Researchers collected the SOL (soleus muscle) muscle samples from 60-d and 112-d hibernation group, both were those from the ground squirrels that had already spent 2 months or more than 3 months of hibernation inactivity, which was long enough to cause significant atrophy of SOL muscle in non-hibernators. The SOL muscle wet weight decreased less than the body weight in hibernating ground squirrels, which suggested a protective effect that reduces or avoids muscle atrophy during hibernation in Daurian ground squirrels.
The cytoplasm of a myofiber contains a regular array of contractile units (sarcomeres) comprised of actin-containing thin filaments and myosin-containing thick filaments that, along with additional structural and regulatory proteins, are arranged longitudinally as myofibrils. With iTRAQ approach, they identified myosin-2 significantly up-regulated while myosin-3 significantly down-regulated in the 60-d hibernation group compared to the pre-hibernation ground squirrels. Meanwhile, myosin-13 significantly down-regulated in the 112-d hibernation group compared to the pre-hibernation group. Moreover, actin exhibited significantly down-regulated expression in the 112-d hibernation group compared to the pre-hibernation group. The assembly and maintenance of myofibrils is regulated by multiple proteins that interact with the primary sarcomeric proteins actin and myosin. Myosins constitute a large superfamily of proteins that share a common domain which has been shown to interact with actin, hydrolyze ATP and produce movement. Phylogenetic analysis currently places myosins into 15 classes. The conventional myosin such as myosin-2 forms filaments in muscle, however, little is known about the structure, enzymatic properties, intracellular localization and physiology of most unconventional myosin classes including myosin-3 and myosin-13. Seo Y et al. have reported that the disproportional changes in http://www.cusabio.com/ components result in tissue having different levels of actin versus myosin, thus leading to shifts in contractile capacity. Also the actin to myosin ratio is one of the muscular atrophy hallmarks. After 60-d hibernation inactivity, myosin-2 showed a significantly increase while myosin-3 decreased significantly, but actin showed no obvious change, which suggested that the ratio of actin/myosin filaments was likely to remain steady. However, actin and myosin-13 were both decreased significantly in 112-d hibernation group. Because it was not determined whether the degree of their decline was different, it was difficult to determine whether the balance of actin/myosin was destroyed. But our results showed a significant loss of myofibrillar proteins in 112-d hibernation group, which is consistent with the results of our research group that the CSA of SOL muscle fiber significantly decreased after 112-d hibernation (unpublished). Inconsistent with our results, the level of one isoform of actin was significantly higher (P < 0.05) in hibernation group than in summer active group in pectoral or biceps brachii muscles of the bat Murina leucogaster. This might be due to that the pectoral or biceps brachii muscles are involved in bat flight. Although torpid bats showed decreased cytoskeleton plasticity, they selectively upregulated vesicle-associated proteins to retain their basic environmental sensibility.
Obviously, different hibernating animals overcome muscle atrophy by different strategies. But compared with the non-hibernator, loss of myofibrillar proteins in Daurian ground squirrel in hibernation is limited. For example, after three weeks of hindlimb unloading, levels of contractile proteins decreased by 40-70%, and the ratio of actin/myosin filaments decreased by 31%. Reduction in muscle quality caused by alterations in myofilament contractile proteins (myosin and actin) may scale up from the molecular to the single fiber and tissue level to impact muscle performance. Thus, up-regulation of myosin-2 is one of the most important mechanisms to maintain the integrity of the SOL muscle fiber in the 60-d hibernation ground squirrels. Because the function of myosin-3 and myosin-13 in hibernation is unknown, the relationship between the decrease of these two myosin subtypes and skeletal muscle function is not clarified.
Except for the contractile proteins including myosin and actin, other regulatory proteins showed significant change in hibernation. In present study troponin C was found significantly up-regulated while tropomodulin-1 down-regulated in the 60-d hibernation group compared to the pre-hibernation ground squirrels. However, both troponin C and tropomodulin-1 was unaltered in the 112-d hibernation group. Troponin proteins consisting of 3 subunits (troponin I, troponin T and troponin C) are responsible for controlling the contraction of the striated muscles in response to changes in the intracellular calcium concentration. The binding of Ca 2+ to troponin C induces a series of conformational changes in troponin complex and sarcomeric actin thin filament to activate cross bridge cycling between myosin and actin and muscle contraction. Troponin I is the inhibitory subunit of troponin which forms a coiled coil interface with troponin T. Troponin C increased by 59% in soleus of human bed rest study while troponin T showed a significant decrease in rat soleus muscle after 3-week hindlimb suspension, suggesting troponin C showed a increase in SOL of hibernation which was similar as in disuse atrophy. Tropomodulin is another regulatory protein and the only protein known to cap the pointed end of actin filaments and plays important roles in actin-driven processes by controlling the addition and dissociation of actin subunits at filament ends. Calpain-mediated proteolysis of tropomodulin isoforms leads to thin filament elongation in dystrophic skeletal muscle. It appears reasonable to assume that the changes of tropomodulin might be an adaptive factor for inhibiting the contractile activity during hibernation. Proper contraction requires two filament systems (actin-containing thin filaments and myosin-containing thick filaments) to appropriately align with one another. Accordingly, their orientations, spacing and lengths are highly regulated. It is evident that the specification of thin filament lengths requires the coordinated activity of tropomodulin-1, which prevents actin polymerization and depolymerization in vitro. Blockade of the C-terminal tropomodulin-1 actin binding domain results in lengthening of the actin filaments in striated muscle cells in culture. Overexpression of tropomodulin-1 in mouse hearts results in degenerating myofibrils. In present study, tropomodulin-1 down-regulated in the 60-d hibernation group, and actin subsequently exhibited significantly down-regulated expression in the 112-d hibernation compared to the pre-hibernation. Therefore, they assume that the down-regulated tropomodulin-1 in 60-d hibernation might be an crucial component for regulating the length of actin-containing thin filament in soleus during hibernation.
Other sarcomeric proteins such as α-actinin, titin, tropomyosin and desmin were not detected proteomic variations in present study. However, evidence presented by other study clearly showed that long-term disuse causes preferential loss of the giant sarcomere protein titin results in altered muscle function via abnormal sarcomeric organization. The level of titin phosphorylation did not vary between seasons in the skeletal muscles of brown bear (Ursidae, Mammalia) while a decrease in the content of T2 fragments of titin was observed during hibernation. Absence of α-actinin-3 resulted in reduced atrophic response and altered adaptation to disuse. Desmin and titin globally reduced in hibernating myocardium suggested a qualitative cardiomyocyte remodeling. Obviously, homeostasis of most sarcomeric proteins is an important mechanism against disuse atrophy in ground squirrels.
In conclusion, these results suggested that myofibrillar protective remodeling marked by dysregulated contractile proteins (myosins and actin) and regulatory proteins (troponin and tropomodulin), and maintain of most of sarcomeric proteins is a major factor in protecting atrophy in SOL of Daurian ground squirrels during hibernation.
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