The Effects Of IGF-1 On Maintaining Structure And Function Of Sensory Reinnervated Skeletal Muscle Through Mitochondrial Signaling

A balance between protein synthesis and degradation maintains the healthy condition of normal skeletal muscle(SKM).When the balance is disrupted(e.g.,during proximal peripheral nerve injury),muscle atrophy occurs,along with a series of serious clinical problems,especially motor dysfunction.Poor motor function recovery manifests due to prolonged muscle denervation,which leads to a loss of regenerative potential,especially when nerve repair is delayed or when the injury is located at a proximal point.So,it is the principal work to regain the innervation of denervated SKM.However,in some severe situations(e.g.,nerve injuries that occur at highly proximal locations or when many motor axons are destroyed),sensory protection,which reconnects the sensory nerves to the injured motor nerve stumps,has been shown to be a promising technique.However,the donor sensory axons provided only a temporary or minor preservation of the denervated SKM unless the original motor nerves reached the denervated muscle in time.Multiple biological effects of IGF-1 were produced by activating the phosphatidylinositol 3-kinase(PI3K)/Akt signalling pathway.The Akt signalling cascade regulates the growth of many tissues by suppressing protein degradation and improving synthesis.But whether IGF-1 potentiates sensory innervation signalling has not been confirmed.Recently,it has been indicated that Akt activation maintains mitochondrial functions.The energy-generating mitochondria have been the focus of studies on the preservation of cell function.Mitochondria are highly dynamic in morphology continuously undergo fusion and fission.It is well recognized that mitochondrial fusion maintains the function of mitochondria and clears pathogenic mitochondria,as well as maintains the precision of the mitochondrial genome.In contrast,mitochondrial fission leads to loss of mitochondria and results in muscle atrophy.But whether IGF-1 improves mitochondrial function in sensory innervated SKM has not been studied.In the present study,we established relative models to investigate the function and mechanism of IGF-1 administration to sensory protection system.These data in the present study may provide novel therapy and experimental evidence for preventing denervated atrophy.Part ? IGF-1 enhances the protective effects of sensory reinnervation on skeletal muscle in vivoRestoring the contractile function of long-term denervated skeletal muscle(SKM)cells is difficult due to the long period of denervation,which causes a loss of contractility.Although sensory innervation is considered a promising protective approach,its effect is still restricted.IGF-1 has multiple biological effects to SKM and nervous system.But the effects of IGF-1 on sensory protection remain uninvestigated.In the present study,25 rats were divided randomly into 5 groups.The experiments designed as follows:(1)Sham operation group,a longitudinal incision exposed the trifurcation of sciatic nerve to free the tibial,peroneal and sural nerves,and the incision was irrigated thoroughly and closed layer by layer.(2)Denervation group,same incision was performed and the tibial nerve was transected.The proximal stumps were ligated and sewn into a cap to prevent renascent fibers from reconnecting the distal stumps and then the incision was irrigated and sutured in layers.(3)IGF-1 group,same operation in sham operation group were performed and gastrocnemius(GAS)muscles of right hind limbs were injected with 10?g of IGF-1 every 3 days following surgeries until 8 weeks.(4)Sensory protection group,tibial nerve and sural nerve was transected,the distal ending of tibial nerve was anastomosed to the proximal ending of sural nerve in an end-to-end fashion using a 9-0 nylon suture.The proximal stump of tibial nerve was also ligated and sewn into a cap and the incision was irrigated and closed.(5)IGF-1 + sensory protection group,operation was the same with sensory protection group and IGF-1 administration was the same as IGF-1 group.GAS muscle samples were collected at 8th week.After that,muscle wet weight was recorded immediately.Muscle wet weight and cross-sectional area(CSA)were the directly perceived and vivid reflection of the muscle condition.The CSA and wet weight of GAS muscles increased in the IGF-1,sensory protection,and IGF-1 + sensory protection groups.The average area and weight in the sensory protection groups was moderately higher than that in the denervated group.Furthermore,the IGF-1 + sensory protection group achieved a higher improvement than the groups that received sensory protection alone.The contractile protein myosin heavy chain-1(MyHCl)is an important mechanical component of myofilaments.MyHC1 alterations reflect the dynamic balance between protein synthesis and degradation and the functional status of SKM cells.An obvious downregulation of MyHC1 has been demonstrated in denervated muscle.The MyHC1 expression levels of different groups corresponded to CSA and wet weight.The contractile protein downregulation was largely dependent on mitochondrial status.IGF-1-induced a higher mtDNA:nuDNA ratio in the sensory reinnervated SKM,which represent the mitochondrial content level.This result demonstrated that sensory protection with or without IGF-1 prevented both the GAS muscle atrophy and mitochondria loss caused by denervation,and the combination of IGF-1 and sensory protection achieved the best effects.The results of the present study provide new clues and understanding for the function of IGF-1 on sensory protection.Part ? The protective effects of IGF-1 on skeletal muscle cells with sensory innervation in vitroPreventing muscle atrophy and restoring the contractile function of long-term denervated SKM cells are so difficult that the previous studies are not able to figure out for a long time.We have investigated that IGF-1 potentiated the effects of sensory protection in vivo.To better control the experimental conditions and to find the change of mitochondria,we modelled sensory innervated in vitro using SKM cells.In the present study,the SKM cell cultures and neuromuscular cocultures were treated as follows.(1)Control group(SKM group):The SKM cells were cultured for 2 days with DMEM/F-12 supplemented with 20%fetal bovine serum and another 4 days with DMEM/F-12 medium for the coculture;(2)Coculture group:The SKM cells were cultured for 2 days and then were cocultured for another 4 days with DRG explants;(3)SKM + IGF-1 group:The SKM cells were cultured for 2 days with DMEM/F-12 supplemented with 20%fetal bovine serum and 2 days with coculture DMEM/F-12 medium and another 2 days with coculture DMEM/F-12 medium which would contain IGF-1(20 nmol/L);(4)Coculture +IGF-1 group:The SKM cells were cultured for 2 days and another 2 days with coculture DMEM/F-12 medium and then another 2 days with coculture DMEM/F-12 medium which would contain DRG explants plus IGF-1.The morphology of SKM cells and DRG neurons in different cultures with different treatments was observed by double immunofluorescence labelling.The morphology of the SKM cells in cultures with different treatment conditions was similar.However,the average area was higher in the SKM + DRG group compared with SKM group.furthermore,both the average length and area in the SKM + IGF-1 group were increased,and the average length and area in the SKM + DRG + IGF-1 group was even higher.In the neuromuscular cocultures,DRG neurons sent axons across or terminating on the surface of SKM cells.The dense network of axons was observed on the surface of the SKM cells around the DRG explants.IGF-1 incubation could enhance formation of the axon network.Hence,a much denser network was observed on the surface of SKM cells after IGF-1 treatment.Corresponding with the muscle weight and CSA,the SKM +DRG group showed mild upregulation of the expression of MyHC1 content,with the highest elevation in the SKM + DRG + IGF-1 group,as indicated by comparison to the SKM group via Western blot analysis.IGF-1 incubation increased the mtDNA/nuDNA ratio in each SKM cell in both SKM cells cultured alone and SKM cells with DRG explants.However,IGF-1 may have a large latent capacity to increase the number of mitochondria in SKM cells that received sensory innervation.More filamentous and tubular or thread-like mitochondria shown by MitoTracker Red staining were found in the SKM cells after sensory innervation and IGF-1 treatment.With the application of sensory innervation and IGF-1,cytosolic Cytochrome C(Cyt C)levels decreased markedly.These results suggested that IGF-1 could enhance the effects of sensory innervation,specifically the promotion of SKM cell condition and mitochondrial pattern.This study provides novel data for the effects of IGF-1 on sensory protection,and supplied new evidence for the relationship between SKM cell condition and mitochondria.Part ? The protective effects of IGF-1 on skeletal muscle cells with sensory innervation through mitochondrial signalingContractile function loss of the long-term denervated SKM cells is hard to avoid due to the long period of denervation.Even if sensory innervation is considered a promising protective approach,its effect is still not sufficient enough.Based on our previous study,IGF-1 could potentiated the effects of sensory protection by preventing denervated muscle atrophy,improving the condition of denervated muscle cells as well as the mitochondria status.In this study,the mechanisms of the effects of IGF-1 on the sensory innervation in vitro were investigated by focusing on Akt,Mull,and AMPKa,the downstream targets of IGF-1.There was no difference in the phosphorylation of Akt between the SKM and neuromuscular cocultures.To further exclude a different timing of activation,we tested Akt phosphorylation level at 24h,48h,and 72h after sensory innervation,and there was no difference compared with SKM group.But the pAkt levels in the SKM+ IGF-1 group increased moderately;additionally,the highest activation level was observed in the SKM + DRG + IGF-1 group.To further clarify the role of the PI3K/Akt signalling pathway in improving SKM cell status by Akt phosphorylation in IGF-1-treated neuromuscular cocultures,the PI3K inhibitor LY294002(10?mol/L)was applied to block the effects of IGF-1 on Akt phosphorylation in the SKM cells in neuromuscular cocultures.The further study suggested that IGF-1 treatment improved mitochondria pattern,decreased the atrogin-1 mRNA and MuRF1 mRNA levels and finally promoted MyHC1 protein expression by Akt phosphorylation in neuromuscular cocultures.Mull is a key regulator of SKM mitochondrial morphology.The expression of Mull and its mRNA could be inhibited by the presence of IGF-1.The effects of IGF-1 on the inhibition of Mull expression could be blocked by the PI3K inhibitor LY294002.To further investigate whether Mull gene overexpression could counteract the effect of IGF-1,the rat Mull gene was inserted into a lentiviral vector,which was transfected into SKM cells.The validity of Mull lentivirus transfection was confirmed in Mull lentivirus-infected SKM cells even in the presence of sensory innervation.With the successfully established Mull expression culture system,the effects of Mull on its downstream target or signalling cascade were tested.These results suggested that Mull overexpression could abolish the effects of IGF-1 on mtDNA level elevation,mitochondrial fission inhibition,mitochondrial integrity improvement,atrogin-1 and MuRF1 expression inhibition,and MyHCl expression promotion.To further clarify the mechanisms of IGF-1 signalling for the improvement of myocyte status,adenosine 5'-monophosphate-activated protein kinase a(AMPKa)was measured as a downstream target of mitochondrial energy production.The effects of AMPKa activation on contractile protein expression and its mechanisms were investigated.5-aminoimidazole-4-carboxamide-1-D-ribonucleoside(AICAR)was commonly used to activate AMPK directly without changing cellular ATP,ADP,and AMP concentrations.In the present study,the lower pAMPK? and levels indicated suppression of AMPKa in the SKM + DRG + IGF-1 group compared with the SKM+ DRG group.The further study suggested that AMPK? activation in the neuromuscular coculture system could counteract the effects of IGF-1 on AMPKa inhibition,UPS inhibition,and MyHC1 protein expression promotion.These findings provide rationale and experimental evidence for the mechanisms of IGF-1 on improving myocyte status in neuromuscular cocultures.