Effects And Mechanisms Of Cigarette Smoke Exposure During Pregnancy On Skeletal Muscle Phenotype Similar To Congenital Clubfoot In Fhl1^-/y Offspring Mice

Objectives:Congenital clubfoot?CCF?is a common birth defect that seriously endangers children's health.The abnormal muscle phenotypes of CCF mainly include:muscle weakness,muscle atrophy,increased number of type I fibers,destruction of the Z-band structure,expansion of the sarcoplasmic reticulum and turgidity of the mitochondrion.At present,the pathogenesis of CCF is unclear.However,it is believed that the occurrence of CCF is due to the interaction of genetic factors and environmental agents which cause abnormalities in certain genes and their products related to the development of distant limb bones and neuromuscularity,during the embryonic development.In the previous work,our team mainly conducted a preliminary study on CCF and found that Fhl1 gene was a suscept-ble gene of CCF.The Fhl1 gene is located at Xq26 and highly expressed in skeletal muscle.It is involved in skeletal muscle cytoskeleton maintenance,myoblast differentiation,mechanotransduction,and regulation of muscle fiber size.In order to explore the relationship between Fhl1 gene and muscle tissue abnormality in CCF patients,our team successfully produced Fhl1^-/y mice using TALENs technology,found that Fhl1^-/y/y mice have reproductive ability and have similar symptoms to CCF,such as destruction of the Z-band structure,expansion of the sarcoplasmic reticulum and turgidity of the mitochondrion.Therefore,Fhl1^-/y mice can be used as an appropriate animal model to investigate the mechanisms of the development of CCF phenotype.In addition,genetic epidemiological survey results indicate that smoking during pregnancy is an important risk environmental factor for CCF.Maternal smoking during pregnancy,the harmful components in cigarette can directly cause abnormal fetal development across the placental barrier,and can also indirectly affect embryo development by damaging the placental structure.Therefore we speculate maternal smoking during pregnancy may aggravate skeletal muscle phenotype similar to CCF in Fhl1^-/y offspring mice.However,the mechanisms of how Fhl1 gene knockout causes skeletal muscle phenotype similar to CCF and CS exposure during pregnancy aggravates skeletal muscle phenotype similar to CCF in Fhl1^-/y offspring mice,are unknown.Study has shown that the pathogenesis of CCF was related to cell apoptosis and inflammation.P2RX7 belongs to the P2X receptor family,which expression is increased in certain myopathy and it is used as a target for the treatment of myopathy.P2RX7 can induce cell autophagy,apoptosis and pyroptosis?inflammatory cell death?via cell-specific downstream signaling.Abnormal autophagy is involved in muscle damage in duchenne muscular dystrophy?DMD?patients and mdx mice?a murine model for DMD?,and regulating autophagy can be used as a treatment method for DMD.Apoptosis plays an important role in skeletal muscle atrophy caused by denervation.P2RX7 activates NLRP3 inflammatory bodies by triggering complex downstream signaling,and then promotes the occurrence of pyroptosis.In addition,NLRP3 inflammatory bodies are significantly activated in skeletal muscle of DMD patients and mdx mice.The above results suggest that P2RX7-mediated autophagy,apoptosis and pyroptosis play important roles in skeletal muscle abnormalities.Skeletal muscle is a plastic organ.Effective and precise metabolic regulation is required to maintain skeletal muscle structure and function.Protein synthesis and degradation are closely related to maintaining cell homeostasis.When protein degradation exceeds protein synthesis,skeletal muscle atrophy occurs.Histone deacetylases?HDACs?play an important role in skeletal muscle atrophy by regulating protein synthesis and degradation.The class O of the forkhead transcription factors?FOXOs?is downstream target protein of HDACs.Activation of FOXOs inhibits the expression of protein synthesis-related protein 4EBP,promotes the expression of ubiquitin-protease-related protein TRIM63,and the expression of autophagy-lysosomal degradation-related protein LC3.HDACs promote skeletal muscle atrophy by activating FOXOs-dependent?protein synthesis and degradation-related genes?transcriptional regulation.The above results suggest that P2RX7-mediated skeletal muscle cell autophagy,apoptosis and pyroptosis,and HDACs-regulated protein synthesis and degradation play important role in skeletal muscle development and function maintenance.However,their role in skeletal muscle phenotype similar to CCF in Fhl1^-/y mice and skeletal muscle abnormal phenotypes caused by cigarette smoke?CS?exposure are unclear.After exposure to CS during pregnancy,can harmful substances cross the placental barrier through P2RX7-mediated skeletal muscle cell death to aggravate the skeletal muscle phenotype similar to CCF in Fhl1^-/y offspring mice,which is still unclear.Therefore,this study first investigated the effects of genetic factor?Fhl1gene knockout?and environmental factor?CS exposure?on the skeletal muscle phenotype of adult mice,and mechanisms of P2RX7-mediated skeletal muscle cell autophagy,apoptosis and pyroptosis,and HDACs-regulated protein synthesis and degradation in this process.Second,explore the role of P2RX7-mediated skeletal muscle cell death in aggravating the phenotype similar to CCF of Fhl1^-/y offspring skeletal muscles caused by CS exposed during pregnancy.This study reveals the role of the interaction between genetic factor?Fhl1 gene knockout?and environmental factor during pregnancy?CS exposure?in the pathogenesis of CCF.This study provides a theoretical basis for further understanding the mechanisms of abnormal muscle phenotype in CCF patients,exploring environmental factor for preventing CCF during pregnancy,guiding aristogenesis and exploring potential targets for treatment of CCF.Methods:Part?:Fhl1 gene knockout caused the formation of muscle phenotype similar to CCF1.Fhl1^-/y mice were made using TALENs technology,and 2.5-month-old WT and Fhl1^-/y mice were selected for subsequent experiments.2.Morphological structure of skeletal muscle of WT and Fhl1^-/y mice was observed by hematoxylin-eosin staining?HE?staining.3.The indicators for autophagy?BNIP3,Beclin1,LC3B,P62?,apoptosis?Apaf1,Bcl-2,caspase9,cleaved-caspase3?,pyroptosis?NLRP3,ASC,cleaved-caspase1,IL-1??,muscle fiber regeneration?PAX7,MYOD,MEF2C,MYH?,protein synthesis?mTOR,P70,p-P70?and degradation?TRIM63,FBXO32?,and FHL1,P2RX7,HSP90,HDAC1/2,FOXO1,ac-FOXO1 and p-FOXO1 were detected at the protein level by western blot or immunohistochemistry?IHC?.4.TUNEL staining was used to detect the level of apoptosis in skeletal muscle of WT and Fhl1^-/y mice.5.RT-PCR was used to detect the transcription levels of MYH?,MYH?A,MYH?X and MYH?B in skeletal muscle of WT and Fhl1^-/y mice.Part?:Trichostatin A inhibited skeletal muscle atrophy induced by cigarette smoke exposure in mice1.Making a mouse model of CS exposure.The mice were divided into three groups:room air exposure?Control?,CS exposure?CS?and CS exposure+Trichostatin A?CS+TSA?,at least five mice in each group.Mice in the Control group were placed in a whole-body exposure system for 120 min,twice daily for 40days.Mice in the CS exposure group were placed in a whole-body exposure system and exposed to CS for 40 days?twice a day,6 cigarettes each time,each cigarette exposed for 20min?.Mice in the CS+TSA group were exposed to CS with intraperitoneal injection of TSA?0.6 mg/kg,every other day for 40 days?.2.Morphological structure of skeletal muscle of each group was observed by HE staining.3.Western Blot or IHC techniques were used to detect the protein level of HDACs,FOXO1,p-FOXO1,FOXO3 and p-FOXO3,and the indicators for autophagy?LC3B,P62?,apoptosis?caspase9,cleaved-caspase3?,pyroptosis?NLRP3,ASC,cleaved-caspase1,IL-1??,protein synthesis?4EBP,p-4EBP,P70,p-P70?,protein degradation?TRIM63,FBXO32,MUL1?,muscle fiber differentiation?MEF2C,MYOD,MYH,PITX1?and cytoskeletal proteins?MYBPC2,LDB3?.Part?:Effects and mechanisms of cigarette smoke exposure during pregnancy on skeletal muscle phenotype similar to congenital clubfoot in Fhl1^-/y offspring mice1.Mouse model of CS exposure during pregnancy.Female C57BL/6 mice(Fhl1^+/-)and male C57BL/6 mice?WT?mated at 3 months of age.On the 11th day of pregnancy,pregnant mice were divided into air exposure group?Control?,CS exposure?CS?.Pregnant mice in the Control group were exposed to room air in a whole-body exposure system for 80 min,twice daily,until the offspring were born.Pregnant mice in the CS exposure group were exposed to CS in a whole-body exposure system?twice a day,4 cigarettes each time,each cigarette exposed for20min?,until the offspring were born.Male mice with genotypes of WT and Fhl1^-/y in the offspring of Control group and CS group?9 days of exposure to CS during pregnancy?were selected for subsequent experiments.2.Morphological structure of skeletal muscle of each group was observed by HE staining.3.Western Blot was used to detect the protein level of P2RX7 and the indicators for pyroptosis?NLRP3,ASC,cleaved-caspase1,IL-1??and cytoskeletal proteins?MYBPC2,LDB3?.Results:Part?:Fhl1 gene knockout caused the formation of muscle phenotype similar to CCF1.Compared with WT mice,muscle fibers with central nucleus,atrophic muscle fibers and necrotic muscle fibers were significantly increased in muscle tissue of Fhl1^-/y mice.2.P2RX7 and autophagy-related proteins?HSP90,BNIP3,Beclin1,LC3B,P62?,apoptosis-related proteins?Apaf1,Bcl-2,caspase9,cleared-caspase3?and pyroptosis-related proteins?NLRP3,ASC,cleared-caspase1,IL-1??were significantly higher expressed in muscle tissue of Fhl1^-/y mice than WT mice.3.The expression of muscle fiber regeneration-related proteins?PAX7,MYOD,MEF2C,MYH?were significantly higher in muscle tissue of Fhl1^-/y mice than WT mice.4.The expression levels of HDAC1/2,FOXO1,and protein synthesis and degradation related proteins?TRIM63,FBXO32,mTOR,P70,p-P70?were significantly higher in muscle tissue of Fhl1^-/y mice than WT mice.Part?:Trichostatin A inhibited skeletal muscle atrophy induced by cigarette smoke exposure in mice1.Compared with mice in the Control group,the wet weight of skeletal muscles of mice after CS exposure was significantly reduced,and the muscle fibers with central nucleus were significantly increased.2.Compared with mice in the Control group,the expression levels of HDAC1/2and protein synthesis and degradation related proteins?4EBP,p-4EBP,P70,p-P70,TRIM63,FBXO32,MUL1,LC3,P62?were significantly higher in the muscle tissue of mice after CS exposure.3.Muscle fiber differentiation-related proteins?MEF2C,MYOD,MYH,PITX1?and cytoskeleton-related proteins?MYBPC2,LDB3?were significantly higher in the muscle tissue of mice after CS exposure than mice in the Control group.4.The expression of P2RX7,apoptosis-related protein?caspase9,cleared-caspase3?and pyroptosis-related protein?NLRP3,ASC,cleared-caspase1,IL-1??were significantly higher in muscle tissue of CS exposure mice than in Control group mice.5.Compared with the mice in the CS exposure group,the wet weight of skeletal muscles of mice was significantly increased,and the muscle fibers with central nucleus were significantly decreased after TSA treatment.6.The expression levels of HDAC1/2 and ubiquitination degradation-related proteins?TRIM63,FBXO32,MUL1?in TSA-treated mice's muscle tissue were significantly lower than those in CS group.7.The expression levels of muscle fiber differentiation-related proteins?MEF2C,MYOD,MYH,PITX1?and cytoskeletal proteins?MYBPC2,LDB3?in TSA-treated mice's muscle tissue were significantly lower than those in the CS group.8.The expression levels of P2RX7,apoptosis-related protein?caspase9,cleared-caspase3?and pyroptosis-related protein?NLRP3,ASC,cleared-caspase1,IL-1??were significantly lower in TSA-treated mice's muscle tissue than those in the CS group.Part?:Effects and mechanisms of cigarette smoke exposure during pregnancy on skeletal muscle phenotype similar to congenital clubfoot in Fhl1^-/y offspring mice1.Compared Control-KO group with Control-WT group,there was no significant difference in the weight of offspring.Compared CS-KO group with the CS-WT group,there was no significant difference in the weight of offspring.Compared with Control-WT group,the weight of offspring was significantly decreased in CS-WT group.Compared with Control-KO group,the weight of offspring was significantly decreased in CS-KO group.2.The expression of P2RX7 and cell pyroptosis-related proteins?NLRP3,ASC,cleaved-caspase1,IL-1??was significantly higher in the skeletal muscle of mice in Control-KO group than that in Control-WT group,in CS-KO group than that in CS-WT group,in CS-WT group than that in Control-WT group,in CS-KO group than that in Control-KO group.3.The expression of cytoskeletal protein?MYBPC2,LDB3?was significantly higher in the skeletal muscle of mice in Control-KO group than that in Control-WT group,in CS-KO group than that in CS-WT group,in CS-WT group than that in Control-WT group,in CS-KO group than that in Control-KO group.Conclusions:Part?:Fhl1 gene knockout caused the formation of muscle phenotype similar to CCF1.Fhl1 gene knockout caused the formation of skeletal muscle phenotype similar to CCF.2.P2RX7-mediated abnormal autophagy,apoptosis and pyroptosis,and abnormal muscle fiber regeneration,abnormal protein synthesis and degradation were involved in the formation of skeletal muscle phenotype similar to CCF in Fhl1^-/y mice.Part?:Trichostatin A inhibited skeletal muscle atrophy induced by cigarette smoke exposure in mice1.CS exposure induced skeletal muscle atrophy and myofiber abnormality.2.Abnormal protein synthesis and degradation,abnormal muscle fiber differentiation,abnormal expression of cytoskeleton protein and cell death?apoptosis,pyroptosis?were involved in skeletal muscle atrophy and abnormal muscle fiber structure caused by CS exposure.3.TSA may inhibit HDAC1/2 and thus inhibited ubiquitination degradation,muscle fiber differentiation,cytoskeleton protein,apoptosis and pyroptosis,and finally restored skeletal muscle atrophy and abnormal muscle fiber structure caused by CS exposure.Part?:Effects and mechanisms of cigarette smoke exposure during pregnancy on skeletal muscle phenotype similar to congenital clubfoot in Fhl1^-/y offspring mice1.CS exposure during pregnancy aggravated skeletal muscle phenotype similar to CCF in Fhl1^-/y offspring mice.2.P2RX7-mediated abnormal pyroptosis and abnormal expression of cytoskeleton proteins?MYBPC2,LDB3?participated in the exposure of CS during pregnancy aggravated skeletal muscle phenotype similar to CCF in Fhl1^-/y offspring mice.