Systematic Screening Of Skeletal Muscle Secretory Factors That Regulate The Function Of Skeletal Muscle Stem Cells

Adult skeletal muscle stem cells,also known satellite cells,are located between the plasma membrane of a muscle fiber and the basal lamina.As an immediate niche,myofiber regulates muscle stem cell function by secreting various cytokines.However,it is largely unknown how myofiber-secreted factors are regulated.Interestingly,there are two metabolically distinct myofibers(oxidative and glycolytic)in skeletal muscle tissues.We and others demonstrate the muscle stem cells localized on oxidative and glycolytic myofibers have significant difference in multiple aspects,including stem cell numbers,heterogeneity,and engraftment efficiency.The underlining mechanism remains elusive.We reasoned that oxidative and glycolytic myofibers might metabolically regulate certain secreted factor(s)which act as paracrine signaling to modify cellular behavior or functions of muscle stem cells localized thereon.The specific focus of my project is to systematically identify metabolically-regulated myofiber-secreted factors potentially implicating muscle stem cell function.To test our hypothesis,we firstly analyzed RNA-Seq data on tibialis anterior(TA,predominantly glycolytic)and soleus(Sol,mainly oxidative)muscle.Totally,we found 1301 differentially expressed genes between TA and Sol muscle.GO and KEGG analysis demonstrated that the differentially expressed genes are enriched in muscle contraction and metabolism which is consistent with the physiological function and metabolic features of the two muscle groups.Next,we further identified secreted factors from the differentially expressed genes by searching secreted protein database.Out of 1301 differentially expressed genes,there are 86 genes encoding secreted protein.To further understand whether those factors are regulated by muscle metabolism,we established four kind of metabolically modified experimental system:1)muscle from exercise or sedentary mice control,2)muscle from fasting or feeding mice,3)muscle from PPAR-?/? transgenic(almost oxidative myofibers)and wild type control mice,4)C2C12 myo tube cultured in pyruvate-free medium and normal medium.Using above four kind of metabolically modified experimental system,we demonstrate 24 out of 86 muscle-secreted factors are potentially regulated by muscle metabolism.Physiologically,skeletal muscle is one of the most active metabolic organ,thus,skeletal muscle constantly generate plenty of metabolites.Recently,several reports demonstrate intermediate metabolites play important regulatory roles in a paracrine or endocrine manner.To identify differentially metabolites secreted by TA and Sol muscle,we performed metabolomics analysis on conditional medium from TA and Sol muscle and found that 68 differentially secreted metabolites.Combined the metabolomics data and RNA-Seq analysis,we found that several metabolic enzymes differentially expressed between TA and Sol contribute the concentration difference of those secreted metabolites.We will further perform functional investigation of the differentially metabolites secreted by TA and Sol muscle in regulating muscle stem cells function in the future.Recent studies demonstrate exosomes play important regulatory roles by mediating intracellular trafficking of various cargos,including peptide,protein,RNA,and metabolites.To further investigate whether those muscle-secreted protein and metabolite are mediated by muscle-derived exosomes and have regulatory function on muscle stem cells,we isolated exosomes from muscle-derived conditional medium.Functional assay demonstrate muscle-derived conditional medium and purified exosomes have significantly regulatory roles on muscle stem cell proliferation and differentiation.We will further identify the functional components from exosome by proteomics and metabolomics in the future.Collectively,we systematically identified several candidates potentially regulated by muscle metabolism.Further functional investigation on muscle stem cells will provide valuable evidence for myofiber as a metabolic niche in regulating muscle stem cell behavior and functions.