The Effect Of Skeletal Muscle DNA Damage Caused By Heavy-loaded Exercise On Mitochondrial Membrane Permeability

Objective:This study observes whether heavy-loaded exercise can cause DNA damage in skeletal muscle cells and changes in mitochondrial membrane permeability,and explores whether DNA damage caused by exercise can lead to changes in mitochondrial membrane permeability by regulating the components of the endoplasmic reticulum mitochondrial binding structure（MAM）（Plasma reticulum protein REEP1 protein,endoplasmic reticulum protein EI24 protein and mitochondrial outer membrane protein VDAC2 protein）.Methods:48 healthy 8 weeks male Spraque Dawley（SD）rats,SPF grade,are randomly divided into the control group（C,n=8）,the group immediately after exercise（E0,n=8）,the group 12 hours after exercise（E12,n=8）,the group 24 hours after exercise（E24,n=8）,the group 48 hours after exercise（E48,n=8）,and the group72 hours after exercise（E72,n=8）.The exercise program refers to the Armstrong centrifugal exercise model.The training on is a one-time continuous centrifugal downhill run for each exercise group of rats,with the gradient of-16°,the speed of16m/min and the exercise time of 90 minutes.After training,the soleus muscles are sampled according to the phases of the experimental group.Immunofluorescence detects DNA oxidative damage marker 8-OHd G.Western blot detects DNA damage-related protein p53 in the nucleus and expression of EI24 and REEP1proteins in whole cells,co-localization coefficient of endoplasmic reticulum protein EI24 and mitochondrial protein VDAC2 detects by immunofluorescence double staining,fluorescent detects mitochondrial Ca²⁺concentration in skeletal muscle,and finally,the openness of mitochondrial permeability transition pores（m PTP）is detected by immunofluorescence.The obtained data will be processed by IPP,Image Lab,Image J,etc.,and then imported into SPSS 21.0 for statistical analysis.When P<0.05,the data are defined as significantly different.Result:（1）The content of 8-OHd G,a marker of oxidative damage to DNA in skeletal muscle tissue,exhibits a phasic change after a one-time heavy-loaded centrifugal exercise,which immediately begins to increase after the exercise（P<0.05）,reaches a peak at 12 hours after the exercise（P<0.05）,and then maintains a recorvery till 48 hours after the exercise（P<0.05）.And the expression is lower than that of the control group at 72 hours after exercise（P<0.05）,indicating that one-time heavy-loaded centrifugal exercise caused DNA damage with chronological changes in skeletal muscle.（2）The expression of the regulatory protein p53 in the nucleus in response to DNA damage immediately decreased after exercise（P<0.01）,increases to a peak 12 hours after exercise（P<0.01）,returns to the control group level 48 hours after exercise,and decreases slightly 72 hours after exercise（P<0.05）.（3）As a downstream protein of p53,the EI24 protein and REEP1 protein that increase the endoplasmic reticulum-mitochondrial contact to promote endoplasmic reticulum formation both increase to a peak value then decrease at 12 hours after exercise（P<0.01）.In addition,EI24 is slightly reduces at 72 hours after exercise Compared to the control group（P<0.05）,REEP1 increases slightly and then decreases at 48 hours after exercise（P<0.05）.（4）The colocalization coefficients of endoplasmic reticulum protein EI24 and mitochondrial calcium channel protein VDAC2 at the same MAM grade and the Mander’s coefficient of the two proteins within 72 hours after exercise are generally consistent with each other,that is,increses first then recovers（P<0.05）.（5）Mitochondrial Ca²⁺begins to increase immediately after exercise,and reach a peak at 12 hours after exercise,then decreases in subsequent phases.（6）The opening amount of mitochondrial permeability conversion pores begins to increase significantly immediately after exercise（P<0.05）,and m PTP opens the most 12 hours after exercise（P<0.05）,and then the opening amount gradually decreases.Conclusion:（1）The heavy-loaded exercise causes DNA damage in skeletal muscle cells.At the same time mitochondria showes the Ca²⁺overload and mitochondrial membrane permeability increases.（2）The DNA damage caused by heavy-loaded exercise may promote the transcriptional activation of REEP1 and EI24,enhance the interaction between EI24 and VDAC2 through p53,and then lead to the increase of Ca²⁺in skeletal muscle mitochondrial and the increase of opening of membrane permeability transition pores.