Paraventricular Nucleus P2X7 Receptors Aggravate Acute Myocardial Infarction Injury via ROS-Induced Vasopressin-V1b Activation in Rats

Abstract

The present study was designed to investigate the mechanisms by which P2X7 receptors (P2X7Rs) mediate the activation of vasopressinergic neurons thereby increasing sympathetic hyperactivity in the paraventricular nucleus (PVN) of the hypothalamus of rats with acute myocardial ischemia (AMI). The left anterior descending branch of the coronary artery was ligated to induce AMI in rats. The rats were pretreated with BBG (brilliant blue G, a P2X7R antagonist), nelivaptan (a vasopressin V1b receptor antagonist), or diphenyleneiodonium (DPI) [an nicotinamide adenine dinucleotide phosphate (NADPH) oxidase inhibitor]. Hemodynamic parameters of the heart were monitored. Myocardial injury and cardiomyocyte apoptosis were assessed. In the PVN of AMI rats, P2X7R mediated microglial activation, while reactive oxygen species (ROS) and NADPH oxidase 2 (NOX2) were higher than in the sham group. Intraperitoneal injection of BBG effectively reduced ROS production and vasopressin expression in the PVN of AMI rats. Moreover, both BBG and DPI pretreatment effectively reduced sympathetic hyperactivity and ameliorated AMI injury, as represented by reduced inflammation and apoptosis of cardiomyocytes. Furthermore, microinjection of nelivaptan into the PVN improved cardiac function and reduced the norepinephrine (AE) levels in AMI rats. Collectively, the results suggest that, within the PVN of AMI rats, P2X7R upregulation mediates microglial activation and the overproduction of ROS, which in turn activates vasopressinergic neuron-V1b receptors and sympathetic hyperactivity, hence aggravating myocardial injury in the AMI setting.

Keywords: Myocardial ischemia; P2X7 receptor; PVN; Reactive oxygen species; Vasopressin; c-fos.

Fig. 1

P2X7R expression and microglial activation in the PVN of AMI rats. A Representative image of immunohistochemistry of Iba-1-ir cells (brown) (scale bars, left 200 μm; right 50 μm). B Fluorescence images of microglia immunostained for anti-OX42 (cd11b/c) in the PVN from Sham, AMI, and BBG + AMI rats (scale bars, 50 μm). C Numbers of Iba-1-ir cells in the PVN. D Mean densitometry of OX42 (cd11b/c) immuno-positivity. E Representative immunoblot bands of P2X7R, pro-IL-1β, and IL-1β in the PVN in the different groups. F–H Optical density analysis of P2X7R (F), pro-IL-1β (G) and IL-1β (H) immunoblotting bands. I Double immunofluorescence showing co-localization of P2X7R and Iba-1 analyzed by confocal fluorescence microscopy (scale bars, 20 μm). J Quantitative analysis of fluorescence intensity of P2X7R and Iba-1 staining in microglia from the groups. K Levels of co-localization of P2X7R and Iba-1 assessed using the Pearson coefficient. Data are expressed as the mean ± SEM. **P < 0.01 versus Sham group, ^#P < 0.05 versus AMI group.

Fig. 2

Effects of the P2X7R antagonist BBG on NOX2 expression and ROS production in the PVN of AMI rats. A Representative images of western blots showing protein expression of NOX2. B Histogram of protein levels relative to GAPDH levels. C Representative images showing the enhanced DHE fluorescence in the PVN of AMI compared with sham rats and those with other treatments (scale bars, 20 μm). D Mean NADPH-dependent O²⁻ production in the PVN of different groups. Data are presented as the mean ± SEM. **P < 0.01, *P < 0.05 versus Sham group, ^##P < 0.01, ^#P < 0.05 versus AMI group.

Fig. 3

Pretreatment with BBG or an NADPH inhibitor (DPI) reduces VPergic cell activation. A Confocal microscopy images of double immunofluorescence showing co-localization of VP and NeuN (scale bars, 25 μm). B Confocal microscopy images of double immunofluorescence showing co-localization of c-Fos (red) and VP (green); nuclei stained with DAPI (blue) (scale bars, 20 μm). C Statistics of mean fluorescence intensity of c-Fos-ir and VP-ir in the PVN among groups. D Levels of co-localization of c-Fos and VP assessed using Pearson coefficients. Data are presented as the mean ± SEM. **P < 0.01, *P < 0.05 versus Sham group; ^##P < 0.01, ^#P < 0.05 versus AMI group. Ir, immunopositivity.

Fig. 4

Cardiac function and heart histopathology. A Typical cMRI images from a cine study of the rat heart. Short-axis images spanning the base to apex are depicted in 5 slices, showing the radiological features in different groups of rats. Arrows indicate enhancement of the subendocardial late gadolinium. B, C LIVP recording and analysis before and after LAD ligation in rats. D Analysis of ±dp/dtmax. E HE staining of heart tissue showing histopathological changes post-AMI [scale bars, 200 μm (insert), 50 μm (below)]. F, G Representative images and analysis of apoptotic cardiomyocytes in myocardial sections using the TUNEL method (scale bars, 10 μm in F). H Expression of caspase-3, caspase-9, and HSP27 assessed by RT-PCR. I Agarose gel electrophoresis of the PCR products for caspase-3, caspase-9, HSP27, and GAPDH from individual samples (left lane: 500-bp molecular weight marker). Data are expressed as the mean ± SEM. **P < 0.01, *P < 0.05 versys Sham group; ^##P < 0.01, ^#P < 0.05 versus AMI group.

Fig. 5

Protective effects of pretreatment with BBG on HR and MAP of AMI rats. A HR and blood pressure (BP) real-time recordings. B, C Statistics showing the protective effects of BBG on HR (B) and MAP (C) in AMI rats. Data are expressed as the mean ± SEM. **P < 0.01 versus Sham group, ^##P < 0.01 versus AMI group.

Fig. 6

Protective effects of BBG and DPI on myocardial injury via increased sympathetic activity. A Serum levels of NE as measured by ELISA. B HE staining showing histopathological changes in heart tissue after AMI [(scale bars, 200 μm (insets), 50 μm (below)]. C Gene expression of caspase-3, caspase-9, and HSP27 mRNA by RT-PCR analysis. D Agarose gel electrophoresis of PCR amplicons for caspase-3, caspase-9, HSP27, and GAPDH from individual samples. Data are expressed as the mean ± SEM. **P < 0.01, *P < 0.05 versus Sham group; ^##P < 0.01, ^#P < 0.05 versus AMI group.

Fig. 7

Inhibition of V1b receptors attenuates the hemodynamic dysfunction and reduces the NE level in AMI rats. A Analysis of change in LVEF in both groups. B, C Analysis of hemodynamic parameters LVEDP and dP/dtmax. D NE concentration in serum. Data are expressed as the mean ± SEM. **P < 0.01, *P < 0.05 versus Sham group, ^##P < 0.01, ^#P < 0.05 versus AMI group.

Fig. 8

Schematic displaying that PVN P2X7R activation aggravates acute myocardial injury via ROS-induced vasopressin-V1b activation in AMI rats.