Extracellular and Intracellular Angiotensin II Regulate the Automaticity of Developing Cardiomyocytes via Different Signaling Pathways.

Tao Wang,Heinrich Sauer,Zhi-Feng Chen,Zongwei Cai,Chun Kit Wong,Xiaoqiang Yao,Cheng Long,Xiangmao Chen,Suk Ying Tsang,Zenghua Qi,Qianqian Ding

doi:10.3389/fmolb.2021.699827

Abstract

Angiotensin II (Ang II) plays an important role in regulating various physiological processes. However, little is known about the existence of intracellular Ang II (iAng II), whether iAng II would regulate the automaticity of early differentiating cardiomyocytes, and the underlying mechanism involved. Here, iAng II was detected by immunocytochemistry and ultra-high performance liquid chromatography combined with electrospray ionization triple quadrupole tandem mass spectrometry in mouse embryonic stem cell–derived cardiomyocytes (mESC-CMs) and neonatal rat ventricular myocytes. Expression of AT1R-YFP in mESC-CMs revealed that Ang II type 1 receptors were located on the surface membrane, while immunostaining of Ang II type 2 receptors (AT2R) revealed that AT2R were predominately located on the nucleus and the sarcoplasmic reticulum. While extracellular Ang II increased spontaneous action potentials (APs), dual patch clamping revealed that intracellular delivery of Ang II or AT2R activator C21 decreased spontaneous APs. Interestingly, iAng II was found to decrease the caffeine-induced increase in spontaneous APs and caffeine-induced calcium release, suggesting that iAng II decreased spontaneous APs via the AT2R- and ryanodine receptor–mediated pathways. This is the first study that provides evidence of the presence and function of iAng II in regulating the automaticity behavior of ESC-CMs and may therefore shed light on the role of iAng II in fate determination.

Highlights

The renin–angiotensin system (RAS) plays a central role in the regulation of water balance and blood pressure; in addition, it has an important role in the cardiovascular system (Harada et al, 1999)
The intracellular Ang II (iAng II) was detected by immunocytochemistry and our newly developed method by UHPLC-ESI-MS/MS in mESCCMs and NRVMs, respectively
Our main findings include the following: 1) iAng II could be detected in both NRVMs and mESC-CMs, 2) Ang II type 1 receptor (AT1R) is located on the plasma membrane, while Ang II type 2 receptors (AT2R) is predominately located on the nucleus and the sarcoplasmic reticulum (SR) of developing CMs, 3) extracellular Angiotensin II (Ang II) increases action potentials (APs) in a transient manner through the AT1R-dependent pathway, 4) iAng II decreases APs in a persistent manner through the AT2R-dependent pathway, and 5) iAng II regulates the automaticity of developing CMs by decreasing the activity of ryanodine receptor isoform 2 (RyR2)

Summary

Introduction

The renin–angiotensin system (RAS) plays a central role in the regulation of water balance and blood pressure; in addition, it has an important role in the cardiovascular system (Harada et al, 1999). Two distinct Ang II receptor subtypes, namely, the Ang II type 1 receptor (AT1R) and the Ang II type 2 receptor (AT2R), have been identified Both AT1R and AT2R belong to the G protein–coupled receptor (GPCR) superfamily, but they are found to induce different cellular signaling pathways (Akazawa et al, 2013; Karnik et al, 2015). AT2R has been proven to be a GPCR, with all the classical motifs and signature residues of a GPCR (Lokuta et al, 1994; Gallinat et al, 1999; Asada et al, 2018) It is still unclear whether AT2R can activate a classical G protein signaling pathway

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