The Role And Mechanism Of Ach In NE-mediated C2C12 Cell Differentiation

Background:Skeletal muscle is not only the basis of human health,but also guarantee of human physical activity and exercise capacity.Muscle atrophy has been the difficult problem of human health to overcome,skeletal muscle atrophy not only bring trouble to the patient’s own treatment,but also to the community a heavy burden.Previous studies have shown that skeletal muscle atrophy is associated with denervation,malnutrition,disuse,and cAchexia such as cancer,diabetes,age-related aging,kidney disease,heart failure,chronic obstructive pulmonary disease（COPD）,sepsis,and severe burns.Further research found that:abnormal sympathetic regulation can cause diabetes,while diabetes is often accompanied by sympathetic nerve damage.These results suggest that the abnormality of sympathetic regulation may also be related to the atrophy of skeletal muscles.However,there are few related studies.However,in fact,in addition to the innervation of the motor nerves,the skeletal muscle itself is innervated by the sympathetic and vasodilator nerves.However,when the sympathetic and vasodilatory nerves are excited,the main neurotransmitter that exerts a biological effect is acetylcholine.However,it is not clear whether acetylcholine is involved in the proliferation and differentiation of skeletal muscle stem cells,thereby affecting skeletal muscle self-renewal or repair.Objective:to investigate the effect and possible mechanism of acetylcholine（Ach）on C2C12 cells differentiation into skeletal muscle cells and muscle atrophy mediated by norepinephrine（NE）.Methods:Muscarinic acetylcholine receptors（m-AchRs）expressions in C2C12 cells were detected using immunofluorescence cytochemistry staining,QPCR and Westernblot,and the cultured C2C12 cells that reAched 70%confluence in vitro were used to establish the model of skeletal muscle stem/progenitor cells differentiation under 2%horse serum with high glucose DMEM.CCK8 assay were used to evaluate C2C12 proliferation.And then,immunofluorescence staining and QPCR were used to detect the changes of muscle fiber types of C2C12 cells differentiation,and associated regulators such as Myogenin（MyoG）,Myod and Myf5.Finnally,the effects of Ach,or Ad-Kpc1/Ad-shKpc1 on NE-mediated C2C12 cells differentiation inhibition using the above-mentioned method.Results:（1）immunofluorescence staining,RT-PCR and Western blot showed that all mAchRs subtypes including M1,M2,M3,M4 and M5 were expressed in C2C12 cells,and M2-AchR highest expression and M3-AchR lowest expression.（2）CCK8 assay showed that different concentrations acetylcholine had not significant effect on on C2C12 cell proliferation.QPCR and immunofluorescence staining showed that 10^-5M acetylcholine inhibited C2C12 cell differentiation.QPCR and immunofluorescence staining showed that low concentration acetylcholine had not significant effect on the changes of fiber types derived from C2C12 cells.However,high concentration acetylcholine inhibited the formation of fast muscle fibers and slow muscle fibers in C2C12 cells.（3）.Compared with control group,10^-55 M Ach group and 10^-55 M NE group showed the reduced numbers of myotubes under light microscopy,and NE group greater decreased than Ach group.Similarly,QPCR and immunofluorescence staining showed the decreased numbers of fast and slow muscle fibers in Ach group or NE group,compared to control group.However,in combination of Ach and NE group,the slow muscle fibers were reduced,but maintained greater than NE group.（4）.Compared with control group,Ad-Kpc1 promoted myotube differentiation of C2C12cells under under light microscopy;however,Ad-sh Kpc1 reduced its effects.And Kpc1 overexpression obviously abolished the inhibitory effects of NE on C2C12 cells differentiation.Knock down of Kpc1 did not markedly enhance the NE’effects.Using QPCR and immunofluorescence staining,C2C12 cells with and without the forced Kpc1expression showed the increase in fast and slow muscle fibers,mainly in slow muscle fibers.By contrast,Knock down of Kpc1 by shRNA reduced the muscle fibers,mainly in slow muscle fibers.Furthermore,Ad-Kpc1 obviously abolished the inhibitory roles in NE-mediated muscle fibers formation of C2C12 cells,especially in slow muscle fibers.Inversely,Ad-sh Kpc1 showed the decrease of fast and slow muscle fibers following the stimulation of NE,especially in slow muscle fibers.Conclusion:（1）.Low concentration of Ach facilitates differentiation of C2C12 cells mainly through binding to M2 receptors on C2C12 cells,only high dose 10^-55 M Ach inhibite C2C12 cells differentiation,mainly in slow muscle fibers.（2）.NE inhibite C2C12 cells differentiation of both fast and slow muscle fibers,greatest decrease in slow muscle fibers.（3）.Ach partially abolished the inhibitory effects of NE-mediated C2C12 cells differentiation.（4）.Kpc1 induced C2C12 cells differentiation,and markedly abolished the inhibitory effects of NE-mediated C2C12 cells differentiation.