Exploring The Relationship Between Porcine Myofibers Subtypes And Intramuscular Fat Deposition Through Spatial Transcriptome

With the improvement of residents’ living standards,high-quality pork is the future consumption trend.The type of muscle fiber and the fat content in skeletal muscle are two core factors that affect meat quality.According to the type of myosin MYH,the skeletal muscle fibers of pigs can be divided into four types: type I,type IIA,type IIX,and type IIB.Type I muscle fibers are the thinnest and contain higher levels of myoglobin,while type II muscle fibers are thicker and have relatively lower levels of myoglobin.Therefore,the higher the proportion of type I muscle fibers,the redder the meat color,and the more delicate the taste,while the intramuscular fat content will affect the meat quality,flavor,and juiciness.Deeply exploring the relationship and mechanism between type I muscle fiber ratio and intramuscular fat deposition can provide a molecular basis for improving pork quality and provide direction for the breeding of high-quality pork.However,currently,the relationship between the proportion of muscle fiber subtypes and intramuscular fat deposition in skeletal muscles is still unclear.Spatial transcriptome is a newly developed transcriptome sequencing technology,which can provide transcriptional information and spatial location of muscle fiber subtypes in skeletal muscle without separating the precursors of various muscle fiber subtypes.Combining transcriptome information with spatial location information,it provides a new means for studying the composition and gene expression of muscle fiber subtypes in different skeletal muscles.Therefore,this study used pig soleus muscle and longissimus dorsi muscle as the research model,and explored the characteristics of fat deposition and metabolism of muscle fiber subtypes in skeletal muscle at the living level through spatial transcriptome sequencing,immunofluorescence,Elisa and other technical methods.The main results obtained are as follows:1.Skeletal muscle fiber subtypes are related to the content of intramuscular fat.The results of immunofluorescence triple staining of extensor digitorum longus,soleus,longissimus dorsi,tibialis anterior and gastrocnemius muscle showed that soleus and gastrocnemius muscle were mainly composed of oxidized muscle fibers;The extensor digitorum longus,longissimus dorsi,and tibialis anterior muscles are mainly composed of glycolytic muscle fibers,with type IIB muscle fibers having the highest content.Bodypy and Elisa showed that the lipid stored in soleus,longissimus dorsi and gastrocnemius muscle was significantly higher than that in extensor digitorum longus and tibialis anterior muscles.Combined analysis showed that in slow muscles such as soleus and gastrocnemius muscle,fat drops were more easily stored in type I muscle fibers,and in fast muscles such as extensor digitorum longus,longissimus dorsi,and tibialis anterior,fat drops were more easily stored in type II muscle fibers.2.A set of pig skeletal muscle spatial transcriptome sequencing sample preparation process was established.The results of HE staining showed that freezing and fixing pig skeletal muscles in a liquid nitrogen isopentane metal bath can ensure the morphological integrity and RNA integrity of pig skeletal muscles;The results of immunofluorescence showed that the optimal permeation times for soleus and longissimus dorsi were 18 and 12 minutes,respectively;The results of database construction and sequencing indicate that a slice thickness of 16 um is necessary to ensure that the chip captures sufficient genetic information.3.Different muscle fiber subtypes were identified by spatial transcriptome.In LD and Sol skeletal muscles,four muscle fiber subtypes were identified in the space transcriptome.The spatial distribution characteristics of skeletal muscle fibers show that type I fibers are surrounded by type II fibers in skeletal muscle,with type I fibers in clusters and type II fibers in rings.4.The energy metabolism of muscle fiber subtypes is influenced by the type of skeletal muscle.The results of spatial transcriptome sequencing showed that Sol type I muscle fibers had stronger glucose aerobic oxidation and fatty acids than LD type I muscle fibers β The oxidative capacity of LD type I muscle fibers is stronger than that of Sol type I muscle fibers in glycolysis and lactate transport oxidation.In addition,LD type II muscle fibers have stronger glycolytic ability than Sol type II muscle fibers.In conclusion,this study explored the heterogeneity and spatial distribution characteristics of pig skeletal muscle fibers through spatial transcriptome.In addition,our results indicate that the presence of more lipid droplets in type I muscle fibers in slow muscles is due to the lack of type II muscle fibers in slow muscles,which cannot transport enough lactate for glucose aerobic oxidation.Therefore,type I muscle fibers in slow muscles require stronger fatty acids β Oxidation.The results of this study provide new research ideas for improving meat quality.