Mechanical stress-evoked but angiotensin II-independent activation of angiotensin II type 1 receptor induces cardiac hypertrophy through calcineurin pathway

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Abstract

Mechanical stress can induce cardiac hypertrophy through angiotensin II (AngII) type 1 (AT1) receptor independently of AngII, however, the intracellular mechanisms remain largely indeterminate. Since calcineurin, a Ca2+-dependent phosphatase, plays a critical role in pressure overload-induced cardiac hypertrophy, we therefore, asked whether calcineurin is involved in the AT1 receptor-mediated but AngII-independent cardiac hypertrophy. Mechanical stretch failed to elicit hypertrophic responses in COS7 cells co-transfected with plasmid of AT1 receptor and siRNA of calcineurin. Mechanical stresses for 2 weeks in vivo and for 24 h in vitro significantly induced upregulation of calcineurin expression and hypertrophic responses, such as the increases in cardiomyocytes size and specific gene expressions, in cardiomyocytes of angiotensinogen gene knockout (ATG−/−) mice, both of which were significantly suppressed by a specific calcineurin inhibitor FK506, suggesting a critical role of calcineurin in mechanical stress-induced cardiac hypertrophy in the ATG−/− mice. Furthermore, an AT1 receptor blocker Losartan not only attenuated cardiac hypertrophy but also abrogated upregulation of cardiac calcineurin expression induced by mechanical stresses in the AngII-lacking mice, indicating that calcineurin expression is regulated by AT1 receptor without the involvement of AngII after mechanical stress. These findings collectively suggest that mechanical stress-evoked but AngII-independent activation of AT1 receptor induces cardiac hypertrophy through calcineurin pathway.

Introduction

It was generally believed that the activation of angiotensin II (AngII) type 1 (AT1) receptor was induced by the increased secretion of AngII resulting from various pro-hypertrophic stimuli mainly consisting of the biochemical and biomechanical factors [1], [2]. In pressure overload-induced cardiac hypertrophic models, significantly increased cardiac local and circulating AngII was detected, and treatment with angiotensin-converting enzyme (ACE) inhibitors or AT1 receptor blockers (ARBs) effectively prevented the hypertrophic responses, indicating AngII plays a critical role in the development of cardiac hypertrophy induced by mechanical stress [3]. Significant hypertrophic response as well as enhanced activation and expression of calcineurin, a Ca2+-dependent phosphatase characterized by high conservation in evolution, wide distribution in tissues and sensitive responding to the intracellular Ca2+/calmodulin, were observed in the hearts of wild type mice imposed with transverse aorta constriction (TAC), which were much less remarkable in the hearts of the transgenic mice artificially over-expressed the dominant negative mutant of calcineurin specifically in the heart, indicating a critical role calcineurin plays in the development of pressure overload-induced cardiac hypertrophy [4], [5]. Kohzo Nagata et al. reported that AT1 receptor blocker (ARB) attenuated the development of cardiac hypertrophy and the activation of calcineurin, without an antihypertensive effect, in rats with salt-sensitive hypertension [6]. Thus calcineurin was considered as a crucial intracellular signaling molecule in AT1 receptor-mediated cardiac hypertrophy in the presence of AngII. We ever considered that it is the increased AngII after pressure overload that activates AT1 receptor, and then leads to the activation of calcineurin and the resultant cardiac hypertrophy. In another study, however, we proved that mechanical stress activated AT1 receptor without the involvement of AngII and therefore induced cardiac hypertrophy [7]. Moreover, mechanical stress led to the conformation switch of AT1 receptor and possibly resulted in different intracellular signaling transduction pathways in the development of cardiac hypertrophy [8], [9]. Thus there raise a presumption whether calcineurin still functions as a central effector in transducing the intracellular signals of AT1 receptor-mediated but AngII-independent cardiac hypertrophy elicited by mechanical stress.

In the present study, we found that mechanical stress could upregulate calcineurin protein through AngII-independent activation of AT1 receptor, leading to the development of cardiac hypertrophy.

Section snippets

COS7 cells culture, transfection and treatment

COS7 cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) with 10% fetal bovine serum (FBS) and then incubated in serum and antibiotic-free conditions in silicon-based plates pre-coated with collagen for 24 h. Transfection of AT1 receptor or calcineurin plasmids was performed by using lipofectamine 2000™ transfection reagent (Invitrogen, 11668-019) according to the manufacturer’s instructions. Forty-eight hours after transfection, COS7 cells were mechanically stretched for 24 h and

Mechanical stress-evoked but AngII-independent activation of AT1 receptor failed to induce hypertrophic responses without calcineurin

We firstly detected the expression of AT1 receptor protein and calcineurin in COS7 cells transfected with plasmids of AT1 receptor and calcineurin or siRNA of calcineurin, respectively. AT1 receptor and calcineurin were over-expressed in COS7 cells transfected with AT1 receptor and calcineurin. After the COS7 cells were transfected with the selected siRNA silencing the gene of calcineurin specially, the expression of calcineurin was downregulated significantly (Fig. 1A).

Mechanical stretch

Discussion

AT1 receptor plays an essential role in the development of mechanical stress-induced cardiac hypertrophy [14], [15], [16]. Mechanical stress increases the secretion of both local and circulating AngII and resultantly enhances the activity of AT1 receptor which activates the intracellular signaling molecules, inducing the reprogramming of fetal type genes and acceleration of the protein synthesis [1], [17]. One such involved intracellular signaling molecule is calcineurin, which implicates the

Acknowledgments

We thank Dr. Issei Komuro at Chiba University, Japan for kindly providing the AT1 receptor and calcineurin plasmids and ATG−/− mice, and Guoliang Jiang, Jingyi Ge, Yong Ye for providing technical supports in carrying out these experiments. This work was supported by the grants from National Science Fund for Distinguished Young Scholars from the National Natural Science Foundation of China (30525018), National Basic Research Program of China (2007CB512003), Project Sponsored by the Scientific

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These authors contributed equally to this work.

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