| OBJECTIVE:Mammalian hibernators undergo a remarkable state of little to no activity. Although such inactivity would cause muscular atrophy in non-hibernating animals, hibernating mammals experience no significant muscle atrophy during several months of hibernation. Research on non-hibernating animals have shown that muscle atrophy mainly induced increase of muscle protein degradation and cell apoptosis. Cytosolic calcium overload can not only cause the activation of Calpain signaling pathway trigging protein degradation, and can activate apoptotic signaling pathway, causing apoptosis. Therefore, to explore the molecular mechanism of anti disuse muscle atrophy, this study investigated the regulation of apoptosis, protein degradation and the regulation of calcium homeostasis in different periods of soleus muscle cells in hibernation ground squirrels (Spermophilus dauricus).Calcium homeostasis is an important part of homeostasis. Disequilibrium of calcium homeostasis is one of the main mechanisms that activate Calpain system, then follow the protein degradation, and result in disuse muscle atrophy. There are two main factors for regulation of intracellular calcium homeostasis:rise and fall of calcium concentration. There are three main factors for the fall of calcium concentration, the first is SERCA2(Ca2+ATPase), an important factor in reducing the intracellular calcium concentration, which is located in the sarcoplasmic and pumps cytoplasm calcium back to the sarcoplasmic reticulum; the second is NCX (Na+-Ca2+exchanger) which located on the cell membrane, transferred the sodium ions into the cell and the calcium ion out of the cell, and thus serve to reduce the concentration of intracellular calcium effect; the third is the calcium-binding protein CALM1(calmodulin), which can combine with calcium ions in the cytoplasm, thereby reducing the intracellular free calcium concentration. And there are two main factors for calcium rise. One is the L-type calcium channels on the cell membrane. Since the channel is voltage-gated channels, membrane potential is the main factor affecting the channel, this study did not study its gene expression. LETAL (EF hand-containing transmembrane protein1) is H+-Ca+exchanger on mitochondria. Mitochondrial calcium can release into the cytoplasm by LETAL. It is an important factor to avoid mitochondrial calcium overload and increase cytoplasmic calcium concentration. Calcium homeostasis of skeletal muscle cells is homeostasis of cytoplasmic Ca+, sarcoplasmic reticulum Ca2" and the mitochondrial matrix Ca2+, mainly implemented by a collaborative work of calcium channels, calcium pumps, calcium-binding proteins and ion exchangers mentioned above. This study systematicly researched effects of hibernation on skeletal muscle calcium homeostasis by selecting NCX on cell membrane, sarcoplasmic reticulum SERCA2, LETAL on mitochondrial and cytosolic calcium binding protein CALM1, from the perspective of gene expression.Apoptosis inhibitory factor Bcl-2, pro-apoptotic factor Bax and cysteine Caspase-3were selected for apoptotic signaling pathway in.Both Bcl-2and Bax belong to the Bcl-2gene family. Apoptosis regulator Bax promotes apoptosis by binding to and antagonizing the Bcl-2protein. The ratio of Bax to Bcl-2determines the direction of survival or death of cells. Therefore changes in mRNA and protein expression were examined, and the ratio of Bax to Bcl-2. Caspase-3is a member of the cysteine-aspartic acid protease (caspase) family. Sequential activation of caspases plays a central role in the execution-phase of cell apoptosis. Caspase-3exists as an inactive proenzyme that undergoes proteolytic processing at conserved aspartic residues to produce two subunits, large and small, that dimerize to form the active enzyme.Calpains of Calpain-1, Calpain-2and Calpain inhibitor Calpastatin were selected for proteolytic signaling pathway. Calpain is the Ca2+-dependent cysteine protease in the cytoplasm. Previous studies have shown that in skeletal muscle protein degradation process, Calpain was activated firstly, and then it degrades cytoskeletal proteins, following myofibrillar off from the cytoskeleton. Calpastatin Calpain inhibitor can specifically inhibit the activity of Calpain Calpain-1and Calpain-2. As it has been studied on its protein expression previously in our laboratory, it was only studied from the perspective of gene expression.METHODS: Animals were matched for body mass and were randomly assigned to fur groups (n=6): PRE:Control (no hibernation) animals investigated in late-autumn; HIB:Animals after two months hibernation; IBA:interbout arousal; POST:Animals two days after arousal from90±12days hibernation; Gene expression were determined by the SYBR Green RT-PCR, Protein expression were determined by western blotting, Z-disk ultrastructure was observed by transmission electron microscopy.RESULTS:The following results were found in the fellow group compared to the PRE group:1. In comparison with the PRE group, the body weights were decreased24%(p<0.05) and38%(p<0.05) in HIB and POST, respectively; SOL muscle wet weights were decreased9%(p<0.05) and16%(p<0.05) in HIB and POST, respectively. The SOL muscle weight (mg) to total body weight (g) ratio was significantly higher in the HIB increased33%,(p<0.001) and POST59%(p<0.001) groups in comparison with that of the PRE%roup.2. Gene expression of Bcl-2increased by74.4%(p<0.001),106.6%(p<0.001) and48.8%(p<0.001), respictively, in the HIB, IBA and POST group compared to the PRE group; Gene expression of Bax increased by43%(p<0.001), and55%(p<0.001), respictively, in the HIB and IBA group compared to the PRE group;The ration of Bax to Bcl-2gene expression decreased by18%(p<0.001),26%(p<0.001), respictively, in HIB and IBA group compared to PRE group.3. Protein expression of Bcl-2increased by500%(p<0.001),262%(p<0.05) and385%(p<0.001), respictively, in the HIB, IBA and POST group compared to the PRE group; Protein expression of Bax decreased by58%(p<0.001) in the HIB group compared to the PRE group compared to the PRE group; Protein expression of Caspase-3increased by29%(p <0.001) in the IBA group compared to the PRE group; The ration of Bax to Bcl-2protein expression decreased by92%(p<0.001) in HIB group compared to PRE group.4. There is not significantly change in Calpain-1and Calpain-2gene expression; Gene expression of Calpastatin increased by61%(p<0.001),111.3%(p<0.001) and69.6%(p<0.001), respictively, in the HIB, IBA and POST group compared to the PRE group; 5. Gene expression of SERCA2increased by50.3%(p<0.05),131.5%(p<0.001) and36.6%(p<0.001), respictively, in the HIB, IBA and POST group compared to the PRE group; gene expression of CALM1increased by18.2%(p<0.05),33.9%(p<0.001) and32.5%(p <0.001), respictively, in the HIB, IBA and POST group compared to the PRE group; Gene expression of LETA1increased by43%(p<0.05) in the IBA group compared to the HIB group; Gene expression of NCX decreased by19.7%(p<0.05) in the HIB group compared to the PRE group.6. The myofilaments were well-organized, and the width of the sarcomere and the Z-disk both appeared visually similar among the pre-hibernation, hibernating and post-hibernation animals.CONCLUSION:1. Wet weight of the soleus muscle did not significantly decrease in ground squirrels during hibernation and the ratio of muscle weight to body weight significantly increased, which suggests that the soleus muscle only exhibited slight atrophy during hibernation in ground squirrels.2. The Z-line ultrastructure in the soleus muscle cells of ground squirrels did not change significantly during hibernation.3. The gene and protein expression of the Bcl-2gene significantly increased in the pre-hibernation, hibernating and post-hibernation groups. The gene and protein expression of Bax and Caspase-3did not change significantly. The ratio of Bax to Bcl-2was significantly reduced. These results suggest that ground squirrels inhibit apoptosis during hibernation.4. The gene expression of calpastatin significantly increased in the pre-hibernation, hibernating and post-hibernation groups, while the gene expressions of Calpain-1and Calpain-2did not change significantly. These results suggest that ground squirrels inhibit muscle atrophy during hibernation by the over-expression of calpastatin, which in turn inhibits the activities of Calpain-1and Calpain-2.5. Gene expression of SERCA2and CALM increased significantly in the hibernating and inter-bout arousal ground squirrels. Gene expression of LETAL increased and LETA1released calcium from the mitochondria to the cytoplasm, which may be one reason for the higher concentration of intracellular calcium ions during inter-bout arousal of ground squirrels.These results show that hibernating squirrels can actively regulate intracellular calcium concentrations in skeletal muscle, thus avoiding activation about the muscle atrophy signaling pathways, and thereby preventing apoptosis and muscle atrophy and allowing the Z-line ultrastructure to remain intact. |
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