Early Activation Of P160rock By Pressure Overload In Rat Heart

We investigated the effects of acute pressure overload on activation of p160ROCK in rat myocardium. Constriction of transverse aorta, controlled to increase peak systolic pressure of ascending aorta by -40 mmHg, induced a rapid association of RhoA with Dbl-3 and p160ROCK. The binding of p160ROCK to RhoA was rapidly increased, peaking at 30 min (∼3.5-fold), but reduced to lower levels (∼1.9-fold) by 60 min of pressure overload. The activity of immunoprecipitated p160ROCK toward myosin light chain increased ∼2.5-fold within 10 min but decreased to lower levels (∼1.6-fold) after 60 min of pressure overload. Confocal microscopic analysis indicated that pressure overload induced the formation of aggregates of p160ROCK and RhoA along the longitudinal axis of cardiac myocytes. Immunoelectron microscopic analysis showed that pressure overload induced the association of p160ROCK and RhoA to Z-line, T-tubule, and subsarcolemmal areas. 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Arhgap21 Associates With Fak And Pkcζ And Is Redistributed After Cardiac Pressure Overload

ARHGAP21 is highly expressed in the heart, which demonstrates activity over Cdc42 and interacts with proteins of the cytoskeleton and adherent junctions. The main cause of cardiac hypertrophy is mechanical stimulus; therefore we analyzed ARHGAP21 expression after acute mechanical stress in the myocardium and its association with FAK and PKCζ. We demonstrated that ARHGAP21 is relocated to Z-lines and costameres after pressure overload, and interacts with PKCζ and FAK in control rats (sham), rats submitted to aortic clamping and spontaneously hypertensive rats (SHR). Co-transfection using ARHGAP21 and PKCζ constructions demonstrated that ARHGAP21 associates with PKCζ-GST and endogenous FAK. Pulldown assay showed that ARHGAP21 binds to the C-terminal region of FAK. Moreover, ARHGAP21 binds to PKCζ phosphorylated on Thr410 in sham and SHR. However, ARHGAP21 only binds to FAK phosphorylated on Tyr925 of SHR. Additionally, PKCζ is phosphorylated by mechanical stimuli. 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