Fig 1.
Alteration of Muscle Fiber Cross-Sectional Areas: Pre-Hibernation vs. Post-Hibernation.
Fiber cross-sectional areas (CSAs) were significantly decreased following hibernation. The sartorius muscle was collected from bear legs both at pre-hibernation (late November) and post-hibernation (early April). Due to the limitation of animal availability, different individual bears were used at each time point (N = 3 in each group). (A) Typical images of cross sections with Hematoxylin-Eosin staining and immunohistochemistry (dystrophin localization) are shown. The scale bar shows 100 μm. (B) The mean size of the fibers was obtained for each individual sample followed by the calculation of group data. Data are expressed as mean ± standard error. Significant differences: #, p < 0.05.
Fig 2.
Gene expression of protein breakdown pathways.
Gene expression levels of the ubiquitin-proteasome system (atrogin1 and murf1) and autophagy-lysosome system (atg7, beclin1, and map1lc3) were quantified by real-time PCR. RPL26 was used as an internal control for the 2–ΔΔCT method. N = 3 in each group. Data are expressed as mean ± standard error. Significant differences: #, p < 0.05.
Fig 3.
Enhanced mTORC1 signaling and suppression of myostatin expression following hibernation.
(A) Representative images of Western blotting for phosphorylated (Thr389 and Thr421/Ser424) and total expression of S6K1. (B) Phosphorylation status of S6K1 at Thr421/Ser424 sites were quantified. (C) Gene expression levels of myostatin were determined by real-time PCR. RPL26 was used as an internal control for the 2–ΔΔCT method. N = 3 in each group. Data are expressed as mean ± standard error. Significant differences: #, p < 0.05.
Fig 4.
Expression of mitochondria-related genes following hibernation.
(A) Typical images of cross sections with NADH-tetrazolium reductase (NADH-TR) staining are shown. The scale bar shows 200 μm. (B) Expression levels of mitochondria-related gene transcripts (pgc1a, pgc1b, ucp3, cycs, cox4, and cpt1b) were quantified by real-time RT-PCR. RPL26 was used as an internal control for the 2–ΔΔCT method. N = 3 in each group. Data are expressed as mean ± standard error. Significant differences: #, p < 0.05.
Fig 5.
Hypothetical schema of skeletal muscle adaptation in hibernating bear.
Protein anabolism in skeletal muscle of hibernating bears is potentially activated by mTORC1 activation and down regulation of myostatin expression for counteracting to the corresponding enhancement of protein degradation, which can then lead to a net balance of protein metabolism and prevention of excessive muscle loss. Muscle phenotypes shifting toward slow-oxidative fiber and mitochondrial biogenesis are also induced and likely contribute to the more efficient utilization of energy resources for muscle contraction during winter survival.