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.

Nerve growth factor 1 (NRG1) and its receptor Erb-B2 Receptor Tyrosine Kinase 4 (ERBB4) play pivotal roles in cardiac physiology and pathology. NRG1, an extracellular ligand, interacts with the ErbB4 receptor to activate a cascade of signaling pathways that are crucial for heart development, maintaining structural integrity, and responding to cardiac injuries⁵. Emerging evidence highlights the role played by the NRG1/ErbB4 signaling pathway in heart diseases. Under conditions of myocardial ischemia-reperfusion, the expression and function of NRG1 may be altered⁶. Previous studies have suggested that NRG1 promotes myocardial cell survival and inhibits excessive apoptosis by activating ErbB4. Additionally, the NRG1/ErbB4 signaling pathway may regulate the oxidative stress response in the heart and mitigate cell damage caused by free radicals. Therefore, elucidating the function of the NRG1/ErbB4 signaling pathway in MIRI is vital for understanding the biological basis of this injury and developing novel therapeutic strategies^7,8.

This study speculates that the NRG1/ErbB4 signaling pathway plays a protective role in MIRI. Therefore, in this study, a rat model of myocardial ischemia-reperfusion was constructed to systematically investigate the mechanism of the NRG1/ErbB4 signaling pathway in MIRI. By regulating the concentration of NRG1 and observing the changes in ErbB4 activation status, myocardial cell apoptosis rate, and oxidative stress level. This study attempts to reveal the molecular mechanism by which the NRG1/ErbB4 signaling pathway exerts a protective effect in MIRI. The novelty of this study lies in revealing the significance of the NRG1/ErbB4 signaling pathway in myocardial ischemia-reperfusion injury, and also providing a new perspective for the treatment strategies of heart diseases based on this pathway.

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 the neck and chest areas were shaved and disinfected with 75% ethanol. A midline cervical incision (~2 cm) was made to expose the trachea, followed by oral intubation using a 16 G catheter to maintain airway patency during surgery. Subsequently, the left pectoralis major muscle was carefully dissected to expose the third and fourth intercostal spaces. A thoracotomy was performed by making an incision between these spaces, and the pericardium was opened to fully expose the heart. To induce ischemia, the left anterior descending coronary artery (LAD) was ligated using an 8-0 nylon suture. A polyethylene tubing (PE-10) was inserted between the LAD and the ligature to enable reversible occlusion. Ischemia was maintained for 45 min, confirmed visually by myocardial blanching and ECG changes. Reperfusion was initiated by releasing the ligature, allowing blood flow to resume for 3 h. The ligation was removed 45 min later.

Experimental grouping
The rats were randomly assigned to five groups (n = 6 each). (1) Control group: rats received standard care without any surgical intervention or treatment. (2) Sham operation group: rats underwent thoracotomy and threading of the left anterior descending coronary artery (LAD) without ligation, serving as surgical controls. (3) MIRI group: rats underwent myocardial ischemia-reperfusion injury induction and were administered normal saline intragastrically as a vehicle control. (4) Recombinant NRG1 group: rats received an intravenous injection of recombinant human NRG1 (rh-NRG1) at a dose of 5 mg/kg, 30 min before thoracotomy, followed by the MIRI procedure. (5) AG1478 group: rats were administered intravenous rh-NRG1 (5 mg/kg) combined with AG1478 (20 mg/kg), an ErbB receptor kinase inhibitor, 30 min prior to thoracotomy, and then underwent the MIRI procedure.

Enzyme-linked immunosorbent assay (ELISA)
Animals were euthanized by intraperitoneal injection of pentobarbital sodium (150 mg/kg). Heart tissues were collected immediately after reperfusion and homogenized on ice in an appropriate volume of ice-cold PBS. The homogenates were then centrifuged at 1200 x g for 10 min at 4 °C to separate the supernatant. The concentration of NRG1 in the supernatant was quantified using a commercially available NRG1 ELISA kit, following the manufacturer's instructions precisely. Briefly, samples and standards were added to the ELISA plate wells, incubated at room temperature for the recommended time, followed by washing and addition of detection antibodies. The absorbance was measured at the specified wavelength using a microplate reader, and NRG1 levels were calculated based on the standard curve. The sample volume was 100 µL, the dilution of the antibody was 1:100, and the wavelength for measuring absorbance was set at 450 nm.

Western blot analysis
Cardiac tissues were excised from anesthetized rats and rinsed in ice-cold PBS to remove residual blood. For each heart, approximately 0.5 g of left ventricular tissue was weighed and placed in 500 µL of freshly prepared RIPA lysis buffer. The tissue was then homogenized on ice using a mechanical tissue grinder until fully lysed, followed by incubation on ice for 30 min to ensure complete protein extraction. Lysates were centrifuged at 12,000 × g for 15 min at 4 °C to remove debris. Protein concentrations were determined using a BCA assay kit. Equal amounts of total protein (e.g., 30 µg per lane) were loaded and separated by SDS-PAGE. Proteins were then transferred onto polyvinylidene fluoride (PVDF) membranes using a wet transfer system at 100 V for 90 min. Membranes were blocked with 5% non-fat dry milk in Tris-buffered saline with 0.1% Tween-20 (TBST) for 1 h at room temperature. Subsequently, membranes were incubated overnight at 4 °C with primary antibodies against phosphorylated ErbB4 (p-ErbB4) and total ErbB4, each diluted 1:1000. After washing, membranes were incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies for 1 h at room temperature. Protein bands were visualized using enhanced chemiluminescence (ECL) substrate and captured using a gel imaging system. Band intensities were quantified using ImageJ software, and the ratio of p-ErbB4 to total ErbB4 was calculated to evaluate phosphorylation levels.

Flow cytometry
Cardiac myocyte apoptosis was assessed by flow cytometry. Heart tissues were finely minced and enzymatically digested using a mixture of collagenase type II (1 mg/mL) and protease (e.g., trypsin, 0.25%) at 37 °C with gentle agitation for 20-30 min. Enzymatic digestion was terminated by adding an equal volume of fetal bovine serum (FBS). The cell suspension was filtered through a 70-µm nylon mesh and centrifuged at 300 × g for 5 min at 4 °C to collect the cells. The cell pellet was resuspended in binding buffer, then incubated with Annexin V-FITC and propidium iodide (PI) according to the manufacturer's protocol. After incubation for 15 min at room temperature in the dark, samples were immediately analyzed by flow cytometry. Early apoptotic cells were identified as Annexin V-positive and PI-negative, whereas late apoptotic or necrotic cells were positive for both markers. Apoptosis rates were quantified and compared among experimental groups. The gating strategy adopts a cross-gate/rectangular gate setting. When conducting on-machine detection, the cross gate was set as a dotted line, and all parameters were applied to ensure the consistency of data screening. The flow rate was adjusted according to the cell concentration (maintained at 1000 cells/s), and low-speed sample loading can avoid nozzle clogging.

Detection of reactive oxygen species (ROS) levels
Cardiomyocytes were isolated from heart tissues, followed by centrifugation at 300 × g for 5 min and washing twice with phosphate-buffered saline (PBS). The cells were then incubated with 10 µM 2',7'-dichlorofluorescein diacetate (DCFDA) dye at 37 °C for 30 min in the dark, following the manufacturer's instructions. After staining, cells were washed twice with PBS to remove excess unbound dye. Intracellular ROS levels were measured immediately by flow cytometry, detecting the fluorescence intensity emitted upon oxidation of DCFDA. The mean fluorescence intensity (MFI) of each sample was quantified using FlowJo software to compare ROS levels across experimental groups. The gating strategy is as described above (flow cytometry section).

Statistical analysis
Data analysis was conducted using SPSS software, presented as the mean ± standard deviation (SD). One-way analysis of variance with Tukey's post hoc test was performed to compare multiple groups, and Student's t-test was used for pairwise comparisons. A p-value < 0.05 was considered statistically significant.

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