Effects Of The PLIN1 Gene On Muscle Development Using Cellular And Mouse Models

Lipid droplet encapsulated protein 1 (PLIN1) is the most abundant structural protein on the surface of lipid droplets and plays an important role in regulating lipid synthesis and lipolysis.In our research group,we used different pig models to study the differences in body size and muscle development,and screened a number of genes with significant differences in expression in different muscle tissues,including the PLIN1 gene.In this study,we aim to investigate the effects of PLIN1 gene on skeletal muscle satellite cells (SMSCs) at the cellular level and to construct a conditional knocKOut mouse model with specific knocKOut of PLIN1 gene in skeletal muscle tissues to study the phenotypic changes and functional effects of PLIN1 gene on skeletal muscle development.The effect of PLIN1 gene on skeletal muscle development was studied in a conditional knocKOut mouse model.In this way,the specific effects of PLIN1 gene on muscle development and meat quality can be comprehensively investigated,which will help to promote the improvement of meat production traits in domestic pigs.Study 1 Investigating the effects of PLIN1 gene on myogenic and lipogenic differentiation functions using cellular modelsIn this study,we investigated the effect of PLIN1 gene on myogenic and lipogenic differentiation by measuring the expression of myogenic differentiation markers MYH1 and MyoDl in each group of cells after myogenic induction.The effect of PLIN1 gene on myogenic differentiation was investigated by detecting the expression of PPARy,a marker of myogenic differentiation,and PRKAG2,an inhibitor of myogenic differentiation,in each group of cells after induction of myogenic differentiation;by quantifying the amount of fat in the cells using oil red O staining;and by measuring the content of triglyceride (TG) in the cells.The results showed that after 4 days of induction of myogenic differentiation in all groups,the mRNA expressions of MYH1 and MyoD1 in the cells of NC group were highly significant higher than those of PLIN1-KO and PLIN1-EX groups (P<0.01);MYH1 and MyoD1 protein expressions were also highly significant higher in NC group cells than in PLIN1-KO and PLIN1-EX groups (P<0.01);cellular immunofluorescence The results of detecting MYH1 and MyoD1 were consistent with the above trend.After lipogenic differentiation in all groups,the levels of oil red O staining and quantification in the cells of NC group were significantly higher than those of PLIN1-KO and PLIN1-EX groups(P<0.01),and the expression of PPARy gene in the cells of NC group was significantly higher than those of PLIN1-KO and PLIN1-EX groups (P<0.05);the expression of PRKAG2 gene in NC group was significantly lower than that in PLIN1-KO and PLIN1-EX groups (P<0.01),and the TG level in NC group cells was significantly higher than that in PLIN1-KO and PLIN1-EX groups (P<0.01).In conclusion,the stable expression of PLIN1 gene is very important for the differentiation function of skeletal muscle satellite cells,and overexpression or knockdown of PLIN1 gene resulted in a significant decrease of myogenic and lipogenic differentiation ability of porcine skeletal muscle satellite cells.Study 2:Investigating the effects of PLIN1 gene on muscle development using a mouse modelIn this study,we designed 2 sgRNA sequences at both ends of exon 3 of the mouse PLIN1 gene,constructed Donor vectors containing homologous arms of PLIN1 exon3 with loxp sequences at both ends,prepared PLIN1 Flox mice by microinjection of embryos at prokaryotic stage,and screened skeletal muscle tissues by mating with mice specifically expressing Cre recombinase in Gene editing mice with specific knocKOut of PLIN1 gene were screened by mating with mice expressing Cre recombinase specifically in skeletal muscle tissue.Using wild-type C57BL/6J mice as control (WT),the PLIN1 gene edited mice were firstly characterized at the DNA,RNA and protein levels for molecular biology;the mice were phenotypically examined by plotting growth curves,measuring organ coefficients and muscle morphology of quadriceps and gastrocnemius in 8-week-old mice.The gastrocnemius muscle was collected from two groups of mice,and the intramuscular fat content was measured by soxhlet extraction,the lipid content of muscle tissue was measured by oil red O after preparation of frozen sections,the content of TG in muscle tissue was measured by using kits,and the cross-sectional area of muscle fibers in muscle tissue was observed by HE staining;the myogenic marker genes MyoD1 and MYH and lipogenic marker genes were detected in skeletal muscle tissue by qRT-PCR and WB,respectively PPARγ,LPL mRNA and protein expression were detected by qRT-PCR and WB to examine the effect of PLIN1 gene on the differentiation function of muscle tissue.The mitochondria of skeletal muscle tissues from two groups of mice were extracted to detect the mitochondrial membrane potential,reactive oxygen species and ATP levels,to comprehensively examine the effects of PLIN1 gene on muscle metabolism.The results showed that PLIN1 mRNA expression in heart,liver,spleen,lung,kidney.skin and adipose of PLIN1 cKO group mice was not significantly different from WT group mice,and was significantly lower in skeletal muscle pole than WT group mice (P<0.01),PLIN1 cKO group mice in skeletal muscle The relative expression of PLIN1 protein in the skeletal muscle of mice in the PLIN1 cKO group was extremely significantly lower than that in the WT group (P<0.01).The growth weight of the mice was measured at 8 weeks after birth,and there was a significant increase in the WT group compared with the PLIN1 cKO group from week 2 onwards,with significant differences in body weight between the two groups (P<0.05);the organ coefficients of the gastrocnemius and quadriceps muscles were significantly higher in the WT group than in the PLIN1 cKO group (P<0.05);the results of HE staining of histological sections showed that The results of HE staining of histological sections showed that the cross-section of muscle fibers in the WT group was positively significantly higher than that in the PLIN1 cKO group (P<0.01);The results of oil red O staining of frozen sections of gastrocnemius muscle tissues showed that the relative amount of lipids in muscle tissues in the WT group was extremely significantly higher than that in the PLIN1 cKO group (P<0.01).The lipid content in the gastrocnemius muscle of WT group was significantly higher than that of PLIN1 cKO group (P<0.05);the expression levels of MYH,MyoD1,PPARγ and LPL mRNA in the muscle tissue of WT group were significantly higher than that of PLIN1 cKO group (P<0.01),and the expression of myogenic differentiation marker proteins MyoD1,Myog,MYH and lipogenic differentiation marker proteins PPARγ and LPL were significantly higher in the WT group than in the PLIN1 cKO group by WB (P<0.05).The results of muscle tissue metabolism-related indexes showed that compared with the WT group,the skeletal muscle membrane potential decreased by 42%,reactive oxygen species increased by 22% and ATP level decreased by 29% in the PLIN1 cKO group.The above results indicate that this study successfully prepared a mouse model with specific knockdown of PLIN1 gene in skeletal muscle and demonstrated that PLIN1 knockdown had significant inhibitory effects on muscle fiber and fat production and metabolism in muscle tissue.Conclusion:This study demonstrates for the first time that the PLIN1 gene has an important effect on skeletal muscle development and metabolism,using both cellular and mouse models.In this study,we demonstrated that the steady-state expression of PLIN1 gene helps to maintain the normal differentiation function of porcine SMSCs at the cellular level,while over-or under-expression of PLIN1 gene inhibits the differentiation function of SMSCs;in a mouse model,we demonstrated that knockdown of PLIN1 gene in skeletal muscle tissues directly inhibits the production of fat and muscle fibers in skeletal muscle tissues and suppresses muscle tissue metabolism.This study will be followed by an analysis of the association between PLIN1 expression levels and muscle development in different pig populations,which will help to advance the genetic selection and breeding of pig breeds in China.