P2X7 receptor regulates sympathoexcitatory response in myocardial infarction rats via NF-κB and MAPK pathways

Abstract

Previous studies have provided evidence for the regulatory effect of P2X7 receptor (P2X7R) on cardiovascular activities. Our study focused on exploring the function and fundamental mechanism of microglial P2X7R in controlling sympathoexcitatory response using rats with acute myocardial infarction (AMI). Coronary artery ligation was used in rats to cause AMI. And before that, rats were administrated with P2X7R siRNA that targeted P2X7R mRNA into paraventricular nucleus (PVN) or BBG (Brilliant Blue G, a P2X7 receptor antagonist). Increased expression levels of P2X7R and adenosine triphosphate (ATP) were observed in the hypothalamic PVN of AMI rats. Moreover, the knockdown of P2X7R expression by P2X7-siRNA or suppression of P2X7 receptor by BBG attenuated the elevation of both vasopressin and oxytocin levels in the PVNs of AMI rats. There was also a decrease in renal sympathetic nerve activity (RSNA) by P2X7-siRNA and BBG. Besides, inflammation was alleviated by P2X7-siRNA and BBG through suppressing pro-inflammatory cytokines IL-1β and IL-6 in PVN of AMI rats. Furthermore, blockade of P2X7R moderated the process of cardiac remodeling. This was achieved due to the regulatory effect of P2X7R on sympathoexcitatory response by influencing NF-κB and mitogen-activated protein kinase (MAPK) signaling. These findings suggest that P2X7R can act as a new regulator of sympathoexcitatory response via NF-κB and MAPK signaling pathways in AMI rats.

Keywords: AMI; MAPK; NF-κB; P2X7 receptor; PVN; microglia.

Figure 1

Changes of P2X7R expression and ATP levels in the PVN of different ischemia time groups. Rats underwent coronary artery ligation to induce AMI for 5, 10, 15 days. (A) P2X7R protein levels at indicated time point after AMI were measured by Western blot analysis. (B) P2X7R signal intensity in (A) was quantified by IMAGEJ software. (C) P2X7R mRNA levels at indicated time point after AMI were measured by qRT-PCR. (D) Brain tissue sections were processed and performed immunohistochemical analysis for staining P2X7R and representative staining sections at day 10 were shown. Scale bars are 100 μm. (E) ATP levels at indicated time point after AMI were determined using the ATP bioluminescence assay kit. Data are presented as mean ± SD (6 rats per group). **, P<0.01 compared with the control (Sham) group.

Figure 2

Expression of oxytocin and vasopressin immunoreactive neurons, RSNA responses to P2X7R-siRNA and BBG. Before myocardial ischemia, rats were administrated with P2X7R siRNA that targeted P2X7R mRNA into paraventricular nucleus (PVN) or BBG (Brilliant Blue G, a P2X7 receptor antagonist). Representative oxytocin staining (A) and the number of oxytocin immunoreactive neurons (B) in AMI + siRNA, AMI + BBG and AMI groups. Representative vasopressin staining (C) and the number of vasopressin immunoreactive neurons (D) in AMI + siRNA, AMI + BBG and AMI groups. Scale bar, 100 μm. (E) Mean changes of RSNA in AMI + siRNA, AMI + BBG and AMI groups. Data are presented as mean ± SD (6 rats per group). **, P<0.01 compared with the control group.

Figure 3

Withdrawal of P2X7R inhibited inflammation against AMI. Rats were administrated with P2X7R siRNA that targeted P2X7R mRNA into paraventricular nucleus (PVN) and undergoing coronary artery ligation to induce AMI. Proinflammatory cytokines (IL-1β and IL-6) expression levels in the PVN of AMI control group or P2X7R-siRNA AMI group were determined by Western blot (A) and qRT-PCR (B) analysis. All values are shown as mean ± SD (n=6). **, P<0.01 in comparison with the control group.

Figure 4

Effect of P2X7R deletion on NF-κB pathway. Rats were administrated with P2X7R siRNA that targeted P2X7R mRNA into paraventricular nucleus (PVN) and undergoing coronary artery ligation to induce AMI. Representative western blots (A) and statistical analysis (B) of p65 and IκBa in the PVN of sham, AMI or P2X7R-siRNA AMI rats (6 rats per group). Data are presented as mean ± SD. **, P<0.01 compared with the control group. (C) Brain tissue sections were processed and performed immunohistochemical analysis for staining p-IκBα and p-p65, and representative staining sections were shown. Scale bars are 100 μm.

Figure 5

P2X7R gene knockout stimulated cardiac remodeling post-AMI. Rats were administrated with P2X7R siRNA that targeted P2X7R mRNA into paraventricular nucleus (PVN) and undergoing coronary artery ligation to induce AMI. A: Ratios of HW/BW, LW/BW, and HW/TL in AMI control group and P2X7R-siRNA AMI group. B: Hematoxylin and eosin staining (H&E) was used in the hearts of both AMI and P2X7R-siRNA AMI rats. C: Statistical analysis of the cross-sectional area (CSA) of cardiomyocytes with the heart tissues of AMI and P2X7R-siRNA AMI rats. D: Hypertrophy mRNAs levels in heart slices of both AMI and P2X7R-siRNA AMI rats group. E: Fibrosis mRNAs in the heart tissues of AMI and P2X7R-siRNA AMI group. All values are shown as mean ± SD (n=6). **, P<0.01 in comparison with the control group.

Figure 6

P2X7R decrease may stimulate cardiac remodeling through MAPK pathway regulation. Rats were administrated with P2X7R siRNA that targeted P2X7R mRNA into paraventricular nucleus (PVN) and undergoing coronary artery ligation to induce AMI. Western-blot assay (A) and statistical analysis (B) of JNK1/2, p38 and ERK1/2 in the PVN of AMI and P2X7R-siRNA AMI rats. All values are shown as mean ± SD (n=6). **, P<0.01 in comparison with the control group. (C) Brain tissue sections were processed and performed immunohistochemical analysis for staining p-ERK1/2, p-JNK1/2 and p-p38, and representative staining sections were shown. Scale bars are 100 μm.