ß₁ Integrins Participate in the Hypertrophic Response of Rat Ventricular Myocytes

From the Departments of Physiology (R.S.R., C.P., S.-Y. S.), Medicine (R.S.R., C.P., S.-Y. S., J.I.G.), and The Cardiovascular Research Laboratories, UCLA School of Medicine, Los Angeles, Calif; Department of Vascular Biology (C.F., M.H.G.), Scripps Research Institute, La Jolla, Calif; The Molecular Biology Institute and Department of Biology, San Diego State University (C.C.G.), San Diego, Calif; and Mayo Clinic Scottsdale (J.C.L.), Scottsdale, Ariz.

Correspondence to Robert S. Ross, Department of Physiology, University of California–Los Angeles School of Medicine, Center for the Health Sciences, Room 53–231, 10833 Le Conte Ave, Los Angeles, CA 90095-1751. E-mail rross{at}physiology.medsch.ucla.edu

	Abstract

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Abstract—Multiple signaling pathways have been implicated in the hypertrophic response of ventricular myocytes, yet the importance of cell-matrix interactions has not been extensively examined. Integrins are cell-surface molecules that link the extracellular matrix to the cellular cytoskeleton. They can function as cell signaling molecules and transducers of mechanical information in noncardiac cells. Given these properties and their abundance in cardiac cells, we evaluated the hypothesis that ß₁ integrin function is involved in the ₁-adrenergic mediated hypertrophic response of neonatal rat ventricular myocytes. The hypertrophic response of this model required interaction with extracellular matrix proteins. Specificity of these results was confirmed by demonstrating that ventricular myocytes plated onto an anti–ß₁ integrin antibody supported the hypertrophic gene response. Adenovirus-mediated overexpression of ß₁ integrin augmented the myocyte hypertrophic response when assessed by protein synthesis and atrial natriuretic factor production, a marker gene of hypertrophic induction. DNA synthesis was not altered by integrin overexpression. Transfection of cultured cardiac myocytes with either the ubiquitously expressed ß_1A integrin or the cardiac/skeletal muscle–specific ß₁ isoform (ß_1D) activated reporter expression from both the atrial natriuretic factor and myosin light chain-2 ventricular promoters, genetic markers of ventricular cell hypertrophy. Finally, suppression of integrin signaling by overexpression of free ß₁ integrin cytoplasmic domains inhibited the adrenergically mediated atrial natriuretic factor response. These findings show that integrin ligation and signaling are involved in the cardiac hypertrophic response pathway.

Key Words: hypertrophy • myocardium • extracellular matrix • integrin • myocyte

	Introduction

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Mechanical stress on the heart from either pressure or volume loading can cause hypertrophy of the myocardium to optimize cardiac performance. Results from in vivo studies and cell culture models have indicated that morphological hypertrophy coincides with a cascade of intracellular biochemical signals.^1–8 These signals include rapid expression of immediate-early genes (eg, c-fos and c-jun), reexpression of fetal genes or isoforms (ANF¹, -skeletal actin, -ß myosin heavy chain isoform switching), or downregulation of calcium regulatory genes (sarcoplasmic reticulum calcium ATPase). However, the mechanisms by which hypertrophic stimuli, including mechanical stress, lead to signaling events in the cardiac cell are poorly understood.

The ECM influences cardiac myocyte morphology.⁹ Furthermore, components of the ECM, such as fibronectin^10,11 or collagens I and III,¹¹ are upregulated with in vivo hypertrophy. Integrins comprise a large family of heterodimeric cell-surface receptors that link the ECM and the intracellular cytoskeleton.¹² Although integrins were initially thought of solely as molecules necessary for adhesive interactions between cells and the ECM, recent work has indicated that integrins are bidirectional signaling molecules.¹³ Integrins transduce their biochemical signals via tyrosine phosphorylation of intracellular proteins, including FAK. Intracellular events modulated by integrins include immediate-early gene induction,¹⁴ modification of intracellular pH,¹⁵ cytosolic Ca²⁺,¹⁶ and activation of p21Ras, MAP, and MAP kinases.^17,18 In noncardiac cells, integrins can act as mechanotransduction receptors, and their stimulation has been shown to modulate cellular growth and gene expression.¹⁹ In the cardiac cell, expression of several members of the ß₁ integrin family has been identified, including ₁, ₃, ₅, ₆, and _7B.²⁰ Notably, upregulation of ₁, ₅, and ß₁ integrin protein levels is seen after induction of hypertrophy in the adult rat, suggesting that integrins play a role in this process.²¹ Recent observations that components of the integrin signaling cascade are modulated after pressure overload of the cat right ventricle further support this concept.²²

Adrenergic stimulation of primary cultures of NRVMs induces a hypertrophic response biochemically and morphologically similar to that which occurs in the intact heart.²³ Numerous groups have used this in vitro model to study hypertrophic signaling events of the cardiac myocyte.^24–26 When stimulated by PE, these cells show increases in protein synthesis, larger cellular spread area and volume, and induction of various markers of hypertrophy, including ANF and MLC-2_V. Furthermore, like their endogenous genes, induction of chimeric promoter/reporter plasmids such as ANF (3100 bp)/luciferase or MLC-2_V (2700 bp)/luciferase is also seen after adrenergic stimulation.^25,27

Because ß₁ integrins are abundant in the cardiac cell, we examined the regulatory role of ß₁ integrins in the hypertrophic response of adrenergically stimulated NRVMs. We report that the hypertrophic response was dependent on cell-ECM interaction. Overexpression of ß₁ integrins in the cardiac myocyte increased hypertrophic marker gene expression and protein synthesis but had no effect on cell DNA synthesis. Attachment of myocytes to substrate via antibody ligation of ß₁ integrins was sufficient to allow hypertrophic marker gene induction, suggesting a requirement for ß₁ integrins in this response pathway. Furthermore, both ß₁ integrin isoforms present in the heart, including the skeletal/cardiac muscle–specific isoform ß_1D, modulated hypertrophic marker genes. Finally, inhibition of integrin signaling downregulated the adrenergically stimulated hypertrophic gene response. Taken together, these results suggest that integrins play a significant role in the hypertrophic response of cardiac myocytes.

	Materials and Methods

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Cell Culture
NRVMs from ventricles of 1- to 2-day-old Sprague-Dawley rats were cultured as previously described.²⁸ Cell cultures with >95% myocytes, as assessed by immunofluorescence with MLC-2_V antisera, were obtained by discontinuous Percoll gradient purification. Myocytes were plated on various substrata as indicated at a density of 300 cells/mm². Plates were coated overnight with substrates at 4°C and then blocked with 2% BSA for 2 hours before plating. Cells were cultured in serum-free media containing antibiotics (ampicillin 34 µg/mL and gentamicin 3 µg/mL) and L-glutamine (2 mmol/L) or antibiotics/glutamine plus 100 µmol/L PE. After isolation of the NRVMs, cells were plated and either maintained in the uninfected state or infected with various recombinant adenoviral constructs as noted.

To plate NRVMs on antibody-coated wells, cells were isolated as above and maintained in serum-free media on 2% BSA–coated plates, either with or without viral infection, for 24 hours. Cells were then transferred to new culture plates coated with the anti-human ß₁ antibody P5D2, control antibodies, or polylysine at concentrations of 10 µg/mL. Cells were maintained for an additional 36 hours in either serum-free medium or serum-free medium supplemented with 100 µmol/L PE.

Transformed 293 human embryonic kidney cells (ATCC No. CRL-1573) and CHO cells (ATCC No. CCL-61) were cultured as advised by the supplier.

Volume Measurements
Volumes were determined by use of a modification of the method of Satoh et al.²⁹ Briefly, cells were cultured onto fibronectin-coated coverslips and incubated in appropriate media for 36 hours. Cells were then loaded for 20 minutes at room temperature in serum-free medium containing 10 µmol/L calcein acetoxymethylester and 25% wt/wt pluronic (Molecular Probes). Cells were then washed, and digital confocal images were acquired by use of an Odyssey XL laser-scanning confocal imaging system (Noran Instruments) attached to a Zeiss Axiovert 100 TV inverted microscope fitted with a Zeiss C-Apochromat 40x water immersion objective lens (1.2 numerical aperture). Optical sections through the full depth of cells were obtained at 0.75-µm intervals. We minimized bleaching by attenuating the laser light to <10 mW and by exposing the specimen to light only during focusing and acquisition. Light was shuttered during movement of the objective lens through the Z-series. Slit width was set to 15 nm to achieve maximum confocalization, and pixel dwell was set to 1600 ns. No frame averaging was used. Images were reconstructed with the use of a Silicon Graphics Indy workstation using Intervision 1.5 software (Noran Instruments). Image size was set to 640x480 pixels, and pixel area was calibrated by use of a stage micrometer. Pixel resolution for the system was 0.19 µm. Offset and gain of the photomultiplier were chosen to represent noncellular background near zero and fluorescence intensities from cellular elements to cover the full range of 256 gray levels. Cell volumes were determined off-line. Elimination of out-of-focus fluorescence and noncellular contributions from voxels at the cell border was achieved by photometric thresholding of the fluorescence images. The lower level of thresholding was determined from a central section of the cardiac myocyte and was defined as the lowest level that included all cellular voxels but that excluded >99% of extracellular voxels. The central section was used to calculate cell area. The same threshold values were then applied to all the optical sections in the Z-series from a single myocyte. Three-dimensional reconstruction, cell volume, and cell areas were determined by use of Intervision software.

cDNAs and Antibodies
A full-length human ß_1A integrin cDNA was obtained from Y. Takada (The Scripps Research Institute). To construct the ß_1D expression vector, the ß_1D cytoplasmic domain was amplified from human heart mRNA by reverse-transcriptase polymerase chain reaction with the use of primers based on published sequences.³⁰ The resulting fragment was cloned as an HindIII-BamHI fragment into pcDNA3 (Invitrogen). An HindIII fragment containing the extracellular and transmembrane portions of ß₁ was cloned 5' of the ß_1D cytoplasmic domain. The resulting full-length ß_1D clone was assayed for protein expression in CHO cells by fluorescent activated cell sorting. Chimeric constructs consisting of the extracellular and transmembrane domains of the TAC subunit of the human IL-2 receptor fused to the cytoplasmic domain of ß_1A (TAC-ß_1A) or integrin ₅ (TAC-₅) were the kind gift of S. LaFlamme and K. Yamada (National Institute of Dental Research, National Institutes of Health, Bethesda, Md).³¹ Reporter gene constructs consisting of a 3003-bp rat ANF promoter or a 2700-bp rat MLC-2_V promoter fused to a firefly luciferase cDNA have been described previously.^27,32 The anti-human ß₁ monoclonal antibodies P5D2 and 102DF5 were obtained from the Developmental Studies Hybridoma Bank at the University of Iowa, Iowa City and from I. Virtanen (University of Helsinki, Helsinki, Finland), respectively. Anti-myosin monoclonal antibody MF-20 was also obtained from the Developmental Studies Hybridoma Bank. The monoclonal anti-human IL-2 receptor antibody 7G7B6 was obtained from the ATCC (No. HB-8784). Rabbit polyclonal anti-rat ANF and sheep anti-BrdU were obtained from Research and Diagnostic Antibodies. Monoclonal anti-ANF (102–126) has been described previously.³³ Rhodamine-conjugated phalloidin was obtained from Molecular Probes. DAPI was from Sigma Chemical Co. FITC, rhodamine, and Cy-5–labeled secondary antibodies were obtained commercially (Jackson ImmunoResearch Labs, Inc).

Recombinant Adenoviral Expression Constructs
The full-length ß_1A fragment was cloned into the BamHI site of the E1-deficient shuttle vector pacCMVpLpA.³⁴ TAC-₅ and TAC-ß_1A cDNAs were excised from the parent vector with SnaB1 and XbaI and ligated into pacCMVpLpA digested with these same restriction enzymes. pHCMVsp1LacZ was obtained from Dr F.L. Graham (McMaster University, Hamilton, Ontario, Canada). Construct integrity was confirmed by restriction enzyme and sequencing analyses.

Constructs in pacCMVpLpA vectors were cotransfected by the standard calcium-phosphate technique³⁵ with the adenoviral plasmid JM17 into the E1-transformed cell line 293.³⁶ Virus was clonally isolated. Recombination was verified by polymerase chain reaction analysis with oligonucleotide primer sets present in the adenoviral sequences, the foreign gene of interest, or both. Viral production of recombinant protein was assayed by infection of CHO or NRVM cells for 48 hours, followed by immunostaining or flow cytometry. All viral stocks were titered by use of plaque assays. Cells were infected at matched multiplicities of infection ranging from 1 to 50.

Immunofluorescent Studies
Cellular immunostaining was performed as described previously.³⁷ Microscopic analysis was performed with either a Nikon Diaphot microscope equipped with epifluorescent optics alone or as a component in a Bio-Rad MCR-1000 Laser Scanning Confocal Microscope System (Bio-Rad Laboratories).

Measurement of Protein Content/Synthesis and ANF Peptide
Protein content was determined by use of a Lowry assay,³⁸ whereas relative synthetic rates were assessed as described previously.³⁹ ANF secretion from NRVMs was assessed by radioimmunoassay of media collected from each culture condition, as described previously.⁴⁰

Monitoring of DNA Synthesis
Assessment of cellular DNA synthesis was performed as described previously with minor modification.⁴¹ BrdU at a final concentration of 10 µmol/L was added to the control or infected cultures for the final 16 hours of the culture period. Immunofluorescent staining was performed as described above, with DAPI used to locate all cell nuclei, anti-myosin antibody MF20 to localize myocytes, and an anti-BrdU antibody to evaluate BrdU incorporation into the cells. BrdU-positive myocytes were scored by visual determination of the number of BrdU-positive cells that also stained positively with the anti-myosin antibody. Scoring of control, control-infected, and integrin-infected groups was then compared.

Flow Cytometry
Surface expression of full-length ß₁ or TAC chimeras in NRVMs was analyzed by flow cytometry with specific antibodies as described previously.⁴² Briefly, 5x10⁵ cells were incubated on ice for 30 minutes with primary antibody, washed, and then incubated on ice for an additional 30 minutes with fluorescein-conjugated goat anti-mouse second antibody (Tago). Cells were washed, resuspended in PBS/1% paraformaldehyde, and fixed for 30 minutes at 4°C in the dark. They were then pelleted, resuspended, and analyzed on a FACScan (Becton Dickinson).

Transfection
Primary cultures of myocytes were cotransfected with full-length ß_1A or ß_1D and luciferase reporter constructs by the calcium-phosphate technique as described previously.⁴³ Luciferase reporter activity was determined in a luminometer (Analytical Luminescence) as previously described,²⁷ and luciferase activity was normalized to cellular protein concentration.

Statistical Analyses
Student's t test was performed, and a value of P<0.05 was considered significant.

	Results

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Validation of the Adrenergically Stimulated NRVM as a Model of Cardiac Hypertrophy
The initial studies that measured cellular area and volume of the adrenergically induced rat ventriculocyte were performed on trypsinized myocytes.²³ To validate that adrenergic stimulation causes hypertrophy of the intact, living neonatal ventricular cell, we used laser-scanning confocal microscopy to obtain cellular volumes and areas of myocytes plated on fibronectin.²⁹ Cells were loaded with the fluorescent indicator calcein, which distributes with cell water. Confocal images were recorded from above the cell, through the entire depth of the cell, to below where the cell attached to a fibronectin-coated glass coverslip. Three-dimensional reconstructions of the recorded images were rendered using the three-dimensional module of the Intervision program as noted in Methods, with appropriate photometric thresholding of the image. Analysis indicated a significant increase in cell volume for the adrenergically stimulated cells compared with untreated cells (P<0.035, Table). Planar area measurements were also made and revealed a significant correlation of volume versus area (r²=0.88, P<0.025; Table). These studies provide the first direct measurement of cell volume and area of the living, adrenergically stimulated NRVM and validate the adrenergically stimulated cell model as one of in vitro hypertrophy.

Adrenergic Stimulation of ANF Expression by NRVMs Requires ECM
Because PE stimulation caused cellular hypertrophy of cultured NRVMs, we used this model to examine the effect of attachment to ECM proteins on the hypertrophic response. Previous data from our laboratory and others have shown that ANF is a marker of the hypertrophic phenotype.^27,44 Freshly isolated NRVMs were plated onto dishes coated with fibronectin (10 µg/mL), laminin (10 µg/mL), or BSA (2 mg/mL). Cells were incubated in either serum-free medium or serum-free medium plus 100 µmol/L PE and, after 36 hours of stimulation, were fixed and immunostained with antibodies for MLC-2_v, a myocyte-specific marker, and ANF (Figure 1). ANF induction, as assessed via immunofluorescent staining, was observed in cells attached to fibronectin or laminin but not in cells plated on BSA. Similar results were obtained when ANF secretion was assessed by radioimmunoassay data not shown). These results indicate that ANF induction requires both adrenergic stimulation and myocyte adhesion to ECM proteins.

View larger version (40K): [in this window] [in a new window]   Figure 1. Attachment substrate influences expression of ANF, a marker of the hypertrophic phenotype in NRVMs. NRVMs were plated onto dishes coated with fibronectin, laminin, or BSA in either serum-free medium or serum-free medium plus 100 µmol/L PE. After 36 hours of stimulation, cells were fixed and immunostained as discussed in Methods. More than 300 cells from each group were observed, and the number of ANF-positive myocytes (compared with MLC-2v–positive cells, a myocyte-specific marker) was determined. Observations were repeated in duplicate from 3 separate experiments. Induction of ANF was only detected in cells plated onto fibronectin or laminin compared with BSA (P<0.005 versus BSA).

ß₁ Integrin Overexpression Augments Adrenergic Induction of the Hypertrophic Program
Because myocyte-matrix interaction was important for the adrenergically mediated ANF response, we examined the capability of integrins to modulate this response program. Previous studies have shown that ß₁ integrins are abundant in the neonatal rat ventriculocyte.²¹ To assess the effect of ß₁ overexpression on NRVM hypertrophic signaling, a recombinant adenoviral vector was constructed to express the human ß_1A integrin subunit. The human ß₁ subunit is similar to ß₁ integrins from other species, including rat,⁴⁵ and dimerization of integrin subunits from divergent species (eg, mouse and chick) has been observed previously.⁴⁶ Human ß₁ was differentiated from the endogenous rat ß₁ with species-specific antibodies. A ß-galactosidase–expressing recombinant adenovirus served as a control virus for these experiments.

NRVMs were plated onto fibronectin-coated slides and infected with control or integrin viruses. Overexpression of ß₁ integrin in cells maintained in serum-free medium modestly increased the number of ventricular cells that expressed ANF (green perinuclear staining in Figure 2A compared with Figure 2C). PE stimulation of the cells in combination with ß₁ overexpression visibly increased the area that individual cells spread across and augmented the intensity and number of cells with ANF staining compared with PE stimulation alone (Figure 2B versus 2D). Enumeration of the myocytes with perinuclear ANF staining confirmed these qualitative results (Figure 2E). To quantitatively assess effects of integrin expression on cellular growth, relative rates of protein synthesis were measured by incorporation of L-[³H]phenylalanine.³⁹ Protein synthesis was increased by ß₁ integrin expression in both the presence and absence of PE (Figure 3A). Whereas control infection caused an increase in relative protein synthesis compared with uninfected cells, ß₁ integrin overexpression augmented protein synthesis significantly above this control infection (P<0.05). Furthermore, overexpression of ß₁ integrin also resulted in increased ANF secretion by NRVMs in the presence and absence of adrenergic stimulation (Figure 3B). Notably, ß₁ overexpression in combination with PE stimulation resulted in synergistic augmentation of ANF secretion. We also determined whether ß₁ integrin overexpression would alter DNA synthesis of NRVM. No increase in BrdU incorporation was evidenced in myocytes infected with the ß₁ integrin virus compared with uninfected cells or titer-matched control-infected cells, either with or without the presence of adrenergic stimulation (data not shown). Thus, as assessed by qualitative and quantitative assays, ß₁ integrin overexpression augmented the hypertrophic response to PE and did not alter cell replication.

View larger version (53K): [in this window] [in a new window]   Figure 2. ANF expression is increased by overexpression of ß1 integrin. Cells were cultured on fibronectin and infected with a recombinant adenovirus expressing ß1A integrin (A and B) or maintained in the uninfected state (C and D). As also indicated, they were cultured in serum-free medium alone (A, C) or in the presence of 100 µmol/L PE (B, D). After culture, cells were fixed and immunostained to detect ß1 integrin (red; lissamine-rhodamine staining) and ANF (green; FITC staining) expression. ß1 integrin overexpression in the unstimulated cells (A) only modestly increased ANF expression by NRVMs, yet a combination of ß1 integrin overexpression with adrenergic stimulation (B) resulted in both greater spreading and ANF expression by myocytes than by uninfected cells (C, D) or cells infected with matched concentrations of control viruses (not shown). E, Immunostaining was performed with an ANF polyclonal antibody, and expression of ANF in myocytes was visibly determined. The percentage of ventricular cells that expressed ANF was increased by ß1 integrin overexpression compared with uninfected or control (lacZ) infected conditions. *P<0.001 versus no infection, serum-free medium. #P<0.001 versus no infection or control infection, serum-free medium+100 µmol/L PE. Initial cell plating density was identical for all panels. n=5–8 experiments performed in triplicate.

View larger version (26K): [in this window] [in a new window]   Figure 3. Overexpression of ß1 integrin results in increased protein synthesis and ANF secretion by rat ventricular myocytes. Relative rates of protein synthesis were measured by [H3]phenylalanine incorporation and ANF secretion by radioimmunoassay. A, Relative rates of protein synthesis presented as mean±SEM of 3 separate experiments. B, Relative ANF secretion as assessed by radioimmunoassay. ß1 overexpression increased protein synthesis and ANF secretion in cells maintained in serum-free medium. When infection was combined with PE stimulation, overexpression of ß1 integrin synergistically increased relative protein synthesis and ANF secretion compared with other groups. A and B, *P<0.05 versus no infection; #P<0.05 versus lacZ control.

Direct Ligation of ß₁ Integrins Augments Adrenergic Induction of the Hypertrophic Program
The above results show that integrin overexpression modulates myocyte protein synthesis and ANF production when cells are attached to fibronectin. To prove that this effect occurs as a result of ß₁ integrin ligation, we examined whether cells attached to a ß₁ integrin antibody–coated substrate could initiate ANF expression. Myocytes that had been infected with a human ß₁ integrin recombinant adenovirus attached and spread onto anti-human ß₁ antibody–coated plates (Figure 4, A and B). Few of the uninfected or lacZ-infected cells attached to the anti-human ß₁–coated plates, and none of these cells spread; no cells attached to the control antibody-coated plates data not shown). After 48 hours in culture, cells were fixed and immunostained to detect both ß₁ integrin and ANF expression. The number of cells expressing both ANF and human ß₁ integrin was determined. Addition of PE augmented cell spreading and induced an ANF response in cells attached to this anti-human ß₁ antibody (Figure 4C and 4D; Figure 5). This response was dependent on integrin ligation rather than cell adhesion per se, because adhesion to polylysine-coated plates failed to support ANF expression, and cells showed no significant spreading on this substrate (Figure 5).

View larger version (102K): [in this window] [in a new window]   Figure 4. Ligation of ß1 integrin results in cell spreading and ANF induction. NRVMs that had been infected with a human ß1 integrin recombinant adenovirus were attached to plates coated with an anti-human ß1 integrin antibody. A and B, Phase-contrast microscopy after cell capture onto the antibody. C and D, Immunofluorescent staining of cells for virus (red; rhodamine) and ANF (green; FITC). Serum-free conditions (A, C) permitted cell spreading and ANF induction, both of which were augmented when the cells were also stimulated with 100 µmol/L PE (B, D).

View larger version (29K): [in this window] [in a new window]   Figure 5. ANF induction by PE in neonatal ventricular cells requires integrin engagement. NRVMs infected with a human ß1 integrin recombinant adenovirus were captured onto ß1 antibody–coated plates or nonspecifically onto polylysine-coated dishes. Induction of ANF expression in cells stimulated by PE was effective only when integrins were engaged by antibody capture but not by attachment to polylysine. n=>300 cells from 3 independent experiments. Results are mean±SEM.

ß_1A and ß_1D Isoforms Augment Promoter Activity of ANF and MLC-2_v Genes Stimulated by PE
The ß_1D isoform of ß₁ integrin is preferentially expressed in skeletal and cardiac muscle.^30,47 This isoform differs from ß_1A in the sequence of its cytoplasmic domain. The role of ß_1D in the cardiac hypertrophic response pathway was examined by cotransfection experiment with reporter constructs. Similarly to their respective endogenous genes, activity of ANF (3003 bp) and MLC-2_v (2700 bp) luciferase reporter constructs is induced by pharmacological or other treatments that evoke the hypertrophic response in NRVMs.²⁷ Therefore, we cotransfected full-length ß_1A or ß_1D expression vectors with either ANF or MLC-2_v–luciferase plasmids. Coexpression of either the ß_1A or ß_1D expression vector, along with adrenergic stimulation, caused significant augmentation of luciferase activity of both ANF and MLC-2_v, with no significant difference between the two ß₁ isoforms (Figure 6). These results confirm that ß₁ integrin overexpression can modulate the expression of ANF as well as a second marker (MLC-2_v) of the hypertrophic phenotype. Moreover, these results indicate that both the A and D isoforms can act on the hypertrophic signaling pathway.

View larger version (31K): [in this window] [in a new window]   Figure 6. ß1A and ß1D isoforms augment promoter activity of ANF and MLC-2v genes stimulated by PE. NRVMs were transfected with ANF-luciferase or MLC-2v–luciferase reporter plasmids and either control, ß1A, or ß1D integrin expression vectors. After cotransfection, cells were incubated for an additional 36 hours in serum-free medium or serum-free medium+100 µmol/L PE. Luciferase activity was normalized to micrograms of protein for each sample. Data are mean±SEM for 3 to 9 independent samples. Both ß1 integrin isoforms significantly augmented baseline adrenergic induction of ANF and MLC-2V promoters. No significant differences were noted between ß1A and ß1D isoforms. *P<0.05 relative to control/PE.

Overexpression of Isolated ß₁ Integrin Cytoplasmic Domains Inhibits Adrenergic Induction of ANF in NRVMs
To examine the role of integrin signaling in the hypertrophic gene response pathway, we sought to alter integrin-mediated signaling in the cardiac myocyte. Previous studies have shown that overexpression of free ß₁ integrin cytoplasmic domains disrupts bidirectional integrin signaling in noncardiac cells.^48–50 An adenoviral construct that expressed the TAC-ß_1A chimera was used for this purpose. A similarly constructed TAC-₅ virus served as a control. PE stimulation of uninfected cells or cells infected with the TAC-₅ chimera induced ANF expression (Figure 7, A through D). In contrast, cells infected with the TAC-ß_1A chimera expressed little ANF after adrenergic stimulation (Figure 7, E and F). Expression of TAC-ß_1A did not affect cell spreading or organization as assessed by actin immunostaining. The effect of TAC-ß_1A expression on PE-induced secretion of ANF protein by myocytes was quantified by radioimmunoassay (Figure 8). Expression of TAC-ß_1A depressed ANF secretion, whereas infection with an equal quantity of the control TAC-₅ virus had no effect. This inhibitory effect could not be offset by an increased dose of the adrenergic agonist, because raising the PE concentration 10-fold to 10^-3 mol/L resulted in no change in cellular ANF in NRVMs, as assessed by both direct cellular immunostaining and radioimmunoassay of ANF secretion (data not shown). Increases in PE concentration in the medium >10^-3 mol/L were cytotoxic. The TAC-ß_1A mutant did not cause generalized depression of immunolocalizable proteins. As shown in Figure 9, expression of YB-1, a DNA-binding protein critical for normal transcription of the contractile protein MLC-2_v,⁵¹ is unchanged by infection with the TAC-ß_1A virus. Thus, disruption of integrin-mediated signaling can significantly alter adrenergic induction of ANF.

View larger version (137K): [in this window] [in a new window]   Figure 7. Adrenergic induction of ANF expression by NRVMs is disrupted by infection with a recombinant adenovirus expressing a dominant-negative ß1 integrin. NRVMs were plated onto a fibronectin-coated substrate and maintained in either serum-free medium (A, C, and E) or in serum-free medium plus 100 µmol/L PE (B, D, and F). Groups were then maintained in the uninfected state (A, B) or infected with either TAC-5 control virus (C, D) or TAC-ß1A virus, which alters integrin signaling (E, F). After culture, cells were fixed and immunostained with antibodies to F-actin (red; lissamine-rhodamine staining), virus (green; FITC staining), and ANF (blue; CY-5 staining). Immunofluorescence was detected with confocal microscopy. Although significant numbers of cells in either uninfected or TAC-5 virus groups had induction of ANF by PE (B versus A, D versus C), essentially no ANF induction could be detected in TAC-ß1A–infected cells (F versus E) despite adequate cellular spreading and organization.

View larger version (32K): [in this window] [in a new window]   Figure 8. ANF secretion by adrenergically stimulated rat ventriculocytes is reduced by alteration of integrin signaling. Cells were cultured as described in the text, and medium was assayed by radioimmunoassay for ANF protein. ANF secretion is markedly reduced in the TAC-ß1A group compared with the uninfected or control (TAC-5) virally infected cells, despite stimulation with PE.

View larger version (54K): [in this window] [in a new window]   Figure 9. Dominant-negative ß1 integrin infection of NRVMs has no effect on YB-1 expression. Cells were cultured in serum-free medium plus 100 µmol/L PE and harvested after 48 hours in culture/infection. Cells were fixed and stained for virus (red; lissamine rhodamine) and YB-1 (green; FITC). A, TAC-ß1A–infected cells; B, uninfected cells. Despite significant expression of the TAC-ß1A mutant virus, cells spread and had high-level expression of YB-1, a binding protein important for regulation of MLC-2V transcription.

	Discussion

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In the present study, we report that integrin-mediated adhesion and signaling play significant roles in the cardiac hypertrophic response pathway. The major findings are as follows: First, adhesion of ventricular myocytes to ECM proteins was necessary for adrenergic induction of ANF, a key marker of the hypertrophic phenotype. Second, overexpression of ß₁ integrins caused upregulation of ANF expression and secretion, as well as protein synthesis, both indicators of hypertrophy in the NRVM model. No alteration of DNA synthesis was found. Third, these myocyte responses required ß₁ integrins, because antibody ligation of ß₁ integrins induced ANF expression. Fourth, both the ubiquitously expressed ß_1A isoform and the skeletal and cardiac muscle–specific ß_1D isoform modulated promoter activity of 2 markers of the hypertrophic phenotype in NRVMs. Finally, disruption of integrin signaling in myocytes blocked PE-induced ANF expression.

Adrenergic stimulation of NRVMs has been used by numerous investigators as an in vitro model of cardiac myocyte hypertrophy.^{23,27,39,52–56} In the current study, confocal microscopy was used to prove that adrenergic stimulation caused significant increases in the cell volume of cultured NRVMs. This hypertrophic phenotype was absolutely dependent on adhesion to ECM. Previous studies have shown that the cardiac myocyte phenotype can be modulated by the binding of myocytes to collagen, fibronectin, and laminin but not by attachment to substances such as BSA or polylysine.^9,57 The current results extended these findings and demonstrated that induction of the hypertrophic marker gene ANF in ventricular myocytes required adhesion to fibronectin or laminin. These results are consistent with data obtained in noncardiac cells in which cell adhesion is required for growth factor activation of the Na⁺/H⁺ antiporter.⁵⁸

Overexpression of human ß₁ integrin increased myocyte protein synthesis and ANF secretion, both of which are indicative of the hypertrophic phenotype, but did not change DNA synthesis. Thus, cell replication was not altered. Ligation of ß₁ integrins in the ventricular myocytes by plating on anti-ß₁ antibody–coated dishes was sufficient to induce ANF expression in the absence of serum, indicating a direct role for ß₁ integrins in the response pathway. ANF expression was further augmented when myocytes plated on anti-ß₁ antibodies were simultaneously stimulated by PE. Interestingly, ANF expression and secretion were significantly higher in myocytes overexpressing human ß₁ plated on fibronectin than in those plated onto anti-ß₁ antibody. A potential explanation for this result is that when cells expressing human ß₁ were plated onto the anti-ß₁ antibody, only the human/rat heterodimers would be ligated, whereas fibronectin would ligate both the endogenous rat ß₁ integrins as well as rat-/human-ß₁ heterodimers. Alternatively, attachment to the anti-human ß₁ antibody could activate signaling pathways distinct from those induced by binding to fibronectin.⁵⁹ In addition, adhesion of myocytes to fibronectin could also be mediated by non-ß₁ integrins, leading to activation of signaling pathways different from those activated by ß₁ integrins.

Recent work has identified a unique integrin isoform, ß_1D, that is preferentially expressed in heart and skeletal muscle.^30,60 Our results suggest that ß_1D as well as the ubiquitously expressed ß_1A isoform both participate in the hypertrophic response. ß_1D may strengthen cytoskeleton-matrix linkages, which could be necessary when the cardiac cell is hemodynamically stressed as hypertrophy is induced.⁶¹ Although expression of ß_1A and ß_1D in nonmuscle cells can activate FAK,⁶⁰ it is unknown if ß_1A and ß_1D activate similar downstream pathways in the cardiac cell. In this regard, preliminary work by our laboratory and others has shown that FAK overexpression in the cardiac cell upregulates hypertrophic marker genes.⁶² However, whether ß_1A and ß_1D activate hypertrophic markers via FAK-dependent or -independent mechanisms is presently unknown.

Disruption of integrin signaling in the cardiac cell inhibited PE-induced ANF expression. For these experiments, we used well-characterized chimeric molecules that cannot bind ECM.^48–50,63 In noncardiac cells, the mutants did not alter expression of endogenous ₁, ₃, ₅, _v, ß₁, or ß₅ integrins or prevent heterodimerization of /ß subunits. Low-level mutant expression reduced FAK phosphorylation, whereas high-level expression increased FAK phosphorylation and caused altered cell adhesion.^49,50 At the level expressed in the present study, we saw altered ANF expression without changes in adhesion or cytoskeletal organization. These data suggest that adrenergic induction of ANF expression requires downstream molecules that are components of the integrin signaling pathway. Modulation of FAK phosphorylation by the mutant could be one mechanism that alters hypertrophic gene expression. Alternatively, the freely expressed ß₁ cytoplasmic domain mutants could bind and deplete factors that are essential for both hypertrophic and integrin-mediated signaling. These results support the concept that cross talk occurs between integrin and hypertrophic signaling pathways in the cardiac cell.

-Adrenergic–mediated hypertrophic gene induction in the cardiac myocyte is complex and modulated by at least two distinct signaling pathways, G_q and Ras/MAP kinase.^64–66 Studies suggested that activation of MAP kinase pathways, particularly MKK-6–p38, was sufficient for the full hypertrophic response and that blockade of the p38 pathway could block -adrenergic induction of the hypertrophic response.^26,67–69 G_q-mediated hypertrophic gene expression has been suggested to require Rho, which can regulate organization of the actin cytoskeleton in many cell types.^54,70 Rho is also a component of integrin-mediated signaling pathways.⁷¹ Studies have shown distinct separation of signaling molecules that impact morphological hypertrophy of the NRVM, as opposed to hypertrophic marker gene responses. Examples include inhibition of adrenergically stimulated ANF expression by the Rho inhibitor C3 transferase without change in the cellular actin organization⁷⁰ and Raf-1 overexpression, which induced activation of the ANF-luciferase marker gene without affecting cardiac cell morphology.⁷² These results are in agreement with those in the present study that showed that the TAC-ß_1A mutant inhibited adrenergic induction of ANF without alteration in myocyte cytoskeletal organization. Thus, it is possible that disruption of normal integrin signaling by use of our Tac-ß_1A mutant might indirectly alter Rho or Rac signaling. Furthermore, inhibition of tyrosine phosphorylation by genistein was found to prevent PE-induced activation of hypertrophic marker genes, including ANF.⁴⁴ Multiple proteins are stimulated to undergo tyrosine phosphorylation by integrin clustering in noncardiac cells.⁷³ Although the specific tyrosine-phosphorylated protein critical for integrin-meditated signaling in cardiac cells is unknown, adrenergic and integrin-mediated hypertrophic signaling could function through similar tyrosine kinase–dependent pathways.

Hypertrophic induction of NRVMs by adrenergic stimulation is also modulated by activation of phospholipase-C-ß (PLC-ß). PLC-ß signals through diacylglycerol, inducing multiple protein kinase C isoforms to cause expression of hypertrophic marker genes such as ANF.⁷⁴ It has been proposed that protein kinase C may serve an important function to mediate cross talk between growth factor and integrin signaling pathways in noncardiac cells.⁷⁵ It is therefore possible that ß₁ integrin and hypertrophic signaling pathways in the myocyte may intersect and synergize through other known mediators.

Cardiac myocyte stretch can cause hypertrophy and induction of gene expression.^5,76–78 Several recent studies have linked mechanical perturbation of noncardiac cells through ß₁ integrins with induction of gene expression.^19,79 However, no data have directly linked cell-matrix interactions or integrin engagement/signaling to the hypertrophic process. Because the present results show that integrin ligation and ECM interaction of the myocyte are necessary for the hypertrophic response, a component of the adrenergically mediated hypertrophic response may act by mechanical activation of integrins.

Hypertrophy and induction of molecular hypertrophic markers in this cell system can also be induced by humoral factors such as endothelin-1 or angiotensin-II, which act through MAP kinase.^66,80,81 In noncardiac cells, endothelin-1 has been shown to cause tyrosine phosphorylation of FAK.⁸² Furthermore, mechanically mediated stress activation of the myocyte hypertrophic response is partially mediated via angiotensin II.⁸³ However, the direct role that ß₁ integrin signaling plays in these other response pathways in the myocyte is presently unknown and will need to be explored.

In summary, this study is the first to directly link integrin signaling to the hypertrophic pathway in a cellular model using adrenergic stimulation of NRVMs. Integrins may initiate intracellular signals either by organization of the cytoskeleton and alteration of cell shape or through mechanisms akin to growth factor signaling.⁸⁴ This signaling mechanism could provide a link between the integrins and adrenergically mediated neonatal ventricular cell hypertrophy. Given that integrins have been implicated as mechanotransducers¹⁹ and that cardiac hypertrophy can be modulated through mechanical means, this cell model should allow investigation of the convergence of these two pathways. Future studies are under way to evaluate the mechanism through which ß₁ integrins influence hypertrophic responses both in this in vitro system as well as in vivo.

	Selected Abbreviations and Acronyms

 

ANF = atrial natriuretic factor ATCC = American Type Culture Collection BrdU = 5-bromo-2'-deoxyuridine CHO = Chinese hamster ovary ECM = extracellular matrix FAK = focal adhesion kinase IL-2 = interleukin-2 lacZ = ß-galactosidase MAP = mitogen-activated protein MLC-2v = myosin light chain-2ventricular NRVM = neonatal rat ventricular myocyte PE = phenylephrine

	Acknowledgments

 
The authors wish to thank Ken Chien for continued support of this work. Dr Ross was supported by National Institutes of Health Award HL-57872 and the Laubisch Cardiovascular Research Fund. Drs Ginsberg and Loftus were supported by the National Institutes of Health Award HL-48428. Components of this work were performed during the tenure of an Established Investigatorship from the American Heart Association (Dr Loftus) and a Veterans Administration Research Associate Award (Dr Ross).

Received November 10, 1997; accepted March 25, 1998.

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