Focal Adhesion Kinase Is Activated and Mediates the Early Hypertrophic Response to Stretch in Cardiac Myocytes

Abstract

Abstract-Previously we reported that the rapid activation of the Fak/Src multicomponent signaling complex mediates load-induced activation of growth and survival signaling pathways in adult rat heart. In this study, we report that 5% to 20% (10-minute) cyclic stretch (1 Hz) of neonatal rat ventricular myocytes (NRVMs) was paralleled by increases of Fak phosphorylation at Tyr-397 (from 1.5-to 2.8-fold), as detected by anti-Fak-pY 397 phosphospecific antibody. Moreover, 15% cyclic stretch lasting from 10 to 120 minutes increased Fak phosphorylation at Tyr-397 by 2.5-to 3.5-fold. This activation was accompanied by a dramatic change in Fak localization in NRVMs from densely concentrated in the perinuclear regions in nonstretched cells to aggregates regularly distributed along the myofilaments in stretched cells. Furthermore, a 4-hour cyclic stretch enhanced the activity of an atrial natriuretic factor (ANF) promoter-luciferase reporter gene by 2.7-fold. Disrupting endogenous Fak/Src signaling either by expression of a dominant-negative Fak mutant with phenylalanine substituted for Tyr-397 or by treatment with a c-Src pharmacological inhibitor (PP-2) markedly attenuated stretch-induced Fak activation and clustering at myofilaments and inhibited stretch-induced ANF gene activation. On the other hand, overexpression of wild-type Fak potentiated the stretchinduced Fak phosphorylation but did not enhance either baseline or stretch-induced ANF promoter-luciferase reporter gene activity compared with the responses of nontransfected NRVMs. These findings identify Fak as an important element in the early responses induced by stretch in cardiac myocytes, indicating that it may coordinate the cellular signaling machinery that controls gene expression program associated with load-induced cardiac myocyte hypertrophy. Key Words: focal adhesion kinase Ⅲ mechanotransduction Ⅲ cell signaling Ⅲ hypertrophy M echanical overload is both cause and consequence of most heart diseases. 1 Cardiac myocytes respond to increased mechanical load by hypertrophic growth, but mechanical stress is also an important stimulus for triggering the initial steps toward cardiac myocyte degeneration and death, which play a critical role in the maladaptive myocardial remodeling and heart failure. 1,2 A major goal in this field is to decipher the mechanisms that link biomechanical forces to the activation of signaling pathways that mediate the hypertrophic as well as maladaptive responses of cardiac myocytes to mechanical stress.The mechanistic pathways that link mechanical stimuli to biochemical signals in cardiac myocytes are presently unclear, but a growing body of evidence indicates that costameres (complex structures constituted by integrins and cytoskeletal proteins at the junction of sarcolemma and Z-discs) have a critical role in sensing and transducing mechanical stress into biochemical signals that coordinate growth responses to hypertrophic stimuli in both cardiac and skeletal muscle. [3][4][5][6][7] The prominent location of integrins at the junc...

Mechanical Strain on Osteoblasts Activates Autophosphorylation of Focal Adhesion Kinase and Proline-rich Tyrosine Kinase 2 Tyrosine Sites Involved in ERK Activation

“…Furthermore, we observed a similar FAK-Tyr-397 phosphorylation in rat primary osteoblasts and MC3T3 osteoblastic cells, demonstrating that ROS17/2.8 cells are a valid model. These data are in line with previous reports that showed a mechanical strain-induced FAK-Tyr-397 phosphorylation in vitro and in vivo (72,73) in fibroblasts and in the heart, respectively. Upon integrin binding, FAK autophosphorylation at tyrosine residue 397 is followed by phosphorylation by other tyrosine kinases at other sites, including FAK-Tyr-925.…”

Cell Adhesion on Amyloid Fibrils Lacking Integrin Recognition Motif

“…FAK is known to be involved as a primary integrin effecter and is considered as a measure of cell spreading and migration (59 -61). FAK and other focal adhesion proteins generally show a well distributed cytoplasmic staining pattern in adhered cells (62)(63)(64). A similar distribution of FAK was also observed in our study for the cells cultured on amyloid surfaces, indicating strong cell adhesion.…”

αB-crystallin interacts with and prevents stress-activated proteolysis of focal adhesion kinase by calpain in cardiomyocytes

“…The myocytes were isolated by digestion with pancreatin and type-2 collagenase and then purified on a discontinuous Percoll gradient, suspended in plating medium containing 10% horse serum, 5% fetal bovine serum and 0.5% penicillin-streptomycin, and plated in type-I collagen Bioflex plates or in Petri plates (25 mm) and incubated for 44 h under 95% air-5% CO 2 . The medium was then replaced by serum-free DMEM and the cells were incubated for 24 h before use 16 . The purity of cardiomyocyte cultures was estimated using 4 0 ,6-diamidino-2-phenylindole (DAPI) and anti-a-actinin antibody (1:100) for staining of nuclei and the cardiomyocyte sarcomeres, respectively.…”

“…Focal adhesion kinase (FAK) is a mediator of the hypertrophic growth and survival of cardiomyocytes during mechanical stress [16][17][18][19] . However, the regulation of FAK in this particular cell type remains unclear.…”