NBQX mediates ventricular fibrillation susceptibility in rat models of anxiety via the Nrf2/HO-1 pathway

Abstract

Objective: Anxiety disorder (AD) is a common mental disorder related to cardiovascular disease morbidity. Oxidative stress plays a crucial role in the anxiety state and can lead to cardiac remodeling. Over-activation of the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR) in cardiomyocytes and neurons can cause oxidative stress. Additionally, the AMPAR inhibitor-2,3-dihydroxy-6-nitro-7-sulfamoyl-benzoquinoxaline-2,3-dione (NBQX) plays an important role in ameliorating oxidative stress. This study aimed to explore the anti-arrhythmic effects of NBQX in a rat model of anxiety.

Methods: The AD model was induced using empty bottle stimulation. Male Sprague Dawley rats were randomly divided into four groups: control + saline, control + NBQX, AD + saline, and AD + NBQX. Open field test was conducted to measure anxiety-like behavior. Electrophysiological experiments, histological analysis, biochemical detection and molecular biology were performed to verify the effects of NBQX on the amelioration of electrical remodeling and structural remodeling. Furthermore, the nuclear factor erythroid 2-related factor 2 (Nrf2) inhibitor (ML385) was used in vitro to demonstrate the signaling pathway.

Results: Oxidative stress levels increased with AMPAR over-activation in AD rats, leading to heightened vulnerability to ventricular fibrillation (VF). NBQX reverses anxiety and VF susceptibility. Our results showed that NBQX activated the Nrf2/heme oxygenase-1 (HO-1) pathway, leading to a decline in oxidative stress levels. Connexin 43 and ion channel expression was upregulated. NBQX treatment attenuated fibrosis and apoptosis. This effect was diminished by ML385 treatment in vitro.

Conclusion: NBQX can alleviate VF susceptibility in rat models of anxiety by alleviating electrical remodeling, structural remodeling via regulating the Nrf2/HO-1 pathway to some extent.

Keywords: AMPAR; Anxiety disorder; NBQX; Oxidative stress; Ventricular arrhythmia.

Fig. 1

NBQX alleviated anxiety behaviors in AD rats. (A) Schematic representation of the behavioral test (n = 9 per group). (B) Representative heat map images highlighting the motion trails of SD rats in the open field test (n = 9 in each group). (C) Frequency of rats entering the central zone (n = 9 per group). (D) Cumulative duration of rats in the center zone (n = 9 per group). (E) Number of rearing instances in 5 mi of testing (n = 9 per group). Data are expressed as mean ± SD, ****p < 0.0001, **p < 0.01.

Fig. 2

NBQX treatment alleviated heart rate variability (HRV). (A–B) Statistical analysis of RR and RMSSD (n = 9). (C–E) Statistical data of HF, LF, and LF/HF in all groups (n = 9). *p < 0.05, **p < 0.01, ***p < 0.001.

Fig. 3

Effects of NBQX on ventricular electrical remodeling. (A) Typical record of APD50/90 at PCL = 100 ms. (B) Representative images of the effective refractory period (ERP) in different groups. (C) Typical record of ventricular fibrillation (VF) in each group. (D, E) Incidence rate and duration of VF in the AD and ADN groups. (F, G) Statistical analysis of APDs at PCL = 100 ms. (H) Statistical analysis of ERP in the four groups. n = 9 per group. *p < 0.05, **p < 0.01, ***p < 0.001.

Fig. 4

NBQX enhanced the expression of CX43 and ion channel proteins. (A) Representative immunoblotting images of protein CX43, KV4.2, KV4.3, AMPAR. (B–E) Immunoblotting quantitative analysis of KV4.2, KV4.3, CX43, and AMPAR expression（n = 3 per group）. (F, G) Typical images of CX43 in immunohistochemical staining and quantitative analysis of the CX43 area; scale bar = 50 μm (n = 5 in each group). (H, I) Immunofluorescence staining and quantitative analysis of CX43 expression; scale bar = 50 μm (n = 5 per group)., **p < 0.01, ***p < 0.001.

Fig. 5

Effects of NBQX on ameliorating ventricular fibrosis. (A) Representative images of tissues stained with Masson's trichrome stain (n = 5 in each group); scale bar = 50 μm. (B) Representative images of Sirius red staining (n = 5 in each group); scale bar = 50 μm. (C) Percentage of fibrotic area. (D–F) Typical images of immunoblotting and quantitative analyses of collagen Ⅰ and collagen Ⅲ (n = 3 per group). *p < 0.05, **p < 0.01, ***p < 0.001. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Fig. 6

NBQX reduced oxidative stress levels and apoptosis in vivo and in vitro. (A) Representative images of reactive oxygen species (ROS) in left ventricle (LV) tissues. n = 5 per group. (B) Relative fluorescence intensity of ROS in LV. n = 5 per group, bar = 50 μm. (C, D) Concentration of malondialdehyde (MDA) and superoxide dismutase (SOD) activity in the serum. n = 12 per group. (E) Representative images of ROS in H9C2 cells. n = 5 per group; scale bar = 50 μm) (F) Relative fluorescence intensity of ROS in H9C2 cells. n = 5 per group. (G, H) Concentration of malondialdehyde (MDA) and superoxide dismutase (SOD) activity in H9C2 cell. n = 5 per group. (I, J) Typical images of TUNEL immunohistochemical staining and quantitative analysis of the positive area. n = 5; scale bar = 50 μm. (K–N) Immunoblotting and quantitative analysis of BAX, Bcl-2 and Capase-3 in LV. n = 3. (O) Representative images of electron microscopy: In the myocardium of AD rats, mitochondrial was swollen and dissolved, which was reversed following NBQX treatment. n = 5; scale bar = 500 nm *p < 0.05, **p < 0.01, ***p < 0.001.

Fig. 7

NBQX downregulated oxidative stress by regulating the Nrf2/HO-1 pathway. (A–D) Immunoblotting and quantitative analyses of Nrf2, HO-1, and NOX4 in LV, n = 3 per group. (E–F) Representative immunofluorescence staining images and positive rate of Nrf2 in LV. n = 5, bar = 50 μm. (G–I) Immunoblotting and quantitative analysis of Nrf2 and HO-1, n = 3. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Fig. 8

Summary of the effects of NBQX on reducing ventricular fibrillation susceptibility in rat models of anxiety and its underlying mechanisms.