Hydrogen Sulfide Signaling And Stress Response In Skeletal Muscle Functions

Hydrogen sulfide(H2S),a multifunctional gasotransmitter,participates in a wide range of cellular signal transduction and pathophysiological processes.In mammals,H2 S promotes vasodilation to regulate blood pressure,protects nerves or myocardium from oxidative damage,regulates metabolic balance or ion homeostasis in mitochondria,participates in the synthesis of proteins and lipids in endoplasmic reticulum,and mediates hormone secretion.It is found that the endogenous H2 S producing enzymes mainly include cystathionine ?-lyase(CSE),cystathionine ?-synthase(CBS)and 3-mercaptopyruvate sulfurtransferase(3MST),among which CSE is the key enzyme for the production of H2 S in peripheral organs or tissues.Skeletal muscle is a kind of plastic organ which is involved in the regulation of multiple systems,and it is responsible for the process of skeletal movement,posture maintenance and nutrition storage.In this study,skeletal muscle was taken as the object to study the effects of H2 S S-sulfhydration on metallothionein-1(MT-1)under oxidative stress,the involvement of H2 S in Golgi stress response,and the effect of H2 S metabolism on calcium(Ca2+)homeostasis during the physiological or pathological process of myogenesis or muscle atrophy.The main results are as follows.1.The viability of mouse skeletal myoblasts(C2C12)decreased in a dose-dependent manner with the increase of Cd Cl2 concentration.The treatment with Cd Cl2(30 ?mol·L-1)resulted in the increase of ROS level,the decrease of thiol content and the apoptotic cell death in C2C12 cells.At the same time,the expression of CSE and the production of H2 S increased significantly,but the protein level of 3MST did not change.After PPG or 2-KA incubation,it was found that 3MST was not involved in cell death induced by cadmium(Cd).Na HS treatment can reduce cell death or skeletal muscle damage,decrease ROS level,restore the content of intracellular thiol,and prevent cell necrosis.Cd can also increase the expression of MTF-1 as well.And the decrease of MT-1 expression enhanced Cd-stimulated oxidative damage or cell death.Under Cd stress,the expression of CSE protein increased,which was inhibited in si RNA-MT-1 cells.However,the expression of MT-1 in si RNACSE cells showed almost the same compared with normal cells.Exogenous Na HS treatment upregulated the expression level of S-sulfhydrated MT-1.Cd Cl2 treatment significantly increased the ionic strength of intracellular labile zinc(Zn2+),which was reversed by exogenous H2 S.It is indicated that H2 S modified MT-1 through S-sulfhydration or stable Zn-protein complex.2.Monensin(Mone)or Brefeldin A(BFA)induced the expressions of GM130 and ATP2C1 in C2C12 cells,impaired Golgi integrity,stimulated oxidative damage or cell apoptosis,induced the expressions of cleaved-caspase 3 and-caspase 7,increased intracellular Ca2+ and ROS levels,upregulated CSE expression,and induced endogenous H2 S production.Supplement of H2 S alleviated Golgi stress-induced oxidative damage and protected Golgi integrity.On the contrary,inhibitory CSE/H2 S system enhanced Golgi stress and upregulated Mu RF-1 or Atrogin-1 expression.Chelated intracellular Ca2+ with BAPTA could alleviate the situation above and induce ATP2C1 expression as well.ATF4 deficiency inactivated CSE in response to Golgi stress.Angiotensin II(Ang II)or BFA treatments can cause Golgi stress or skeletal muscle atrophy in mice.The symptoms of CSE knockout(CSE-KO)mice worsened significantly after administration,while the physiological concentration of Na HS could alleviate the stress response.3.Compared with 10-week old mice,the weight of 51-week old mice decreased,the expression of CSE in tibialis anterior(TA)muscle decreased,and the production of H2 S decreased significantly.Myogenin expression in muscle regulatory factors(MRFs)decreased by about 50%,PAX7 and Vimentin increased by about 50% and 45%,respectively.The lack of CSE worsened the phenomenon of muscle atrophy in the process of aging,and the expression of MRFs increased significantly.Cardiactoxin(CTX)was used to induce and construct skeletal muscle injury model in WT or CSE-KO mice.Muscle degradation occurred in varying degrees,in accompany with upregulating the expression of Myogenin,Myo D and Myosin.Compared with WT,the downregulation of Myogenin and the upregulation of PAX7 were enhanced in CSE-KO mice,while exogenous Na HS could normalize the muscle structure and the transcription levels of these 4 genes.4.In the differentiation medium,the expression of MEF2 c or MRF4 were significantly induced.Although exogenous Na HS treatment can induce cell differentiation,it cannot further enhance the expression level of the above 2 proteins.On the one hand,the specific antibody of MEF2 c can precipitate the MRF4 protein,indicating that the additional Na HS has ability to promote the formation of heterodimer MEF2c-MRF4 complex.On the other hand,both MRF4 and MEF2 c can combine with the promoter of Myogenin gene,and H2 S treatment can enhance the stability of the complex and promote muscle differentiation.In the process of myogenesis,inhibitory Cyclin D1 and Cyclin D3 protein expressions,increased F-actin protein level and cell multinucleation appeared.Exogenous Na HS treatment can further strengthen these results,and inhibit cell migration as well.To sum up,Ang II,BFA or CTX are performed in this paper to construct muscle injury or oxidative injury model of mice.And we also use MTT assay,si RNA transfection,realtime PCR,immunoblotting,Co IP or Ch IP assay,methylene blue-HPLC,cell or tissue section staining,biotin switch method and other in vitro experiments to prove the positive significance of CSE/H2 S signaling pathway in Cd stress resistance,maintaining Golgi integrity or intracellular Ca2+ homeostasis,protecting skeletal muscle from aging-dependent atrophy or CTX infusion injury,and promoting muscle regeneration.It provides a candidate factor and therapeutic regimen for the treatment of acute and chronic myopathy.