Role Of MFN2 In Exercise-induced Mitochondria Adaptation In Skeletal Muscle

Skeletal muscle is the main exercise organ and the demand for energy in skeletal muscle varies during exercise, while mitochondria respond to the increasing demand for oxygen and ATP induced by exercise, its morphology, structure and function change positively as well. While exercise promotes mitochondria biogenesis and increases mitochondria function, a negative adaptation of autophagy and mitophagy is also activated by exercise at the same time, accelerating the turnover of mitochondria network. Mitochondria are highly dynamic organelles that undergo fusion and fission frequently. Mitochondria fusion and fission plays an important role in regulating mitochondria morphology and function as well as the response to apoptosis stimulus. And mitochondria fusion and fission is also closely connected with mitochondria biogenesis and mitophagy. MFN2 not only controls the fusion of mitochondria outer membranes, but also plays an important role in regulating the interaction between mitochondria and endoplasmic reticulum, thus influencing uptake of Ca²⁺ by mitochondria and its energy metabolism. Recent studies showed that MFN2 is an important receptor for E3 ubiquitin lignase Parkin-mediated mitophagy, ablation of MFN2 imparied the location of Parkin to damaged mitochondria. And MFN2 deficiency also initiates the expression of PGC-la and mitochondria biogenesis. However, the role of MFN2 in exercise-induced skeletal muscle mitochondria adaptation is not clear. In this paper, the effect and mechanism of MFN2 on exercise-induced skeletal muscle mitochondria adaptation is explored mainly from views of mitochondria biogenesis, mitophagy as well as mitochondria fusion and fission. OBJECTIVEIn this study, MCK-Cre MFN2flox/flox mice were used to explore the role of MFN2 on skeletal muscle mitophagy and mitochondria biogenesis. The effect of MFN2 deficiency on exercise-induced skeletal muscle mitochondria adaptation was also studied. Here, we discussed the possible molecular mechanisms of skeletal muscle mitochondria adaptation induced by exercise training from views of mitochondria biogenesis, mitophagy as well as mitochondria fusion and fission. METHODSCre/Loxp system was used to construct conditional skeletal muscle MFN2 gene knockout mice (MCK-Cre MFN2^flox/flox) and littermates (MFN2^flox/flox).?1? The role of MFN2 gene in skeletal muscle mitophagy and mitochondria biogenesis was studied in animals. Group:3-month-old MCK-Cre MFN2^flox/flox mice and littermates MFN2^flox/flox mice were divided into knockout group ?K, n=24? and control group ?C, n=24?.8 mice from each group were randomly picked and killed to collect quadriceps, gastrocnemius and tibialis anterior muscle. The left samples were used for RT-qPCR experiment while the right samples were used for Western blotting experiment. Another 8 mice from each group were randomly picked and killed to collect gastrocnemius and quadriceps. The left samples were fixed in 4% paraformaldehyde and used for paraffin section, HE staining and immunohistochemical staining while the right samples were kept in 2.5% glutaraldehyde for electron microscopy observation to detect mitochondrial morphology and distribution, autophagosomes. Finally,8 mice from each group were randomly selected and killed to collect gastrocnemius for mitochondria extraction. Mitochondria were used to detect membrane potential, citrate synthase ?CS? activity, ROS. And some tissue samples were used to determine oxidative stress indicators.?2? The role of MFN2 on skeletal muscle mitochondrial adaptation to exercise training was explored.Group:2-month old MCK-Cre MFN2^flox/flox mice and littermate MFN2^flox/flox mice were randomly divided into control group ?C, n=8?, exercise group ?E, n=8?, knockout group ?K, n=8? and knockout exercise group ?KE, n=8?. E and KE group were subjected to 4 week endurance treadmill running program at a speed of 14 m/min while C and KC group were free access to static treadmill. And 12h after the last bout of training, all mice were killed to collect gastrocnemius. The left samples were used for RT-qPCR experiment while the right samples were used for western blotting and oxidative indicators measurement.The indexes of this study measured in this study are as follows:1) Metabolic indicators:Body weight, epididymal fat mass and its ratio to body weight, heart and liver mass.2) Mitochondria fusion and fission related indicators:RT-qPCR was used to detect MFN2, MFN1, Drpl, Fisl, OPA1, MFF mRNA expression; Western blotting was used to detect MFN2, MFN1,OPA1, Drp1, MFF protein content.3) Mitophagy and autophagy related indicators:RT-qPCR was used to detect Atg5 Atg12, ULK1, Parkin, PINK1, LC3, p62, BNIP3, FUNDC1, LAMP2 mRNA; Western blotting was used to detect ULK1, p-ULK1Ser⁵⁵⁵, Parkin, PINK1, LC3, BNIP3, p62, AMPK?, p-AMPK?Thr¹⁷² protein content.4) Mitochondria biogenesis-associated indicators:RT-qPCR was used to detect PGC-1?, TFAM, NRF1, TFB1M, ATP5a, COXI, COX5b, NDUFS8 mRNA expression and mtDNA content; Western blotting was used to determine PGC-1?, TFAM, NRF1, TOM20 protein content.5) Mitochondrial function indicators:mitochondrial membrane potential, CS activity, COX and SDH staining.6) Morphological indexes:HE staining was used to observe the morphology of muscle fibers while transmission electron microscope was used to observe ultrastructure of muscle fibers, mitochondria morphology and autophagosomes.7) Oxidative stress indicators:H₂O₂ release from isolated mitochondria and skeletal muscle H₂O₂, MDA and T-SOD activity.RESULTS1) Cre/Loxp system was used to successfully construct a conditional skeletal muscle MFN2 gene knockout mice model, with MFN2 mRNA and protein content decreased significantly than the control group ?p<0.01?.2) Compared with group C, abnormal morphological mitochondria increased in skeletal muscle of group K, some mitochondria are swollen and contain loosely packed cristae, and irregular vacuoles appeared in these mitochondria. In addition, Drpl protein content was significantly increased in group K compared to group C ?p<0.05?. Compared with group C, mitochondrial membrane potential was significantly reduced in skeletal muscle of group K while ?p<0.05?. In addition, H₂O₂ release from mitochondria of group K was significantly higher than group C ?p<0.05?. H₂O₂ and MDA levels in skeletal muscle of group K were significantly higher than group C ?p<0.05?. Compared with group C, PGC-1?, TFAM and TOM20 protein content was significantly increased in skeletal muscle in group K?p<0.05?. 3) Compared with group C, FUNDC1 and BNIP3 mRNA expression in skeletal muscle of group K was significantly increased ?p<0.05?. In addition, compared with group C, BNIP3, Parkin and p62 protein content as well as LC3-?/? ration was significantly increased in skeletal muscle of K group?p<0.05?. Phosphorylation of AMPKa Thr¹⁷² and ULK1 Ser⁵⁵⁵ levels was significantly increased in group K ?p<0.05?.4) Compared with group C, MFN1, MFN2, Drp1 and FIS1 mRNA expression and Drp1 and MFN1 protein content in skeletal muscle of group K was significantly increased ?p<0.05?. Compared with group E, MFN1, MFN2, Drp1 and FIS1 mRNA expression and Drp1 and MFN1 protein content in skeletal muscle of group KE was significantly increased ?p<0.05?. Drp1 protein content in skeletal muscle of group KE was significantly higher than group E ?p<0.05?.5) Compared with group C, PGC-1?, NRF1, TFAM, TFB1M, COXI, COX5b mRNA and PGC-la, TFAM, TOM20 protein content in skeletal muscle of group E was significantly increased ?p<0.01or p<0.05?, mtDNA content was also significantly increased ?p<0.05?. Compared with group K, PGC-1?, NRF1, TFAM, TFB1M, COXI, COX5b mRNA and PGC-1?, TFAM, TOM20 protein content in skeletal muscle of group KE was significantly increased ?p<0.05 or p<0.05?. In addition, TFAM protein content in skeletal muscle of group KE was significantly higher than group E.6) Compared with group C, LC3, Atg5, ULK1, BNIP3, Parkin, FUNDC1 mRNA expression in skeletal muscle of group E was significantly increased?p<0.05 or P<0.01?, LC3-?/? ratio and Parkin, PINK1, BNIP3, ULK1 protein content in group E was significantly increased?p<0.05?, phosphorylation of AMPKaThr¹⁷² and ULK1Ser⁵⁵⁵ was significantly increased?p<0.05?. Compared with group K, LC3, Atg5, ULK1, BNIP3, FUNDC1 mRNA expression in skeletal muscle of KE group was significantly increased ?p<0.05?, Parkin, BNIP3, phosphorylation of AMPKaThr¹⁷² and ULK1Ser⁵⁵⁵ in KE group was significantly increased ?p<0.05?. BNIP3, FUNDC1 mRNA expression and Parkin, BNIP3, p62 protein content was significantly higher than group E ?p<0.05?.7) Compared with C group, MDA in skeletal muscle of group E was significantly reduced ?p<0.05? while T-SOD activity was significantly increased ?p<0.05?. Compared with group K, T-SOD activity in group KE was significantly increased ?p<0.05?. However, H2O2, MDA in skeletal muscle of KE group was significantly higher than E group ?p<0.05 or p<0.01?.CONCLUSIONS1) MFN2 deficiency disturbed the dynamic balance of skeletal muscle mitochondria fusion and fission which led to an increase of abnormal mitochondria, an increase of ROS generation and oxidative stress in skeletal muscle.2) MFN2 deficiency triggers an increase of mitochondria biogenesis in skeletal muscle which is helpful to maintain mtDNA content and mitochondrial respiratory function.3) MFN2 deficiency impaired autophagy and Parkin-mediated mitophagy in skeletal muscle, but initiated a compensatory increase of BNIP3-dependent mitophagy.4) Endurance training established higher level of mitochondria fusion and fission which was accompanied with an increase of mitochondrial biogenesis and autophagy, mitophagy. This suggests that exercise training have positive effects on skeletal muscle mitochondria quality control.Although MFN2 deficiency impaired autophagy and Parkin-mediated mitophagy induced by exercise training, mitochondria biogenesis and BNIP3-mediated mitophagy was further increased.5) Endurance training cannot improve skeletal muscle autophagy by activating AMPK-ULK1 pathway under the conditions of MFN2 deficiency.