Molecular Mechanism Study Of CircMYLK4 Promoting The Transition From Glycolytic To Oxidative Fiber Type In Porcine Skeletal Muscle

China is the world’s largest producer and consumer of pork,with the country’s pig farming volume and pork consumption accounting for approximately 50% of the global total.As living standards continue to improve,there is an increasing demand for higher quality meat products.Therefore,improving the quality of pork and understanding the molecular mechanisms that influence meat quality has become areas of research focus.The composition of muscle fiber types determines the overall biochemical and functional characteristics of muscle tissue,which in turn determines the quality of fresh meat.In our previous work,high-throughput sequencing was conducted on pig fast and slow muscle fibers,and circMYLK4 was identified and preliminarily validated as a novel regulatory factor for pig skeletal muscle fast/slow fiber types.Therefore,elucidating the molecular mechanisms of circMYLK4 is of significant importance for improving the quality of pork.This study elucidated the functional role of circMYLK4 in muscle development through integrated analyses of transcriptomics,proteomics,phosphoproteomics,and energy metabolism.Molecular biology techniques were employed to demonstrate the impact of circMYLK4 on muscle cell metabolism in porcine skeletal muscle satellite cells.Furthermore,the mechanisms of circMYLK4 were elucidated through multimer analysis,dual-luciferase reporter assays,RNA pull-down,and RNA immunoprecipitation methods.Finally,the conservation of circMYLK4 function was confirmed by exogenous injection of circMYLK4-AAV in mouse skeletal muscle.The following key findings were obtained:1.circMYLK4 inhibits glycolysis and promotes oxidative phosphorylation in porcine skeletal muscle.Integrated analyses of transcriptomics,proteomics,and phosphoproteomics revealed that overexpression of circMYLK4 led to upregulation of genes primarily enriched in fatty acid metabolism,while downregulating genes primarily involved in glycolysis.Further analysis showed that circMYLK4 did not affect the expression of the glucose metabolism key enzyme HK2 but inhibited the expression of glycogenolysis key enzymes PYGM and PGM1,thereby suppressing glycolysis.Moreover,circMYLK4 promoted the expression of genes involved in fatty acid oxidation,TCA cycle,and mitochondrial electron transport chain.Consistently,energy metabolism analysis demonstrated a significant decrease in intermediate metabolites of glycolysis and a significant increase in intermediate metabolites of the TCA cycle.Furthermore,analysis of genes related to muscle contraction revealed an increase in the expression of slow muscle fiber marker genes MYH7,TNNI1,TNNT1,and TNNC1,while the expression of fast muscle fiber marker genes MYH4 and TNNT3 was decreased.2.circMYLK4 inhibits glycolysis and enhances mitochondrial metabolism in porcine satellite cells.Overexpression of circMYLK4 in porcine skeletal muscle satellite cells significantly suppresses the m RNA and protein levels of key glycolysis genes(P<0.05),leading to a significant reduction in cellular lactate levels(P<0.05).Furthermore,circMYLK4 significantly promotes the m RNA and protein levels of genes involved in fatty acid oxidation and the TCA cycle(P<0.05),resulting in a significant increase in cellular succinate levels(P<0.05).Additionally,the m RNA and protein levels of key genes involved in mitochondrial oxidative phosphorylation are significantly increased(P<0.05),leading to a significant elevation in cellular ATP levels(P<0.05)and enhanced mitochondrial activity.3.circMYLK4 does not encode peptides or sequester miRNAs but instead interacts with the protein CACNA2D2.Bioinformatics analysis revealed that circMYLK4 contains three ORFs,and polyribosome analysis showed that circMYLK4 is predominantly enriched in the monosome fraction,indicating its lack of coding potential.Subsequently,RNA pull-down was performed to enrich miRNAs bound to circMYLK4.Accumulated distribution curves and dual-luciferase assays revealed that the top four enriched miRNAs did not bind to circMYLK4.Further investigation using AGO2 immunoprecipitation demonstrated that circMYLK4 does not interact with miRNAs.Additionally,RNA pull-down was conducted to enrich proteins that interact with circMYLK4,and WB and RIP assays confirmed that the auxiliary subunit of the calcium channel DHPR,CACNA2D2,can bind to circMYLK4.4.CACNA2D2 is involved in the regulation of calcium homeostasis mediated by circMYLK4.Overexpression of CACNA2D2 in porcine skeletal muscle satellite cells significantly promotes glycolysis and mitochondrial oxidative phosphorylation.MitoTracker staining,mitochondrial copy number,and increased cellular ATP levels indicate that CACNA2D2 enhances mitochondrial metabolism.In-depth analysis of calcium signaling-related genes in the proteome revealed that circMYLK4 suppresses the protein level of the calcium release channel RYR1,significantly reduces the protein level of the calcium-binding protein PVALB(P<0.05),and significantly increases the protein level of the mitochondrial calcium uniporter MCU(P<0.05).Calcium ion fluorescence probe experiments demonstrated that circMYLK4 decreases cytosolic calcium ion concentration(P<0.05),which can be restored by CACNA2D2,counteracting the inhibitory effect of circMYLK4 on calcium ion concentration.Furthermore,CACNA2D2 can counteract the inhibitory effect of circMYLK4 on the key enzymes PHKB and PHKG1 involved in glycogen breakdown(P<0.05).5.circMYLK4 promotes the conversion of mouse fast-twitch muscle fibers to slow-twitch muscle fibers.Analysis of circular RNAs generated by MYLK4 in the circ Atlas 3.0 database for pig,mouse,and human revealed no circRNA derived from the pig MYLK4 gene.Furthermore,it was found that the mouse mmu-Mylk4＿0004 and human hsa-MYLK4＿0016 sequences share high similarity with the circMYLK4 sequence,indicating that circMYLK4 is a novel and conserved circRNA.Injection of exogenous circMYLK4-AAV into the gastrocnemius muscle of mice resulted in a significant increase in the protein and m RNA levels of the slow-twitch muscle marker gene MYH7(P<0.05),while significantly suppressing the protein and m RNA levels of the fast-twitch muscle marker gene MYH4(P<0.05).H&E staining and immunofluorescence staining demonstrated that circMYLK4 reduced the cross-sectional area of muscle fibers and increased the proportion of slow-twitch muscle fibers.In summary,this study revealed that circMYLK4,through its interaction with CACNA2D2,reduces cytoplasmic calcium ion concentration,thereby inhibiting the activity of key enzymes involved in glycogen breakdown,namely PHK and PYGM,leading to the suppression of glycolysis.On the other hand,circMYLK4 enhances fatty acid oxidation,tricarboxylic acid cycle,and mitochondrial oxidative phosphorylation.Furthermore,this study identified circMYLK4 as a highly conserved molecule that promotes the conversion of mouse fast-twitch muscle fibers to slow-twitch muscle fibers.Therefore,circMYLK4,as a novel regulatory factor for pig skeletal muscle fast/slow fiber composition,holds significant implications for improving the quality of pork.