Heat treatment attenuates activities of ET‐1 signaling pathway and enhances the protective HSP72‐SOD2 in ischemic skeletal muscle of rats with peripheral artery disease

Abstract

Background The arterial blood pressure (BP) response is augmented during walking in patients with peripheral artery disease (PAD) and in simulated PAD animal models during static exercise. The exercise pressor response (EPR) is a contributor to the exaggerated BP response and heating treatment is effective to attenuate exaggeration of the EPR. However, the underlying mechanisms leading to the inhibitory effects of heat treatment on the EPR, especially on the essential alteration originating within the ischemic skeletal muscles of PAD, is poorly understood. Aims We targeted if the repeated heat treatment protocol (~1.5oC muscle temperature evaluation, 30 minutes per time, twice per day in 72 hours after the femoral artery occlusion) alters some of the fundamental molecular mediators involved in ischemic insult and oxidative stress in the skeletal muscles of the hindlimb. Based on the existing literature, we determined the levels of endothelin-1 (ET-1), and the protein expression of heat shock protein 72 (HSP72) and superoxide dismutase 2 (SOD2) in the skeletal muscles of control rats and PAD rats with 72 hours of femoral artery occlusion. Methods The red and white components of the gastrocnemius muscles were obtained in groups of control, PAD and PAD + heat rats. ET-1 was measured by the method of colorimetric immunometric enzyme immunoassay. The expression of HSP 72 and SOD2 was measured by western blotting. To elevate the muscle temperature, a heating pad was placed locally on the hindlimb and the Tm was increased by ~1.5 °C. Then the Tm was stabilized and maintained for 30 min. One way ANOVA with LSD post-hoc test was applied to determine the difference among groups. Results Compared with control rats, in PAD rats a significant increase in ET-1 was observed in both of the red muscle (0.91±0.28 pg/ml/µg total protein in PAD/n=5 vs. 0.52±0.05 pg/ml/µg total protein in control/n=4; P<0.05); and the white muscle (0.71±0.14 pg/ml/µg total protein in PAD rats/n=5 vs. 0.47±0.09 pg/ml/µg total protein in control rats/n=4; P<0.05). Notably, following heat treatment the amplification of ET-1 in the skeletal muscle of PAD rats was significantly attenuated (red muscle: 0.53±0.08 pg/ml/µg total protein in PAD + heat rats n=5 vs. 0.91±0.28 pg/ml/µg total protein in PAD rats/n=5; P<0.05; white muscle: 0.52±0.10 pg/ml/µg total protein in PAD + heat rats n=5 vs. 0.71±0.14 pg/ml/µg total protein in PAD rats/n=5; P<0.05). Note that no significant difference in the ET-1 levels was seen in skeletal muscle of control rats and PAD + heat rats (P>0.05). In addition, heat treatment increased expression of HSP72 and SOD2 in the red muscle of PAD rats (HSP72: 1.22±0.39 in PAD + heat rats/n=4 vs. 0.68±0.27 in PAD rats/n=4, P<0.05; SOD2: 1.42±0.45 in PAD + heat rats/n=5 vs. 0.92±0.12 in PAD rats/n=4, P=0.062). Conclusion Heat treatment attenuates the activities of ET-1 signaling pathway and enhances the levels of HSP 72 and SOD2, potential protective mediators, in the skeletal muscle of PAD rats under ischemic situations.