Muscle afferent ASIC3 upregulation mediates the exacerbated exercise pressor reflex in male rats following hindlimb ischemia–reperfusion

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  FIGURE 1. Blockage of ASIC3 during evoking exercise pressor reflex (EPR) in the sham and IR rats. The EPR was evoked by a protocol of ~30s static muscle contraction, with a transductor continuously monitoring the muscle tension during the contraction. Mean arterial pressure (MAP) and heart rate (HR) were recorded. During each experiment, three sessions were designed: (1) Baseline control: Static muscle contraction was performed without arterial injection of ASIC3 antagonist, APETx2; (2) Blockage: Static muscle contraction was performed following the arterial injection of APETx2 (100 μg/kg); and (3). Recovery: Static muscle contraction was performed after the APETx2 was washed out. The interval among sessions was ~30 min. The white bars represent the sham group, and the gray bars represent the IR group. Two‐way [group (sham vs. IR) × time (baseline vs. blockage vs. recovery)] ANOVA was used to test the differences among groups. Upper panel: MAP responses to static muscle contraction in sham and IR rats. Compared with the sham group, the MAP response in IR rats was amplified during the baseline control (p = 0.031). In IR rats, the blockage of ASIC3 with APETx2 significantly reduced the MAP…

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  FIGURE 2. MAP response to lactic acid (LA) injection in the sham and IR rats. Different doses of LA (1, 2, and 4 μmol/kg body weight) were arterial injected into the hindlimb to activate ASIC3 in the muscle afferent terminals of the decerebrated rats. A total of 12 limbs from 6 Sham rats and 16 limbs from 8 IR rats were tested. Mean arterial pressure (MAP) and heart rate (HR) were recorded. Before and after the injection of LA, vehicle solution, 0.9% saline was injected as baseline and recovery control. As indicated in the figure, the white color bar indicates saline control during the baseline and recovery session. The light blue color bar indicates 1 μmol/kg body weight LA injection, the dark blue color indicates 2 μmol/kg body weight LA injection, and the purple color indicates 4 mM LA injection. Two‐way [group (sham vs. IR) × time (baseline vs. LA vs. recovery)] ANOVA was used to test the differences among groups. Compared with the sham, the MAP response to LA was higher in IR rats, regardless of the LA concentration. Except for 4 μmol/kg LA injection, no significant difference was found between sham and IR regarding the HR responses to LA injection. Data were presented as mean ±…

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  FIGURE 3. Blockage of ASIC3 during lactic acid (LA) injection in the sham and IR rats During each experiment, three sessions were designed: (1) Baseline control: LA (4 μmol/kg body weight) was injected without arterial injection of APETx2 (100 μg/kg); (2) Blockage: LA was injected following the arterial injection of APETx2 (100 μg/kg); and (3) Recovery: LA was injected after the APETx2 was washed out. Upper panel: MAP responses to LA injection in sham and IR rats. Two‐way [group (sham vs. IR) × time (baseline vs. blockage vs. recovery)] ANOVA was used to test the differences among groups. Compared with the sham group, the MAP response in IR rats was amplified during the baseline control (p < 0.05). In IR rats, the blockage of ASIC3 with APETx2 significantly reduced the MAP responses (baseline vs. blockage, p < 0.01). Lower panel: HR responses to LA injection in sham and IR rats. No significant difference was found among groups (all p > 0.05). Data are presented as mean ± standard deviation (SD). The round dots in the figure bar indicate individual data points in the respective group. The sample size was indicated in the bracket below the group name. Minor variations in sample size between…

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  FIGURE 4. Delineating the role of ASIC3 in L4‐6 DRG of sham and IR rats by ex vivo and in vitro studies. (a) Western blot detected the protein expression of ASIC3 in the protein extraction of L4‐6 DRG. The optical density was normalized by the β‐actin protein expression in the respective samples. Western blot quantification of ASIC3 expression in L4–L6 DRGs (Sham: n = 8 DRGs from 4 rats; IR: N = 10 DRGs from 5 rats). An independent t‐test was applied to analyze the difference between the sham and IR groups. The dark bar indicates the sham group, and the gray bar indicates the IR group. Compared with sham (n = 8), the ASIC3 protein expression was higher in L4‐6 DRG of IR rats (n = 10) (p = 0.012). The molecular band indicates that ASIC3 was detected near the band of 60 kDa (molecular weight ~67 kDa), and β‐actin was detected near the band of 40 kDa (molecular weight ~42 kDa). The numbers in the data bar indicate the sample size of each group. (b) Co‐localization of the ASIC3 and peripherin (marker of C‐fiber) in rat L4‐6 DRG. Upper panel: ASIC3 (Green) and co‐expressed with peripherin (red). The white arrows in the merged pictures indicate the location of the neurons co‐expressed with ASIC3…