Molecular Mechanism Of Musclular Quality Control Induced By Resistance Exercise

SKELETAL muscle is pivotal for daily locomotion accounting for 1/3-1/2 of our body mass and aging and aging related diseases induce skeletal muscl atrophy,in term of sarcopenia,which not only compromises life quality but also exacerbates disease condition.Exercise,especially resistance exercise/strength training,prevents aging-induced or pathological amyotrophy by ptomoting skeletal muscular hypertrophy and improving contractile function.Although accumulated researches have illustrated benefits of exercise on resistance exercise induced health promotion,the concealed mechanism is still unknown.mTOR signaling pathway plays a critical central role in controlling protein synthesis,but in which manner during resistnce exercise induced muscular remodeling?Satellite cell has been reported as the vital unipotent stem cell in skeletal muscle regeneration,is it required in resistance exercise induced muscle hypertrophy?To clarify these scientific questions,there should have been a feasibly,reliable,and physiological resistance exercise animal model to begin with.In the last 40 years,tenotomy,electrical stimulation,ladder climbing,passive jumping,and some resistance specific facilities have been applied in this area.But based on the limitation of invansive injury,human resource comsuption,and other stress,all the above animal models are not representative faithfully resistance exercise under physiological condition like voluntary wheel running mimicking endurance or lifelong exercise.In the present study,we developed a novel weighliting mouse model facilitating mouse to do a sqush-like exercise(including heel and hindlimb extention)by food reward and reported transcriptional,translational,contratile and metabolic functional changes in mice by acute,long-term and short-term weightlifting.Our data validated the weightlifting indued skeletal muscle hypertrophy,hyperfucntion and metabolic attenuation.We next answer the behind mechanism by utilizing weightlifting exercise intervention on a Dexamethasone induced muscle hypotrophy animal model and a satellite cell deletion(Pax7CreER-ROSADTA176)transgenic animal model,and our data showed contratile functional protection on DEX-induced mucle atophy and satellite cell deletion mice could not acquire as same amount of the adaptation in muscle mass and muscular contractile function as wildtype mice by long-term weightlifting.OBJECTIVEIntroduce the novel weightlifting model,including the design,validation,and parameters;investigate the transcriptional effects by one single bout of acute weightlifting in skeletal muscle and the specific muscle groups recruited in this process;study the influences on muscle mass,contractile and metabolic functional changes induced by short-term and long-term weightlifting,and reveal the role of protein synthesis related mTOR signaling pathway and the activity of satellite cells during resistance exercise induced muscular remodeling;detect the effects of the prevention on Dexamethasone induced muscular atrophy;explore that is satellite cell necessary in resistance exercise induced muscle hypertrophy.METHOD1)The development of weightlifting model and the training protocols.The design of the weightlifting model was designed to train mice to use the facility to do a squat and heel-exenteion like movement by food rewards.A magenic sensor was attached to the facility to record the weightlifting acitivities.All the cages were calibrated to generate the linest equtiont to calculate the output work resistance on mice and the added weight on the weightlifting lever arm.The resistance was presented by the percentage of the mice body weight.We tried several mice for acute and long-term to define the acute,short-term,and lont-term weightlifting training protocols.2)The sigle bout of acute,long-term and short-term weightlifting training 10-12 week old wildtype C57BL/6 male mice were trained to utilize the weightlifting facility.In the single bout of acute weightlifting,mice pushed resistance of 150%of BW for one night(AWL,n=5),while in the long-(LWL,n=8)and short-(SWL,n=5)term study mice were trained for 8 weeks and 2 weeks by pushing resistance from 100-240%of BW by daily increasing 20%.Except for the acute weightlifting,mice were fasted 2 hours before been put into weightlifting cages and the training session started at 6:00 pm to next 9:00 am with one day rest after 2 or 3 days trainging.The fasting strategy was manipulated as same as weightlifting mice in control groups.After exercise intervention,physiological parameters were collected and mice were sacrificed by dislocation to further tissue harvesting.Samples were strored at-80? for future detection.RNA sequencing was acquired by Illumina system in gastrocnemius muscle after acute weightlifting,and semi-quantitive PCR was applied to validate the target genes Tweak,Fn14,Dscrl,Nr4a3,and CytbRNA expression changes in recruited muscle and other tisusues.Protein synthese,protein degradation,satellite cell proliferation,and mitochondrial content related protein expressions were measured by western blotting in acute,long-and short-term weightlfitng studies,including p-AKT,AKT,p-AMPK,AMPK,Notch 1,p-4e-Bp-1,4e-Bp-1,Ubiqutin,mitochondrial complex I-V,Lc3,Cox4,and p62.Protein synthesis was detected in recruited skeletal muscles,heart,and liver in short-and long-term studies by recently revealed SUnSET technique.Cell proliferation was measured by EdU injection for labelling new synthesized DNA in skeletal muscle after short-tem weightlifting training.Immunochemistry detected pax7 positive puncture in short-term weightlifting study.Aurora system was applied to assess the contractile function of skeletal muscle after long-and short-term weightlifting training.Echo-MRI detected body composition,and hindlimb MRI assessed muscle cross-sectional area in long-term training.IGTT evaluated glucose tolerance and treadmill endurance test measured endurance capacity after long-term weightlifting.Micro-CT assessed femour parameters and Echocardiography evaluated heart contractile function.3)The effects of long-term weightlifting on muscle mass and contractile function in satellite cell deletion transgenic mice(Pax7CreER-ROSADTA176,mSCD)10-12 week-old male mSCD and littermate wildtype mice were obtained by cross-fertilization and divided into 4 groups mSCD-Sed(n=5),mSCD-WL(n=5),WT-Con(n=5),and WT-WL(n=5).Mice in weightlifting groups were training for 8 week long-term weightlifting by increasing workload from 100-240%of body weight and maintaining in 240%of body weight until training session was due.Hindlimb skeletal contractile function was measured by Arura system and IGTT detected glucose clearance capacity in mice.Mice were sacrificed to sample collections for the future detection.4)The effects of short-term weightlifting on glucocorticoide induced muscle atrophy 10-12 week-old male wildtype C57BL/6 mice were divided randomly into three groups,including sedentary control group(Con,n=5),Dexamethasone injection group(Dex,n=4),and weightlifting plus Dexamethasone injection group(Dex+WL,n=4).Mice in Dex and Dex+WL group were intraperitoneally injected with Dexamethasone by 25 mg/Kg body weight for 7 consecutitive days and Dex+WL mice were trained for short-term weightlifting by increase workload from 100-240%of body weight.After skeletal muscle contractile function assessment,mice were sarcrificed for sample collection for future detection.Protein synthesis was measured by SUnSET technique mentioned above.RESULTS1)Pilot study showed that 10-12 week-old male wildtype mice could be trained to employ the weightlifting facility in the correct manner with hindlimb and heel extension and according to the increase workload the repetition of weighlting decreased.With the pilot study,all the weightlifting setting up protocols were published.The acute weightlifting training workload was set to 150%of BW for one single night and the long-and short-term weightlifting training protocol set the workload increasing from 100-240%of BW and maintaining in 240%of BW for long-term training,with duration of 8 and 2 weeks.2)The acute single bout of weightlifting was accomplished under 150%of body weight and mice pushed 422±64 repititons with no body weight loss in exercise group.RNA-seq data screened 490 genes up-(341)and down-(149)regulated in gastrocnemius muscle.Semi-quantitve PCR data validated plantaris and quadriceps muscle were recruited in the weightlifting model besides gastrocnemius muscle by targeting Fn14 and Dscrl transcriptional expression.Tibil antenor muscle was not resiponsible for changing on transcriptional changes' expressions.Heart was appreciated increase in Fn14 mRNA expression in weightlifting group.There was no changes on Akt?4e-Bpl protein expression and phosphorylation modification,as same as Notch1 and mitochondrial content marker Cox4 protein expression.Compared to Con,Lc31 protein content was showed increase tendency with no changes on Lc3?/Lc3? ratio.3)In long-term weightlifiting training study,mice showed negtive-relationship pattern of the weightlifting activity with repetition decreased according to the increase workload and maintaining about 200 repititions under 240%of body weight resistance afterwards.All the body weight of mice increased after 8 weeks while compared to Con,mice in LWL showed significantly increase on body weight at the end of the training.Weightlifting training increased hindlimb tetanic force and twitch force in either absolute or specific value normalized by body weight and muscle mass,with no changes on Force-Frequency analysis.The Fatigue sequenc curve was shown right shifted in LWL,and there was no change on recover sequence.Echo-MRI measured no stwiching on body composition while tissue harvesting detected mass mass increase significantly in soleus,plantaris,gastrocnemius,and extensor digitorum longus muscle as well a obvious decrease in epididymal fat weight with no changing on tibialis anterior muscle.Hindlimb MRI showed a significantly increase in skeletal muscle cross-sectional area with increase anterpposterior dimension(AP)and no change on medial-lateral length.IGTT test measured s significantly improvement of glucose clearance capacity in LWL mice and the area under curve showed significantly decrease compared to Con.There was no changes on treadmill endurance tolerance test and cox4 protein content.SUnSET assay detected significantly increases on protein synthesis in plantaris,gastrocnemius,and quadriceps mucle as well as tibialis anterior muscle and heart,and there was no change in liver which could serve as the negative control.For the molecular mechanism,we detected higher Akt,p70S6k,and 4e-Bp-1 activity by showing the phosphorylation to total protein ratios changing in skeletal muscle of mice in LWL comparing to Con.Stimulated by insulin,Akt phosphorylation was high in WL whereas Glut4 protein expression remained unchanged between these two groups.Long-term weightlifting promoted autophagy initiation protein Lc3?protein expression with no changing on Lc3?/Lc3? ratio,while the autophagy degradation marker p62 was shown significant lower in LWL with Cox4 protein conten unchanged.The ubiquitin content was slightly higher in LWL compared to mice in Con.4)In short-term weightlifting study,pushing activity of weightlifting mice decreased according to the increased workload from 100-240%of body weight.During these two weeks intervention,there was no changes on body weight and mass/tissue mass between mice in SWL and Con.Hindlimb mucle contractile function showed a tendency of increase in tetanic force with no significantly changing.Specific Force-Frequency sequence detected significantly increases force output of 80,100,and 125 Hz stimulations in SWL compared to mice in Con group.A significantly increase of protein synthesis was detected in plantaris musle of mice in SWL group with no changing in liver tissue by SUnSET assay.Compared to Con,Lc3I protein content was shown significantly increase in muscle of SWL mice,with significantly decrease on the ratio of Lc3?/Lc3? ratio and no change on p62,Cox4,and mitochondrial complex ?-? protein expression.H&E staining showed an increase trend of myofiber cross-sectional area in SWL mice compared to Con and EdU staining showed the same increase tendency by weightlifting with P value equaling 0.09.5)For the long-term weighliting intervention on mSCD mice study,we detected a big variation on the pushing activity compared mice in mSCD-WL and WT-WL,which was inconsistent with the priouse result as shown before in long-term weightlifting mainly because of different animal background.Transgenic and weightlifting had no effects on body weight of mice.IGTT assay showed improved glucose clearance capacity in WT-WL compared to WT-Con,as well as mSCD-WL compared to mSCD-Con,with no changing compared with WT-Con and mSCD-Con.Compared to WT-Sed,there was no change on muscle mass but a decrease trend of heart weight and weightlifting did not affect muscle mass,whereas mSCD-WL showed decreased muscle mass in soleus,plantaris,gastrocnemius muscle as well as epididymal fat with opposite effect on heart weight.Contractile function assay detected no obviouse changes absolute tetanic and twtch force but there was an increase trend of specific tetanic force normalized by muscle mass compared with WT-WL and WT-Sed,as well as compared with mSCD-WL and mSCD-Sed,indicating the potential promotion on contractile function induced by long-term weightlifting in mSCD mice.Other than the present study,we had a previous pilot study with 5 transgenic mice including 3 mice(TG-WL)injected with tamoxifen to induce cre expression and 2 mice(Con-WL)injected with placebo,and all these 5 mice underwent 4 weeks weighliting training.The results indicated that,compared to Con-WL,plantris and gastrocnemius muscle were significantly lower in TG-WL with significantly decreasing epididymal fat mass.Compared to Con-WL,the hindlimb muscle tetanic force was lower with P value equaling 0.08.6)Dexamethason injection drived mice in Dex and Dex+WL body weight decrease only showing siginificant in Dex+WL group.Compared to Con,mice in Dex showed muscle mass drop significantly in plantaris,gastrocnemius,tibialis anterior,and extensor digitorum longus musce,and mice in Dex+WL were shown musle mass decrease even worse.Heart mass was shown increase in Dex,with a little decrease in Dex+WL mice while there was an increase trend of epididymal fat weighlt in Dex and Dex+WL mice showing a tendency of decrease compared to mice in Dex.No change on absolute tetanic force was detected among these three groups whereas Dex+WL mice showed high mean value on specific tetanic force compared to Dex.Protein synthesis did not appreciate changes among these three groups.CONCLUSION1)In the present study,we developed a novel resistance weightlifting animal model by encouraging mice to do a squat-like(including heel and hindlimb extention)exercise by food rewards.Our pilot study validated that mice could be trained to utilize this facility in the correct way and all the parameters were justified and reported.This novel weightlifting model was under physiological condition compared to previou invasive tenatomy and electrical stimulation models and was easy to set up compared to human resource comsumption models including ladder climbing,passive jumping and other resistant facilities.3D-printing technique was applied in the updated weightlifting model and the present weightlifting could combine with voluntary wheel running model to estabolish cross-fit exercise model.2)A single bout of acute weightlifting exercise intervention drived 490 genes transcriptional profile modification in gastrocnemius muscle,including exercise sensitive bio-marker Dscrl,reissitance specific sensitive hallmarker Fnl4,and a nulear ophan receptor Nr4a3 was the most responsible changed gene which had been reported related to the skeletal muscle mass control.Not all the hindlimb muscle groups were recruited in the acute weightlifting stress and except soleus,plantaris,gastrocnemius,and gastrocnemiuse muscle,tibialis anterior muscle was not involved in weightlifting recruited muscle.In summary,the present weightlifting model induced resistance exercise specific transcriptional effects in recruited skeletal muscle.3)Long-term weightlifting intervention was sufficient to induced muscular hypertrophy and contractile function improvention and mTOR related protein synthesis pathway was responsible involving in this process.Equally,weightlifting promoted metabolic adaptation by increase fast blood glucose clearance capacity in which Akt phosphorylation was detected contributed to the remodeling but not Glut4 signal.Autophagy was detected invlolving in long-term weightlifting induced adaptation by increasing autophay capacity(Lc3I content)and autophagy flux(p62 content).In summary,long-term weightlifting induced muscular hypertrophy phenotype,improved contractile and metabolic function by promoting protein synthesis and autophy capacity,which further validated the resistance effects of the present weightlifting model.4)The effects of short-term weightlifting on muscle mass were not detected,but there was an increase trend on type I myofiber riched solues muscle mass.Short-term weightlifting aggrandized specific tetanic force normalized by muscle mass and autophay capacity was activated during this process.A trend of increased cell proliferation and accelerated protein synthesis was activated contributing to the short-term weightlifting induced adaptation.5)mSCD mice did not show muscular atrophy,contractile,or metabolic functional defect but could not gain the same amount of long-term weightlifting induced adaptation in muscle mass increase and contractile functional improvention,with no differences on glucose clearance capacity.Satellite cells were necessary in resistance induced muscular hypertrophy and hyperfunction,but not required for metabolic adaptaion.6)Dexamethason injection induced skeletal muscle hypertrophy,and short-term weightlifting intervention did not show protection on muscle mass control but restored specific muscular contractile function.The inconsistent data comparing with the previous report were mainly dependant on the drug information and store condition.Food intake should be considered in the future study and repeatition of the present study was emergencily necessary.