Sinalização celular durante aumento de tensão no coração : implicações no desenvolvimento de hipertrofia cardiaca : estudos em corações isolados

AUTOR(ES)
DATA DE PUBLICAÇÃO

2002

RESUMO

The mechanical input plays a major role in cardiac adaptive responses to hemodynamic overload. In addition of triggering the force-Iength mechanism and the homeometric autoregulation, the mechanical input activates signaling mechanisms involved on hypertrophic growth of cardiac myocytes, the hal1mark of themyocardial structural adaptation in response to sustained hemodynamic overload. How mechanical input is converted to biochemical signals that induce the myocardial responses is still not completely understood. However, increasing evidence support the notion that the living cells transduce and transmit forces into biochemical signals through specialized focal sites of the membrane whereby integrins connect the cytoskeleton to the extracellular matrix. This includes cardiac and skeletal myocytes, where the costameres, the counterpart of focal adhesion compleXoconnect the sarcolemma to sarcomere -Z lines through cytoskeletal proteins. Such structures provide a continuous path for mechanical signal transfer ttom extracellular matrix to internal organelles, sarcomere and nucleus. In addition, many signaling molecules are immobilized and have their function dependent on the anchorage to the cytoskeleton, which provide the physical basis for mechano-biochemical transduction. Among the various signaling molecules involved on integrin-mediated signaling, focal adhesion kinase (Fak) has received much attention, because experimental evidence indicate that it plays a key role in the celIular effects elicited by the integrinlcytoskeletal system, such as celI growth and gene expression. Fak activation has been demonstrated in isolated cardiac myocytes subjected to pulsatile mechanical stretch and overloaded feline and rat myocardium.The close relationship between increased load and Fak/c-Src activation, Fak/PI-3 kinase/Grb2 association and the concurrent activation of Akt and Erk1/2, two potential downstream effectors of the Fak multicomponent-signaling compleXoindicated that it may play a role in the earlier myocardial responses to increased workload. Thus, this study W@Sdesigned to examine, in an isolated perfused heart preparation, the separated influence of mechanical stimulus, tension development and contractile activity on Fak activation during increases in myocardial workload. In addition, the Erk1/2 activation, a downstream effector ofFak, was studied by using an anti-phosphospecific antibody against Erk1/2. The relative importance of myocardial passive tension and contractile activity on load-induced Pak and Erk1l2 activation were examined by perfusing isolated hearts with cardioplegic solution and by increasing calcium concentration in the perfusate, respectively. Purthermore, the locations of Pak and Erkl/2 in cardiac myocytes were confirmed by immunohistochemical analysis of myocardial sections. Increases of diastolic pressure from ~O up to ~15 mmHg rapidly (within 10 min) increased Pak tYfosine phosphorylation (maximum: 2.3-fold) and binding to c-Src (mx: 2.8-fold) and Grb2 (mx:3.6-fold). This was paralleled by a load-induced activation of Erk1l2 (mx; 2.8-fold) as assessed with phospho-specific antibodies, and also binding of Erk1l2 and phospho-Erk1l2 to Pak. Pak and Erk1l2 protein were immunolocalized at sarcolemmal sites of cardiac myocytes, but consistent Pak and Erk1l2 immunoreactivity were also detected regularly distributed at sarcoplasma and in the case of Erkl/2 at nuclei. Balloon inflation to raise ventricular pressure from ~O to ~15 mmHg in hearts perfused with cardioplegic solution activated Pak and Erk1l2 to levels comparable to those seen in beating hearts. However, increases in contractile activity induced by the heart perfusion with increasing calcium concentration (from 0.5 to 5 mM) did not activate Pak multicomponent signaling complex or Erk1l2 in the myocardium. Over all, these findings demonstrated tOOtmechanical stretch rather than contractile activity, triggers Pak and Erk1l2 activation during increased myocardium workload. In conc1usion, our results provide further argument for a role ofPak in the early myocardial responses to mecOOnicalstimuli. The demonstration here tOOtstretch or passive tension alone instead of increases in contractile activity can activate Pak and its downstream effectors is compatible with the appealing hypothesis that load induced Pak activation in myocardial cells could be linked to the integrin/citoskeletal signaling complexo Purthermore, experiments showing tOOtstretch or passive tension-induced binding of Pak to Grb2, Erk1l2, and activated Erkl/2, and also a common localization ofthese eI1ZYIlles at the sarcolemma and sarcoplasma of cardiac myocyts, indicate a linkage between load-induced Pak and Erk1/2 activation during mechanical overload

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