Exaggerated Activities of P2X3 and ASIC3 Signaling Pathway in Muscle Afferent Following Himblimb Muscle Ischemia‐Reperfusion

Abstract

BackgroundPeripheral artery disease (PAD) is one of the major cardiovascular concerns and dramatically reduces the blood flow supply in the affected limbs. A combination of revascularization surgery in the femoral artery and supervised exercise intervention is among the most general approaches for the treatment of PAD. The approach of revascularization inevitably induces ischemia‐reperfusion (IR) injury in the tissues with the reperfusion of blood flow. Notably, following muscle IR in rats, we have observed an exaggerated exercise pressor reflex (EPR) indicating a risk of cardiovascular diseases, as the increased blood pressure (BP) response during exercise. In specific, a time‐course study (18, 66 and 114 hours post‐IR) showed that an increment in BP response was most profound in 18 hours post‐IR (IR 18h) among all the time points. However the underlying molecular mechanism leading to the IR‐induced exaggerated EPR has yet poorly understood.AimsWe targeted if the hindlimb muscle IR alters the activities of two key signaling pathway in muscle afferent nerves in regulating the EPR, namely P2X3 and ASIC3.MethodsWestern blot technique was used to determine the protein expression of P2X3 and ASIC3 in L4‐6 dorsal root ganglion (DRG) of sham rats and IR 18h rats. Meanwhile, the mean arterial pressure (MAP) responses to α‐β‐me‐ATP and lactic acid intra‐arterially injected into the hindlimb were examined to assess the P2X3 and ASIC3‐mediated EPR, respectively. Whole‐cell patch clamp was applied to evaluate the P2X and ASIC3 currents in the isolated muscle DRG neurons in both sham and IR 18h rats. All data were presented as mean ± standard deviation.ResultsCompared with sham rats, there were significant increases in both P2X3 and ASIC3 protein expression in L4‐6 DRGs in IR 18h rats (P2X3: 1.56±0.10 in IR 18h rats/n=4 vs. 1.00±0.10 in sham rats/n=6; P<0.05; and ASIC3: 1.42±0.47 in IR 18h rats/n=8 vs. 1.00±0.07 in sham rats/n=10; P<0.05). In addition, compared with sham rats, the MAP responses following the injection of α‐β‐me‐ATP (0.125 mM) and lactic acid (2 and 4 µmol/kg) were significantly amplified in IR 18h rats (α‐β‐me‐ATP: 29±8 mmHg in IR 18h rats/n=5 vs. 20±5 mmHg in sham rats/n=2; P<0.05; Lactic acid: 2 µmol/kg, 28±8 mmHg in IR 18h rats/n=15 vs. 21±7 mmH in sham rats/n=8, P<0.05; 4 µmol/kg, 37±10 mmHg in IR 18h rats/n=15 vs. 25±6 mmH in sham rats/n=9, P<0.01). In the isolated DRG neurons, amplitude of both P2X and ASIC3 currents was significantly higher in IR 18h rats than that in sham rats. i.e., P2X transient currents: 1281.64±776.74 pA in IR 18h rats/n=25, vs. 717.91±556.2 pA in sham rats/n=9, (P<0.05); and sustain currents: 466.94±168.68 pA in IR 18h rats/n=11, vs. 271.49±200.90 pA in sham rats/n=8 (P<0.05).ConclusionThe activities of the P2X3 and ASIC3 signaling pathways in the muscle afferents are exaggerated following muscle IR. Results of this study shed light upon the future potential intervention strategy to minimize the IR‐induced exaggerated EPR response in the post‐revascularization PAD patients.