Biochemical and Biophysical Research Communications
ARHGAP21 associates with FAK and PKCζ and is redistributed after cardiac pressure overload
Section snippets
Materials and methods
Experimental animal model. Male Wistar rats (160–200 g) underwent acute pressure overload induced by controlled constriction of transversal aorta as previously described [13]. Left ventricle (LV) of male spontaneously hypertensive rats (SHR) at 8 weeks of age were used to extract total protein for co-immunoprecipitation and Pulldown assays. Three independent experiments were carried out for each procedure. The animals were obtained from the Animal Breeding Center of the University of Campinas.
Relocation of ARHGAP21 to Z-lines and costameres after pressure overload
We separated LV into cytoplasmic (CF), membrane (MF), and nuclear (NF) fractions. ARHGAP21 was weakly expressed in CF (Fig. 1A) and expression decreased in cardiomyocyte NF after aortic clamping (AoCo) when compared with control animals (sham). In contrast, a progressive increase of ARHGAP21 occurred in MF.
Confocal microscopy demonstrated that ARHGAP21 is hardly expressed in myocyte nucleus from AoCo hearts (Fig. 1B; lower), whereas sham cardiomyocytes had their nucleus completely labeled (Fig.
Discussion
In the present study, we demonstrated that ARHGAP21 is recruited from the nuclei and myofilaments to costameres, Z-lines, and intercalated disks of cardiomyocytes after AoCo. These data suggest that ARHGAP21 is modulated by mechanical stimuli and can develop an important role during cardiac stress and early hypertrophy.
Moreover, ARHGAP21 associates with PKCζ in cardiac cells of rats submitted to AoCo, sham and SHR. We also demonstrated that ARHGAP21 interacts with PKCζ phosphorylated on Thr410,
Acknowledgments
This work was supported by Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) and Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq). We thank Juvani L. Saturno for immunoelectron microscopy and Raquel S. Foglio for English revision. We are also thankful to Dr. Gail Fraser, Dr. Pascale Cossart, Dr. Jun-Lin Guan, and Dr. Michael Schaller for the constructions, and to the students and employees involved in the study.
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