Focal Adhesion Kinase as a RhoA-activable Signaling Scaffold Mediating Akt Activation and Cardiomyocyte Protection

Abstract

RhoA a small G-protein that has an established role in cell growth and in regulation of the actin cytoskeleton. Far less is known about whether RhoA can modulate cell fate. We previously reported that sustained RhoA activation induces cardiomyocyte apoptosis (Del Re, D. P., Miyamoto, S., and Brown, J. H. (2007) J. Biol. Chem. 282, 8069–8078). Here we demonstrate that less chronic RhoA activation affords a survival advantage, protecting cardiomyocytes from apoptotic insult induced by either hydrogen peroxide treatment or glucose deprivation. Under conditions where RhoA is protective, we observe Rho kinase-dependent cytoskeletal rearrangement and activation of focal adhesion kinase (FAK). Activation of endogenous cardiomyocyte FAK leads to its increased association with the p85 regulatory subunit of phosphatidylinositol-3-kinase (PI3K) and to concomitant activation of Akt. Treatment of isolated perfused hearts with sphingosine 1-phosphate recapitulates this response. The pathway by which RhoA mediates cardiomyocyte Akt activation is demonstrated to require Rho kinase, FAK and PI3K, but not Src, based on studies with pharmacological inhibitors (Y-27632, LY294002, PF271 and PP2) and inhibitory protein expression (FAK-related nonkinase). Inhibition of RhoA-mediated Akt activation at any of these steps, including inhibition of FAK, prevents RhoA from protecting cardiomyocytes against apoptotic insult. We further demonstrate that stretch of cardiomyocytes, which activates endogenous RhoA, induces the aforementioned signaling pathway, providing a physiologic context in which RhoA-mediated FAK phosphorylation can activate PI3K and Akt. We suggest that RhoA-mediated effects on the cardiomyocyte cytoskeleton provide a novel mechanism for protection from apoptosis.

The small G-protein RhoA is a transducer of signals from the plasma membrane and mediates a range of cellular processes. There is abundant evidence supporting a requirement for RhoA signaling in regulating the cytoskeleton, cell cycle progression, and gene expression (1–3). There is, in contrast, far less known regarding the involvement of RhoA in cell survival or apoptosis. Some published reports implicate RhoA as a mediator of survival (4–6), whereas others link it to cell death pathways (7–9). Recently, our laboratory demonstrated that prolonged expression of activated RhoA in cardiomyocytes up-regulates the proapoptotic protein Bax and triggers a mitochondrial death pathway and apoptosis (10). Interestingly, we noted that RhoA expression initially induced a classic hypertrophic response with associated changes in gene expression, cell enlargement, and organization of the actin cytoskeleton.

It is well established that the engagement of the cytoskeleton with cell surface integrins and the extracellular matrix influences cell survival (11–13). Detachment of cells can induce programmed cell death (14–16). Conversely, increased cell adhesion and extracellular matrix interactions promote cell survival through a variety of signaling mechanisms in which activation of focal adhesion kinase (FAK)2 plays a central role (17–19). FAK is a nonreceptor tyrosine kinase that serves as a scaffold for multiple signaling cascades, stimulating activation of tyrosine kinases, mitogen-activated protein kinases, and phosphatidylinositol-3-kinase (PI3K) (20). Integrin engagement is an established mechanism for FAK activation (21, 22). Additionally, FAK has been shown to be activated through a RhoA/Rhokinase-dependent pathway in response to agonist treatment, stretch, or mechanical stress in cardiomyocytes (23, 24) and other cell types (25–27). However, the biological significance of FAK activation in cardiomyocyte survival and the involvement of RhoA and the cytoskeleton in this process have not been explored.

In this report, we demonstrate that RhoA activation affords a survival advantage to cardiomyocytes, and this occurs through FAK phosphorylation, association of FAK with PI3K, and, consequently, activation of Akt. Akt is a well accepted mediator of survival, protecting the heart from apoptotic insult (28, 29), but the precise molecular events leading to its activation in the heart are not fully established. This is, to our knowledge, the first report demonstrating that cytoskeletal events and the downstream cascade initiated by RhoA activate Akt and mediate protection of cardiomyocytes.

EXPERIMENTAL PROCEDURES

Cell Culture—Neonatal rat ventricular myocytes were prepared from hearts of 2–3-day-old Sprague-Dawley rat pups, as described previously (30). Briefly, hearts were digested with collagenase and myocytes purified over a Percoll gradient. Myocytes were plated at a density of 0.4 × 10⁶ cells/3.5-cm dish, 1.2 × 10⁶ cells/6-cm dish, or 4 × 10⁶ cells/10-cm dish in serum-containing medium overnight. For stretch experiments, cells were plated at a density of 1.5 × 10⁶ onto silicon membranes coated with collagen type I (0.5 μg/cm²), which were assembled in a biaxial stretch device, as described previously (31). Cells were serum-starved for 24 h and then stretched for the times indicated.

Adenoviral Infection—Cells cultured in serum-free medium overnight were subsequently infected with (L63)RhoA, FAK-related nonkinase (FRNK), or AdCMV control adenovirus for 2 h. Cells were washed and maintained in serum-free medium. Inhibitors were added at infection and replaced following virus washout unless otherwise described. The Rho kinase inhibitor Y-27632, PI3K inhibitor LY294002, Src inhibitor PP2, and cytochalasin D were purchased from Calbiochem. The 2-deoxyglucose was purchased from Sigma. The cell-permeable exoenzyme C3 transferase was purchased from Cytoskeleton. The GTPase-deficient mutant (L63)RhoA adenovirus was generated as described previously (32).

Western Blot Analysis—Cells were harvested in lysis buffer (20 mm Tris, 250 mm NaCl, 3 mm EDTA, 3 mm EGTA, 20 mm β-glycerophosphate) supplemented with sodium vanadate, leupeptin, aprotinin, p-nitrophenyl phosphate, phenylmethysulfonyl fluoride, and 0.5% Nonidet P-40. Bradford analysis was performed to determine protein concentration. Equal amounts of protein (10–20 μg) were separated by SDS-PAGE and transferred to an Immobilon membrane, and the resulting blot was probed using the following antibodies. The total Akt, phospho-Akt (threonine 308 and serine 473), and p85 phosphorylated binding motif antibodies were purchased from Cell Signaling Technologies. The RhoA, FRNK, p85, total Src, and actin antibodies were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). The phospho-Src (tyrosine 416) antibody was purchased from Upstate Biotechnology, Inc. The phospho-FAK (tyrosine 397) antibody was purchased from Calbiochem. The total FAK antibody was purchased from BD Transduction Laboratories.

Immunocytochemistry—Following viral infection, cardiomyocytes were fixed in 4% paraformaldehyde, permeabilized in 0.1% Triton X-100, and blocked in 5% bovine serum albumin for 1 h at room temperature. Cells were then incubated with anti-phospho-FAK (tyrosine 397) rabbit polyclonal antibody (BIOSOURCE) overnight at 4 °C and blocked for 30 min in 10% normal goat serum before incubating with fluorescein isothiocyanate anti-rabbit (Molecular Probes, Inc., Eugene, OR) and rhodamine-conjugated phalloidin for 90 min at room temperature.

Cell Death Enzyme-linked Immunosorbent (POD) Assay—DNA fragmentation indicative of apoptosis was assayed using the Cell Death Detection ELISA^PLUS (Roche Applied Science) according to the manufacturer's instructions. Briefly, lysates were incubated with anti-histone-biotin and anti-DNA-POD in a streptavidin-coated microplate for 2 h and washed, colorimetric substrate was added, and absorbance was measured at 405 nm.

Co-Immunoprecipitation Assay—Following intervention, cells were collected in lysis buffer (supplemented with sodium vanadate, leupeptin, aprotinin, p-nitrophenyl phosphate, phenylmethysulfonyl fluoride) and cleared, and equal amounts of total protein were incubated with anti-FAK (BD Transduction Laboratories) primary antibody at 4 °C overnight. Complexes were pulled down using protein A/G-Sepharose beads (Santa Cruz Biotechnology) and resolved by SDS-PAGE.

RhoA Activation Assay—Activated RhoA was determined using a glutathione S-transferase fusion protein of the RhoA binding domain of the RhoA effector rhotekin, as described previously by our group (33). Briefly, cells were rinsed with ice-cold Tris-buffered saline, lysed, clarified by brief centrifugation, and then incubated with the Sepharose-bound glutathione S-transferase-rhotekin-RhoA binding domain for 40 min at 4 °C. The beads and precipitated proteins were washed, boiled, and separated by SDS-PAGE. The precipitated GTP-bound RhoA was normalized to total RhoA present in the whole cell lysate.

Langendorff Perfused Heart—Adult male C57B6 mice (8 weeks old, weighing 28–32 g) were heparinized (500 units/kg, intraperitoneally) and anesthetized (2% isofluorane with 0.8% oxygen). Hearts were rapidly excised, washed in ice-cold Krebs-Henseleit solution, and cannulated via the aorta on a 20-gauge stainless steel blunt needle. Hearts were perfused at 80 mm Hg on a Langendorff apparatus using Krebs-Henseleit solution at 37 °C. Hearts were perfused for 30 min to allow for equilibration, followed by 10 min of perfusion with sphingosine 1-phosphate (S1P) or vehicle.

Statistical Analysis—All results are reported as mean ± S.E. Comparison of two groups was accomplished using an unpaired Student's t test. Data from experiments with more than two groups were compared by one-way analysis of variance followed by the Tukey post hoc test for comparison between groups.

RESULTS

RhoA has been shown to mediate hypertrophic growth of cultured rat cardiomyocytes as indicated by increased cell size, cytoskeletal organization, and gene expression (34–36). RhoA has also been implicated in mediating activation of FAK in response to endothelin and mechanical stretch (23, 24). To confirm that RhoA signaling pathways increase cytoskeletal organization and to directly determine whether FAK is a downstream RhoA target, we infected neonatal rat cardiomyocytes with constitutively active (L63)RhoA and visualized the cells using confocal microscopy. Cells were infected with control adenovirus (AdCMV) or RhoA adenovirus for 2 h, washed, and examined 6–24 h later. RhoA transgene expression was demonstrated to be maximal, attaining a modest 2–3-fold increase in RhoA protein expression compared with control-infected cells, by 12 h following virus washout (data not shown). By 24 h, RhoA-expressing cells were noticeably larger, appeared more spread, and showed increased actin filament organization relative to control-infected cells. RhoA-expressing cells also showed a robust increase in activated FAK, which colocalized with actin filaments (Fig. 1A). Thus, RhoA-induced cytoskeletal changes are temporally and spatially associated with FAK activation.

FIGURE 1.

RhoA expression induces cytoskeletal reorganization and activates FAK. A–C, neonatal rat ventricular myocytes were infected with constitutively activated (L63)RhoA adenovirus or control (AdCMV) adenovirus at 4 MOI. A, cells were treated with 10 μm Y-27632 or vehicle at virus addition, fixed, and stained 24 h after infection. Rhodamine-conjugated phalloidin was used to visualize actin filaments (a, d, g, and j), and rabbit polyclonal anti-FAK (Tyr(P)³⁹⁷; pY397) and fluorescein isothiocyanate anti-rabbit were used to visualize phosphorylated FAK (P-FAK; b, e, h, and k). Bar,20 μm. B, lysates were prepared 24 h after infection, and levels of phosphorylated FAK (Tyr³⁹⁷; Y397), total FAK, phosphorylated Src (Tyr³⁹⁷; Y416), and total Src were determined by Western blot. C, lysates were obtained from cardiomyocytes infected for 24 h and treated with either 10 μm Y-27632 (at infection) or 10 μm cytochalasin D (treatment for 1 h prior to cell collection). Values from Western blot analysis were quantitated by densitometry and are shown as averages ± S.E. (n = 3). ^***, p < 0.001 versus AdCMV. #, p < 0.001 versus RhoA.

Based on the immunocytochemical results described above we sought to confirm and quantify the effect of RhoA on FAK activation using Western blot analysis. These studies demonstrated a maximal 3-fold increase in FAK phosphorylation at tyrosine 397 (indicative of FAK activation), following 24 h of RhoA expression (Fig. 1B), with lesser but significant increases at earlier (12 h) and later (48 h) times (data not shown). No significant difference in total FAK expression was observed at any of these times. Since FAK associates with and activates the nonreceptor tyrosine kinase, Src (37), we asked whether Src phosphorylation was also increased in cells expressing RhoA. Notably, Western analysis carried out using phosphoantibody directed to the tyrosine 416 site (indicative of activation) showed no change in Src phosphorylation in RhoA-infected cells versus control cells (Fig. 1B).

Rho kinase has been demonstrated to mediate many of the effects of RhoA on the cytoskeleton; thus, we asked whether Rho kinase was involved in FAK activation. The ability of the Rho kinase selective inhibitor, Y-27632, to block FAK phosphorylation was examined. As shown by both immunofluorescence (Fig. 1A) and Western blot analysis (Fig. 1C), treatment with 10 μm Y-27632 significantly reduced RhoA-induced FAK phosphorylation at tyrosine 397. To examine the role of RhoA-induced cytoskeletal changes in FAK activation, cells were treated with cytochalasin D to disrupt actin polymerization. Treatment with 10 μm cytochalasin D completely abolished the RhoA-induced increase in FAK phosphorylation (Fig. 1C). Taken together, these data provide evidence that Rho kinase, as well as cytoskeletal integrity, are required for RhoA-mediated FAK activation.

FAK serves as a protein scaffold that mediates activation of signaling molecules, including PI3K (20). An interaction between heterologously expressed FAK and p85, a regulatory subunit of the catalytic p110 subunit of PI3K, has been shown to increase PI3K activity in fibroblasts (38–40). The p85 subunit contains a Src homology 2 domain known to bind the consensus sequence Y^*XXM, where X is any amino acid and Y^*isa phosphorylated tyrosine residue (41). A sequence scan of FAK revealed three corresponding motifs conserved between mice, rats, and humans. To test the possibility that RhoA increases phosphorylation of this p85 recognition motif in FAK, we immunoprecipitated FAK from control- and RhoA-infected cells and probed for this phosphorylated motif. RhoA expression markedly increased phosphorylation of this sequence in FAK, and this increase was mediated by Rho kinase, since it was completely blocked by Y-27632 treatment (Fig. 2A). To test for an interaction between FAK and p85 in cardiomyocytes, we performed co-immunoprecipitation assays using a total FAK antibody. These experiments demonstrated increased association of endogenous FAK and p85 in cells expressing RhoA (Fig. 2B). This increased association was completely abolished by treatment with either Y-27632 (10 μm) or PF271 (1 μm), a pharmacological inhibitor of FAK, indicating that both Rho kinase and FAK activity are required to elicit this interaction (Fig. 2B).

FIGURE 2.

RhoA increases the association of endogenous FAK and p85. A and B, cardiomyocytes were infected with (L63)RhoA or control (AdCMV) adenovirus for 2 h, washed, and harvested at 24 h. A, cells were treated with 10 μm Y-27632 or vehicle at time of infection and lysates prepared. Total FAK protein was immunoprecipitated (IP) using mouse monoclonal anti-FAK antibody, separated by SDS-PAGE, and probed using a selective p85 recognition phosphomotif antibody. B, cells were treated with 10 μm Y-27632, 1 μm PF271, or vehicle at the time of infection. Co-immunoprecipitation was performed using mouse monoclonal anti-FAK antibody, and complexes were separated by SDS-PAGE and probed with a pan-p85 antibody to recognize endogenous p85 protein. Blots are representative of three independent experiments.

Based on our findings that RhoA increases the association of FAK with PI3K, we asked whether RhoA activates the well characterized cardioprotective kinase, Akt. Western blot analysis demonstrated that expression of RhoA increased Akt phosphorylation at serine 473 as well as threonine 308 (data not shown), inducing a nearly 4-fold increase versus control cells (Fig. 3, A and B). Akt phosphorylation was increased significantly within 12 h of RhoA infection, was maximal at 24 h, and then declined by 48 h postinfection (data not shown), paralleling the time course of FAK activation. Treatment with the PI3K inhibitor, LY294002 (10 μm), completely abolished the increase in Akt phosphorylation, indicating involvement of PI3K (Fig. 3, A and B).

FIGURE 3.

RhoA expression activates Akt, which is mediated by PI3K. A and B, cardiomyocytes were infected with (L63)RhoA or control (AdCMV) adenovirus in the presence or absence of 10 μm LY294002 for 24 h. Lysates were prepared and levels of phosphorylated Akt (Ser⁴⁷³; S473) were determined by Western blot analysis. A, representative blot showing LY294002 inhibits Akt phosphorylation. B, quantified densitometry results. Values represent averages ± S.E. (n = 5). ^**, p < 0.01 versus AdCMV. #, p < 0.001 versus RhoA.

To prove that Akt activation results from the recruitment of PI3K by FAK, we infected cells with adenovirus encoding the FAK-related nonkinase, FRNK. FRNK is an endogenously expressed truncated form of FAK that contains the C-terminal domain of FAK but lacks its kinase domain and therefore acts as a dominant inhibitory regulator (42). Expression of FRNK adenovirus prevented RhoA-induced FAK phosphorylation at tyrosine 397, evidence that the construct acted as intended (Fig. 4A). Importantly, expression of FRNK fully inhibited the phosphorylation of Akt elicited by RhoA expression. Western blot analysis was also performed on cells treated with Y-27632 (10 μm), the FAK inhibitor, PF271 (1 μm), or the Src-selective inhibitor, PP2 (1 μm), and levels of phosphorylated Akt were determined. Fig. 4B quantifies Akt activation in the absence or presence of these inhibitors or FRNK adenovirus (10 MOI). The blockade of Akt activation by Y-27632 is consistent with the data above showing that Rho kinase is required for FAK phosphorylation and for PI3K association with FAK (Figs. 1C and 2B). The lack of inhibition by PP2 is consistent with our observation that Src is not activated by RhoA expression (Fig. 1B). The data with PF271 and FRNK, taken with the data above, implicate FAK, acting independently of Src, as a RhoA/Rho kinase-dependent mediator of PI3K and Akt activation.

FIGURE 4.

RhoA-induced activation of Akt is mediated by FAK. A and B, cardiomyocytes were infected with (L63)RhoA or control (AdCMV) adenovirus and co-infected with increasing amounts of FRNK adenovirus. Lysates were prepared 24 h after infection, and Western blot analysis was performed. B, values from Western blot analysis for phosphorylated Akt (Ser⁴⁷³; S473) with 10 μm Y-27632, 10 MOI FRNK, 1 μm PF271, 1 μm PP2, or vehicle treatment were quantitated by densitometry and are shown as averages ± S.E. (n = 3). ^***, p < 0.001 versus AdCMV. #, p < 0.001 versus RhoA. n.s., not significant.

To examine the possibility that RhoA signaling pathways mediate a protective response, control- and RhoA-infected cells were treated with 100 μm hydrogen peroxide for 16–18 h. Myocytes infected with control adenovirus showed a roughly 5-fold increase in DNA fragmentation. This increase was significantly attenuated in cells expressing RhoA (Fig. 5A). To demonstrate the involvement of PI3K/Akt in RhoA-mediated protection, we examined the effect of LY294002. Inhibition of PI3K had no effect on DNA fragmentation induced by hydrogen peroxide in control-infected cells (data not shown). However, the ability of RhoA to protect against DNA fragmentation elicited by hydrogen peroxide was fully blocked in the presence of LY294002 (Fig. 5A), indicating that Akt activated by RhoA serves a protective function.

FIGURE 5.

RhoA expression protects cells from apoptotic insult. A, cardiomyocytes were infected with (L63)RhoA or control (AdCMV) adenovirus. Following virus removal, cells were pretreated with 10 μm LY, 1 μm PF271, or vehicle for 30 min prior to treatment with 100 μm H₂O₂ for 16–18 h. B, cardiomyocytes were infected with (L63)RhoA or control (AdCMV) adenovirus and co-infected with FRNK (10 MOI) or control virus. Following virus removal, cells were treated with 100 μm H₂O₂ for 16–18 h or cultured in low glucose medium containing 4 mm 2-deoxyglucose for 30 h. Lysates were prepared, and nucleosomal fragmentation was quantified by a POD assay. Values represent averages ± S.E. (n = 5). #, p < 0.001 versus AdCMV + H₂O₂. ^***, p < 0.001 versus RhoA + H₂O₂.†, p < 0.05 versus AdCMV + 2-deoxyglucose. %, p < 0.05 versus RhoA + 2-deoxyglucose.

The data above suggest that RhoA and Akt-mediated protection occurs via increased FAK phosphorylation. To prove that FAK is the mediator of cardiomyocyte protection, we tested the ability of FRNK adenoviral expression and of pharmacological FAK inhibition to block RhoA-mediated protection. Blocking FAK through either means precluded RhoA from protecting cardiomyocytes from apoptosis induced by hydrogen peroxide or glucose deprivation (Fig. 5, A and B). Thus, not only is FAK activated by RhoA/Rho kinase, but this response is critical for protection against apoptosis.

To test whether a more physiologic intervention would activate this same signaling pathway, we employed a well characterized model of biaxial static stretch (43). Our laboratory previously reported that stretching cardiomyocytes leads to rapid activation of RhoA (31), and the data presented here confirm that finding (Fig. 6A). Furthermore we examined the effect of stretch on FAK and Akt and found phosphorylation of these kinases to be significantly increased (∼2- and 5-fold, respectively; Fig. 6B). To implicate RhoA in stretch-induced FAK and Akt activation, cardiomyocytes were treated with C3 exoenzyme prior to stretching. Blocking RhoA activation was sufficient to completely abolish the increased phosphorylation of FAK and Akt induced by stretch (Fig. 6C). To determine whether stretch also increases the association of FAK with the p85 subunit of PI3K, total FAK protein was immunoprecipitated from lysates of stretched and unstretched cardiomyocytes. We observed a robust increase in the association of endogenous FAK and p85 following stretch (Fig. 6D). Taken together, these data demonstrate that stretch leads to activation of Akt via a RhoA-mediated FAK/p85 interaction.

FIGURE 6.

Stretch-induced FAK and Akt activation are mediated by RhoA. A–D, cardiomyocytes were plated onto silicon membranes and maintained overnight in serum-free medium. Cells were then subjected to 10% equibiaxial static stretch for the times indicated. A, following 5 min of stretch, cardiomyocytes were harvested to determine RhoA activation using the glutathione S-transferase-RhoA binding domain pull-down assay as described under “Experimental Procedures.” B, cardiomyocytes were stretched for 2 h and harvested, and FAK and Akt phosphorylation were determined by Western blot analysis. Values were quantitated by densitometry and are shown as averages ± S.E. (n = 3). ^***, p < 0.001 versus unstretched. C, cardiomyocytes were treated with C3 exoenzyme (1 μg/ml) or vehicle for 4 h prior to 2 h of stretch. Western blot analysis was performed to assess phosphorylated FAK and Akt. D, cardiomyocytes were stretched for 2 h, and lysates were prepared. Co-immunoprecipitation (IP) was performed using mouse monoclonal anti-FAK antibody, and complexes were separated by SDS-PAGE and probed with a pan-p85 antibody to recognize endogenous p85 protein. Blots are representative of three independent experiments. Ctrl, control.

Our group previously reported that the G-protein-coupled receptor ligand, S1P, protects the heart from ischemia/reperfusion injury in vivo (44). S1P₂ and S1P₃ receptors activate RhoA through coupling to Gα_12/13 (45). Using the Langendorff isolated perfused mouse heart, we demonstrate that S1P treatment leads to activation of RhoA, concomitant with increased phosphorylation of FAK and Akt (Fig. 7A). In other studies, we immunoprecipitated endogenous FAK from lysates of S1P and vehicle-treated perfused hearts. S1P treatment led to increased association of the p85 subunit of PI3K and FAK (Fig. 7B), implicating FAK in S1P-mediated Akt activation in the intact heart.

FIGURE 7.

S1P activates RhoA, FAK, and Akt in the heart. Adult mouse hearts were isolated and perfused for 30 min to allow for equilibration using a Langendorff preparation as described under “Experimental Procedures.” Hearts were then perfused with S1P (1 μm) or vehicle (Veh) (control) for 10 min. Tissue was snap-frozen and homogenized, and lysate was prepared for analysis. A, Western blot analysis of whole heart lysate was carried out to assess activation of RhoA and phosphorylation of FAK and Akt. B, immunoprecipitation of endogenous FAK was carried out using antibody to total FAK, and complexes were separated by SDS-PAGE and probed for FAK and co-immunoprecipitated p85. GAPDH, glyceraldehyde-3-phosphate dehydrogenase.

A model summarizing the proposed pathway for protective signaling is illustrated in Fig. 8. RhoA, acting though Rho kinase-dependent changes in the cytoskeleton, induces phosphorylation of FAK at tyrosine 397. FAK serves as a scaffold, binding the p85 regulatory subunit of PI3K and recruiting it to the plasma membrane, where increased PI3K activity leads to activation of Akt. This signaling scaffold initiated by RhoA serves to protect cardiomyocytes from apoptotic insult.

FIGURE 8.

Proposed schema illustrating RhoA-mediated protection. Mechanical stretch and the G-protein-coupled receptor ligand S1P activate RhoA in cardiomyocytes. Activated RhoA signals through its effector Rho kinase to induce cytoskeletal rearrangement, resulting in the activation of FAK. FAK binds the p85 regulatory subunit of PI3K, increasing PI3K activity and promoting Akt activation and cardiomyocyte protection.

DISCUSSION

RhoA acts as a transducer of signals from a subset of G-protein-coupled receptors as well as mechanical stretch and integrin engagement (1, 2). Canonical RhoA signaling pathways alter the cytoskeleton via activation of the RhoA effector, Rho kinase, leading to increased myosin phosphorylation and contraction (3). RhoA/Rho kinase also initiate actin stress fiber formation via activation of LIM kinase or phosphorylation of ERM proteins, both of which promote actin assembly (46). The ability of RhoA to stimulate actin myofilament rearrangement in neonatal rat cardiomyocytes has been established (32, 34, 36). However, the relationship between RhoA-mediated changes in cytoskeletal structure and cell fate has not been explored. Because increased focal adhesion formation and cell attachment are known to promote cell survival, we hypothesized that there might be a role for RhoA in cardiomyocyte protection. This speculation was triggered by our observation that, whereas sustained RhoA activation leads to apoptosis, the early hypertrophic response of cardiomyocytes was accompanied by increased Akt activation (10), a response that could function as a mediator of cell protection by RhoA signaling.

Several published papers implicate RhoA signaling in Akt regulation, but these reports suggest that RhoA activation negatively regulates Akt. Studies using neutrophils and fibroblasts described the ability of RhoA to increase activity of the phosphatidylinositol 3,4,5-trisphosphate phosphatase PTEN (phosphatase and tensin homolog) and negatively regulate PI3K/Akt activation (47, 48). Work with endothelial cells and aortic tissue suggested a similar conclusion, demonstrating that Akt activation was increased when RhoA and/or Rho kinase were inhibited (49, 50). In a similar vein, expression of activated RhoA in human umbilical vein endothelial cells was shown to prevent Akt activation and negatively regulate NO synthesis (51). The ability of RhoA to regulate Akt activation in a terminally differentiated or nonmigratory cell, such as the cardiomyocyte, had not, to our knowledge, been investigated. Our studies contrast with those cited above and are the first to demonstrate that RhoA can positively regulate Akt activity through cytoskeletal events.

RhoA has been previously implicated in cytoskeletal regulation and FAK activation in cardiomyocytes (23, 24, 32). We demonstrate here that FAK activation links RhoA signals to Akt. A mechanism for this was suggested by earlier reports demonstrating that FAK can physically interact with the regulatory p85 subunit of PI3K in fibroblasts (39), human glioblastoma cells (52), and the pressure-overloaded heart (53). The current studies show that RhoA expression, stretch, and S1P all increase the association of endogenous p85 with FAK. Remarkably, only a fraction of the total cellular p85 precipitates with FAK, yet this is sufficient to markedly increase Akt activation in response to each of these stimuli. These data support our hypothesis that FAK serves as a scaffold to facilitate downstream signaling. We previously reported that RhoA activation and S1P signaling are compartmentalized in cardiomyocytes (31, 54) and suggest that the cytoskeleton and FAK scaffold serve as points of convergence by which these stimuli signal Akt activation and cardioprotection.

We further implicated FAK in PI3K/Akt activation using adenoviral overexpression of FRNK, which has been shown to reduce phosphorylation of FAK and its downstream substrates and to promote cell detachment (42, 55). Our studies are the first to demonstrate that FRNK not only prevents FAK activation but blocks Akt activation and cardiomyocyte protection elicited by RhoA. Notably, although Src has been implicated in FAK-mediated activation of Akt in other cell types (21, 56), we find that Src is neither activated by RhoA nor required for RhoA and FAK-mediated Akt activation, suggesting cell type specificity of this signaling paradigm. These data provide a strong and heretofore undescribed link between FAK and Akt activation in the cardiomyocyte while further confirming FAK as a prosurvival signaling molecule.

Two mouse models have been generated using inducible and cardiomyocyte-specific FAK ablation (57, 58). Both implicate FAK in the development of cardiac hypertrophy and suggest the possibility of altered extracellular signal signaling in this response. These reports are consistent with the earlier finding that FAK is involved in the hypertrophic response of cultured cardiomyocytes (59, 60). More recent work using small interfering RNA to knock down FAK expression in the adult mouse heart further implicated FAK signaling in the development of pressure overload-induced hypertrophy (61). These studies did not, however, examine Akt activation or cell survival. Thus, the question of whether FAK signaling confers cardiac protection in vivo has not been addressed. Our studies suggest that imposing ischemia/reperfusion injury or myocardial infarct on cardiac specific FAK null animals could reveal an additional role for FAK signaling and demonstrate that FAK-mediated activation of PI3K/Akt normally plays a protective role in the in vivo heart.

Although we suggest that RhoA/Rho kinase signaling is protective in cardiomyocytes, several published papers suggest a maladaptive role of ROCK (RhoA/Rho kinase) signaling in the cardiovascular system. For example, inhibition of Rho kinase with Y-27632 or fasudil (HA-1077) decreased fibrosis in response to myocardial infarct (62) and reduced infarct size following both short and long term ischemia/reperfusion injury (49, 63). Moreover, it has been reported that both ROCK1 null and ROCK1^+/- haploinsufficient mice respond better to increased cardiovascular demand, exhibiting more compensatory hypertrophy and less cardiac fibrosis (64, 65). Similarly, recent work from Shi et al. demonstrates increased cardiac function in mice lacking ROCK1 in the Gα_q model of dilated cardiomyopathy (66). Although these studies suggest that RhoA/Rho kinase signaling in the heart is maladaptive rather than protective, the site of inhibition of RhoA/Rho kinase signaling responsible for its salutary effects may not be the cardiomyocyte. Indeed, a host of other cell types, including fibroblasts, endothelial cells, and macrophages, all present in the in vivo system, respond to RhoA with proliferative or inflammatory responses, and inhibition of these responses by RhoA/Rho kinase inhibition may be beneficial in a chronic pathophysiological setting. Our data suggest, however, that acute RhoA/Rho kinase signaling in cardiomyocytes affords protection via its profound effect on the cytoskeleton and consequent activation of FAK and Akt.

Our previous publication provided, to our knowledge, the only evidence for a role for RhoA in determining cardiomyocyte fate (10). This work demonstrated that extended RhoA activation led to up-regulation of Bax and subsequent apoptosis. Interestingly, as we now demonstrate, shorter durations of RhoA activation provide a striking protective advantage from apoptotic insult. It is possible that the transition from cardiomyocyte survival to apoptosis reflects the transient nature of RhoA-induced FAK and Akt activation, ultimately shifting the balance from protection to cell death. We conclude that RhoA is a pleiotropic molecule that can modulate cell survival in response to G-protein-coupled receptor stimulation and stretch and affords acute cardiomyocyte protection by activating FAK and Akt.