Given the central position of the focal adhesion complex, both physically in coupling integrins to the interstitium and biochemically in providing an upstream site for anabolic signal generation, we asked whether the recruitment of non-receptor tyrosine kinases to the cytoskeleton might be a mechanism whereby cellular loading could activate growth regulatory signals responsible for cardiac hypertrophy. Analysis revealed cytoskeletal association of c-Src, FAK, and ␤3-integrin, but no Fyn, in the pressure-overloaded right ventricle. This association was seen as early as 4 h after right ventricular pressure overloading, increased through 48 h, and reverted to normal in 1 week. Cytoskeletal binding of nonreceptor tyrosine kinases was synchronous with tyrosine phosphorylation of several cytoskeletal proteins, including c-Src. Examination of cytoskeleton-bound cSrc revealed that a significant portion of the tyrosine phosphorylation was not at the Tyr-527 site and therefore presumably was at the Tyr-416 site. Thus, these studies strongly suggest that non-receptor tyrosine kinases, in particular c-Src, may play a critical role in hypertrophic growth regulation by their association with cytoskeletal structures, possibly via load activation of integrin-mediated signaling.

Cardiac hypertrophy is characterized by both remodeling of the extracellular matrix (ECM) and hypertrophic growth of the cardiocytes. Here we show increased expression and cytoskeletal association of the ECM proteins fibronectin and vitronectin in pressureoverloaded feline myocardium. These changes are accompanied by cytoskeletal binding and phosphorylation of focal adhesion kinase (FAK) at Tyr-397 and Tyr-925, c-Src at Tyr-416, recruitment of the adapter proteins p130Cas , Shc, and Nck, and activation of the extracellular-regulated kinases ERK1/2. A synthetic peptide containing the Arg-Gly-Asp (RGD) motif of fibronectin and vitronectin was used to stimulate adult feline cardiomyocytes cultured on laminin or within a type-I collagen matrix. Whereas cardiocytes under both conditions showed RGD-stimulated ERK1/2 activation, only collagen-embedded cells exhibited cytoskeletal assembly of FAK, c-Src, Nck, and Shc. In RGD-stimulated collagenembedded cells, FAK was phosphorylated only at Tyr-397 and c-Src association occurred without Tyr-416 phosphorylation and p130Cas association. Therefore, cSrc activation is not required for its cytoskeletal binding but may be important for additional phosphorylation of FAK. Overall, our study suggests that multiple signaling pathways originate in pressure-overloaded heart following integrin engagement with ECM proteins, including focal complex formation and ERK1/2 activation, and many of these pathways can be activated in cardiomyocytes via RGD-stimulated integrin activation.Cardiovascular diseases such as hypertension, valvular defects, and myocardial infarction are often associated with the development of cardiac hypertrophy. This hypertrophy occurs in response to an increased mechanical (hemodynamic) load on the heart in the form of pressure or volume overload, which is characteristic of hypertension and valvular defects, or to a decrease in functional heart tissue as seen in myocardial infarction. The initial hypertrophic response of the heart is compensatory but frequently deteriorates into heart failure and increased morbidity/mortality (1, 2). This transition from compensation to failure occurs when further hypertrophy of the heart cannot normalize wall stress and maintain contractile function in the face of its hemodynamic load. Although mechanical load appears to directly regulate mass and associated phenotypic changes at the level of the cardiocyte (for a review see Ref.3), the mechanisms that couple load to the hypertrophic growth initiation and to the transition into heart failure have yet to be delineated. Whereas several key players including G-proteins (4), calcineurin (5, 6), mitogen-activated protein kinase (MAPK) 1 family members, namely, extracellular-regulated kinases (ERK1/2) (7) and p38 MAPK (8), as well as protein kinase C (9) and p70/85 S6 kinase (10, 11) have been implicated in the pathways that connect load to hypertrophic growth, the complexity of interaction between signaling pathways make deciphering them a difficult task in hypertrophic research.In an...

Increased microtubule density, for which microtubule stabilization is one potential mechanism, causes contractile dysfunction in cardiac hypertrophy. After microtubule assembly, α-tubulin undergoes two, likely sequential, time-dependent posttranslational changes: reversible carboxy-terminal detyrosination (Tyr-tubulin ↔ Glu-tubulin) and then irreversible deglutamination (Glu-tubulin → Δ2-tubulin), such that Glu- and Δ2-tubulin are markers for long-lived, stable microtubules. Therefore, we generated antibodies for Tyr-, Glu-, and Δ2-tubulin and used them for staining of right and left ventricular cardiocytes from control cats and cats with right ventricular hypertrophy. Tyr- tubulin microtubule staining was equal in right and left ventricular cardiocytes of control cats, but Glu-tubulin and Δ2-tubulin staining were insignificant, i.e., the microtubules were labile. However, Glu- and Δ2-tubulin were conspicuous in microtubules of right ventricular cardiocytes from pressure overloaded cats, i.e., the microtubules were stable. This finding was confirmed in terms of increased microtubule drug and cold stability in the hypertrophied cells. In further studies, we found an increase in a microtubule binding protein, microtubule-associated protein 4, on both mRNA and protein levels in pressure-hypertrophied myocardium. Thus, microtubule stabilization, likely facilitated by binding of a microtubule-associated protein, may be a mechanism for the increased microtubule density characteristic of pressure overload cardiac hypertrophy.

Hypertrophic cardiac growth is a major compensatory response of the heart to an increased mechanical (hemodynamic) load in the form of either pressure or volume overload. Although this response is initially compensatory, a transition from this state to failure occurs when further growth of the heart is not sufficient to normalize the wall stress and maintain contractile function (1). Therefore, a major research interest in cardiovascular disease is to understand how the increase in hemodynamic load is transmitted intracellularly for mediating hypertrophic growth. Although the mechanical load appears to directly regulate the hypertrophic growth initiation, the signaling mechanism that connects load to such growth is not well understood.A major cellular event during cardiac hypertrophy is increased protein synthesis (1-5). Enhanced protein synthesis can occur via accelerated protein translation, increased biogenesis of translational components, or both. A significant amount of mRNA of vertebrate cells possesses a unique 5Ј-terminal oligopyrimidine (5Ј-TOP) 1 sequence in the 5Ј-untranslated region (5Ј-UTR), and these mRNA species generally code for specific ribosomal proteins (6, 7). Their translation is largely controlled via phosphorylation of the 40 S ribosomal S6 protein (S6 protein) at its C terminus (8) by p70/85 S6 kinase (S6K1) (9 -12). There are two isoforms of S6K1: the 70-kDa isoform was first isolated from mouse 3T3 cells (13), and the 85-kDa isoform of this kinase was then identified (14). The p85 isoform is expressed from the same transcript as the p70 isoform through an alternative translational initiation start site, which adds a 23-amino acid nuclear localization signal to the N terminus (15,16). Therefore, the 85-kDa isoform is predominantly in the nucleus, whereas the 70-kDa isoform is present mostly in the cytoplasm. Both the S6K isoforms are collectively called p70/85S6K, p70S6K, or S6K1 and have been shown to phosphorylate the S6 protein and mediate the biogenesis of the translational components, including several of the ribosomal proteins and elongation factors (12). The p85 isoform has been shown to have additional roles in translational control, G 1 to S phase transition, and increased DNA synthesis (17). Recent studies using S6K1 knockout mice (18) demonstrate no appreciable change in S6 protein phosphorylation, 5Ј-TOP mRNA translation, or cell growth, although these mice exhibited a small mouse phenotype. These studies (18) and other independent studies (19 -21) resulted in the discovery of another S6K (S6K2), which possesses 70% homology with the p70 isoform of

Developing a biodegradable scaffold remains a major challenge in bone tissue engineering. This study was aimed at developing novel alginate-chitosan-collagen (SA-CS-Col)-based composite scaffolds consisting of graphene oxide (GO) to enrich porous structures, elicited by the freeze-drying technique. To characterize porosity, water absorption, and compressive modulus, GO scaffolds (SA-CS-Col-GO) were prepared with and without Ca-mediated crosslinking (chemical crosslinking) and analyzed using Raman, Fourier transform infrared (FTIR), X-ray diffraction (XRD), and scanning electron microscopy techniques. The incorporation of GO into the SA-CS-Col matrix increased both crosslinking density as indicated by the reduction of crystalline peaks in the XRD patterns and polyelectrolyte ion complex as confirmed by FTIR. GO scaffolds showed increased mechanical properties which were further increased for chemically crosslinked scaffolds. All scaffolds exhibited interconnected pores of 10-250 μm range. By increasing the crosslinking density with Ca, a decrease in the porosity/swelling ratio was observed. Moreover, the SA-CS-Col-GO scaffold with or without chemical crosslinking was more stable as compared to SA-CS or SA-CS-Col scaffolds when placed in aqueous solution. To perform in vitro biochemical studies, mouse osteoblast cells were grown on various scaffolds and evaluated for cell proliferation by using MTT assay and mineralization and differentiation by alizarin red S staining. These measurements showed a significant increase for cells attached to the SA-CS-Col-GO scaffold compared to SA-CS or SA-CS-Col composites. However, chemical crosslinking of SA-CS-Col-GO showed no effect on the osteogenic ability of osteoblasts. These studies indicate the potential use of GO to prepare free SA-CS-Col scaffolds with preserved porous structure with elongated Col fibrils and that these composites, which are biocompatible and stable in a biological medium, could be used for application in engineering bone tissues.

Osteoclasts are terminally differentiated, multinucleated, and highly motile cells responsible for bone resorption. The adhesion of osteoclasts to bone leads to the formation of the osteoclast clear zone, an actin-rich ring-like adhesion zone (sealing ring) circumscribing an area of bone resorption. Sealing ring formation has been considered to be a marker of osteoclast activation for bone resorption (1, 2). Bone resorption is mediated by the dynamics of an actin cytoskeleton ring. Distinct pathways and signaling molecules (c-Src, PYK2, c-Cbl, p130 Cas, leupaxin, and phosphatidylinositol 3-kinase) have been described to play roles in the organization of the sealing ring during bone resorption (3-10). Despite success in identifying the role of these kinases in osteoclast sealing ring formation in the clear zone of resorbing osteoclasts, the potential target protein of these kinases involved in the sealing ring formation is poorly understood. Calle et al. (11) have demonstrated that osteoclasts from WASP 2 knock-out mice failed to demonstrate sealing ring, and these osteoclasts are bone resorption-disabled. Expression of WASP restores normal cytoarchitecture in these osteoclasts. More recently, we have demonstrated that sealing ring formation and bone resorption are enhanced by interaction of phosphatidylinositol 4,5-biphosphate (PIP 2 ) and Cdc42 with the WASP (12).WASP integrates signals from Rho, Cdc42, and kinase(s) to bind to the Arp2/3 complex and stimulate Arp2/3-dependent actin polymerization (13-15). Phosphorylation of WASP has been reported in several cell systems. Tyrosine or serine phosphorylation of WASP increases the actin polymerization activity of WASP through the Arp2/3 complex (15-19). The phosphorylation of WASP at Tyr-291 is regulated by several kinases, including c-Src, FAK, Hck, and Nck (17,18,[20][21][22][23]. Efficient phosphorylation and dephosphorylation of WASP at amino acid Tyr-291 within the GTPase binding domain (GBD) of

Identifying the molecular mechanisms activated in compensatory hypertrophy and absent during decompensation will provide molecular targets for prevention of heart failure. We have previously shown enhanced ubiquitination (Ub) during the early growth period of pressure overload (PO) hypertrophy near intercalated discs of cardiomyocytes, where integrins are important for mechanotransduction. In this study, we tested the role of integrins upstream of Ub, whether enhanced Ub contributes to survival signaling in early PO, and if loss of this mechanism could lead to decreased ventricular function. The study used a beta(3) integrin (-/-) mouse and a wild-type mouse as a control for in vivo PO by transverse aortic constriction (TAC) and for cultured cardiomyocytes in vitro, stimulated with the integrin-activating peptide RGD. We demonstrate beta(3) integrin mediates transient Ub of targeted proteins during PO hypertrophy, which is necessary for cardiomyocyte survival and to maintain ventricular function. Prosurvival signaling proceeds by initiation of NF-kappaB transcription of the E3 ligase, cIAP1. In PO beta(3)(-/-) mice, absence of this mechanism correlates with increased TUNEL staining and decreased ventricular mass and function by 4 wk. This is the first study to show that a beta(3) integrin/Ub/NF-kappaB pathway contributes to compensatory hypertrophic growth.

Because we find persistent increases both in microtubules and in their biosynthetic precursors in pressure-hypertrophied myocardium, the mechanisms for this cytoskeletal abnormality must be sought through studies of the control both of microtubule stability and of tubulin synthesis.