FERM domain interaction with myosin negatively regulates FAK in cardiomyocyte hypertrophy.

Focal adhesion kinase (FAK) regulates cellular processes that affect several aspects of development and disease. The FAK N-terminal FERM (4.1 protein-ezrin-radixin-moesin homology) domain, a compact clover-leaf structure, binds partner proteins and mediates intramolecular regulatory interactions. Combined chemical cross-linking coupled to MS, small-angle X-ray scattering, computational docking and mutational analyses showed that the FAK FERM domain has a molecular cleft (~998 Å(2)) that interacts with sarcomeric myosin, resulting in FAK inhibition. Accordingly, mutations in a unique short amino acid sequence of the FERM myosin cleft, FP-1, impaired the interaction with myosin and enhanced FAK activity in cardiomyocytes. An FP-1 decoy peptide selectively inhibited myosin interaction and increased FAK activity, promoting cardiomyocyte hypertrophy through activation of the AKT-mammalian target of rapamycin pathway. Our findings uncover an inhibitory interaction between the FAK FERM domain and sarcomeric myosin that presents potential opportunities to modulate the cardiac hypertrophic response through changes in FAK activity.

A role for focal adhesion kinase in cardiac mitochondrial biogenesis induced by mechanical stress.

We studied the implication of focal adhesion kinase (FAK) in cardiac mitochondrial biogenesis induced by mechanical stress. Prolonged stretching (2-12 h) of neonatal rat ventricular myocytes (NRVM) upregulated the main components of mitochondrial transcription cascade [peroxisome proliferator-activated receptor coactivator-1 (PGC-1α), nuclear respiratory factor (NRF-1), and mitochondrial transcription factor A]. Concomitantly, prolonged stretching enhanced mitochondrial biogenesis [copy number of mitochondrial DNA (mtDNA), content of the subunit IV of cytochrome oxidase, and mitochondrial staining-green fluorescence intensity of Mitotracker green] and induced the hypertrophic growth (cell size and atrial natriuretic peptide transcripts) of NRVM. Furthermore, the stretching of NRVM enhanced phosphorylation, nuclear localization, and association of FAK with PGC-1α. Recombinant FAK COOH-terminal, but not the NH(2)-terminal or kinase domain, precipitated PGC-1α from nuclear extracts of NRVM. Depletion of FAK by RNA interference suppressed the upregulation of PGC-1α and NRF-1 and markedly attenuated the enhanced mitochondrial biogenesis and hypertrophic growth of stretched NRVM. In the context of energy metabolism, FAK depletion became manifest by a reduction of ATP levels in stretched NRVM. Complementary studies in adult mice left ventricle demonstrated that pressure overload upregulated PGC-1α, NRF-1, and mtDNA. In vivo FAK silencing transiently attenuated the upregulation of PGC-1α, NRF-1, and mtDNA, as well as the left ventricular hypertrophy induced by pressure overload. In conclusion, activation of FAK signaling seems to be important for conferring enhanced mitochondrial biogenesis coupled to the hypertrophic growth of cardiomyocytes in response to mechanical stress, via control of mitochondrial transcription cascade.

FAK mediates the activation of cardiac fibroblasts induced by mechanical stress through regulation of the mTOR complex.

AIMS: Cardiac fibroblasts are activated by mechanical stress, but the underlying mechanisms involved remain poorly understood. In this study, we investigated whether focal adhesion kinase (FAK) plays a role in the activation of cardiac fibroblasts in response to cyclic stretch. METHODS AND RESULTS: Neonatal (NF-P3/80--third passage, 80% confluence) and adult (AF-P1/80--first passage, 80% confluence) rat cardiac fibroblasts were exposed to cyclic stretch (biaxial, 1 Hz), which enhanced FAK phosphorylation at Tyr397. Proliferation (anti-5-bromo-2'-deoxyuridine and anti-Ki67 nuclear labelling), differentiation into myofibroblasts (expression of alpha-smooth muscle actin--alpha-SMA), and the activity of matrix metalloproteinase-2 were equally enhanced in stretched NF-P3/80 and AF-P1/80. Treatment with the integrin inhibitor RGD peptide impaired FAK phosphorylation and increased apoptosis (TUNEL) in non-stretched and stretched NF-P3/80, whereas FAK silencing induced by small interfering RNA modestly enhanced apoptosis only in stretched cells. RGD peptide or FAK silencing suppressed the activation of NF-P3/80 invoked by cyclic stretch. In addition, NF-P3/80 depleted of FAK were defective in AKT Ser473, TSC-2 Thr1462, and S6 kinase Thr389 phosphorylation induced by cyclic stretch. The activation of NF-P3/80 invoked by cyclic stretch was prevented by pre-treatment with the mammalian target of rapamycin (mTOR) inhibitor rapamycin, whereas supplementation with the amino acid, leucine, activated S6K and rescued the stretch-induced activation of NF-P3/80 depleted of FAK. CONCLUSIONS: These findings demonstrate a critical role for the mTOR complex, downstream from FAK, in mediating the activation of cardiac fibroblasts in response to mechanical stress.

MEF2C silencing attenuates load-induced left ventricular hypertrophy by modulating mTOR/S6K pathway in mice.

BACKGROUND: The activation of the members of the myocyte enhancer factor-2 family (MEF2A, B, C and D) of transcription factors promotes cardiac hypertrophy and failure. However, the role of its individual components in the pathogenesis of cardiac hypertrophy remains unclear. METHODOLOGY/PRINCIPAL FINDINGS: In this study, we investigated whether MEF2C plays a role in mediating the left ventricular hypertrophy by pressure overload in mice. The knockdown of myocardial MEF2C induced by specific small interfering RNA (siRNA) has been shown to attenuate hypertrophy, interstitial fibrosis and the rise of ANP levels in aortic banded mice. We detected that the depletion of MEF2C also results in lowered levels of both PGC-1alpha and mitochondrial DNA in the overloaded left ventricle, associated with enhanced AMP:ATP ratio. Additionally, MEF2C depletion was accompanied by defective activation of S6K in response to pressure overload. Treatment with the amino acid leucine stimulated S6K and suppressed the attenuation of left ventricular hypertrophy and fibrosis in the aforementioned aortic banded mice. CONCLUSION/SIGNIFICANCE: These findings represent new evidences that MEF2C depletion attenuates the hypertrophic responses to mechanical stress and highlight the potential of MEF2C to be a target for new therapies to cardiac hypertrophy and failure.

Shp2 negatively regulates growth in cardiomyocytes by controlling focal adhesion kinase/Src and mTOR pathways.

The aim of this study was to investigate whether Shp2 (Src homology region 2, phosphatase 2) controls focal adhesion kinase (FAK) activity and its trophic actions in cardiomyocytes. We show that low phosphorylation levels of FAK in nonstretched neonatal rat ventricular myocytes (NRVMs) coincided with a relatively high basal association of FAK with Shp2 and Shp2 phosphatase activity. Cyclic stretch (15% above initial length) enhanced FAK phosphorylation at Tyr397 and reduced FAK/Shp2 association and phosphatase activity in anti-Shp2 precipitates. Recombinant Shp2 C-terminal protein tyrosine phosphatase domain (Shp2-PTP) interacted with nonphosphorylated recombinant FAK and dephosphorylated FAK immunoprecipitated from NRVMs. Depletion of Shp2 by specific small interfering RNA increased the phosphorylation of FAK Tyr397, Src Tyr418, AKT Ser473, TSC2 Thr1462, and S6 kinase Thr389 and induced hypertrophy of nonstretched NRVMs. Inhibition of FAK/Src activity by PP2 {4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine} abolished the phosphorylation of AKT, TSC2, and S6 kinase, as well as the hypertrophy of NRVMs induced by Shp2 depletion. Inhibition of mTOR (mammalian target of rapamycin) with rapamycin blunted the hypertrophy in NRVMs depleted of Shp2. NRVMs treated with PP2 or depleted of FAK by specific small interfering RNA were defective in FAK, Src, extracellular signal-regulated kinase, AKT, TSC2, and S6 kinase phosphorylation, as well as in the hypertrophic response to prolonged stretch. The stretch-induced hypertrophy of NRVMs was also prevented by rapamycin. These findings demonstrate that basal Shp2 tyrosine phosphatase activity controls the size of cardiomyocytes by downregulating a pathway that involves FAK/Src and mTOR signaling pathways.

Targeting focal adhesion kinase with small interfering RNA prevents and reverses load-induced cardiac hypertrophy in mice.

Hypertrophy is a critical event in the onset of failure in chronically overloaded hearts. Focal adhesion kinase (FAK) has attracted particular attention as a mediator of hypertrophy induced by increased load. Here, we demonstrate increased expression and phosphorylation of FAK in the hypertrophic left ventricles (LVs) of aortic-banded mice. We used an RNA interference strategy to examine whether FAK signaling plays a role in the pathophysiology of load-induced LV hypertrophy and failure. Intrajugular delivery of specific small interfering RNA induced prolonged FAK silencing ( approximately 70%) in both normal and hypertrophic LVs. Myocardial FAK silencing was accompanied by prevention, as well as reversal, of load-induced left ventricular hypertrophy. The function of LVs was preserved and the survival rate was higher in banded mice treated with small interfering RNA targeted to FAK, despite the persistent pressure overload. Studies in cardiac myocytes and fibroblasts harvested from LVs confirmed the ability of the systemically administered specific small interfering RNA to silence FAK in both cell types. Further analysis indicated attenuation of cardiac myocyte hypertrophic growth and of the rise in the expression of beta-myosin heavy chain in overloaded LVs. Moreover, FAK silencing was demonstrated to attenuate the rise in the fibrosis, collagen content, and activity of matrix metalloproteinase-2 in overloaded LVs, as well as the rise of matrix metalloproteinase-2 protein expression in fibroblasts harvested from overloaded LVs. This study provides novel evidence that FAK may be involved in multiple aspects of the pathophysiology of cardiac hypertrophy and failure induced by pressure overload.

Increased expression and phosphorylation of focal adhesion kinase correlates with dysfunction in the volume-overloaded human heart.

FAK (focal adhesion kinase) has been shown to mediate the hypertrophic growth of the left ventricle. Experimental results also suggest that FAK may contribute to the structural and functional deterioration of the chronically overloaded left ventricle. In the present study, we postulated that FAK expression and phosphorylation may be altered in the volume-overloaded heart in humans. FAK expression and phosphorylation at Tyr(397) were detected by Western blotting and immunohistochemistry in samples from endomyocardial biopsies from patients with MR (mitral regurgitation; n=21) and donor subjects (n=4). Hearts from patients with MR had degenerated cardiac myocytes and areas of fibrosis. In this group, the myocardial collagen area was increased (18% in MR hearts compared with 3% in donor hearts respectively) and correlated negatively with left ventricular ejection fraction (r=-0.74; P>0.001). FAK expression and phosphorylation at Tyr(397) (a marker of the enzyme activity) were increased in samples from MR hearts compared with those from donor hearts (3.1- and 4.9-fold respectively). In myocardial samples from donor hearts, anti-FAK staining was almost exclusively restricted to cardiac myocytes; however, in myocardial samples from MR hearts, staining with the anti-FAK antibody was found to occur in myocytes and the interstitium. There was a positive correlation between collagen and the interstitial areas stained with the anti-FAK antibody (r=0.76; P>0.001). Anti-FAK and anti-vimentin staining of the interstitial areas of samples from MR hearts were extensively superimposed, indicating that most of the interstitial FAK was located in fibroblasts. In conclusion, FAK expression and phosphorylation are increased and may contribute to the underlying structural and functional abnormalities in the volume-overloaded heart in humans.

Differentiation of C2C12 myoblasts is critically regulated by FAK signaling.

This study examined whether focal adhesion kinase (FAK) plays a role in the differentiation of C(2)C(12) myoblasts into myotubes. Differentiation of C(2)C(12) myoblasts induced by switch to differentiation culture medium was accompanied by a transient reduction of FAK phosphorylation at Tyr-397 (to approximately 50%, at 1 and 2 h), followed by an increase thereafter (to 240% up to 5 days), although FAK protein expression remained unchanged. FAK and phosphorylated FAK were found at the edge of lamellipodia in proliferating cells, whereas the later increase in FAK phosphorylation in differentiating cells was accompanied by its preferential location at the tip of well-organized actin stress fibers. Hyperexpression of FAK autophosphorylation site (Tyr-397) mutant (MT-FAK) reduced FAK phosphorylation at Tyr-397 in proliferating cells and was accompanied by reduction of cyclin D1 and increase of myogenin expression. These cells failed to progress to myotubes in differentiation medium. In contrast, hyperexpression of a wild-type FAK construction (WT-FAK) increased baseline and abolished the transient reduction of FAK phosphorylation at Tyr-397 in serum-starved C(2)C(12) cells. Cells transfected with WT-FAK failed to reduce cyclin D1 and to increase myogenin expression, as well as to progress to terminal differentiation in differentiation medium. These data indicate that FAK signaling plays a critical role in the control of cell cycle as well as in the progression of C(2)C(12) cells to terminal differentiation. Transient inhibition of FAK phosphorylation at Tyr-397 contributes to trigger the myogenic genetic program, but its later activation is also central to terminal differentiation into myotubes.