The effects of heat stress on morphological properties and intracellular signaling of denervated and intact soleus muscles in rats

Abstract

The effects of heat stress on the morphological properties and intracellular signaling of innervated and denervated soleus muscles were investigated. Heat stress was applied to rats by immersing their hindlimbs in a warm water bath (42°C, 30 min/day, every other day following unilateral denervation) under anesthesia. During 14 days of experimental period, heat stress for a total of seven times promoted growth-related hypertrophy in sham-operated muscles and attenuated atrophy in denervated muscles. In denervated muscles, the transcription of ubiquitin ligase, atrogin-1/muscle atrophy F-box (Atrogin-1), and muscle RING-finger protein-1 (MuRF-1), genes was upregulated and ubiquitination of proteins was also increased. Intermittent heat stress inhibited the upregulation of Atrogin-1, but not MuRF-1 transcription. And the denervation-caused reduction in phosphorylated protein kinase B (Akt), 70-kDa heat-shock protein (HSP70), and peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α), which are negative regulators of Atrogin-1 and MuRF-1 transcription, was mitigated. In sham-operated muscles, repeated application of heat stress did not affect Atrogin-1 and MuRF-1 transcription, but increased the level of phosphorylated Akt and HSP70, but not PGC-1α Furthermore, the phosphorylation of Akt and ribosomal protein S6, which is known to stimulate protein synthesis, was increased immediately after a single heat stress particularly in the sham-operated muscles. The effect of a heat stress was suppressed in denervated muscles. These results indicated that the beneficial effects of heat stress on the morphological properties of muscles were brought regardless of innervation. However, the responses of intracellular signaling to heat stress were distinct between the innervated and denervated muscles.

Keywords: Atrophy; heat stress; hypertrophy; neural innervation; skeletal muscle.

Figure 1

Time‐dependent changes in intramuscular temperature in soleus during heat stress under anesthesia (Heat) or anesthesia only (Cont). Hindlimbs of rats in the Heat group were immersed in a warm water bath (42°C) for 30 min during heat stress, while Cont group rats received anesthesia only. Means ± SD (n = 5 per group).

Figure 2

Effects of intermittent heat stress and/or 14 days of denervation on the percent changes in muscle wet weight/body weight (BW) versus the preexperimental control (Pre‐exp, A) and water content (B) in the sham‐operated and denervated soleus muscles. Cont: nonheated and Heat: heat stress applied groups. Mean ± SD (n = 8 per group). H and D: main effect of heat stress and denervation, respectively.

Figure 3

Effects of intermittent heat stress and/or 14 days of denervation on the morphological properties of soleus muscle fibers. Mean (± SD, n = 8 per group) fiber cross‐sectional areas (CSAs) are shown in A. Representative images of cross‐sections in sham‐operated and denervated muscles are shown in B. Muscle sections were stained for laminin to analyze fiber CSAs. The frequency distribution of fibers with different CSAs in sham‐operated (C) and denervated (D) muscles are also shown. At least 600 fibers in each muscle were analyzed. H and D: main effect of heat stress and denervation, respectively. See Figure 2 for other abbreviations.

Figure 4

Effects of intermittent heat stress and/or 14 days of denervation on the protein expression levels in sham‐operated and denervated soleus muscles. The phosphorylated (p) and total (t) levels of protein kinase B (Akt), ribosomal protein S6 (S6), and AMP‐activated protein kinase α (AMPK α); the expression level of 70‐kDa heat‐shock protein (HSP70), peroxisome proliferator‐activated receptor‐γ coactivator‐1α (PGC‐1α), ubiquitinated proteins, and glyceraldehyde 3‐phosphate dehydrogenase (GAPDH) were evaluated by immune blotting. Representative results of immunoblotting are shown in A and N. The expression level of each protein was calculated after normalization using the expression levels of GAPDH (B, C, E, F, H, I, K, L). The phosphorylation rate of the protein was calculated by dividing the expression level of phosphorylated protein by the expression level of total protein (D, G, M). The relative values pertaining to the sham‐operated Cont muscles are also shown. Means ± SD (n = 8 per group). H and D: main effect of heat stress and denervation, respectively; H×D: interaction of effects of heat stress and denervation; *: the result of Tukey's multiple comparisons test was P < 0.05. See Figure 2 for other abbreviations.

Figure 5

Effects of intermittent heat stress and/or 14 days of denervation on the transcription of atrogin‐1/muscle atrophy F‐box (Atrogin‐1, A) and muscle RING‐finger protein‐1 (MuRF‐1, B) genes in soleus muscles were evaluated by real‐time polymerase chain reaction (RT‐PCR). The expression levels of Atrogin‐1 and MuRF‐1 mRNA were obtained by calculation after normalization using the expression level of TATA box‐binding protein (Tbp). The relative expression levels pertaining to the sham‐operated Cont muscles are also shown. Mean ± SD (n = 8 per group). D: main effect of denervation, respectively; H×D: interaction of effects of heat stress and denervation; *: the result of Tukey's multiple comparisons test was P < 0.05. See Figure 2 for other abbreviations.

Figure 6

Effects of a single heat stress and/or 1 day of denervation on the protein expression levels in sham‐operated and denervated soleus muscles. The phosphorylated (p) and total (t) levels of Akt, Akt1, Akt2, and S6; the expression level of HSP70 and GAPDH was evaluated by immune blotting. Representative results of immunoblotting are shown in A. The expression level of each protein was calculated after normalization using the expression levels of GAPDH (B, C, E, F, H, I, K, L, N). The phosphorylation rate of the protein was calculated by dividing the expression level of phosphorylated protein by the expression level of total protein (D, G, J, M). The relative values pertaining to the sham‐operated Cont muscles are also shown. Means ± SD (n = 5 per group). H and D: main effect of heat stress and denervation, respectively; H×D: interaction of effects of heat stress and denervation; *: the result of Tukey's multiple comparisons test was P < 0.05. See Figures 2 and 4 for other abbreviations.

Figure 7

Effects of a single heat stress and/or 14 days of denervation on the protein expression levels in sham‐operated and denervated soleus muscles. The phosphorylated (p) and total (t) levels of Akt, Akt1, Akt2, and S6; the expression levels of HSP70 and GAPDH were evaluated by immune blotting. Representative results of immunoblotting are shown in A. The expression level of each protein was calculated after normalization using the expression levels of GAPDH (B, C, E, F, H, I, K, L, N). The phosphorylation rate of the protein was calculated by dividing the expression level of phosphorylated protein by the expression level of total protein (D, G, J, M). The relative values pertaining to the sham‐operated Cont muscles are also shown. Means ± SD (n = 5 per group). H and D: main effect of heat stress and denervation, respectively; H×D: interaction of effects of heat stress and denervation; *: the result of Tukey's multiple comparisons test was P < 0.05. See Figure 2 and 4 for other abbreviations.