Targeting to C-Terminal Myosin Heavy Chain May Explain Mechanotransduction Involving Focal Adhesion Kinase in Cardiac Myocytes

Abstract

Abstract-Focal adhesion kinase (Fak) has been implicated as a signaling molecule involved in the early response of cardiac myocytes to mechanical stress. The mechanism of Fak activation by mechanical stimuli is not clear. In this study, we report the load-induced Fak activation and its association with myosin heavy chain in cardiac myocytes. Pressure overload lasting from 3 to 60 minutes was shown to induce Fak phosphorylation at Tyr-397, -576/7, -861, and -925 as detected by phosphospecific antibodies. This was paralleled by increases of Fak/Src association and Src activity (Tyr-418 phosphorylation). Yeast two-hybrid screening of an adult rat cDNA library revealed an interaction between Fak and C-terminal coiled-coil region of ␣-myosin heavy chain. This was confirmed by pulldown assay with GST-C-terminal myosin fragment and native Fak from rat left ventricle. Such interaction was confirmed by coimmunoprecipitation assay with anti-Fak and anti-heavy chain cardiac myosin antibodies, confocal microscopy of double-labeled isolated cardiac myocytes and immunoelectron microscopy with anti-Fak antibody. Fak activation by mechanical stress was accompanied by a reduction of Fak/myosin heavy chain association and its relocation at subcellular sites such as costameres, Z-discs, and nuclei. Thus, our present data identify Fak interaction with C-terminal region of myosin heavy chain adding comprehensive data on Fak activation by mechanical stress and mechanotransduction in cardiac myocytes. Key Words: focal adhesion kinase Ⅲ mechanotransduction Ⅲ cell signaling Ⅲ hypertrophy Ⅲ myosin M echanical stress is a major factor involved in the development of myocardial adaptive and maladaptive changes in heart diseases. Local mechanical forces activate signaling mechanisms in cardiac myocytes inducing the expression of specific genetic programs linked to myocardial structural and functional remodeling. 1,2 Although mechanical forces might directly trigger signaling mechanisms in cardiac myocytes, the mechanism by which they are sensed and converted to biochemical signals remains elusive.Structures such as sarcomeric lattice, cytoskeleton, and the extracellular matrix operate in the transmission of either passive or active forces in cardiac myocytes. 3,4 Studies have confirmed the critical importance of the molecular integrity of Z-disc and cytoskeleton to the expression of genetic program induced by mechanical stress in cardiac myocytes. Z-disc structure is organized by N-terminal titin Z repeats linked to ␣-actinin and associated proteins such as MLP, ALP, telethonin (T-cap), cypher/Zasp, and myotilin. 4,5 Notably, MLP null mice were shown to fail to upregulate brain and atrial natriuretic factors mRNA in response to stretch. 6 Human titin mutations as well as deletion of the ␣-actinin binding proteins such as ALP or MLP in the mouse causes dilated cardiomyopathy. 6 -10 However, the mechanisms by which these structures and proteins detect physical forces and initiate biochemical signals are yet unclear.The link of Z-discs to sarco...

Targeting cell motility in pulmonary arterial hypertension

“…FAK has been shown to play a role in cardiomyocyte hypertrophy, being activated by either mechanical stress [59][60][61] (which causes a translocation of FAK in the cardiomyocyte nucleus [62][63][64]) or agonists like ET-1 and Ang-II [65,66]. Increased phosphorylation of FAK has also been described in hypertrophied RV of rats after MCT injection [67].…”

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

“…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.…”

“…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.…”

“…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).…”

Dynamic interactions between cells and their extracellular matrix mediate embryonic development

“…Interestingly, FAK appears to be involved in the formation of strong, longlasting CMACs that mediate muscle-tendon adhesion. FAK activation, or autophosphorylation on pY397, can also be induced by mechanical stress in cardiac myocytes (Fonseca et al, 2005). The activation of FAK correlates with many events during myotube formation in cell culture.…”