Mechanism Underlying Spermidine-induced Myofiber Atrophy And Satellite Cell Activation In Adult Mice

Spermidine is a naturally occurring polyamine abundantly found in dry soybeans green peas,mushrooms,chicken liver and cheese,which is involved in multiple biological processes,such as DNA metabolism,autophagy and aging.It was found that diets supplemented with spermidine stimulates autophagy,attenuates D-galactose-induced muscle atrophy in mice,also ameliorates the myopathic defects of collagen ?-null mice.However,little is known about the effect of spermidine on non-pathological skeletal muscle.In this study,healthy adult mice were used as models to explore the effects of long-term spermidine injection on skeletal muscle growth and satellite cells activation.1 Effect of intraperitoneal injection spermidine on the growth of skeletal muscle in mice.Twelve adult healthy C57BL/6 mice were randomly divided into the control and spermidine groups.The control group was injected with saline(Con)and the spermidine(Spd)group was injected with 100 mg/kg of spermidine every other day for 4 weeks.The mice were fasted for 8 h before sampling.After that,the mice were weighed and anesthetized by i.p.administration of 80 mg/kg doses of pentobarbital sodium.The blood was collected for the detection of biochemical parameters.The gastrocnemius muscle was collected for morphological and molecular biology analysis.After 4 weeks of i.p.injection,spermidine significantly decreased the body weight(P<0.05)compared with the control group.When mice were sampled,the weight of gastrocnemius muscle was recorded.The muscular weight was significantly reduced by spermidine administration(P<0.05)compared with the control group.Blood biochemical assay indicated that glucose,urea nitrogen,lactate dehydrogenase levels increased significantly(P<0.05),triglyceride levels decreased significantly(P<0.05),but creatine kinase content did not change.In addition,spermidine treatment resulted in a remarkable decrease in muscle fiber size,as well as in the distribution of the cross-sectional area of muscle fibers.The distribution of small myofibers was increased by the spermidine treatment,whereas the distribution of large myofibers was decreased.Additionally,the ultrastructure of gastrocnemius muscle was detected by the transmission electron microscopy.It was shown that spermidine administration increased the length of I bands,but decreased the length of A bands.Therefore,we detected the expression of myosin heavy chain(MyHC)isoforms in the muscle at mRNA and protein levels.MyHC-?a(P<0.05)gene expression was significantly decreased in a mRNA level by the spermidine treatment,and the protein contents of both MyHC-? and MyHC-? isoforms(P<0.05)were significantly reduced in the spermidine group compared with the control group.2 Effects of spermidine on the activation of skeletal muscle satellite cells in mice.In adults,muscle size is controlled by the homeostasis of protein synthesis and breakdown.Spermidine administration did not affect the protein expression of mammalian target of mTOR,which is a critical molecule in muscle protein synthesis.Muscle atrophy involves the shrinkage of myofibers due to a net loss of proteins.The ubiquitin-proteasome system and the autophagy-lysosome pathway are the degradation systems involved in this process.Spermidine administration significantly induced the expression of atrogin-1,MuRF-1,Atg7 and LC3B at both mRNA and protein levels.Immunofluorescence showed that the MuRF-1 protein were mainly located in the cytoplasm of myofiber cells by spermidine administration,while the LC3B protein was mainly located in the nucleus.Pax7 and MyoD were used as specific markers for satellite cells.It was observed that LC3B and Pax7/MyoD were co-located in satellite cells.The population of LC3B+/Pax7+and LC3B+/MyoD+satellite cells was increased in skeletal muscle of the spermidine group compared with the control group.In addition,it was observed that spermidine administration significantly increased expression of Pax7,Myf5,MyoD,and MyoG at mRNA and protein levels.To determine the distribution of ACVR?B in skeletal muscle,myofibers were isolated and CD34 was selected as a cell surface marker for satellite cells.It was observed that ACVR?B+ is more obviously located in satellite cells than in myocytes.In addition,spermidine administration significantly decreased ACVRIIB expression(P<0.05)at both mRNA and protein levels.Phosphorylated Smad2/3(P<0.05),the downstream proteins of ACVRIIB signaling,were significantly reduced in the spermidine group compared with the control group.3 The effects of spermidine on transcription of ACVRIIB and MRFs.Since spermidine is an acetyltransferase inhibitor and a specific inducer of autophagy,UPLC was used to determine the polyamine levels in blood.It was found that spermidine administration significantly increased the concentration of spermidine in plasma(P<0.05).In addition,increasing spermidine levels inhibited the protein expression of EP300(P<0.05),which is a histone acetyltransferase.In flies and humans,it was found that histone H3 on lysine 56(H3K56)is acetylated by EP300.The ChIP assay indicated that H3K56 was hypoacetylated in the promoter of ACVRIIB(P<0.05)in the spermidine group compared with the control group.Furthermore,Smad3 binding sites in the promoters of autophagy and MRFs were predicted through JASPAR(http://jaspar.genereg.net).Spermidine administration did not affect the Smad3 binding to the promoters of Atg5,Atg7 and LC3B genes,were significantly decreased the enrichment of Smad3 in the promoters of Myf5(P<0.05)and MyoD(P<0.05).