Abstract 266: Novel Sites of Angiotension II Type 1 Receptor are Identified for Direct Activation by Mechanical Stretch Independent of Angiotension II

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The angiotensin II type 1 receptor (AT1R) has a crucial role in cardiac hypertrophy induced by pressure overload. In the previous study, we found a novel mechanism for mechanical stress-induced AT1R activation without the involvement of Ang II. However, few reports focus on how AT1R senses mechanical stress and translates it into biochemical signals inside the cells to induce cardiomyocyte hypertrophy. Here, we constructed different site-directed mutagenesis of AT1R and transfected them to COS7 cells and ATG–/– (Angiotensinogen knockout) cardiomyocytes, respectively, to observe the activation of downstream signaling to identify functional site of AT1R. The results showed AT1R-WT, AT1R-K199Q, AT1R-L212F,AT1R-Q257A and AT1R-C289A plasmids or adenovirus were overexpressed at high level in plasma membrane of COS7 or cardiomyocytes respectively. There was no obvious difference in subcellular expression of wt-AT1R and all the mut-AT1Rs. The further study revealed that Ang II-induced-phosphorylation of ERK, Jak2 and the redistribution of Gαq11 were dramatically decreased in COS7 cells expressing AT1R-K199Q or AT1R-Q257A, while these effects induced by mechanical stretch were greatly suppressed in COS7 cells expressing AT1R-L212F,AT1R-Q257A or AT1R-C289A compared to these in COS7 cells expressing AT1R-WT. We then transfected the adenovirus of wt-AT1R or different mut-AT1Rs to ATG–/– cardiomyocytes to exclude the influence of endogenous Ang II. The results were consistent with these results in COS7 cells. Moreover, ATG–/– cardiomyocytes overexpressing AT1R-K199Q or AT1R-Q257A parlty abolished hypertrophic response induce by Ang II, while the cardiomyocytes overexpressing AT1R-L212F,AT1R-Q257A or AT1R-C289A greatly inhibited the hypertrophic response induced by mechanical stretch. The present study indicated that Leu212, Gln257 and Cys289 are critical sites for AT1R activation by mechanical stretch without Ang II but Lys199 and Gln257 play important role in AT1R activation with Ang II.

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