embryonic stem cells, differentiation of specialized cardiac cell types was delayed and organization of the cytoarchitecture was severely impaired, suggesting that cell–matrix interactions via β1 integrins is important for maintenance of myofibrillar organization [26,34,35].

As a consequence of the switch in β1 isoforms from A to D, there is a change in the associated {alpha} partners. Immunoprecipitation with β1 antibody from heart extracts showed that the β1-subunit is associated with {alpha}3, {alpha}5, {alpha}7B, and {alpha}V monomers [3]. When immunoprecipitation was carried out with an antibody specific for β1D, however, the β1 isoform was only associated with {alpha}7B [3]. There is a similar switch from the A to D isoforms of β1 integrin during blast to tube transformation in skeletal muscle. In this case, β1A and {alpha}7B integrin isoforms are present in proliferating myoblasts [2,3,13]. After myoblast cell fusion, expression of the contractile phenotype and cell cycle withdrawal occurs during the period when β1D is expressed in parallel with {alpha}7A and {alpha}7C isoforms. The {alpha}7A isoform is not expressed in cardiomyocytes in neonatal cells, but there is an increase in {alpha}7B and {alpha}7B(X2), an extracellular domain integrin isoform. The integrin monomeric subtypes, {alpha}1 and {alpha}3, and the heterodimeric integrin receptors, {alpha}1β1 and {alpha}6β1, have also been shown to be down regulated during heart development from the fetal to the adult state [36].

Brancaccio et al. [20] found that the {alpha}6A isoform was the predominant protein expressed during early ventricular heart development, displaying a gradient of increasing expression from the outer to the inner layers of the myocardium [37]. By contrast, in our study levels of {alpha}6A and {alpha}6B were virtually undetectable during fetal development. A transient increase in {alpha}6B was seen shortly after birth, whereas the level of {alpha}6A diminished at the adult stage. The differences between our work and that by Brancaccio et al. [20], may be due to the methods of detection (immunohistochemistry versus RT-PCR), species (mouse versus rat), or cell type (tissue versus primary culture).

In contrast to results in skeletal muscle lines, the {alpha}5β1 fibronectin receptor was not down regulated during the fetal-to-neonatal transition in heart. This is consistent with the results of Terracio et al. [36] who found that fetal and neonatal cardiomyocytes possessed {alpha}1,{alpha}3, and {alpha}5, whereas adult myocytes lacked {alpha}1 and {alpha}5. Presence of the {alpha}-chains correlated with the ability of myocytes to attach to immobilized components of ECM. Neonatal and fetal myocytes attached with high affinity to fibronectin, laminin and collagen. Adult cells did not attach well to collagen type I and fibronectin, and did not possess {alpha}1 and {alpha}5. The present results together with these earlier results clearly indicate that down regulation of the fibronectin receptor in the adult myocyte is caused by decreased expression of the {alpha}5 subunit, since the total amount of integrin β1 appears to increase during development (Fig. 1).

Fibronectin and laminin have been shown to play contrasting roles in skeletal muscle differentiation [22,38–40], which is reflected in opposing roles for their respective receptors, {alpha}5β1 and {alpha}6β1. Ectopic expression of {alpha}5 in myoblasts produces cells that remain proliferative and differentiation inhibited, whereas cells transfected with {alpha}6 do not proliferate but are able to differentiate [22]. It has been concluded from these studies that the ratio of {alpha}5 to {alpha}6 affects the proliferative/differentiation decision and that integrin cytoplasmic domains mediate the required signal. The increase in {alpha}6 integrin relative to {alpha}5 in neonatal cardiomyocytes is consistent with this hypothesis. Also, it has been found that ligation of integrin {alpha}5β1 with fibronectin reverses growth inhibition and gas-1 gene induction with a concomitant activation of immediate early gene transcription in colon carcinoma cells [41]. In ventricular cardiomyocytes a developmentally regulated gene related to gas1 has been identified that blocks the G1/S transition [42]. These results suggest that {alpha}5β1A may play an essential role in the proliferation of fetal cells that is eventually lost during early neonatal development when the laminin receptors, {alpha}6β1D and {alpha}7β1D, become increasingly expressed. A decrease in integrin β1A or an increase in β1D may cause changes in cellular attachment and intracellular signaling that are responsible for the diminished proliferative potential of cardiomyocytes.

The role of multiple {alpha}-chains during development of ventricular cardiomyocytes is difficult to assess completely, but may be the result of rapid changes of the ECM involving fibronectin [43], laminin [44], collagen type IV, and the interstitial collagens [45] during fetal development. Immunological and biochemical data have shown that {alpha}-chains confer integrin ligand specificity towards individual ECM components. Isoforms of {alpha} chains may be important in differentiation of various regions of the heart, and in sustaining the increased hemodynamic load after birth.

In summary, the changes observed in integrin β1 and its associated subunits occur within the same time interval as the switch from p107 to a predominance of p130 and pRb associated with E2F/DP1 [21]. The latter pattern is associated with cell cycle withdrawal or terminal differentiation. Further studies are required to determine the intracellular signaling pathways stimulated by {alpha} subunit isoforms and the accompanying role β1A and β1D play during the withdrawal of cardiomyocytes from the cell cycle.

Time for primary review 31 days.


   Acknowledgements
 
The authors thank Yewen Wu and Anne Fritz for technical assistance. This study was supported by National Institutes of Health Grant PO1-HL 20984, the Gustavus and Louise Pfeiffer Research Foundation, and the Arizona Disease Control Research Commission (Grant No. 9708).


   References
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
 4 Discussion
 References
 

  1. Hayashi Y., Haimovich B., Reszka A., Boettiger D., Horwitz A.F. Expression and function of chicken integrin β1 subunit and its cytoplasmic domain mutants in mouse NIH3T3 cells. J. Cell. Biol. (1990) 110:175–182.[Abstract/Free Full Text]
  2. Zhidkova N.I., Belkin A.M., Mayne R. Novel isoform of β1 integrin in skeletal and cardiac muscle. Biochem. Biophys. Res. Commun. (1995) 214:279–285.[CrossRef][Web of Science][Medline]
  3. Belkin A.M., Zhidkova N.I., Balzac F., Altruda F., Tomatis D., Mair A., et al. β1D integrin displaces the β1A isoform in striated muscles: localization at junctional structures and signaling potential in nonmuscle cells. J. Cell. Biol. (1996) 132:211–226.[Abstract/Free Full Text]
  4. Ross R.S., Pham C., Shai S.-Y., Goldhaber J.I., Fenczik C., Glembotski C.C., et al. β1 integrins participate in the hypertrophic response of rat ventricular myocytes. Circ. Res. (1998) 82:1160–1172.[Abstract/Free Full Text]
  5. Van der Flier, Kuikman I., Baudoin C., Van der Neut R., Sonnenberg A. A novel β1 integrin produced by alternative splicing: unique expression in cardiac and skeletal muscle. FEBS Lett (1995) 369:340–344.[CrossRef][Web of Science][Medline]
  6. Van Kuppevelt T.H.L., Languino J.O., Gailit S., Suzuki S., Ruoslahti E. An alternative cytoplasmic domain of the integrin beta3 subunit. Proc. Natl. Acad. Sci. USA (1989) 86:5415–5418.[Abstract/Free Full Text]
  7. Altruda F., Cervella P., Tarone G., Botta C., Balzac F., Stefanuto G., et al. A human integrin beta subunit with a unique cytoplasmic domain generated by alternative processing. Gene (1990) 95:261–266.[CrossRef][Web of Science][Medline]
  8. Languino L.R., Ruoslahti E. An alternative form of the integrin beta 1 subunit with a variant cytoplasmic domain. J. Biol. Chem. (1992) 267:7116–7120.[Abstract/Free Full Text]
  9. Tamura R.N., Rosso C., Starr L., Chambers J., Reichardt L., Cooper H.M., et al. Epithelial integrin alpha6/beta4: complete primary structure of alpha6 and variant forms of beta4. J. Cell. Biol. (1990) 111:1593–1604.[Abstract/Free Full Text]
  10. Tamura R N., Cooper H.M., Collo Q., Quaranta V. Cell-type specific integrin variants with alternative alpha chain cytoplasmic domains. Proc. Natl. Acad. Sci. USA (1991) 88:10183–10187.[Abstract/Free Full Text]
  11. Hogervorst F., Kuikman I., Van Kessel A.G., Sonnenberg A. Molecular cloning of the human alpha6 integrin subunit: alternative splicing of alpha6 mRNA and chromosomal localization of the alpha6 and beta4 genes. Eur. J. Biochem. (1991) 199:425–433.[Web of Science][Medline]
  12. Velling T., Collo G., Sorokin L., Durbeej M., Zhang H., Gulllberg D., et al. Distinct {alpha}7Aβ1 and {alpha}7Bβ1 integrin expression patterns during mouse development: {alpha}7A is restricted to skeletal muscle but {alpha}7B is expressed in striated muscle, vasculature, and nervous system. Dev Dynamics (1996) 207:355–371.[CrossRef][Web of Science][Medline]
  13. Song W.K., Wang W., Sato H., Bielser D.A., Kaufman S J. Expression of {alpha}7 integrin cytoplasmic domains during skeletal muscle development: alternate forms, conformational change, and homologies with serine/threonine kinases and tyrosine phosphatases. J. Cell. Sci. (1993) 106:1139–1152.[Abstract]
  14. Ziober B.L., Vu M.P., Waleh N., Crawford J., Lin C.-S., Kramer R.H., et al. Alternative extracellular and cytoplasmic domains of the integin {alpha}7 subunit are differentially expressed during development. J. Biol. Chem. (1993) 268:26773–26783.[Abstract/Free Full Text]
  15. Hynes R.O. Integrins: a family of cell surface receptors. Cell (1987) 48:549–554.[CrossRef][Web of Science][Medline]
  16. Ruoslahti E. Integrins. J. Clin. Invest. (1991) 87:1–5.[Web of Science][Medline]
  17. Fang F., Orend G., Watanabe N., Hunter T., Ruoslahti E. Dependence of cyclin E-cdk2 kinase activity on cell anchorage. Science (1996) 271:499–502.[Abstract]
  18. Zhu X., Ohtsubo M., Bohmer R., Roberts J.M., Assoian R.K. Adhesion dependent cell cycle progression linked to the expression of cyclin D1, activation of cyclin E-cdk2 activity, and phosphorylation of the retinoblastoma protein. J. Cell. Biol. (1996) 133:391–403.[Abstract/Free Full Text]
  19. Wary K.K., Mainiero F., Isakoff S.J., Marcantonio E.E., Giancotti F.G. The adaptor protein Shc couples a class of integrins to the control of cell cycle progression. Cell (1996) 87:733–743.[CrossRef][Web of Science][Medline]
  20. Brancaccio M., Cabodi S., Belkin A.M., Collo G., Koteliansky V.E., Tomatis D., et al. Differential onset of expression of {alpha}7 and β1D integrins during mouse heart and skeletal muscle development. Cell. Adhes. Commun. (1998) 5:193–205.[Web of Science][Medline]
  21. Flink I.L., Oana S., Maitra N., Bahl J.J., Morkin E. Changes in E2F complexes containing retinoblastoma protein family members and increased cyclin-dependent kinase inhibitor activities during terminal differentiation of cardiomyocytes. J. Mol. Cell. Cardiol. (1998) 30:563–578.[CrossRef][Web of Science][Medline]
  22. Shin E.K., Shin A., Paulding C., Schaffhausen B., Yee A.S. Multiple change in E2F function and regulation occur upon muscle differentiation. Mol. Cell. Biol. (1995) 15:252–2262.
  23. Zhao J., Araki N., Nishimoto S.K. Quantitation of matrix Gla protein mRNA by competitive polymerase chain reaction using glyceraldehyde-3-phosphate dehydrogenase as an internal control. Gene (1995) 155:159–165.[CrossRef][Web of Science][Medline]
  24. Slepian M.J., Massia S.P., Dehdashti B., Fritz A., Whitesell L. β3-Integrins rather than β1-integrins dominate integrin-matrix interactions involved in postinjury smooth muscle cell migration. Circulation (1998) 97:1818–1827.[Abstract/Free Full Text]
  25. Sastry S.K., Lakonishok M., Thomas D.A., Muschler J., Horwitz A F. Integrin {alpha} subunit ratios, cytoplasmic domains, and growth factor synergy regulate muscle proliferation and differentiation. J. Cell. Biol. (1996) 133:169–184.[Abstract/Free Full Text]
  26. Hilenski L.L., Ma X.H., Vinson N., Terracio L., Borg T.K. The role of beta 1 integrin in spreading and myofibrillogenesis in neonatal rat cardiomyocytes in vitro. Cell. Motil. Cytoskeleton (1992) 21:87–100.[CrossRef][Web of Science][Medline]
  27. Bader D., Masaki T., Fischman D.A. Immunochemical analysis of myosin heavy chain during avian myogenesis in vivo. J Cell Biol (1982) 95:763–770.[Abstract/Free Full Text]
  28. Turner C.E., Glenney J.R., Burridge K. Paxillin: a new vinculin-binding protein present in focal adhesions. J. Cell. Biol. (1990) 111:1059–1068.[Abstract/Free Full Text]
  29. Horwitz A., Duggan K., Greggs R., Decker C., Buck C. The cell substrate attachment (CSAT) antigen has properties of a receptor for laminin and fibronectin. J. Cell. Biol. (1985) 101:2134–2144.[Abstract/Free Full Text]
  30. Menko A.S., Boettiger D. Occupation of the extracellular matrix receptor, integrin, is a control point for myogenic differentiation. Cell (1987) 51:51–57.[CrossRef][Web of Science][Medline]
  31. Meredith J. Jr., Takada Y., Fornaro M., Languino L.R., Schwartz M.A. Inhibition of cell cycle progression by the alternatively spliced integrin β1C. Science (1995) 269:1570–1571.[Abstract/Free Full Text]
  32. Baudoin C., Goumans M.J., Mummery C., Sonnenberg A. Knockout and knockin of the beta1 exon D define distinct roles for integrin splice variants in heart function and embryonic development. Genes Dev (1998) 12:1202–1216.[Abstract/Free Full Text]
  33. Fässler R., Rohwedal J., Maltsev V., Bloch W., Lentin S., Guan K., et al. Differentiation and integrity of cardiac muscle cells are impaired in the absence of β1 integrin. J. Cell. Sci. (1996) 09:2989–2999.
  34. Blaschuk K.L., Holland P.C. The regulation of {alpha}5β1 integrin expression in human muscle cells. Dev Biol (1994) 164:475–483.[CrossRef][Web of Science][Medline]
  35. Volk T., Fessler L.I., Fessler J.H. A role for integrin in the formation of sarcomeric cytoarchitecture. Cell (1990) 63:525–536.[CrossRef][Web of Science][Medline]
  36. Terracio L., Rubin K., Gullberg D., Balog E., Carver W., Jyring R., et al. Expression of collagen binding integrins during cardiac development and hypertrophy. Circ. Res. (1991) 68:734–744.[Abstract/Free Full Text]
  37. Collo G., Domanico S.Z., Klier G., Quaranta V. Gradient of {alpha}6A distribution in the myocardium during early heart development. Cell Adhes Commun (1995) 3:101–113.[Web of Science][Medline]
  38. Gullberg D., Sjöberg G., Velling T., Sejersen T. Analysis of fibronectin and vitronectin receptors on human fetal skeletal muscle cells upon differentiation. Exp. Cell. Res. (1995) 20:112–123.
  39. Garcia A.J., Vega M.D., Boettiger D. Modulation of cell proliferation and differentiation through substrate-dependent changes in fibronectin conformation. Mol Biol Cell (1999) 10:785–798.[Abstract/Free Full Text]
  40. Von der Mark K., Ocalan M. Antagonist effects of laminin and fibronectin on the expression of the myogenic phenotype. Differentiation (1989) 40:150–157.[Web of Science][Medline]
  41. Varner J.A., Emerson D.A., Juliano R.L. Integrin {alpha}5β1 expression negatively regulates cell growth: reversal by attachment to fibronectin. Mol. Biol. Cell (1995) 6:725–740.[Abstract]
  42. Zhu H., Qingquan L., Haujun Y., Dawes N.J., Tzeng J. A novel growth suppressor blocks G1/S transition in fetal rat ventricular cardiomyocytes. Circulation (1996) 54(Suppl_I):I298.
  43. Takeda Y., Elices M.J., Crouse C., Hemler M.E. The primary structure of the {alpha}4 subunit of VLA-4: Homology to other integrins and a possible cell-cell adhesion function. EMBO J. (1987) 8:1361–1368.
  44. Sonnenberg A., Modderman P.W., Hogervorst F. Laminin receptor on platelets is the integrin VLA-6. Nature (1988) 336:487–489.[CrossRef][Medline]
  45. Wayner E.A., Carter W.G. Identification of multiple cell adhesion receptors for collagen and fibronectin in human fibrosarcoma cells possessing unique {alpha} and β subunits. J. Cell. Biol. (1987) 105:1873–1884.[Abstract/Free Full Text]

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