αB-Crystallin interacts and attenuates the tyrosine phosphatase activity of Shp2 in cardiomyocytes under mechanical stress.

The small heat shock protein αB-Crystallin (CryAB, HspB5) and SH2 domain-containing tyrosine phosphatase 2 (Shp2) are important molecules in heart response to pathophysiological stress. Here we show that CryAB interacts with and potentially regulates Shp2 catalytic activity in stretched cardiomyocytes. Such an interaction requires CryAB oligomer to attenuate Shp2 activation. Stretched cardiomyocytes show a robust CryAB/Shp2 association accompanied by a reduction in the Shp2 phosphatase activity. Accordingly, CryAB knock-down in cardiomyocytes enhances Shp2 activity induced by mechanical stress. These results revealed a new role for CryAB, as a modulator of Shp2 phosphatase activity during a functionally relevant stimulus in cardiomyocytes.

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.

Rapamycin reverses hypertrophic cardiomyopathy in a mouse model of LEOPARD syndrome-associated PTPN11 mutation.

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.

SHP-2 regulates myogenesis by coupling to FAK signaling pathway.

Transient dephosphorylation of FAK at Tyr-397 is required for cell cycle withdrawal early on during myogenesis. Here, we show that upon serum starvation of C2C12 myoblasts, FAK is transiently dephosphorylated in parallel with SHP-2 activation and association with FAK. SHP-2 knockdown by RNA interference suppressed the transient upregulation of SHP-2 and dephosphorylation of FAK during myogenesis. Furthermore, depletion of SHP-2 retarded the cell cycle withdrawal and the differentiation of serum-starved myoblasts into myotubes. These data provide a mechanistic basis for the reduction in FAK activity in differentiating myoblasts, indicating that myogenesis is critically triggered by FAK/SHP-2 complex.

Calcium and the mechanotransduction in cardiac myocytes.

Mechanical stress is a major triggering stimulus for the installation of cardiac hypertrophy as well as for the structural and functional deterioration occurring in the hypertrophy decompensation. The sensing of mechanical forces and their conversion into biochemical signals depend on the integrity of subcellular structures such as the costameres and Z-disks. Signaling molecules concentrated into these structures are thought to be activated by the stress-induced deformation of structural proteins. Evidence also indicates that Ca2+ may be involved in mediating the mechanical forces conversion into biochemical signals and biological responses. Ca2+ channels, transporters and activated proteins are concentrated at the junctions between the T-tubules and the sarcoplasmic reticulum which are in close proximity to the costameres and Z-disks. This provides a structural basis for the influence of mechanical forces on Ca2+ transport and on the events related to signaling molecules clustered in the costameres and the Z-disks. Emerging data reviewed here are providing insight into how Ca2+ and mechanical mediated signaling are coordinated to modulate the functional and trophic responses of cardiac myocytes to mechanical stress.

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.

RhoA/ROCK signaling is critical to FAK activation by cyclic stretch in cardiac myocytes.

Focal adhesion kinase (FAK) has been shown to be activated in cardiac myocytes exposed to mechanical stress. However, details of how mechanical stimuli induce FAK activation are unknown. We investigated whether signaling events mediated by the RhoA/Rho-associated coiled coil-containing kinase (ROCK) pathway are involved in regulation of stretch-induced FAK phosphorylation at Tyr(397) in neonatal rat ventricular myocytes (NRVMs). Immunostaining showed that RhoA localized to regions of myofilaments alternated with phalloidin (actin) staining. The results of coimmunoprecipitation assays indicated that FAK and RhoA are associated in nonstretched NRVMs, but cyclic stretch significantly reduced the amount of RhoA recovered from anti-FAK immunoprecipitates. Cyclic stretch induced rapid and sustained (up to 2 h) increases in phosphorylation of FAK at Tyr(397) and ERK1/2 at Thr(202)/Tyr(204). Blockade of RhoA/ROCK signaling by pharmacological inhibitors of RhoA (Clostridium botulinum C3 exoenzyme) or ROCK (Y-27632, 10 micromol/l, 1 h) markedly attenuated stretch-induced FAK and ERK1/2 phosphorylation. Similar effects were observed in cells treated with the inhibitor of actin polymerization cytochalasin D. Transfection of NRVMs with RhoA antisense oligonucleotide attenuated stretch-induced FAK and ERK1/2 phosphorylation and expression of beta-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.

Focal adhesion kinase mediates MEF2 and c-Jun activation by stretch: role in the activation of the cardiac hypertrophic genetic program.

OBJECTIVE: We have previously reported that myocyte enhancer factor-2 (MEF2) transcription factors and c-Jun are rapidly activated by pressure overload and that these events are involved in the early activation of the myocardial hypertrophic genetic program. In this study, we investigated whether focal adhesion kinase (FAK) mediates the activation of MEF2 and c-Jun by mechanical stress in isolated neonatal rat ventricular myocytes (NRVMs). METHODS: NRVMs were subjected to cyclic stretch up to 4 h and studied by immunoblotting, reverse transcriptase-polymerase chain reaction, laser confocal analysis, and reporter gene and electrophoretic mobility shift assays. Analysis was extended to NRVMs transfected with FAK-antisense oligodeoxynucleotide, treated with FAK/Src inhibitor PP2 or JNK/c-Jun inhibitor SP600125. RESULTS: Cyclic stretch increased c-Jun expression, JNK/c-Jun phosphorylation, and MEF2-DNA binding activity in NRVMs. Reporter gene assays indicated that the MEF2 site is critical to c-jun transcription in stretched cells. FAK-antisense transfection abolished MEF2 and c-jun promoter activation, while either FAK-antisense or PP2 treatment inhibited the stretch-induced c-Jun expression and JNK/c-Jun phosphorylation. Finally, treatment of NRVMs with the specific JNK/c-Jun inhibitor SP600125 significantly reduced the stretch-induced increase of atrial natriuretic factor promoter activity. CONCLUSION: 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.