Published online before print June 12, 2003, doi: 10.1161/01.RES.0000081595.25297.1B
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
© 2003 American Heart Association, Inc.
Cellular Biology |
Focal Adhesion Kinase Is Activated and Mediates the Early Hypertrophic Response to Stretch in Cardiac Myocytes
From the Department of Internal Medicine (A.S.T, S.S.C., W.N., K.G.F.), School of Medicine, State University of Campinas, SP, Brazil, and Department of Cell and Developmental Biology (S.K.H.), Vanderbilt University School of Medicine, Nashville, Tenn.
Correspondence to Kleber G. Franchini, MD, PhD, Departamento de Clínica Médica, Faculdade de Ciências Médicas, Universidade Estadual de Campinas, Cidade Universitária "Zefferino Vaz," 13081-970 Campinas, SP, Brasil. E-mail franchin{at}obelix.unicamp.br
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Previously we reported that the rapid activation of the Fak/Src multicomponent signaling complex mediates load-induced activation of growth and survival signaling pathways in adult rat heart. In this study, we report that 5% to 20% (10-minute) cyclic stretch (1 Hz) of neonatal rat ventricular myocytes (NRVMs) was paralleled by increases of Fak phosphorylation at Tyr-397 (from 1.5- to 2.8-fold), as detected by anti-Fak-pY397 phosphospecific antibody. Moreover, 15% cyclic stretch lasting from 10 to 120 minutes increased Fak phosphorylation at Tyr-397 by 2.5- to 3.5-fold. This activation was accompanied by a dramatic change in Fak localization in NRVMs from densely concentrated in the perinuclear regions in nonstretched cells to aggregates regularly distributed along the myofilaments in stretched cells. Furthermore, a 4-hour cyclic stretch enhanced the activity of an atrial natriuretic factor (ANF) promoter-luciferase reporter gene by 2.7-fold. Disrupting endogenous Fak/Src signaling either by expression of a dominant-negative Fak mutant with phenylalanine substituted for Tyr-397 or by treatment with a c-Src pharmacological inhibitor (PP-2) markedly attenuated stretch-induced Fak activation and clustering at myofilaments and inhibited stretch-induced ANF gene activation. On the other hand, overexpression of wild-type Fak potentiated the stretch-induced Fak phosphorylation but did not enhance either baseline or stretch-induced ANF promoter-luciferase reporter gene activity compared with the responses of nontransfected NRVMs. These findings identify Fak as an important element in the early responses induced by stretch in cardiac myocytes, indicating that it may coordinate the cellular signaling machinery that controls gene expression program associated with load-induced cardiac myocyte hypertrophy.
Key Words: focal adhesion kinase • mechanotransduction • cell signaling • hypertrophy
|
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Mechanical overload is both cause and consequence of most heart diseases.1 Cardiac myocytes respond to increased mechanical load by hypertrophic growth, but mechanical stress is also an important stimulus for triggering the initial steps toward cardiac myocyte degeneration and death, which play a critical role in the maladaptive myocardial remodeling and heart failure.1,2 A major goal in this field is to decipher the mechanisms that link biomechanical forces to the activation of signaling pathways that mediate the hypertrophic as well as maladaptive responses of cardiac myocytes to mechanical stress.
The mechanistic pathways that link mechanical stimuli to biochemical signals in cardiac myocytes are presently unclear, but a growing body of evidence indicates that costameres (complex structures constituted by integrins and cytoskeletal proteins at the junction of sarcolemma and Z-discs) have a critical role in sensing and transducing mechanical stress into biochemical signals that coordinate growth responses to hypertrophic stimuli in both cardiac and skeletal muscle.3–7 The prominent location of integrins at the junction of extracellular matrix to Z discs makes them candidates for acting as biomechanical sensors in cardiac myocytes. Accordingly, overexpression of ß1-integrins in cardiac myocytes induces hypertrophic gene expression, whereas disruption of integrin function by conditional Cre-loxP gene targeting in adult mice results in intolerance to hemodynamic overload and abnormal cardiac function.8,9 In addition, Fak, a primary integrin effector that is known to play a key role in the responses of cells to mechanical stimuli through focal adhesion sites,10–12 is rapidly activated by mechanical stimuli in cultured neonatal rat ventricular myocytes (NRVMs)13,14 and in overloaded myocardium of adult animals.15–18 Data obtained in NRVMs have shown that Fak is also involved in the regulation of early gene transcription in response to hypertrophic agonists,19–22 indicating that this kinase may function as a node for the convergence of multiple signaling pathways involved in the hypertrophic growth of cardiac myocytes. However, a clear demonstration that Fak plays a role in the control of early gene expression in response to mechanical stimuli in cardiac myocytes is still lacking.
Thus, in this study we investigated the activation of Fak/Src complex by cyclic stretch and its influence on the early activation of atrial natriuretic factor (ANF) promoter reporter gene (ANF-LUC) in response to cyclic stretch in cultured neonatal rat ventricular myocytes. By disrupting Fak/Src signaling, either through expression of a dominant-negative Fak mutant (Phe-397) or through the inhibition of Src activity with the pharmacological inhibitor PP-2, we showed that Fak plays an important role in the stretch-induced ANF transcriptional activation in cardiac myocytes.
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Detailed methods are described in the online data supplement and in published studies, as referenced.
Cell Culture
Primary cultures of NRVMs (1- to 2-day-old Wistar rats) were prepared as previously reported.8 Briefly, the myocytes were purified on a discontinuous Percoll gradient, suspended in a plating media containing 10% horse serum, 5% fetal serum, and 0.5% penicillin/streptomycin, and plated in type I collagen Bioflex plates (Flexcell International Corp) coated with gelatin at 500 000/well. After 24 hours, the medium was replaced with serum-free DMEM and incubated for 24 to 48 hours under 95% air plus 5% CO2 before being used for study.
Cell Stretching
NRVMs cultured in Bioflex plates were stretched in a Flexercell FX-3000 strain unit to 115% of resting length at a frequency of 1 Hz (0.5-s stretch/0.5-s relaxation) for variable periods, depending on the protocol. Control nonstretched NRVMs were also cultured in Bioflex plates and incubated in serum-free medium. At the conclusion of the experimental protocol, cells were either scraped from membranes and lysed for immunoblot analysis or fixed for confocal immunofluorescence analysis.
Plasmid Transfection and Dual Reporter Gene Assays
Constructions of murine Fak pRc/CMV-FAKwt (wild-type [WT-Fak]) and pRc/CMV-FAKF397 (mutant [MT-Fak]) were described previously.23 WT-Fak and MT-Fak constructs were subcloned into pRc/CMV cytomegalovirus promoter-driven eukaryotic expression vector containing c-myc epitope tag (Invitrogen). Rat ANF promoter luciferase reporter gene (NP328, 700 bp of ANF flanking sequences containing luciferase reporter gene into pXP2) was obtained from Dr Mona Nemer (Institut de Recherches Cliniques de Montréal, Canada). NRVMs were cotransfected with 2 µg of ANF-LUC, 0.1 µg of the internal control SV40-renilla luciferase, and 2 µg of WT-Fak, MT-Fak, or empty plasmid, and 48 hours after transfection they were stretched for 4 hours. All firefly luciferase values were normalized to renilla activities.
Subcellular Fractionation
This procedure was performed as previously reported.28 Briefly, NRVMs were homogenized in lysis buffer and centrifuged (100 000g, 1 hour). The supernatant (S) fraction was concentrated to 10% of original volume. The particulate (P) fraction was resuspended in a buffer with 1% Triton X-100 and 0.1% SDS and recentrifuged (10 000g, 20 minutes). S and P fractions were separated by SDS-PAGE.
Immunoblotting
NRVMs homogenated in lysis buffer were resolved on SDS-PAGE and transferred to nitrocellulose membranes. For immunoprecipitation, normalized samples were incubated with anti-c-myc monoclonal antibody and collected after addition of 25 µL of protein G-Sepharose beads. The membranes were incubated with primary antibodies (anti-Fak, anti-Fak-pY397, anti-Fak-pY577, anti-c-Src, or anti-c-Src-pY418). Band intensities were quantified by optical densitometry of the developed autoradiographs.
Laser Confocal Analysis
NRVMs were fixed with 4% paraformaldehyde/sucrose and incubated with anti-Fak primary antibody. This was followed by incubation with biotin-conjugated secondary anti-rabbit antibody and then with streptavidin-Cy2 and rhodamine-conjugated phalloidin. Images were obtained with laser confocal microscope (Zeiss LSM510).
Statistical Analysis
Data are presented as mean±SEM. Differences between the mean values of the densitometric or luciferase readings were tested by ANOVA and Bonferroni multiple-range test. P<0.05 indicated statistical significance.
An expanded Materials and Methods section can be found in the online data supplement available at http://www.circresaha.org.
|
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Stretch-Induced Fak Activation in NRVMs
The tyrosine residue Tyr-397 has been shown to be phosphorylated via an autophosphorylation process, being critical for FAK activation.23–25 Phosphorylated Tyr-397 then recruits Src family kinases, which lead to an increase in Fak enzymatic activity. We examined the effects of the amplitude and duration of cyclic stretch (1 Hz) on Fak activity by Western blotting NRVM extracts with phosphospecific antibody directed against the autophosphorylation site of Fak [anti-Fak-Tyr(P)-397 antibody (anti-Fak-pY397)]. As shown in Figure 1A, 5% cyclic stretch enhanced the phosphorylation of Fak at Tyr-397 to
150%. Additional increases were seen in NRVMs subjected to 10%, 15%, and 20% stretch (to 210%, 230%, and 280%, respectively). The time course of Fak phosphorylation at Tyr 397 was examined in NRVMs subjected to 15% cyclic stretch up to 120 minutes (Figure 1B). The amount of Fak detected with the anti-Fak-pY397 antibody increased 2.5-fold within 10 minutes to up to 3.5-fold within 120 minutes of cyclic stretch compared with nonstretched cells. No change was observed in the amount of protein detected with anti-Fak antibody in both stimulus intensity and time course experiments.
|
The subcellular distribution of Fak in nonstretched and stretched NRVMs was first examined in soluble (S) and particulate (P) fractions obtained with differential centrifugation of NRVMs extracts. As shown in Figure 2A, in nonstretched cells, most of Fak was present in the S fraction, whereas stretch resulted in a marked reduction of Fak in S fraction and an increase in P fraction.
|
Fak distribution in NRVMs was also evaluated by confocal microscopy in cells doubled-stained with anti-Fak/streptavidin-Cy2 and rhodamine-conjugated phalloidin. In nonstretched NRVMs, Fak was densely concentrated in the perinuclear regions, but less markedly at the cellular periphery, where it was diffusely distributed (Figures 2B and 2C). We used rhodamine-conjugated phalloidin, which labels sarcomeric actin, to define the precise localization of Fak. NRVMs staining with this procedure revealed the typical sarcomeric pattern of repetitive striations, with the labeled structure representing the actin array of two adjacent sarcomeres, where presumably Z discs and costameres are located. At higher magnification (Figure 2D), it was possible to see that at cell periphery, although Fak was detected close to myofilaments, it was not possible to define any distribution pattern or a consistent superimposition to myofilaments. In 30-minute stretched cells, anti-Fak staining was clearly reduced at perinuclear regions and increased at the cell periphery, where it distributed regularly along the myofilaments (Figures 2E and 2F). At higher magnification (Figure 2G), Fak-specific staining was seen as clusters overlapping the regions stained with phalloidin, consistent with the localization of costameres.
Angiotensin II–Induced Fak Activation in NRVMs
FAK has been shown to be activated in response to GPCR agonists, including angiotensin II (Ang II).27 Furthermore, Ang II may act as autocrine/paracrine mediator of stretch-induced cardiomyocyte hypertrophy.5 Thus, we tested the possibility that cyclic stretch induces Fak activation via an Ang II–mediated mechanism. As shown in Figure 3A, Ang II induced a concentration-dependent increase of FAK phosphorylation at Tyr-397 in NRVMs. This effect was completely inhibited by addition to the medium of the AT1-specific receptor antagonist (DUP-753; 10 µmol/L, 1-hour preincubation), indicating that Ang II–induced FAK phosphorylation occurred via AT1 receptor–dependent signaling (Figure 3B). As shown in Figure 3B, cyclic stretch still activated Fak in NRVMs treated with the AT1-antagonist DUP-753, indicating that the stretch and Ang II activate Fak by distinct mechanisms.
|
The effect of Ang II on Fak localization in NRVMs was then evaluated by confocal microscopy of cells double-stained with anti-Fak/streptavidin-Cy2 and rhodamine-conjugated phalloidin. Distinct from cyclic stretch that induced Fak to aggregate at myofilaments, treatment with Ang II did not change the distribution of Fak in NRVMs, except for the augmentation of spot areas around the edge of the cells, consistent with the localization of focal adhesions (Figure 3C, arrows).
Effects of WT-Fak and MT-Fak on Stretch- Induced Expression of ANF-LUC Reporter Gene
To test the role of Fak on the stretch-mediated NRVM hypertrophic response in vitro, we transiently cotransfected NRVMs with Myc-tagged WT-Fak or MT-Fak constructs driven by cytomegalovirus promoter and ANF-LUC/SV40-Renilla reporter genes. Because Fak constructions were Myc-tagged, we initially tested the ability of NRVMs to express WT-Fak or MT-Fak by immunoprecipitation using anti-c-Myc monoclonal and anti-Fak antibodies. As shown in Figure 4A, a considerable amount of Myc-tagged Fak was detected in the immunoprecipitates of both WT-Fak–transfected and MT-Fak–transfected NRVMs but not in nontransfected cells. Furthermore, Fak expression was estimated in whole-cell lysates by immunoblotting with anti-Fak antibody. Figure 4B shows that transfection with 2 µg total DNA of WT-Fak or MT-Fak plasmids (48 hours) enhanced the amount of Fak detected by anti-Fak antibody by 14-fold compared with the amount of Fak detected in control nontransfected NRVMs. Transfection with WT-Fak and MT-Fak did not change the amount of Fak detected with anti-Fak-pY397 antibody in nonstretched NRVMs compared with nontransfected cells (Figure 4C). Nevertheless, transfection with WT-Fak potentiated stretch-induced Fak activation in NRVM, as demonstrated by a marked increase (to 14-fold) in the amount of Fak detected by anti-Fak-pY397 after 30 minutes of cyclic stretch. In contrast, NRVM transfection with MT-Fak abolished the stretch-induced Fak activation.
|
To address the effects of transient transfection with WT-Fak and MT-Fak on cell viability as well as in Fak protein distribution, NRVMs were double-stained with anti-c-Myc antibody/streptavidin-Cy2 and rhodamine-conjugated phalloidin and analyzed by confocal microscopy. In nontransfected and nonstretched NRVMs (Figures 5A and 5B), the anti-c-Myc monoclonal antibody detected only a small amount of c-Myc restricted to perinuclear areas. Cyclic stretch slightly increased the amount of c-Myc in these cells restricted to perinuclear areas (Figures 5C and 5D). Cells transfected with WT-Fak (Figures 5E through 5H) or MT-Fak (Figures 5I through 5L) constructs (48 hours) showed morphological characteristics comparable to those of nontransfected cells. Marked increases were seen in the anti-c-Myc staining of these compared with nontransfected cells. In nonstretched cells, anti-c-Myc staining was observed throughout the cells, although it was densely concentrated in the perinuclear areas. In stretched WT-Fak–transfected cells, anti-c-Myc staining was associate with myofilaments, as was the staining with anti-Fak antibody of stretched nontransfected NRVMs (Figures 5G and 5H), but the perinuclear regions still showed a highly intense anti-c-Myc staining. However, in stretched MT-Fak–transfected cells, the distribution of anti-c-Myc staining remained similar to that of nonstretched cells (Figures 5K and 5L) without a clear association with the myofilaments.
|
We next assessed the effect of cyclic stretch on the activity of transiently transfected ANF-LUC. As shown in Figure 6, a 4-hour stretching protocol increased the normalized luciferase activity of cotransfected NRVMs by 2.7-fold. Overexpression of WT-Fak did not significantly increase the baseline ANF-LUC expression compared with values of nonstretched cells. Likewise, WT-Fak overexpression did not enhance the ANF-LUC activity induced by cyclic stretch compared with nontransfected cells. MT-Fak overexpression did not change the baseline but reduced significantly the stretch-induced increase in ANF expression in NRVMs compared with the responses of nontransfected cells.
|
Effects of PP2 on Stretch-Induced Fak and ANF-LUC Activation
Fak activation in response to cell adhesion is dependent on a reciprocal catalytic activation of Fak and Src family kinases.23–25,29 Autophosphorylation of Fak Tyr-397 creates a high-affinity binding site for the SH2 domain of Src, and Src associated with Fak promotes maximal Fak catalytic activity by phosphorylating Fak Tyr-576 and Tyr-577 located in the kinase catalytic domain. Accordingly, treatment with the selective Src family kinase inhibitor PP-2 has been shown to inhibit the phosphorylation of Tyr-397 and Fak activation induced by integrin engagement.26,27 To additionally evaluate the role of stretch-induced activation of the Fak/Src complex on the early regulation of ANF-LUC activity in NRVMs, we pretreated cells with PP-2. NRVM stretch was accompanied by c-Src activation, as indicated by the increase in the amount of c-Src detected with the phosphospecific antibody directed against c-Src Tyr-418 (Figure 7A). Cyclic stretch also increased Fak phosphorylation at Tyr-576 and Tyr-577 in addition to Fak phosphorylation at Tyr-397. Treatment with PP-2 (1 µmol/L) inhibited stretch-induced c-Src and Fak activation, as indicated by the reduction of c-Src-pY418, Fak-pY397, and Fak-pY577, respectively, additionally suggesting that stretch-induced Fak activation is dependent on integrin engagement and c-Src activation.
|
As shown in the representative examples of Figure 7B, despite the marked inhibition of stretch-induced Fak, treatment with PP-2 for 4 hours did not alter the morphology or viability of NRVM. In addition, image analysis indicated that PP-2 treatment did not change the distribution of Fak in nonstretched cells, because it remained densely concentrated in the perinuclear regions and diffusely distributed at the cell periphery. However, in contrast to what was seen in untreated stretched NRVMs, treatment with PP-2 was not accompanied by a consistent aggregation of Fak at myofilaments.
Experiments performed with ANF-LUC–transfected NRVMs showed that PP-2 treatment markedly attenuated the stretch-induced ANF-LUC activation (Figure 7C). In untreated cells, cyclic stretch increased ANF-LUC expression by 2.7-fold, whereas in PP-2–treated cells, stretch increased ANF-LUC activity by only 1.3-fold.
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
The present study demonstrates that Fak activation plays an important role in the early upregulation of ANF transcription induced by mechanical stress in cardiac myocytes, indicating that its activation by mechanical stress is not simply involved in the initiation of biochemical signals but also that it coordinates cellular signaling machinery that controls gene expression associated with load-induced cardiac myocyte hypertrophy.
Stretch-Induced Fak Activation in NRVMs
Fak was found to be rapidly activated in NRVMs by cyclic stretch, as reported previously.13,14 This activation was shown to be roughly parallel to the amplitude and duration of the stretch. In addition, we have shown here that stretch induced Fak to migrate from soluble to particulate fraction of NRVMs extracts. Additional analysis with confocal microscopy demonstrated that stretch induced Fak to aggregate at myofilaments in a distribution pattern that follows that of costamere sites. Simultaneous with the aggregation at myofilaments, there was a marked reduction of specific Fak staining in the perinuclear regions with no change in the amount of Fak detected by Western blot, supporting the conclusion that cyclic stretch induces Fak to translocate and cluster at costameres sites of NRVMs. These results indicate that activation of Fak by stretch is dependent on its proper location at the costameres, where local mechanical forces are transduced into biochemical signals. This is consistent with our previous demonstration that mechanical stress induces a translocation of Fak to the actin cytoskeletal compartment in rat myocardium.15 Moreover, this generally agrees with data from recent studies that demonstrated that displacement of Fak from focal adhesion sites by overexpressed FRNK (FAK-related non-kinase) in cultured NRVMs impairs Fak activation.19,20,28 However, in contrast to our results, in those studies Fak was found most consistently in classical focal adhesion sites at the edges of cultured myocytes, where it increased in response to agonist treatment. This suggests that displacement of Fak by FRNK might occur preferentially at these sites instead of costameres. The reason for such differences might be related to differences in the nature of stimulus (ie, mechanical versus chemical). This idea is supported by the results of our experiments performed with Ang II. Although Ang II activates Fak in NRVMs, it does not induce the migration of Fak to myofilaments. Instead, Ang II enhanced Fak aggregation at classical focal adhesion sites, indicating that Ang II and stretch activate Fak by distinct mechanisms and sites of NRVMs. This is additionally supported by our demonstration here that AT1 receptor antagonist did not impair the stretch-induced Fak activation although it is able to blockade the Ang II–induced Fak activation. Moreover, this generally agrees with the previous demonstration27 that signaling events leading to Fak activation by GPCR agonists are Src independent whereas those stimulated by integrin receptors require Src activation.
Experiments designed to disrupt stretch-induced activation of FAK/Src signaling, either by the dominant-negative Fak overexpression or pharmacological inhibition of Src, abolished the stretch-induced Fak aggregation at NRVM myofilaments. Because both strategies disrupted Fak signaling by affecting Fak autophosphorylation at Tyr-397, our results indicate that stretch-induced Fak clustering at myofilaments is dependent on autophosphorylation of Tyr-397. Similarly, a critical role of Tyr-397 autophosphorylation to Fak activation and clustering also has been shown at focal adhesion sites of noncardiac cells.29,30 The importance of Tyr-397 to Fak translocation and clustering probably relies on the fact that autophosphorylation of Tyr-397 is responsible for recruiting and activating Src family kinases, which in turn additionally enhance Fak activity by transphosphorylating and recruiting additional Fak molecules to specific sites.23–25 Thus, the impairment of Tyr-397 autophosphorylation would prevent Fak/Src clustering at costameres in cardiac myocytes.
In this regard, one might expect that Fak clustering at costameres optimizes the stretch-induced Fak signaling in cardiac myocytes, not only because of the location at strategic sites that convey mechanical stimuli but also because the molecular proximity in clusters may serve to enhance and sustain Fak signaling. Accordingly, recent studies by Katz et al31 in fibroblasts have shown that Fak clustering enhances and sustains Fak activation, allowing the recruitment and activation of additional cellular signaling pathways such as those involved in the activation of growth and survival pathways. Additional studies are required to dissect the potential role of stretch-induced Fak clustering at costameres of cardiac myocytes to the long-term effect of mechanical stress in cardiac myocytes.
Control of Gene Expression by Stretch-Induced Fak Activation
The impairment of Fak activation markedly attenuated ANF promoter activity induced by stretch in NRVMs, indicating that Fak plays a central role to signaling events involved in the early regulation of gene expression in response to mechanical stress in cardiac myocytes. These results extend to mechanical stimuli previous demonstrations that Fak is required for early gene upregulation in response to hypertrophic agonists in cultured NRVMs.19,20,22 Moreover, the demonstration here that stretch-induced Fak activation in cultured NRVMs occurs at costameres indicates that this is a specific phenomenon directly related to the mechanical forces imposed by stretch on the costameres and also that this phenomenon might be present in cardiac myocytes in intact myocardium.
A role for Fak in cell growth mediated by integrin signaling has been demonstrated in many cell systems.32,33 Early work34 showed that Fak/Src complex activates putative downstream signaling pathways, leading to activation of phosphoinositide 3-kinase, protein kinase C, ERK, Jun N-terminal kinase, and p38 mitogen-activated protein kinase pathways. Although the downstream mediators of the regulation of ANF expression induced by Fak were not explored in the present study, previous evidence indicates that the Fak/Src complex may activate extracellular signal-regulated kinase (ERK) 1 and ERK2 in NRVMs and in rat myocardium,15–17,20 which potentially may mediate the early regulation of ANF expression. Accordingly, the early activation of ERK1/2 has been suggested to contribute to the reexpression of fetal ventricle genes.35 Transfection of constitutively active MEK1 (immediate upstream activator of ERK1/2) has been shown to augment ANF promoter activity in cultured cardiomyocytes, whereas a dominant-negative MEK1 construct attenuated its activity.36 However, the present results do not exclude the possibility that multiple downstream effectors are involved in the influence of Fak on early gene regulation in response to mechanical stress. Thus, additional studies are necessary to clarify the relative importance of the various candidate signaling molecules to Fak influence on early gene regulation in response to mechanical stress.
Interestingly, WT-FAK overexpression in NRVMs did not significantly activate ANF gene expression in nonstretched cells, nor did it enhance stretch-induced ANF-LUC activity despite the fact that it substantially increased the amount of activated Fak in response to stretch in NRVMs. These results might indicate that WT-Fak overexpression alone was not sufficient to activate baseline nor to potentiate the stretch-induced ANF transcription. Alternatively, these results might indicate that Fak-independent mechanisms are responsible for the stretch-induced ANF transcriptional activation. Similarly, previous studies have shown that overexpression of WT-Fak constructs in low-density cultured NRVMs did not enhance Fak activation by endothelin.19 However, transfection of WT-Fak in high-density cultured NRVMs has been shown to stimulate the transcription of fetal genes associated with the hypertrophic phenotype.21,37 The reasons for such discrepancies are not apparent from our results. One may still speculate that under the culture conditions used here, the basal ANF expression was already quite high; thus, despite the potentiation in stretch-induced Fak activation caused by transfection with WT-Fak, ANF transcriptional activity could not increase substantially.
In conclusion, our present results corroborate and extend to isolated cardiac myocytes previous findings in diverse experimental systems that Fak plays a central role as a biomechanical sensor that responds to changes in load in cardiac myocytes. Our data also indicate that signaling by the Fak/Src complex initiated in response to continuous stimulation by mechanical stress coordinates the cellular signaling machinery that controls gene expression program associated with load-induced cardiac myocyte hypertrophy.
|
Acknowledgments |
|---|
This study was sponsored by grants from Fundação de Amparo à Pesquisa do Estado de São Paulo (Proc. 00/05137-4, 01/11698-1) and Conselho Nacional de Desenvolvimento Científico e Tecnológico (Proc. 521098/97-1).
|
Footnotes |
|---|
*Both authors contributed equally to this study.
Original received January 29, 2003; revision received May 15, 2003; accepted June 3, 2003.
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
1. Katz AM. The cardiomyopathy of overload: an unnatural growth response in the hypertrophied heart. Ann Intern Med. 1994; 121: 363–371.
2. Chien KR. Stress pathways and heart failure. Cell. 1999; 98: 555–558.[CrossRef][Medline] [Order article via Infotrieve]
3. Sussman MA, McCulloch A, Borg TK. Dance band on the titanic: biomechanical signaling in cardiac hypertrophy. Circ Res. 2002; 91: 888–898.
4. Terracio L, Rubin K, Gullberg D, Balog E, Carver W, Jyring R, Borg TK. Expression of collagen binding integrins during cardiac development and hypertrophy. Circ Res. 1991; 68: 734–744.
5. Ruwhof C, van der Laarse A. Mechanical stress-induced cardiac hypertrophy: mechanisms and signal transduction pathways. Cardiovasc Res. 2000; 47: 23–37.
6. Borg TK, Goldsmith EC, Price R, Carver W, Terracio L, Samarel AM. Specialization at the Z line of cardiac myocytes. Cardiovasc Res. 2000; 46: 277–285.
7. Ross RS, Borg TK. Integrins and the myocardium. Circ Res. 2001; 88: 1112–1119.
8. Ross RS, Pham C, Shai SY, Goldhaber JI, Fenczik C, Glembotski CC, Ginsberg MH, Loftus JC. ß1 Integrin participates in the hypertrophic response of rat ventricular myocytes. Circ Res. 1998; 82: 1160–1172.
9. Shai S-Y, Harpf AE, Babbitt CJ, Jordan MC, Fishbein MC, Chen J, Omura M, Leil TA, Becker KD, Jiang M, Smith DJ, Cherry SR, Loftus JC, Ross RS. Cardiac myocyte-specific excision of the ß1 integrin gene results in myocardial fibrosis and cardiac failure. Circ Res. 2002; 90: 458–464.
10. Hanks SK, Calalb MB, Harper MC, Patel SK. Focal adhesion protein-tyrosine kinase phosphorylated in response to cell attachment to fibronectin. Proc Natl Acad Sci U S A. 1992; 89: 8487–8491.
11. Wang H-B, Dembo M, Hanks SK, Wang Y-L. Focal adhesion kinase is involved in mechanosensing during fibroblast migration. Proc Natl Acad Sci U S A. 2001; 98: 11295–11300.
12. Li S, Butler P, Wang Y, Hu Y, Han DC, Usami S, Guan J-L, Chien S. The role of the dynamics of focal adhesion kinase in the mechanotaxis of endothelial cells. Proc Natl Acad Sci U S A. 2002; 99: 3546–3551.
13. Seko Y, Takahashi N, Tobe K, Kadowaki T, Yazaki Y. Pulsatile stretch activates mitogen-activated protein kinase (MAPK) family members and focal adhesion kinase [p125(FAK)] in cultured rat cardiac myocytes. Biochem Biophys Res Commun. 1999; 259: 8–14.[CrossRef][Medline] [Order article via Infotrieve]
14. Aikawa R, Nagai T, Kudoh S, Zou Y, Tanaka M, Tamura M, Akazawa H, Takano H, Nagai R, Komuro I. Integrins play a critical role in mechanical stress–induced p38 MAPK activation. Hypertension. 2002; 39: 233–238.
15. Franchini KG, Torsoni AS, Soares PHA, Saad MJA. Early activation of the multicomponent signaling complex associated with focal adhesion kinase induced by pressure overload in the rat heart. Circ Res. 2000; 87: 558–565.
16. Laser M, Willey CD, Jiang W, Cooper C, Menick DR, Zile MR, Kuppuswamy D. Integrin activation and focal complex formation in cardiac hypertrophy. J Biol Chem. 2000; 275: 35624–35630.
17. Domingos PP, Fonseca PM, Nadruz W Jr, Franchini KG. Load-induced focal adhesion kinase activation in the myocardium: role of stretch and contractile activity. Am J Physiol. 2002; 282: H556–H564.
18. Bayer AL, Heidkamp MC, Patel N, Porter MJ, Engman SJ, Samarel AM. PYK2 expression and phosphorylation increases in pressure overload-induced left ventricular hypertrophy. Am J Physiol. 2002; 283: H695–H706.
19. Eble DM, Strait JB, Govindarajan G, Lou J, Byron KL, Samarel AM. Endothelin-induced cardiac myocyte hypertrophy: role for focal adhesion kinase. Am J Physiol. 2000; 278: H1695–H1707.
20. Taylor JM, Rovin JD, Parsons JT. A role for focal adhesion kinase in phenylephrine-induced hypertrophy of rat ventricular cardiomyocytes. J Biol Chem. 2000; 275: 19250–19257.
21. Pham CG, Harpf AE, Keller RS, Vu HT, Shai SY, Loftus JC, Ross RS. Striated muscle-specific ß1D-integrin and FAK are involved in cardiac myocyte hypertrophic response pathway. Am J Physiol. 2000; 279: H2916–H2926.
22. Kovacic-Milivojevic B, Roediger F, Almeida EAC, Damsky CH, Gardner DG, Ilic D. Focal adhesion kinase and p130Cas mediate both sarcomeric organization and activation of genes associated with cardiac and myocyte hypertrophy. Mol Biol Cell. 2001; 12: 2290–2307.
23. Calalb MB, Polte TR, Hanks SK. Tyrosine phosphorylation of focal adhesion kinase at sites in the catalytic domain regulates kinase activity: a role for Src family kinases. Mol Cell Biol. 1995; 15: 954–963.[Abstract]
24. Schlaepfer DD, Hanks SK, Hunter T, van der Geer P. Integrin-mediated signal transduction linked to Ras pathway by GRB2 binding to focal adhesion kinase. Nature. 1994; 372: 786–791.[Medline] [Order article via Infotrieve]
25. Polte TR, Hanks SK. Complexes of focal adhesion kinase (FAK) and Crk-associated substrate (p130Cas) are elevated in cytoskeletal-associated fractions following adhesion and Src transformation: requirements for Src kinase activity and FAK proline-rich motifs. J Biol Chem. 1997; 272: 5501–5509.
26. Hanke JH, Gardner JP, Dow RL, Changelian PS, Brissette WH, Weringer EJ, Pollok BA, Connelly PA. Discovery of a novel, potent, and Src family-selective tyrosine kinase inhibitor. J Biol Chem. 1996; 271: 695–701.
27. Salazar EP, Rozengurt E. Src family kinases are required for integrin-mediated but not for G protein-coupled receptor stimulation of focal adhesion kinase autophosphorylation at Tyr-397. J Biol Chem. 2001; 276: 17788–17795.
28. Heidkamp MC, Bayer AL, Kalina JA, Eble DM, Samarel AM. GFP-FRNK disrupts focal adhesions and induces anoikis in neonatal rat ventricular myocytes. Circ Res. 2002; 90: 1282–1289.
29. Owen JD, Ruest PJ, Fry DW, Hanks SK. Induced focal adhesion kinase (FAK) expression in FAK-null cells enhances cell spreading and migration requiring both auto- and activation loop phosphorylation sites and inhibits adhesion-dependent tyrosine phosphorylation of Pyk2. Mol Cell Biol. 1999; 19: 4806–4818.
30. Schaller MD, Hildebrand JD, Parsons JT. Complex formation with focal adhesion kinase: a mechanism to regulate activity and subcellular localization of Src kinases. Mol Biol Cell. 1999; 10: 3489–3505.
31. Katz B-Z, Miyamoto S, Teramoto H, Zohar M, Krylov D, Vinson C, Gutkind JS, Yamada KM. Direct transmembrane clustering and cytoplasmic dimerization of focal adhesion kinase initiates its tyrosine phosphorylation. Biochim Biophys Acta. 2002; 1592: 141–152.[Medline] [Order article via Infotrieve]
32. Zhao JH, Reiske H, Guan JL. Regulation of the cell cycle by focal adhesion kinase. J Cell Biol. 1998; 143: 1997–2008.
33. Zhao JH, Zheng C, Guan J. Pyk2 and FAK differentially regulate progression of the cell cycle. J Cell Sci. 2000; 113: 3063–3072.[Abstract]
34. Schlaepfer DD, Hauck CR, Sieg DJ. Signaling through focal adhesion kinase. Prog Biophys Mol Biol. 1999; 71: 435–478.[CrossRef][Medline] [Order article via Infotrieve]
35. Sugden PH, Clerk A. "Stress-responsive" mitogen-activated protein kinases (c-Jun N-terminal kinases and p38 mitogen-activated protein kinases) in the myocardium. Circ Res. 1998; 24: 345–352.
36. Gillespie-Brown J, Fuller SJ, Bogoyevitch MA, Cowley S, Sugden PH. The mitogen-activated protein kinase kinase MEK1 stimulates a pattern of gene expression typical of the hypertrophic phenotype in rat ventricular cardiomyocytes. J Biol Chem. 1995; 270: 28092–28096.
37. Eble DM, Qi M, Strait JB, Samarel AM. Contraction-dependent hypertrophy of neonatal rat ventricular myocytes: potential role for focal adhesion kinase. In: Takeda N, Dhalla NJ, eds. The Hypertrophied Heart. Boston, Mass: Kluwer; 2000: 91–107.
This article has been cited by other articles:
 |
|
 |
|
  C. Mercure, A. Yogi, G. E. Callera, A. B. Aranha, M. Bader, A. J. Ferreira, R. A. S. Santos, T. Walther, R. M. Touyz, and T. L. Reudelhuber Angiotensin(1-7) Blunts Hypertensive Cardiac Remodeling by a Direct Effect on the Heart Circ. Res., November 21, 2008; 103(11): 1319 - 1326. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
K. A. Martin and J. Hwa Shp Shape: FAKs About Hypertrophy Circ. Res., October 10, 2008; 103(8): 776 - 778. [Full Text] [PDF]
|
|
|
|
|
|
T. M. Marin, C. F.M.Z. Clemente, A. M. Santos, P. K. Picardi, V. D.B. Pascoal, I. Lopes-Cendes, M. J.A. Saad, and K. G. Franchini Shp2 Negatively Regulates Growth in Cardiomyocytes by Controlling Focal Adhesion Kinase/Src and mTOR Pathways Circ. Res., October 10, 2008; 103(8): 813 - 824. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. Sabri, K. Rafiq, R. Seqqat, M. A. Kolpakov, R. Dillon, and L. J. Dell'italia Sympathetic Activation Causes Focal Adhesion Signaling Alteration in Early Compensated Volume Overload Attributable to Isolated Mitral Regurgitation in the Dog Circ. Res., May 9, 2008; 102(9): 1127 - 1136. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 B.-W. Wang, H. Chang, P. Kuan, and K.-G. Shyu Angiotensin II activates myostatin expression in cultured rat neonatal cardiomyocytes via p38 MAP kinase and myocyte enhance factor 2 pathway J. Endocrinol., April 1, 2008; 197(1): 85 - 93. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. F.M.Z. Clemente, T. F. Tornatore, T. H. Theizen, A. C. Deckmann, T. C. Pereira, I. Lopes-Cendes, J. R. M. Souza, and K. G. Franchini Targeting Focal Adhesion Kinase With Small Interfering RNA Prevents and Reverses Load-Induced Cardiac Hypertrophy in Mice Circ. Res., December 7, 2007; 101(12): 1339 - 1348. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 L. P. Desai, S. E. Sinclair, K. E. Chapman, A. Hassid, and C. M. Waters High tidal volume mechanical ventilation with hyperoxia alters alveolar type II cell adhesion Am J Physiol Lung Cell Mol Physiol, September 1, 2007; 293(3): L769 - L778. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. J. Zhang, G. A. Truskey, and W. E. Kraus Effect of cyclic stretch on beta1D-integrin expression and activation of FAK and RhoA Am J Physiol Cell Physiol, June 1, 2007; 292(6): C2057 - C2069. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 E. N. Dedkova, Y. G. Wang, X. Ji, L. A. Blatter, A. M. Samarel, and S. L. Lipsius Signalling mechanisms in contraction-mediated stimulation of intracellular NO production in cat ventricular myocytes J. Physiol., April 1, 2007; 580(1): 327 - 345. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. K. Quadri and J. Bhattacharya Resealing of endothelial junctions by focal adhesion kinase Am J Physiol Lung Cell Mol Physiol, January 1, 2007; 292(1): L334 - L342. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. Otis and N. Gallo-Payet Differential Involvement of Cytoskeleton and Rho-Guanosine 5'-Triphosphatases in Growth-Promoting Effects of Angiotensin II in Rat Adrenal Glomerulosa Cells Endocrinology, November 1, 2006; 147(11): 5460 - 5469. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
L. A. DiMichele, J. T. Doherty, M. Rojas, H. E. Beggs, L. F. Reichardt, C. P. Mack, and J. M. Taylor Myocyte-Restricted Focal Adhesion Kinase Deletion Attenuates Pressure Overload-Induced Hypertrophy Circ. Res., September 15, 2006; 99(6): 636 - 645. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 T. A. Baudino, W. Carver, W. Giles, and T. K. Borg Cardiac fibroblasts: friend or foe? Am J Physiol Heart Circ Physiol, September 1, 2006; 291(3): H1015 - H1026. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 K. Rafiq, M. A. Kolpakov, M. Abdelfettah, D. N. Streblow, A. Hassid, L. J. Dell'Italia, and A. Sabri Role of Protein-tyrosine Phosphatase SHP2 in Focal Adhesion Kinase Down-regulation during Neutrophil Cathepsin G-induced Cardiomyocytes Anoikis J. Biol. Chem., July 14, 2006; 281(28): 19781 - 19792. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. Brancaccio, E. Hirsch, A. Notte, G. Selvetella, G. Lembo, and G. Tarone Integrin signalling: The tug-of-war in heart hypertrophy Cardiovasc Res, June 1, 2006; 70(3): 422 - 433. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Hoshijima Mechanical stress-strain sensors embedded in cardiac cytoskeleton: Z disk, titin, and associated structures Am J Physiol Heart Circ Physiol, April 1, 2006; 290(4): H1313 - H1325. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. M. Samarel Costameres, focal adhesions, and cardiomyocyte mechanotransduction Am J Physiol Heart Circ Physiol, December 1, 2005; 289(6): H2291 - H2301. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
D. Hakuno, T. Takahashi, J. Lammerding, and R. T. Lee Focal Adhesion Kinase Signaling Regulates Cardiogenesis of Embryonic Stem Cells J. Biol. Chem., November 25, 2005; 280(47): 39534 - 39544. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 E. A. Thackaberry, B. A. Nunez, I. D. Ivnitski-Steele, M. Friggins, and M. K. Walker Effect of 2,3,7,8-Tetrachlorodibenzo-p-Dioxin on Murine Heart Development: Alteration in Fetal and Postnatal Cardiac Growth, and Postnatal Cardiac Chronotropy Toxicol. Sci., November 1, 2005; 88(1): 242 - 249. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
W. Nadruz Jr., M. A.F. Corat, T. M. Marin, G. A. Guimaraes Pereira, and K. G. Franchini Focal adhesion kinase mediates MEF2 and c-Jun activation by stretch: Role in the activation of the cardiac hypertrophic genetic program Cardiovasc Res, October 1, 2005; 68(1): 87 - 97. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. S. Torsoni, T. M. Marin, L. A. Velloso, and K. G. Franchini RhoA/ROCK signaling is critical to FAK activation by cyclic stretch in cardiac myocytes Am J Physiol Heart Circ Physiol, October 1, 2005; 289(4): H1488 - H1496. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C. F. M. Z. Clemente, M. A. F. Corat, S. T. O. Saad, and K. G. Franchini Differentiation of C2C12 myoblasts is critically regulated by FAK signaling Am J Physiol Regulatory Integrative Comp Physiol, September 1, 2005; 289(3): R862 - R870. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Eghbali, R. Deva, A. Alioua, T. Y. Minosyan, H. Ruan, Y. Wang, L. Toro, and E. Stefani Molecular and Functional Signature of Heart Hypertrophy During Pregnancy Circ. Res., June 10, 2005; 96(11): 1208 - 1216. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
P. Martinka, J. Fielitz, A. Patzak, V. Regitz-Zagrosek, P. B. Persson, and H. M. Stauss Mechanisms of blood pressure variability-induced cardiac hypertrophy and dysfunction in mice with impaired baroreflex Am J Physiol Regulatory Integrative Comp Physiol, March 1, 2005; 288(3): R767 - R776. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. Lehoux, B. Esposito, R. Merval, and A. Tedgui Differential Regulation of Vascular Focal Adhesion Kinase by Steady Stretch and Pulsatility Circulation, February 8, 2005; 111(5): 643 - 649. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
P. M. Fonseca, R. Y. Inoue, C. B. Kobarg, D. P. Crosara-Alberto, J. Kobarg, and K. G. Franchini Targeting to C-Terminal Myosin Heavy Chain May Explain Mechanotransduction Involving Focal Adhesion Kinase in Cardiac Myocytes Circ. Res., January 7, 2005; 96(1): 73 - 81. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Y. Boateng, S. S. Lateef, W. Mosley, T. J. Hartman, L. Hanley, and B. Russell RGD and YIGSR synthetic peptides facilitate cellular adhesion identical to that of laminin and fibronectin but alter the physiology of neonatal cardiac myocytes Am J Physiol Cell Physiol, January 1, 2005; 288(1): C30 - C38. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
E. Kardami, Z.-S. Jiang, S. K Jimenez, C. J Hirst, F. Sheikh, P. Zahradka, and P. A Cattini Fibroblast growth factor 2 isoforms and cardiac hypertrophy Cardiovasc Res, August 15, 2004; 63(3): 458 - 466. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
N. Boutahar, A. Guignandon, L. Vico, and M.-H. Lafage-Proust Mechanical Strain on Osteoblasts Activates Autophosphorylation of Focal Adhesion Kinase and Proline-rich Tyrosine Kinase 2 Tyrosine Sites Involved in ERK Activation J. Biol. Chem., July 16, 2004; 279(29): 30588 - 30599. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. E. Saffitz and A. G. Kleber Effects of Mechanical Forces and Mediators of Hypertrophy on Remodeling of Gap Junctions in the Heart Circ. Res., March 19, 2004; 94(5): 585 - 591. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
H. Mansour, P. P. de Tombe, A. M. Samarel, and B. Russell Restoration of Resting Sarcomere Length After Uniaxial Static Strain Is Regulated by Protein Kinase C{epsilon} and Focal Adhesion Kinase Circ. Res., March 19, 2004; 94(5): 642 - 649. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
P. H. Sugden Ras, Akt, and Mechanotransduction in the Cardiac Myocyte Circ. Res., December 12, 2003; 93(12): 1179 - 1192. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||