LEOPARD syndrome (LS) is an autosomal dominant "RASopathy" that manifests with congenital heart disease. Nearly all cases of LS are caused by catalytically inactivating mutations in the protein tyrosine phosphatase (PTP), non-receptor type 11 (PTPN11) gene that encodes the SH2 domain-containing PTP-2 (SHP2). RASopathies typically affect components of the RAS/MAPK pathway, yet it remains unclear how PTPN11 mutations alter cellular signaling to produce LS phenotypes. We therefore generated knockin mice harboring the Ptpn11 mutation Y279C, one of the most common LS alleles. Ptpn11 Y279C/+ (LS/+) mice recapitulated the human disorder, with short stature, craniofacial dysmorphia, and morphologic, histologic, echocardiographic, and molecular evidence of hypertrophic cardiomyopathy (HCM). Heart and/or cardiomyocyte lysates from LS/+ mice showed enhanced binding of Shp2 to Irs1, decreased Shp2 catalytic activity, and abrogated agonist-evoked Erk/Mapk signaling. LS/+ mice also exhibited increased basal and agonist-induced Akt and mTor activity. The cardiac defects in LS/+ mice were completely reversed by treatment with rapamycin, an inhibitor of mTOR. Our results demonstrate that LS mutations have dominant-negative effects in vivo, identify enhanced mTOR activity as critical for causing LS-associated HCM, and suggest that TOR inhibitors be considered for treatment of HCM in LS patients.

Transfection of NRVMs with RhoA antisense oligonucleotide attenuated stretch-induced FAK and ERK1/2 phosphorylation and expression of ␤-myosin heavy chain mRNA. Similar results were seen in cells transfected with FAK antisense oligonucleotide. These findings demonstrate that RhoA/ROCK signaling plays a crucial role in stretch-induced FAK phosphorylation, presumably by coordinating upstream events operationally linked to the actin cytoskeleton. mechanical stress; hypertrophy; cell signaling INCREASED BIOMECHANICAL STRESS can drive changes in cardiac myocytes that are implicated in myocardial hypertrophy and failure (7,19). Numerous signal transduction pathways have been shown to be activated in cardiac myocytes subjected to mechanical stimuli (35). Signals originating from multiple pathways converge intracellularly, leading to altered gene expression and protein synthesis, which result in the hypertrophic growth of cardiac myocytes. However, the mechanism by which mechanical forces are sensed and converted to biochemical signals remains largely unknown. Recent developments in this field indicate that the integrity of structures such as the Z disk, costamere, and intercalated disk is critically important to the ability of cardiac cells to appropriately respond to mechanical stress (4,11,21,32,37,41). It has been hypothesized that such structures participate in monitoring of mechanical force and in communication of strain to signaling molecules in cardiac myocytes (12,29,37).Focal adhesion kinase (FAK), a tyrosine kinase linked to integrin signaling (15,24,42), has been shown to be rapidly activated by mechanical stimuli in cardiac myocytes (2,5,9,12,13,23,34,39). Several lines of evidence support a role for FAK in the regulation of early gene transcription in response to hypertrophic agonists and mechanical stress (10,22,28,38,39), indicating that this kinase may coordinate the convergence of multiple signaling pathways involved in the hypertrophic growth of cardiac myocytes. However, the molecular mechanism responsible for FAK activation by mechanical stress in cardiac myocytes remains elusive. We recently showed (12, 39) that FAK activation by mechanical stress is accompanied by its aggregation at myofilaments, Z disks, and costameres, implying that this kinase might be directly activated by mechanical stress in cardiac myocytes. On the other hand, FAK activation in neonatal rat ventricular myocytes (NRVMs) by agonists such as endothelin has been demonstrated (16) to be dependent on activation of the RhoA/Rho-associated coiled coil-containing protein kinase (ROCK) signaling pathway, which drives the assembly and rearrangement of actin filaments. The demonstration that cytochalasin D, a potent inhibitor of actin polymerization, markedly attenuated endothelininduced FAK phosphorylation (16) revealed the importance of the assembly of actin filaments in FAK activation by this agonist. Similarly, FAK activation by mechanical stress has been suggested to depend on a cooperative interaction with actin filaments (13,39,...

Abstract-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. Key Words: hypertrophy Ⅲ cell signaling Ⅲ cardiomyocytes Ⅲ focal adhesion kinase C ardiomyocytes respond to increases in functional demand by hypertrophic growth. This reactive hypertrophy involves concerted gene expression and the accumulation of myocyte proteins and organelles that are coordinated by signaling cascades activated by mechanical stress and a variety of soluble endocrine, paracrine, and autocrine factors. 1 Focal adhesion kinase (FAK) is involved in the hypertrophic response of cardiomyocytes to biomechanical stress and agonists such as phenylephrine and endothelin. [2][3][4][5][6] In cardiomyocytes, FAK is highly expressed, has a relatively low basal level of activity, and is promptly activated by hypertrophic stimuli. [2][3][4]7 FAK overexpression upregulates marker genes associated with hypertrophy in cardiomyocytes, 8 whereas a loss of FAK function impairs the upregulation of these genes in response to hypertrophic stimuli. 4 -6 The involvement of FAK in reactive hypertrophy has been confirmed in mice with cardiomyocyte-restricted FAK deletion or myocardial FAK silencing, 9 -11 but the mechanistic pathways that link FAK to hypertrophy in cardiomyocytes remain uncertain.Intracellularly, FAK is kept quiescent by intramolecular in...

The present data indicate that FAK regulates the activation of MEF2 and JNK/c-Jun pathways, which in turn have a key role in the early activation of the hypertrophic genetic program by mechanical stress in cardiac myocytes.

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