| Background and Objective: Skeletal muscle atrophy occurs as a result of mechanical unloading,fasting,denervation and ischemia-reperfusion in the non-hibernating rodents and humans.While skeletal muscle is well protected and maintained from inactivity,nutritional deprivation and denervation during hibernation state,as well as ischemia-reperfusion induced stress during “torpor-arousal” cycle in hibernating animals.Thus,it is an interesting and thoughtful research to explore the unique mechanisms and countermeasures of hibernating animals against disuse and fasting induced muscle atrophy.It has been widely reported that endoplasmic reticulum stress(ER stress)mediated unfolded protein response(UPR)plays essential regulating roles in skeletal muscle maintenance under external stimulating conditions.ER stress occurred when the organism exposed in adverse environment which disrupts ER function and results in the accumulation of unfolded protein in the ER lumen.In order to alleviate the stress and restore homeostasis,the GRP78 separated from three transmembrane proteins,PERK,ATF6 and IRE1.Meanwhile,a signaling network called the unfolded protein response(UPR)is activated and regulates the genes related to cell survival and death.Thus,in order to explore whether the ER stress mediated UPR participates in the mechanisms involved in the maintenance of skeletal muscle during hibernation.In this study,the changes of the ER stress marker protein and PERK signaling pathway will be studied in different skeletal muscles of hibernating Daurian ground squirrels.Methods: In order to research whether the gender and body weight have effects on the duration of hibernation-bout,we analyzed the average duration of one hibernation bout through measuring the core temperature of each animal using the infrared scanner.The animals were separated into six groups,summer active group(SA),pre-hibernation group(PRE),early torpor group(ET),late torpor group(LT),inter-bout arousal group(IBA)and post-hibernation group(POST).The plantaris longus(PL),gastrocnemius(GAS)and semitendinosus(ST)were used for all experiments.The muscle fiber type distribution and muscle fiber cross section area(CSA)were measured by frozen section cutting and immunofluorescence.The protein expression of GRP78,p-PERK,p-eIF2?,ATF4 and CHOP were measured through western blot.The mRNA expression of grp78 and atf4 were measured through real-time PCR technique.DNA fragmentation induced by apoptosis was determined by double-labeled fluorometric TUNEL detection.Results: 1.The effect of gender and body weight on the duration of each hibernation bout The duration of each hibernation bout maintained within 7 days,no significance between different gender and body weight.2.The change of morphology index of skeletal muscle in different periods(1)Compared with SA and PRE groups,the squirrels body weight significantly decreased during ET,LT,IBA and POST groups.The MWW of PL,GAS and ST decreased by 11%,13%(P < 0.05)and 20%(P < 0.001),respectively.While,as compared with PRE group,the MWW/BW of PL,GAS and ST significantly increased by 28%(P < 0.001),28%(P < 0.01)and 16%(P < 0.01).(2)Compared with SA and PRE groups,the slow type muscle fiber distribution increased,and recovered to the level in SA or PRE groups.Compared with SA group,the slow and fast CSA of PL decreased by 12% and 13%(P < 0.05)in POST.While the slow and fast CSA in POST decreased by 13% and 13%(P < 0.05)in GAS,12%(P < 0.05)and 17%(P < 0.01)in ST,respectively.3.The change of GRP78 protein and mRNA expression of skeletal muscle in different Periods(1)Protein level: compared with SA and PRE groups,GRP78 protein expression of PL,GAS and ST significantly increased in LT group and recovered to the level in PRE group.Compared with LT group,GRP78 protein expression of PL and GAS decreased 42%(P < 0.001)and 24%(P < 0.05).The decreasing trend also showed in ST with no significance.(2)mRNA level: compared with SA and PRE groups,grp78 mRNA levels significantly increased in ET,LT,IBA and POST groups.While compared with LT group,the mRNA expression of grp78 in PL,GAS and ST declined 89%(P < 0.05),64%(P < 0.05)and 86%(P < 0.001),respectively.4.The change of PERK signaling pathway of skeletal muscle in different periods(1)PL: as compared with SA group,the protein expressions of p-PERK,p-e IF2?,ATF4 and mRNA expression of atf4 increased 80%(P < 0.01),64%(P < 0.05),110%(P < 0.05)and 85%(P < 0.01).While,compared with LT group,the protein expressions of p-PERK,p-eIF2?,ATF4 decreased 32%,40%,37%(P < 0.05),respectively.And the mRNA expression of atf4 significantly declined 73%(P < 0.001).Compared with SA group,the protein expression of CHOP significantly decreased by 21%(P < 0.05)and 24%(P < 0.05)in ET and LT groups,respectively.(2)GAS: as compared with SA group,the protein expression of p-PERK significantly increased 70%(P < 0.001),57%(P < 0.05)and 53%(P < 0.05)in ET,LT and IBA groups,respectively.Similarly,the p-eIF2? protein expression increased 81%(P < 0.01),95%(P < 0.001)and 68%(P < 0.01)in ET,LT and IBA groups,respectively.The ATF4 protein expression increased 82%(P < 0.01)and 74%(P < 0.05)in ET and LT groups.Interestingly,the atf4 mRNA level significantly increased 96%(P < 0.05)in LT group and decreased 72%(P < 0.001)in IBA group compared with LT group.(3)ST: as compared with SA group,the protein expression of p-PERK significantly increased 118%(P < 0.01),76%(P < 0.05)and 84%(P < 0.01)in ET,LT and IBA groups,respectively.The p-eIF2? protein expression increased 97%(P < 0.05),101%(P < 0.01)and 77%(P < 0.05)in ET,LT and IBA groups,respectively.The ATF4 protein expression increased 78%(P < 0.05)and 85%(P < 0.001)in ET and LT groups.The mRNA level of atf4 significantly increased 1.4 folds(P < 0.001)in ET group,while decreased 61%(P < 0.001)in IBA group compared with ET group.Compared with SA group,the protein expression of CHOP significantly decreased by 14%(P < 0.05)and 18%(P < 0.05)in PRE and ET groups,respectively.(4)No cell apoptosis was observed in all types of skeletal muscles during different periods.Conclusions: 1.No significant effects of sex or body weight of squirrels on the duration of each hibernation-bout and the physiological study.2.The different degree of muscle loss were observed in PL,GAS and ST after several months of hibernation,the order is that: PL< GAS < ST.3.During hibernation(especially in LT group),ER stress significantly elevated in all three muscles,and the ER tress mediated PERK signaling pathway was activated,which attenuate the ER stress through two aspects.On the one hand,elevated activation of p-eIF2? decreased the ER stress through inhibiting the protein translation;on the other hand,the up-regulated pro-survival gene grp78 and the down-regulated pro-apoptosis marker(CHOP),which mediated protective role and ultimately prevent ER stress induced skeletal muscle damage.4.The decreased level of ER stress and attenuated activation of PERK signaling pathway plays a key role in the attenuation of stress induced skeletal muscle damage and takes part in the mechanisms involved in the muscle maintenance during hibernation state. |
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