Domingos, Priscila P., Priscila M. Fonseca, Wilson Nadruz, Jr., and Kleber G. Franchini. Load-induced focal adhesion kinase activation in the myocardium: role of stretch and contractile activity. Am J Physiol Heart Circ Physiol 282: H556-H564, 2002; 10.1152/ajpheart.00534.2001.-We investigated the influence of stretch and contractile activity on load-induced activation of focal adhesion kinase (FAK) and extracellular signal-regulated kinase (ERK)1/2 in isolated rat hearts. Increases of diastolic pressure from ϳ0 to ϳ15 mmHg rapidly increased FAK tyrosine phosphorylation (maximum: 2.3-fold) and binding to c-Src (maximum: 2.8-fold) and Grb2 (maximum: 3.6-fold). This was paralleled by activation (maximum: 2.8-fold) and binding of ERK1/2 to FAK. FAK and ERK1/2 were immunolocalized at sarcolemmal sites of cardiac myocytes and in the nuclei, in the case of ERK1/2. Balloon inflation to raise ventricular pressure in hearts perfused with cardioplegic solution also activated FAK and ERK1/2. However, increases in contractile activity induced by increasing calcium concentration in the perfusate (from 0.5 to 5 mM) did not activate the FAK multicomponent signaling complex or ERK1/2 in the myocardium. These results indicate that stretch rather than contractile activity induces FAK and ERK1/2 activation in the myocardium. In addition, the activation and binding of ERK1/2 to FAK suggest that FAK drives the load-induced activation of ERK1/2.
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...
We investigated the effects of acute pressure overload on activation of p160(ROCK) in rat myocardium. Constriction of transverse aorta, controlled to increase peak systolic pressure of ascending aorta by approximately 40 mmHg, induced a rapid association of RhoA with Dbl-3 and p160(ROCK). The binding of p160(ROCK) to RhoA was rapidly increased, peaking at 30 min (approximately 3.5-fold), but reduced to lower levels (approximately 1.9-fold) by 60 min of pressure overload. The activity of immunoprecipitated p160(ROCK) toward myosin light chain increased approximately 2.5-fold within 10 min but decreased to lower levels (approximately 1.6-fold) after 60 min of pressure overload. Confocal microscopic analysis indicated that pressure overload induced the formation of aggregates of p160(ROCK) and RhoA along the longitudinal axis of cardiac myocytes. Immunoelectron microscopic analysis showed that pressure overload induced the association of p160(ROCK) and RhoA to Z-line, T-tubule, and subsarcolemmal areas. The rapid activation of p160(ROCK) by pressure overload and its aggregation in subcellular structures involved in transmission of mechanical force suggest a role for this enzyme in the mechanobiochemical transduction in the myocardium.
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