Original article
Angiotensin II induced proteolytic cleavage of myocardial ACE2 is mediated by TACE/ADAM-17: A positive feedback mechanism in the RAS

https://doi.org/10.1016/j.yjmcc.2013.11.017 Get rights and content

Highlights

  • Ang II mediated proteolytic loss of ACE2 is associated with elevated TACE activity.

  • Inhibition of TACE prevents shedding of ACE2 in vitro and in vivo.

  • AT1 receptor blockade prevents loss of ACE2.

  • NADPH oxidase-mediated ROS plays a key role in ACE2 shedding.

  • These findings illustrate a positive feedback mechanism in the RAS.

Abstract

Angiotensin converting enzyme (ACE) 2 is a key negative regulator of the renin–angiotensin system where it metabolizes angiotensin (Ang) II into Ang 1–7. We hypothesize that Ang II suppresses ACE2 by increasing TNF-α converting enzyme (TACE) activity and ACE2 cleavage. Ang II infusion (1.5 mg/kg/day) in wild-type mice for 2 weeks resulted in substantial decrease in myocardial ACE2 protein levels and activity with corresponding increase in plasma ACE2 activity, prevented by AT1R blockade. Ang II resulted in AT1R-mediated increase in myocardial TACE expression and activity, and membrane translocation of TACE. Ang II treatment in Huh7 cells exhibited AT1R-dependent metalloproteinase mediated shedding of ACE2 while transfection with siTACE prevented shedding of ACE2; cardiomyocyte-specific deletion of TACE also prevented shedding of ACE2. Reactive oxygen species played a key role since p47phoxKO mice were resistant to Ang II-induced TACE phosphorylation and activation with preservation of myocardial ACE2 which dampened Ang II-induced cardiac dysfunction and hypertrophy. In conclusion, Ang II induces ACE2 shedding by promoting TACE activity as a positive feedback mechanism whereby Ang II facilitates the loss of its negative regulator, ACE2. In HF, elevated plasma ACE2 activity likely represents loss of the protective effects of ACE2 in the heart.

Introduction

The renin–angiotensin system (RAS) is a central regulator of the cardiovascular system and plays a fundamental role in the pathophysiology of experimental and human heart failure (HF) [1], [2], [3]. RAS consists of two counter-regulatory arms regulating cardiovascular function. The first arm consists of a series of enzymatic reactions culminating in the generation of angiotensin II (Ang II) which can lead to Ang II type 1 receptor (AT1R) dependent vasoconstriction, inflammation, myocardial fibrosis and hypertrophy and HF [1], [4]. The second arm is constituted by the angiotensin converting enzyme (ACE) 2/Ang 1–7/Mas receptor axis which acts as a physiological antagonist of the ACE/Ang II/AT1R arm [5], [6]. ACE2 as a negative regulator of the RAS, functions to promote the degradation of Ang II into the vasodilator, anti-hypertrophic, and anti-inflammatory heptapeptide, Ang 1–7 [7], [8], [9].

ACE inhibitors and AT1 receptor blockers have revolutionized the treatment of HF and reduce the morbidity and mortality in patients with HF. Interestingly, these drugs also increased myocardial ACE2 levels and activity [10]. Loss of ACE2 is detrimental to the heart as it leads to cardiac hypertrophy and impaired contractility due in part to increased Ang II stimulation of AT1R [8], [9], [11], [12], whereas ACE2 over-expression protects the heart from Ang II-mediated cardiac hypertrophy and myocardial fibrosis [9], [13]. ACE2 is a type I transmembrane protein, with an extracellular N-terminal domain containing the active site [5], [6]. Proteolysis on the cell surface and in the extracellular matrix is an important mechanism leading to cancer, inflammatory and cardiovascular diseases [14], [15], [16]. In this study, we showed that Ang II triggered the cleavage and shedding of a soluble form of ACE2 from the membrane by TNFα-converting enzyme (TACE), also known as a disintegrin and metalloproteinase (ADAM) 17. Ang II-stimulated and TACE-mediated loss of myocardial ACE2 orchestrates a critical link between the RAS, oxidative stress and inflammation in heart disease and represents the first description of a positive feedback mechanism in the RAS.

Section snippets

Experimental animals and protocols

Mice lacking p47phox (p47phoxKO; p47phox −/−) backcrossed into a pure C57BL/6 background were used as previously described [17]. Only C57BL/6 male wild-type (WT) controls were used. An osmotic minipump (model 1002, Alzet Osmotic Pumps, Cupertino, CA) was implanted subcutaneously at the dorsum of the neck to continuously infuse Ang II (1.5 mg/kg/day) or saline (vehicle) for 1 week or 2 weeks. TACEflox/flox mice [18] were crossed with the cardiac-specific α-MHC-Cre transgenic mice to create

Ang II mediates loss of ACE2 with upregulation of TACE/ADAM-17 in the heart: a key role of the AT1R receptor

Western blot analysis showed a marked reduction in heart ACE2 in response to 1 and 2 weeks of in vivo exposure to elevated Ang II (Fig. 1A) with a marked increase in Ace2 mRNA levels (Fig. 1B). Ang II-mediated loss of ACE2 levels was clearly suppressed by AT1R blockade (Fig. 1C) which was mirrored by the assessment of ACE2 activity (Fig. 1D) with reciprocal changes seen in plasma ACE2 activity (Fig. 1E). However, AT1R blockade did not alter the Ang II induced upregulation of Ace2 mRNA expression

Discussion

The RAS plays a central role in the pathophysiology of many cardiovascular diseases and suppression of the ACE/Ang II/AT1R axis of the RAS minimizes cardiovascular complications. ACE2/Ang 1–7/Mas receptor is the counter regulatory axis of the RAS where ACE2 serves as an endogenous negative regulator of the RAS and reduces Ang II levels and increases the generation of Ang 1–7 [7], [8], [9], [11], [12], [33]. We showed that Ang II mediates loss of ACE2 in the heart. Myocardial ACE2 level in

Conflict of interest statement

Vaibhav B. Patel: None.

Nicola Clarke: None.

Zuocheng Wang: None.

Dong Fan: None.

Nirmal Parajuli: None.

Ratnadeep Basu: None.

Brendan Putko: None.

Zamaneh Kassiri: None.

Anthony Turner: None.

Gavin Y. Oudit: None.

Acknowledgments

GYO is a Clinician-Investigator of the Alberta Innovates—Health Solutions (AI-HS) and the Distinguish Clinician Scientist of the Heart and Stroke Foundation of Canada and Canadian Institutes of Health Research (CIHR). ZK is a New Investigator of the Heart and Stroke Foundation of Canada (HSFC) and Scholar of the AI-HS. VBP is supported by AI-HS Post-Doctoral Fellowship and VBP and NP are supported by Heart and Stroke Foundation of Canada Fellowships. We acknowledge the funding support from CIHR

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