Protective Role of NRG1/ErbB4 Signaling in Myocardial Ischemia-Reperfusion Injury

Summary

Here, we present a protocol to evaluate the cardioprotective effects of NRG1/ErbB4 signaling in a rat model of ischemia-reperfusion injury by assessing oxidative stress and apoptotic signaling pathways.

Abstract

Myocardial ischemia-reperfusion injury (MIRI) endures as a substantial impediment to the management of cardiovascular disease. The pathophysiology of MIRI is complex, involving oxidative stress, calcium overload, inflammation, and apoptosis. The NRG1/ErbB4 signaling pathway has been implicated in modulating oxidative stress responses in the heart, potentially reducing cellular damage caused by free radicals. This study aimed to elucidate the molecular mechanisms by which the NRG1/ErbB4 pathway confers protection in a rat model of myocardial ischemia-reperfusion (MIRI) injury. A rat MIRI model was established, involving 30 adult male Sprague-Dawley rats randomly assigned to five groups: control, sham operation, MIRI, recombinant NRG1, and AG1478. NRG1 concentrations in heart tissues were measured by ELISA, while p-ErbB4 and total ErbB4 protein levels were assessed by Western blot. Cardiomyocyte apoptosis and reactive oxygen species (ROS) levels were evaluated by flow cytometry.In the MIRI group, NRG1 levels were significantly reduced compared to the control and sham operation groups (p < 0.05). Recombinant NRG1 treatment led to a marked increase in NRG1 levels in heart tissue, along with an enhanced p-ErbB4/ErbB4 expression ratio (p < 0.05). The AG1478 group exhibited lower NRG1 levels and a reduced p-ErbB4/ErbB4 ratio compared to the recombinant NRG1 group (p < 0.05). In terms of apoptosis and ROS levels, the MIRI group showed significantly higher rates than the control and sham operation groups (p < 0.05). Recombinant NRG1 treatment significantly reduced myocardial cell apoptosis and ROS levels (p < 0.05), while AG1478 also attenuated these effects, but to a lesser extent. The NRG1/ErbB4 signaling pathway plays a pivotal protective role in MIRI in rats. Supplementation with exogenous NRG1 effectively elevates NRG1 levels in the heart, activates the ErbB4 signaling pathway, reduces myocardial cell apoptosis, and decreases ROS levels, thereby mitigating MIRI-induced damage.

Introduction

Myocardial ischemia-reperfusion injury (MIRI) represents a critical challenge in the management of cardiovascular diseases¹. Although restoring blood flow to the ischemic myocardium is essential for treating myocardial infarction, reperfusion itself can exacerbate cardiac tissue damage, thus limiting the therapeutic effect. The mechanisms underlying MIRI are complex and include various pathophysiological processes, among which are oxidative stress, calcium overload, inflammatory responses, and apoptosis^2,3,4.

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Protocol

Experimental animals
Thirty adult male Sprague-Dawley (SD) rats, aged 8 weeks and weighing 180-200 g, were obtained from a commercial vendor. These rats were housed under specific pathogen-free conditions for 1 week prior to the experiments. The experiments received approval from the Ethical Committee of Nanping First Hospital (NO. NPSY202208010).

Animal models of myocardial ischemia-reperfusion injury
Rats were anesthetized via intraperitoneal injection of 3% pentobarbital sodium at a dose of 50 mg/kg. After confirming the absence of reflexes, the animals were placed in a supine position, and t....

Results

NRG1 exerts a protective effect against MIRI-induced cardiomyocyte apoptosis
Flow cytometric analysis indicated a notable elevation in apoptosis rates in the MIRI group when compared to both the control and sham groups (P < 0.05). Both the recombinant NRG1 and AG1478 groups exhibited significantly reduced apoptosis rates compared to the MIRI group (P < 0.05). In contrast, the recombinant NRG1 group exhibited a reduced apoptosis rate compared to the AG1478 group (P < 0.05) (

Discussion

MIRI continues to pose a significant obstacle in cardiovascular disease management, given that the re-establishment of perfusion to ischemic cardiac tissue may paradoxically intensify myocardial injury. The occurrence of MIRI is multifactorial, involving oxidative stress, calcium overload, inflammatory reactions, and mitochondrial dysfunction^9,10.

NRG1, an extracellular ligand, activates downstream signaling pathways by binding to the .......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
AG1478	BioLab Technology	WD102925	Intervention drug AG1478
Annexin V-FITC/PI Apoptosis Detection Kit	Meilune	MA0220	Flow cytometry apoptosis
BCA Protein Assay Kit	Meilunbio	MA0082-2	WB protein quantification
Chemiluminescence Imaging System	Bio-Rad (USA)	ChemiDoc Touch	Western blot imaging
Cyamemazine Hydrochloride	Huamu Animal Health	20220601	Anesthetic for animal modeling
ECL Detection Kit	Meilunbio	MA0186-1	WB chemiluminescence
ERbB4	Proteintech	19943-1-AP	WB primary antibody
Flow Cytometer	BD	FACSCalibur	Flow cytometry apoptosis detection
GAPDH	Proteintech	60004-1-Ig	WB loading control antibody
HRP-conjugated Goat Anti-Mouse IgG(H+L)	Proteintech	SA00001-1	Immunohistochemistry
HRP-conjugated Goat Anti-Rabbit IgG(H+L)	Proteintech	SA00001-2	Immunohistochemistry
Microplate Reader	Thermo	K3	ELISA detection
PAGE Gel Preparation Kit 15%	Meilunbio	MA0384	WB electrophoresis
p-ERbB4	Affinity	AF3445	WB primary antibody
Pre-stained Protein Marker	Meilunbio	MA0342	WB electrophoresis
Rat Neuregulin-1 (NRG-1) ELISA Kit	Meimian	MM-61434R2	ELISA detection
Reactive Oxygen Species (ROS) Assay Kit	Meilune	MA0219	WB assay
rhNRG1	BioLab Technology	WD091127	Intervention drug NRG1
RIPA lysis buffer	Meilunbio	MA0151	Protein extraction for Western blot
SDS-PAGE Loading Buffer (5x)	Beyotime	P0015L	WB electrophoresis
Sodium Dodecyl Sulfate	Sigma	151-21-3	WB electrophoresis
Su Tai 50	Virbac (France)	8X22A	Anesthetic for animal modeling
Vertical Protein Electrophoresis System	Bio-Rad (USA)	POWER PAC 200	Western blot electrophoresis