Overexpression of Activating Transcription Factor 3 Alleviates Cardiac Microvascular Ischemia/Reperfusion Injury in Rats.

Yi Liu,Jingjie Xiong,Yisen Hu,Xiaocong Zeng

doi:10.3389/fphar.2021.598959

Abstract

Activating transcription factor 3 (ATF3) has been confirmed to be responsive to oxidative stress and to negatively regulate the activity of Toll-like receptor 4 (TLR4). However, the effect of ATF3 on cardiac microvascular ischemia/reperfusion (I/R) injury remains unknown. The GEO2R online tool was employed to obtain differentially expressed genes GSE4105 and GSE122020, in two rat I/R injury microarray datasets. We established a rat myocardial I/R model in vivo, and also generated an in vitro hypoxia/reoxygenation (H/R) model of cardiomyoblast H9c2 cells. Overexpression of ATF3 was achieved by adenoviral-mediated gene transfer (Ad-ATF3). Rats were randomly divided into four groups: sham, I/R, I/R + Ad-Lacz (as a control), and I/R + Ad-ATF3. ELISA, CCK-8, DCFH-DA probe, qRT-PCR and Western blotting were used to determine the expression of ATF3, oxidative indices, cellular injury and TLR4/NF-κB pathway-associated proteins. Transmission electron microscopy, immunohistochemistry and immunofluorescence were used to detect the leukocyte infiltration and the alteration of microvascular morphology and function in vivo. Echocardiographic and hemodynamic data were also obtained. Bioinformatics analysis revealed that ATF3 was upregulated in I/R myocardia in two independent rat myocardial I/R models. Cardiac microvascular I/R injury included leukocyte infiltration, microvascular integrity disruption, and microvascular perfusion defect, which eventually resulted in the deterioration of hemodynamic parameters and heart function. Ad-ATF3 significantly restored microvascular function, increased cardiac microvascular perfusion, and improved hemodynamic parameters and heart function. Mechanistically, Ad-ATF3 ameliorated oxidative stress, inhibited TLR4/NF-κB pathway activation and down-regulated the expression of downstream proinflammatory cytokines in I/R myocardium in vivo and in H/R H9c2 cells in vitro. ATF3 overexpression protects against cardiac microvascular I/R injury in part by inhibiting the TLR4/NF-κB pathway and oxidative stress.

Highlights

Primary percutaneous coronary intervention is the standard treatment for reducing myocardial necrosis and improving clinical prognosis in patients undergoing acute ST-segment elevation myocardial infarction (Ibanez et al, 2018)
We investigated the role of Activating transcription factor 3 (ATF3) in I/R-triggered myocardial pathophysiology and the underlying mechanisms
We found that 1) bioinformatics analysis of two GEO datasets related to myocardial I/R showed upregulation of ATF3 in I/R myocardium, 2) ATF3 overexpression inhibited the Toll-like receptor 4 (TLR4)/NF-κB pathway activation, inflammation and oxidative stress during H/R, 3) ATF3 overexpression inhibited the TLR4/NF-κB pathway activation, inflammation and oxidative stress during I/R, and 4) ATF3 overexpression inhibited leukocyte infiltration, maintained microvascular integrity and permeability, restored cardiac microvascular perfusion and effectively protected cardiac microvascular ischemia/reperfusion injury, eventually leading to smaller infarct size, improved hemodynamic indices and heart function in myocardial I/R rats

Summary

Introduction

Primary percutaneous coronary intervention is the standard treatment for reducing myocardial necrosis and improving clinical prognosis in patients undergoing acute ST-segment elevation myocardial infarction (Ibanez et al, 2018). A microvascular reperfusion injury can occur while epicardial blood flow is being restored by reperfusion therapy This event is known as the “no-reflow (NR)” phenomenon (Allencherril et al, 2019) and is regarded as myocardial tissue hypoperfusion. After reperfusion, the extensive microcirculatory dysfunction results in the insufficient availability of blood, energy, oxygen and nutrients to the cardiomyocytes. This occurs even though the normal epicardial flow is restored and results in the further exacerbation of myocardial damage (Zhou et al, 2018d; Wang et al, 2020). Improving microvascular perfusion is considered an efficient therapeutic method to alleviate or abolish the NR phenomenon, and effectively protects the myocardium from I/R injury (O’farrell et al, 2017; Ozawa et al, 2018)

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