The Role And Mechanism Of Transcription Factor ZBED6 In Porcine Skeletal Muscle Growth And Sepsis Induced Muscle Atrophy

Pigs are important agricultural animals.For a long time,pork has accounted for more than 60%of China’s meat consumption,making it crucial for improving the quality of national life.Additionally,pigs share remarkable similarities with humans in terms of their anatomy,physiology,immunology,and genome.This similarity makes them incredibly valuable in the field of medicine,as they can serve as crucial models for medical research and potentially as ideal organ donors for human transplantation.The dual significance of pigs in both farming and medical research makes the use of gene editing technology in pigs exceptionally valuable.This technology not only shortens the genetic breeding cycle and enhances the genetic stability of improved domestic pig breeds but also provides crucial preclinical evidence and insights for the treatment of human diseases.This approach is widely employed in various studies related to human diseases.Research has shown that ZBED6,a transcription factor,plays a vital role in regulating gene programs that control various cellular processes and cell states.It is closely associated with muscle growth in mammals under normal conditions and also holds significance in various disease states.As a result,ZBED6^-/-pigs have the potential to offer dual benefits in the fields of animal husbandry and medical research.This study employed Bama mini pigs with Chinese characteristics as the research subjects and successfully applied CRISPR/Cas9 gene editing technology to create ZBED6^-/-pigs.Utilizing high-throughput sequencing technology,alongside a comprehensive array of imaging,pathological histology,molecular biology,and cell biology methods,we elucidated the molecular mechanism through which ZBED6 regulates skeletal muscle growth and development while also conferring resistance to septic muscle atrophy.Furthermore,we assessed the feasibility of ZBED6 as a dual target for promoting muscle growth and combating septic muscle atrophy in domestic pigs.The key findings of this study are as follows:1.ZBED6 knockout promotes pig muscle growth,muscle fiber hypertrophy,and increases meat production without negative effects on meat quality.This study utilized CRISPR/Cas9 gene editing technology to successfully generate ZBED6^-/-pigs,which were subsequently expanded and bred to the F8 generation for further experiments.Growth trait monitoring indicated that,at 8 months of age,ZBED6^-/-pigs exhibited a 15.18%increase in average weight compared to wild-type pigs（P=0.003）.Additionally,body length,width,and chest circumference significantly increased,improving feed efficiency.Slaughter tests revealed that ZBED6^-/-pigs had a 13.36%increase in carcass lean meat rate and a 6.17%decrease in carcass fat rate,while bone and skin rates between the two genotypes were similar.This suggests that the primary factor contributing to the increased carcass weight in ZBED6^-/-pigs was greater skeletal muscle mass,resulting in increased meat production.Histological analysis further demonstrated that compared to wild-type pigs,the eye muscle area of ZBED6^-/-pigs increased by 22.29%,with a significant increase in average single muscle fiber area and a decrease in average muscle fiber density.Muscle development-related genes MYHC and MYOG protein were significantly elevated in muscle tissue,indicating that the increased meat production in ZBED6^-/-pigs was mainly due to an increase in the size of individual muscle fibers rather than an increase in the number of muscle fibers.Meat quality-related traits（such as redness,cooking loss,amino acid and fatty acid composition,etc.）were assessed in the longissimus thoracic muscle of 8-month-old wild-type and ZBED6^-/-pigs.The results demonstrated that ZBED6 knockout increased meat quantity without affecting meat quality.2.ZBED6 negatively regulates ISLR expression and activates the downstream canonical Wnt pathway,thus promoting myoblast differentiation and myotube formation,which,in turn,promotes muscle growth.In order to explore the potential mechanism by which ZBED6 enhances porcine skeletal muscle growth,this study first conducted a joint analysis of multi-species and multi-tissue transcriptome datasets and ZBED6 gene evolution data.The results indicate that the ZBED6 is conserved across species in mammalian skeletal muscle.This study utilized multi-omics technology for joint analysis to identify genes that can specifically bind to ZBED6 and verified the target genes directly regulated by ZBED6 through dual-luciferase experiments.The results demonstrate that ISLR is a novel evolutionarily conserved target gene directly negatively regulated by ZBED6 and plays a crucial role in ZBED6’s promotion of muscle growth.Furthermore,through transcriptomics,cell biology,and molecular biology experiments,this study discovered that ZBED6 increases the expression of DVL2,a key protein in the Wnt signaling pathway,by negatively regulating ISLR expression.This,in turn,promotes the entry ofβ-catenin into the nucleus,activates the downstream canonical Wnt pathway,and enhances MYOG protein expression,ultimately facilitating myoblast differentiation-mediated muscle growth.3.Increased ZBED6 gene expression in muscle tissue is associated with septic muscle atrophy,and ZBED6 knockout alleviates septic muscle atrophy in pigs.To advance research on muscle atrophy in sepsis,appropriate animal models of sepsis are required.This study introduces a novel cecal ligation and puncture surgical plan for the first time,which significantly reduces the mortality rate and successfully replicates the entire process of the occurrence and development of human sepsis and the symptoms of muscle atrophy in porcine sepsis.Consequently,this model can be employed for further exploration of the mechanisms through which sepsis induces muscle atrophy.To elucidate the relationship between ZBED6 and sepsis-induced muscle atrophy,this study comprehensively employed imaging,histopathology,and various molecular biology methods to conduct a cross-species,high-level study using samples from sepsis patients and sepsis pig models,facilitating a multidimensional joint analysis.The results revealed that,in comparison to the control group,ZBED6 expression levels in the muscle tissue of septic patients and septic pigs were significantly elevated.Further examinations,including muscle ultrasound,serum biochemistry,and molecular biology muscle atrophy indicators,indicated a close association between ZBED6 expression and septic muscle atrophy,underscoring the need for further in-depth research using ZBED6 knockout pigs.To further investigate the role of the ZBED6 in septic muscle atrophy,this study created a ZBED6 knockout pig sepsis model.The results revealed that ZBED6 knockout significantly reduced the weight loss caused by sepsis（P<0.001）and led to a substantial reduction in muscle weight loss（P<0.001）,with no difference in fat weight loss.Further histological examination of muscle tissue in different parts of the bodies of septic pigs demonstrated that ZBED6 knockout markedly alleviated symptoms of various conditions throughout the body,reducing the degree of muscle fiber area atrophy at the site.Additionally,a further analysis using muscle fiber-specific immunofluorescence staining revealed that ZBED6 knockout significantly reduced the degree of atrophy in type II muscle fibers,while the degree of atrophy in type I muscle fibers was smaller and not significantly different.Serum biochemistry,RT-q PCR,western blotting,and SUn SET technology were employed to analyze protein breakdown and anabolic status in pig muscle tissue after sepsis.The results indicated that ZBED6 knockout reduced sepsis-induced protein breakdown,alleviated protein anabolism disorders,and corrected muscle protein metabolism disorders,ultimately mitigating septic muscle atrophy.4.Loss of ZBED6 activates DOCK3-mediated RAC1/PI3K/AKT signaling pathway to protect against sepsis-induced muscle atrophy.To explore the common mechanism of ZBED6 knockout in different muscle parts in sepsis-induced muscle atrophy,this study conducted muscle tests on nine different body parts in wild-type pigs and ZBED6knockout pigs under sepsis.Transcriptome sequencing and Chi P-seq sequencing of the ZBED6 were performed on porcine muscle tissue,followed by joint analysis.The results indicated that four target genes potentially directly regulated by ZBED6 were SOX18,DOCK3,C14orf39,and G0S2.Among them,DOCK3 not only exhibited the highest differential expression fold in RNA-seq but also demonstrated the most significant ZBED6binding in Ch IP-seq.RT-q PCR and Western blot experiments showed a significant increase in DOCK3 protein expression in the muscle tissue of ZBED6 knockout pigs.Further dual-luciferase and Ch IP-PCR experiments revealed that ZBED6 directly binds to the DOCK3promoter sequence,thereby negatively regulating DOCK3 transcriptional activity.KEGG enrichment analysis was conducted using differentially expressed genes from muscle RNA-seq in nine different body parts.The integrated analysis results showed that only the PI3K/AKT signaling pathway was significantly enriched among the top 20 signaling pathways in the nine muscle parts.Additionally,further cell experiments indicated that ZBED6 knockout resulted in the activation of the DOCK3/RAC1/PI3K/AKT signaling pathway.Consistent with the results of cell experiments,the expression of DOCK3 and RAC1-GTP proteins in the skeletal muscle of the ZBED6 knockout pig sepsis group was significantly upregulated compared to the wild-type pig sepsis group,thereby activating the PI3K/AKT signaling pathway.In summary,this study demonstrates,for the first time,that ZBED6 knockout can activate the ISLR-Wnt signaling pathway,thereby promoting pig muscle growth and increasing meat production without negatively affecting meat quality.Additionally,this study pioneered a new surgical plan for a cecal ligation and puncture model,creating a novel porcine sepsis model.Using this model,it was discovered for the first time that the ZBED6 is highly related to muscle atrophy caused by sepsis.Through the application of the new cecal ligation and puncture model,this study demonstrated that ZBED6 deletion activates the DOCK3/RAC1/PI3K/AKT signaling pathway to alleviate muscle atrophy caused by sepsis.By investigating the ZBED6,this study provides crucial insights for molecular targets to enhance pork quality traits and advance research in the treatment of muscular dystrophy.It also offers new perspectives on the’one pig dual use’concept,including pig breed improvement and human disease research,with promising application prospects.