Early Activation of the Multicomponent Signaling Complex Associated With Focal Adhesion Kinase Induced by Pressure Overload in the Rat Heart
Abstract—Mechanical overload elicits functional and structural adaptive mechanisms in cardiac muscle. Signaling pathways linked to integrin/cytoskeleton complexes may have a function in mediation of the effects of mechanical stimulus in myocardial cells. We investigated the tyrosine phosphorylation and the assembly of the multicomponent signaling complex associated with focal adhesion kinase (Fak) and the actin cytoskeleton in the overloaded myocardium of rats. Pressure overload induced a 3-fold increase in Fak tyrosine phosphorylation within 3 minutes after a 60-mm Hg rise in aortic pressure. A pressure stimulus that lasted for 60 minutes was accompanied by a 5-fold increase in the amount of tyrosine-phosphorylated Fak, and a stimulus as low as 10 mm Hg doubled the amount of tyrosine-phosphorylated Fak in the myocardium within 10 minutes. Pressure overload also induced a time-dependent association of actin with Fak and an increase in the amount of Fak detected in the cytoskeletal fraction of the myocardium. These events were paralleled by c-Src activation and binding to Fak and by an association of Grb2 and p85 subunit of phosphatidylinositol 3-kinase with Fak. Erk1/2 and Akt, two possible downstream effectors of Fak via Grb2 and phosphatidylinositol 3-kinase, were also shown to be activated in parallel with Fak. These findings show that pressure overload induced a rapid activation of the Fak multiple signaling complex in the myocardium of rats, which suggests that this mechanism may have a role in mechanotransduction in the myocardium.
Mechanical stress is the major factor responsible for functional and structural adjustments of the myocardium in response to increased workload.1 Mechanosensitive ion channels and the release of autocrine and paracrine factors have been suggested to act as potential mechanisms that link mechanical stimuli to cellular responses in the myocardium.2 3 4
Mechanical stimuli may also trigger cellular signaling mechanisms through the cytoskeleton via the elastic coupling to sites such as plasma membrane, internal organelles, or nucleus.5 6 7 In addition, the filamentous cytoskeletal network provides a scaffold where signaling proteins can anchor and become involved in signal transduction pathways.8 9 The activation of signaling systems associated with cytoskeleton in tissues is fundamentally dependent on the clustering of transmembrane integrins that act as linkers between extracellular matrix proteins and the intracellular cytoskeletal scaffold. Integrins connect to a meshwork of F-actin through bridging proteins such as vinculin, talin, and α-actinin at specialized membrane-bound regions known as focal adhesion complexes. These regions are rich in a variety of signaling molecules, including focal adhesion kinase (Fak), c-Src family kinases, guanine nucleotide exchange factors, Ras family proteins, phosphatidylinositol 3 (PI3)-kinase, and mitogen-activated protein kinases.8 9 After mechanical stimulation, integrin clustering and engagement lead to a marked increase in tyrosine phosphorylation and the recruitment of several cellular proteins to the actin meshwork, in particular including Fak.10 11 The precise mechanism that links integrin to Fak activation is unknown, although it is clear that integrin clustering mediates Fak autophosphorylation, predominantly at Tyr397.12 After autophosphorylation, additional tyrosine residues of Fak are phosphorylated through the action of c-Src family kinases that bind to Fak at Tyr397 via their Src homology 2 (SH2) domains. This leads to the binding of other SH2 domain proteins such as PI3 kinase13 and the Grb2/Sos complex,14 which can then activate signaling pathways that are involved in multiple cellular processes.
Multiple integrins are expressed in the heart and may participate in biological processes such as intracellular pH regulation and hypertrophic growth.15 16 17 In addition, mechanical stimuli such as hypotonic cell swelling and pulsatile stretch have been shown to stimulate Fak tyrosine phosphorylation.18 19 In the myocardium of cats, a 4-hour period of pressure overload leads to tyrosine phosphorylation and activation of c-Src and its redistribution from cytosol to the cytoskeletal compartment.20 However, in this study, the authors were unable to show substantial levels of tyrosine phosphorylation of Fak.
Although this evidence suggests that the Fak signaling complex is activated by and contributes to the adaptive myocardial responses to mechanical stimuli, a clear demonstration of this relationship is still lacking. In the present study, we examined the phosphorylation and activation of the Fak signaling complex in the rat heart during acute pressure overload produced through controlled constriction of the transverse aorta. Moreover, possible downstream pathways mobilized by the Fak signaling complex were explored.
Materials and Methods
Antibodies and Chemicals
Polyclonal rabbit anti-Fak, Grb2, Erk1/2, and monoclonal mouse anti-phosphotyrosine, anti–phospho-Erk1/2 (Thr202/Tyr204), and anti–c-Src antibodies were obtained from Santa Cruz Biotechnology. Polyclonal rabbit anti–phospho-c-Src (Tyr416) was obtained from BioSource International, Inc. Polyclonal rabbit anti-p85 subunit of PI3 kinase was from Upstate Biotechnology. Polyclonal rabbit anti-Akt and anti–phospho-Akt (Ser473) were from New England Biolabs, Inc. Monoclonal mouse anti–nonsarcomeric α-actin was from Zymed. Affinity-purified rabbit anti-mouse IgG was from DAKO. [125I]Protein A and [γ-32P]ATP were from Amersham. Protein A–Sepharose 6 MB was from Pharmacia. All other reagents were from Sigma Chemical Co.
Adult male Wistar rats (n=60; weight 180 to 220 g) were obtained from the animal facilities of the university. All procedures and care of the rats were in accordance with institutional guidelines for the use of animals in research.
Pressure overload was induced in pentobarbital-anesthetized rats (50 mg/kg IP) through constriction of the transverse aorta with an adjustable clamp. After anesthesia was induced, the animals were maintained under controlled temperature and ventilation. The aortic and vagus nerves were sectioned bilaterally to minimize the influence of neural reflex on hemodynamics during aortic constriction. The transverse aorta was accessed through the second left intercostal space. A customized adjustable clamp was placed around this vessel, after which the thoracic cavity was closed. After stabilization (≈20 minutes), pressure overload protocols were started with adjustment of the aortic clamping while blood pressure signals from above and below the constriction were monitored. The experimental protocols included sustained (≈60 mm Hg; 3 to 60 minutes) and stepwise (from 10 to 30 mm Hg; 10 minutes) increases in the ascending aorta blood pressure. At the end of the blood pressure–recording period, the ventricles were rapidly removed, minced coarsely, and homogenized. Sham animals were prepared in the same way except for the aortic constriction.
Protein Analysis With Immunoblotting
Aliquots of whole extracts or immunoprecipitated proteins that contained equal amounts of total protein were treated with Laemmli’s sample buffer and underwent SDS-PAGE. The nitrocellulose membranes with transferred proteins were incubated with specific antibodies and [125I]Protein A. Band intensities were quantified through optical densitometry of the developed autoradiographs.
Isolation of Cardiac Cytoskeleton
Cardiac cytoskeletal preparations were obtained from 100 mg ventricular tissue as described previously.20 Samples of the cytoskeletal fraction and of the soluble fraction obtained through ultracentrifugation underwent SDS-PAGE, were transferred to nitrocellulose membranes, and were blotted with anti-Fak antibody.
Immune Complex Tyrosine Kinase Reactions
Tyrosine kinase activity associated with the immune complex of the anti-Fak antibody was assayed with denatured rabbit muscle enolase used as substrate.21 After SDS-PAGE, the proteins were transferred to nitrocellulose membranes, and the 32P-enolase band (≈46 kDa) was visualized and quantified with autoradiography.
The data are presented as mean±SEM. Differences between the mean values of the densitometric readings were tested with 1-way ANOVA for repeated measurements and Bonferroni’s multiple-range test. A value of P<0.05 indicated statistical significance.
An expanded Materials and Methods section can be found in an online data supplement available at http://www.circresaha.org.
Pressure Overload Induces Tyrosine Phosphorylation and Activation of p125Fak and p60Src
The tyrosine phosphorylation of Fak is closely related to its kinase activity.12 13 Figure 1⇓ shows that acute pressure overload increased the p125Fak phosphotyrosine content mainly in the left ventricle, whereas a comparable amount of this protein was found in both ventricles. Next, we studied p125Fak tyrosine phosphorylation induced with stepwise increases in aortic blood pressure (Figure 2⇓). p125Fak phosphotyrosine content increased to ≈180% in response to pressure increases of 10 mm Hg. Additional increases were seen in hearts when the constriction increased aortic pressure by 20 and 30 mm Hg (to ≈220% and ≈240%, respectively). The time course of p125Fak tyrosine phosphorylation was examined in hearts subjected to sustained increases in aortic pressure of ≈60 mm Hg (Figure 3⇓). p125Fak phosphotyrosine content increased 3-fold within 3 minutes and 5-fold within 60 minutes of continuous and stable pressure increases. Parallel immunoblots with anti-Fak antibody revealed that the amount of p125Fak in the myocardium remained unaltered during this period.
After activation, Fak autophosphorylates Tyr397, creating a binding site for SH2 domain of c-Src. c-Src kinase is then activated with phosphorylation on Tyr416.12 The activation of c-Src also depends on the dephosphorylation of constitutively phosphorylated Tyr527.12 Coimmunoprecipitation assays with anti-Fak and anti–c-Src antibodies showed only a weak binding of p60c-Src to Fak in the myocardium of control rats (Figure 4A⇓). Pressure overload increased the p60c-Src binding to Fak, in parallel with its tyrosine phosphorylation. We next examined whether pressure overload also induces tyrosine phosphorylation and activation of p60c-Src. Immunoprecipitation experiments with anti–c-Src antibodies showed that the tyrosine phosphorylation of p60c-Src increased in overloaded compared with unloaded hearts (Figure 4B⇓). This finding could imply either activation or inactivation, depending on whether the phosphorylation was at Tyr416 or Tyr527.12 We then examined the c-Src tyrosine phosphorylation with a phosphospecific antibody against c-Src (Tyr416). As demonstrated in Figure 4C⇓, pressure overload induced a rapid and sustained increase in phospho-c-Src (Tyr416), indicating that p60c-Src is activated in parallel with Fak. This occurred while the amount of p60c-Src remained constant (Figure 4D⇓).
Further indication of load-induced Fak/c-Src association and c-Src activation was provided by experiments in which the kinase activity of the immune complex of anti-Fak antibody was tested against rabbit denatured muscle enolase, a substrate for c-Src but not for Fak.21 Figure 5⇓ shows that there was negligible tyrosine kinase activity in the immune complex of anti-Fak antibody in unloaded hearts. However, the enolase phosphorylation increased greatly (to ≈150% after 3 minutes and to ≈350% after 60 minutes of pressure overload) when incubated with immunoprecipitates of overloaded hearts.
Redistribution of p125Fak to the Cytoskeletal Compartment Induced by Pressure Overload
Because the migration of Fak to the actin meshwork is a crucial event for Fak activation,8 9 we performed experiments to detect the association of actin with p125Fak. The membranes used to detect p125Fak were stripped and blotted with antibody against nonsarcomeric α-actin. As shown in Figure 6A⇓, overloaded hearts showed a time-dependent coimmunoprecipitation of actin with p125Fak. The pressure-induced migration of p125Fak to the cytoskeletal compartment was further suggested by experiments with cytoskeletal fraction preparations obtained with differential centrifugation. A substantial increase in the amount of p125Fak was detected in the cytoskeletal fraction of homogenates from overloaded myocardial tissue (Figure 6B⇓).
Pressure Overload Induces the Association of Fak With Grb2 and Activation of ERK1/2
Phosphorylation of Fak Tyr925 by c-Src confers a site for Grb2 binding, which potentially links integrin/Fak signaling to the Ras/mitogen-activated protein kinase pathway.14 As shown in Figure 7A⇓, acute pressure overload increased the amount of Grb2 binding to Fak in parallel with activation of the Fak/c-Src complex.
We next investigated whether activation of the Fak/c-Src complex was paralleled by the activation of Erk1/2, a possible downstream effector of Fak via Grb2 binding.21 Western blotting with anti–phospho-Erk1/2 (Thr202/Tyr204)–specific antibody revealed an increase and presumably an activation of this enzyme within 5 minutes (to ≈180%), with a maximal activation being achieved by 10 minutes (to ≈240%) of pressure overload. The values of phospho-Erk1/2 returned toward the baseline values after 60 minutes of pressure overload (Figure 7B⇑). Western blotting with an antibody for Erk1/2 that detects both the phosphorylated and unphosphorylated forms of the kinases showed that myocardial Erk1/2 levels were similar in the heart studied at various periods after aortic constriction (Figure 7C⇑).
Pressure Overload Induces the Association of Fak With p85 Subunit of PI3 Kinase and Activation of Akt
In addition of being a site for c-Src binding, Tyr397 of Fak has been identified as the major site for binding of PI3 kinase, whose inositol lipid products are key mediators of multiple intracellular signaling.13 22 As shown in Figure 8A⇓, pressure overload induced a rapid increase in the association of Fak with p85 subunit of PI3 kinase (to ≈180% within 3 minutes), increasing to ≈360% within 1 hour of aortic constriction. These results indicated that pressure overload induced a recruitment and a possible activation of this enzyme.
One of the multiple downstream signaling molecules regulated by 3′-phosphorylated phosphatidylinositides is the serine/threonine protein kinase Akt.22 In many cases, the activation of Akt is initiated by the binding of 3 phosphoinositides to its pleckstrin homology domain, translocation from the cytoplasm to the plasma membrane, and subsequent phosphorylation by upstream kinases, including PDK1. In the present study, we also examined whether pressure overload activates Akt. Western blotting with anti-Akt (Ser473)-phosphospecific antibody revealed an increase and presumably an activation of this enzyme within 3 minutes (to ≈200%) after the beginning of pressure overload stimulus. The amount of phospho-Akt (Ser473) remained increased to ≈300% up to 1 hour of pressure overload (Figure 8B⇑), whereas the amount of Akt remained unchanged during this period (Figure 8C⇑).
The data presented here support the conclusion that load induces a rapid assembly and activation of the multicomponent signaling complex associated with Fak in the rat heart. The close relationship between increased load and Fak/c-Src activation and the concurrent activation of Erk1/2 and Akt, two potential downstream effectors of the Fak multicomponent signaling complex, indicated that it may play a role in the earlier myocardial responses to increased workload.
Load-Induced Activation and Assembly of the Fak Multicomponent Signaling Complex
Pressure overload induced a rapid increase (3 minutes) in the myocardial p125Fak phosphotyrosine content, preferentially in the left ventricle. Further increases were observed after 1 hour of augmented workload. This was accompanied by a load-induced association of c-Src with Fak, as indicated by the increase in the amount of p60c-Src detected in blots of immunoprecipitated Fak, and c-Src activation, as indicated by the increase in the amount of c-Src detected with phosphospecific antibody against c-Src (Tyr416) in overloaded compared with unloaded hearts. The idea that load induces a rapid activation of c-Src and its association with Fak was strengthened by the increased kinase activity detected in the immune complex of anti-Fak antibody toward the c-Src substrate enolase. Our study also provides evidence that load induces the association of the signaling molecules Grb2 and PI3 kinase to Fak and the migration of p125Fak to the cytoskeletal compartment. Presumably, this p125Fak migration was directed to the actin meshwork, as suggested by the time-dependent association of actin with Fak in overloaded hearts.
These results are in general consistent with data from noncardiac cells that demonstrate that mechanical stimulus activated the Fak/c-Src complex and its recruitment to the actin cytoskeletal meshwork, presumably at sites related to focal adhesion.8 9 Cardiac myocytes contain structures known as costameres, which resemble focal adhesion complexes and have been suggested to be involved in the transduction of mechanical forces from cardiac myocyte surface.23 24 Costameres are also rich in vinculin, talin, integrins, and a meshwork of actin that occur in register with Z lines.23 24 However, a clear demonstration that Fak is localized or migrates to costameres on mechanical stimuli must be confirmed.
The clear relationship between the assembly and activation of the Fak multicomponent signaling complex with the load stimuli suggests that this mechanism may play a central role in the mechanotransduction during increased load in the myocardium. In this context, studies in cultured rat cardiomyocytes have shown that pulsatile stretch can activate Fak within 5 minutes after the beginning of the stretch.19 As has been shown in cultured cells,10 it is possible that this rapid activation of the Fak/c-Src signaling complex is mediated via integrin, but Fak tyrosine phosphorylation and activation may also be elicited through a variety of nonintegrin cell surface receptors, including growth factor tyrosine kinases and G protein–coupled receptors.9 However, Fak activation mediated by soluble factors has been shown to be short in duration, which favors the hypothesis that the activation of Fak seen in the present study is predominantly mediated via integrins.8 9
In contrast with the present results, previous studies20 have failed to detect substantial phosphorylation of Fak in pressure-overloaded myocardium. One possible explanation for this discrepancy may be related to the fact that in previous studies, the tyrosine phosphorylation of Fak was examined after 4 hours of pressure overload, by which time the tyrosine phosphorylation of Fak may have vanished because of the actions of tyrosine phosphatases. However, this explanation is not consistent with data that show a persistent activation of nonreceptor tyrosine kinases for longer periods in overloaded myocardium.20 Experiments with controlled aortic constriction for longer periods could clarify the difference between the results of the present study and those of previous studies. However, the maintenance of a stable and reliable preparation with aortic constriction for periods of >1 hour is difficult in anesthetized rats.
Load-Induced Activation of ERK1/2 and Akt
The activation of Fak has been shown to influence a variety of cellular functions, including the control of cell shape, growth, and survival.8 9 In looking at possible downstream events regulated by the Fak signaling complex, we also showed that Erk1/2 and Akt were activated in parallel with activation of the Fak signaling complex. The rapid association of Fak with Grb2 and PI3 kinase, two intermediate steps between Fak and Erk1/2 and Akt activation, strengthened the idea that Fak/c-Src activation may be the upstream event of such a rapid activation of these kinases, as has been suggested previously.21 25
Erk1/2 regulates an extensive range of cellular processes, including gene transcription, cytoskeletal organization, metabolic homeostasis, cell growth, and survival.26 The activation of Erk1/2 is likely to be an important pathway to the adaptation of myocardial cells to mechanical stimuli. To date, mechanical stretch of neonatal cardiac myocytes has been shown to rapidly (within 5 minutes) activate the Ras/Erk1/2 pathway.27 28 Although the functional roles of these Erks in stretch-induced cardiac hypertrophy are unclear at the present, they may be important in the regulation of the expression of genes such as early responsive genes (eg, c-fos, c-jun, egr-1), the expression of which is rapidly and transiently upregulated in the myocardium and isolated cardiomyocytes subjected to mechanical stress. Moreover, the early activation of Erk1/2 has been suggested to contribute to the reexpression of fetal ventricle genes (eg, atrial natriuretic factor, β-myosin heavy chain, and skeletal muscle α-actin).29
Akt activation transduces signals that regulate multiple biological processes, including glucose metabolism, apoptosis, gene expression, and cell proliferation.22 Studies have shown that Akt mediates the antiapoptosis effects of Fak.25 In cardiac myocytes, it has been shown that Akt mediates β-adrenergic receptor–stimulated atrial natriuretic factor transcription.30 To our knowledge, the present study is the first to show that Akt may be activated during the early response to pressure overload in the myocardium. The importance of the early activation of Akt during pressure overload can only be speculated. Fak-mediated activation of Akt is likely to be important for the overall response to increased load. Akt phosphorylates various intracellular substrates, thereby affecting metabolism,31 protein synthesis,32 cell survival/apoptosis,33 and gene expression through the regulation of transcription factors,34 which could mediate the cellular responses to increased load in the myocardium.
The present results do not exclude the possibility that mechanisms other than Fak/Src activation could be responsible for the activation of Erk1/2 and Akt during the early phase of cardiac response to pressure overload. To date, Erks and Akt have been shown to be activated through protein–tyrosine kinase and G protein–coupled receptors.22 26 Therefore, further studies are necessary to confirm the importance of the Fak signaling complex in the activation of Erk and Akt.
In conclusion, pressure overload induced a rapid assembly of the Fak/Src signaling complex in the myocardium. This activation was shown to be roughly parallel to stimulus intensity and duration and to the activation of possible downstream pathways such as Erk1/2 and Akt activation. In consideration of the potential effects of this signaling system on multiple cellular functions such as ion transport, force transmission, metabolic pathways, intracellular molecular transport, survival, and gene expression, this complex may occupy a central position in the adaptive changes induced by increased load in the myocardium.
This study was sponsored by grants from Fundação de Auxílio à Pesquisa do Estado de São Paulo (FAPESP; Proc. 98/11403-7) and Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq; Proc. 521098/97-1).
- Received March 10, 2000.
- Revision received July 10, 2000.
- Accepted July 28, 2000.
- © 2000 American Heart Association, Inc.
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