The Relationship Between CAⅢ And Muscle Fatigue And The Preliminary Study On Its Anti-fatigue Function

Fatigue,defined as the failure to maintain the required or expected power output,seriously affects the sports ability and physical recovery of sporters.It has been theresearch hotspot in sports medicine and its occurrence mechanism is extremelycomplicated.The obvious reasons are that it is a multifactorial situation and it may becaused by factors within the muscle cells (peripheral fatigue) and diminishedactivation from the central nervous system (central fatigue).The primary sites offatigue appear to be within the muscle cell itself and for the most part do not involvethe central nervous system or the neuromuscular junction in the peripheral fatigue.Inrecent years,it has been suggested that the changes of protein levels and/or activitiesof carbonic anhydraseⅢ(CAⅢ) in skeletal muscles might be related with theoccurrence of muscle fatigue.However,the related research about the levels of CAⅢduring muscle fatigue has not been reported in literature.In addition,how to obtainpurified CAⅢand transfer it into cells to observe its anti-fatigue function? In order tosolve these questions,we established skeletal muscle fatigue models by single bout ofintense treadmill running and low-frequency electric stimulation,and theninvestigated the relationship between CAⅢand muscle fatigue.Furthermore,weconstructed TAT-CAⅢplasmid expression vector,expressed and purified the fusionprotein TAT-CAⅢ,and then observed its transmembrane ability and effect ongastrocnemius myodynamia during the muscle fatigue restoration process.This studywas divided into three parts: PartⅠEstablishment of skeletal muscle fatigue models and expression of CAⅢproteinin skeletal muscles and sera of ratsObjective To establish skeletal muscle fatigue models and observe the expression ofCAⅢprotein in skeletal muscles and sera of rats,in order to investigate therelationship between CAⅢand muscle fatigue.Methods Thirty-six male Sprague-Dawley rats were randomly divided into controlgroup,training group and stimulation group.The training group undertook single boutof intense treadmill running to establish skeletal muscle fatigue model.On the otherhand,the fatigue of gastrocnemius in stimulation group was induced bylow-frequency electric stimulation.The levels of CAⅢprotein in muscles and serawere detected by Western blot.Results ( 1 ) Compared with the control group,the levels of CAⅢin soleus intraining group decreased significantly (P＜0.05 ).While there were no significantdifferences for the CAⅢprotein levels in extensor digitorum longus and sera betweentwo groups (P＞0.05).(2) As the stimulation time prolonging,the peak-to-peakvalues and differential values of gastrocnemius myodynamia decreased gradually,andboth of them revealed very significant differences at 10 min compared with thebeginning of the stimulation (P＜0.01).The fatigue index(FI) increased with theprolonging of stimulation time,and reached 52.14% at 40 min.(3) As thestimulation time prolonging,the peak-to-peak values of EMG amplitude,IEMG,MPFand MF decreased gradually.The significant differences for MPF appeared at 5 min,but at 10 min for IEMG and peak-to-peak values of EMG amplitude compared withthe beginning of the stimulation (P＜0.05).The MF decreased gradually with theprolonging of stimulation time,but there were no significant differences (P＞0.05).(4) ComPared with the control group,the levels of CAⅢin gastrocnemius instimulation group decreased significantly (P＜0.05),while that in soleus and extensordigitorum longus decreased slightly (P＞0.05).Conclusion ( 1 ) The changes of EMG signals during low-frequency electricstimulation were similar to that of sEMG signals during exercise-induced musclefatigue which indicated that the gastrocnemius fatigue was established successfully inrats.(2) The decreased expression of CAⅢin soleus and gastrocnemius could berespectively induced by acute intense treadmill running and low-frequency electric stimulation.It appears that the occurring of muscle fatigue may be related with thedecreased expression of CAⅢin skeletal muscles.Key words carbonic anhydraseⅢ;muscle fatigue;EMG signals;myodynamia;EMG amplitudePartⅡExpression and purification of TAT-CAⅢfusion protein and characterizationof its transmembrane abilityObjective To construct the CAⅢand TAT-CAⅢplasmid expression vectors,express and purify the fusion proteins,and then verify the transmembrane ability ofTAT-CAⅢboth in vitro and in vivo.Methods (1) The CAⅢand TAT-CAⅢgenes obtained by PCR were cloned intoplasmid pET28a and expressed in E.coli BL21(DE3).The transformants wereinduced with IPTG and the fusion proteins with His-tag were purified with aNi-NTA-agarose column.The purified proteins were verified by means ofSDS-PAGE,Western blot and phosphatase activity staining subsequently.(2) TheC2C12 and PC12 cells were treated respectively with serum-free medium containing1μM TAT-CAⅢor 1μM CAⅢfor 1 h and the intracellular distributions of fusionproteins were observed by indirect immunofluorescence.And then the C2C12 cellswere treated respectively with serum-free medium containing 0.1,0.5,1.0,2.0μMTAT-CAⅢfor 1 h or 1μM TAT-CAⅢfor 15,30,60,120min.The relations of proteintransduction efficiency and its incubating concentration or time were examined byindirect immunofluorescence and Hoechst 33342 staining.(3) The TAT-CAⅢandCAⅢwere injected respectively into the external sarcolemma and thegastrocnemiuses were sampled after 30min,60min and 90min.Then the indirectimmunofluorescence staining was performed in order to examine the penetratingability of TAT-CAⅢ.Results (1) The TAT-CAⅢand CAⅢplasmid expression vectors weresuccessfully constructed.The fusion protein TAT-CAⅢand CAⅢwere expressedand purified efficiently,and their relative molecular mass were about 35 000 and 32000.Western blot and phosphatase activity staining showed that the fusion proteinswere obtained successfully.(2) After cultured with TAT-CAⅢor CAⅢfor 1h,greenfuorescence was visible in TAT-CAⅢgroup cells under fluorescence microscope, while no fluorescence was found in CAⅢgroup.The fluorescence intensity increasedobviously with the increase of incubating concentration and prolonging of incubatingtime.And the protein transduction efficiencies of cells in each group were attained100%.(3) Lots of cells with red fluorescence were observed in gastrocnemiuses afterbeing injected with TAT-CAⅢand the fluorescence intensity reached the maximum at60min,while no fluorescence-positive cells were found in CAⅢgroup and negativecontrol group.Conclusion The TAT-CAⅢand CAⅢplasmid expression vectors weresuccessfully constructed and the fusion protein were expressed and purified efficiently.The indirect immunofluorescence showed that TAT could mediate CAⅢtransferringinto cells non-selectively,efficiently and rapidly.PartⅢEffect of TAT-CAⅢon C2C12 cells apoptosis induced byhypoxia/reoxygenation and preliminary study on its anti-fatigue functionObjective To observe the effects of TAT-CAⅢon C2C12 cells apoptosis inducedby hypoxia/reoxygenation and gastrocnemius myodynamia during the muscle fatiguerestoration process,in order to investigate its anti-fatigue function.Methods (1) The effect of TAT-CAⅢon C2C12 cells apoptosis induced byhypoxia/reoxygenation was detected by flow cytometry.(2) The recombinant plasmidpET28a-TAT-CAⅢwas expressed in E.coli BL21(DE3) bylow-temperature-induced method to obtain soluble fusion protein TAT-CAⅢ.Thenthe fusion protein was purified with a Ni-NTA-agarose column and verified by meansof SDS-PAGE,Western blot and phosphatase activity staining subsequently.(3) TheTAT-CAⅢand CAⅢwere injected respectively into the external sarcolemma aftergastrocnemius fatigue which was induced by low-frequency electric stimulation toobserve the effect of TAT-CAⅢon gastrocnemius myodynamia during the musclefatigue restoration process.Results (1) Compared with the OGD group,the apoptosis rate of C2C12 cellsinduced by hypoxia/reoxygenation in CAⅢgroup decreased slightly(P＞0.05 ),whilethe apoptosis rate of C2C12 cells in TAT-CAⅢgroup decreased very significantly (P ＜0.001 ) in a concentration-dependent manner.And there also had significantdifferences between CAⅢgroup and TAT-CAⅢgroup (P＜0.01 ).(2) The solublefusion protein TAT-CAⅢwas obtained by low-temperature-induced method and itsconcentration reached 1.1mg/ml.(3) As the recovery time prolonging,thepeak-to-peak values of gastrocnemius myodynamia increased gradually,and therewere significant differences in 0.2mg TAT-CAⅢgroup at 45 min compared with thecontrol group and saline group (P＜0.05).And at 60 min,the gastrocnemiusmyodynamia of 0.1mg TAT-CAⅢgroup and 0.2mg TAT-CAⅢgroup was close tonormal value and higher than that of control group and saline group (P＜0.05),whilethat of CAⅢgroup increased slightly (P＞0.05 ).Conclusion The TAT-CAⅢcould decrease the apoptosis rate of C2C12 cellsinduced by hypoxia/reoxygenation obviously which indicated that its enzyme activitywas still hold after being transferred into cells.The soluble fusion protein TAT-CAⅢwas successfully obtained by low-temperature-induced method and it had anti-fatiguefunction in some extent,which provides a new method and idea for eliminatingmuscle fatigue.