Effect And Mechanism Of The CNR1Gene Expression On Muscle Fiber Types

One of the biochemical and molecular basis factors determing muscle growth and meat quality is the muscle fiber types.Different muscle fiber types have showed different characteristics of energy metabolism and biochemical characteristics. There are four main muscle fiber types (MyHCI, MyHCIIa, MyHCIIx and MyHCIIb) in mammalian skeletal muscle.Furthermore, different muscle fiber types can be transformed into each other.Taking advantages of the global microarray technology, cannabinoid receptor1(CNR1) have identified as fifferently expression between Jinhua with superior meat quality and Landrace pigs with lower meat quality.In order to inverstgate the effects and mechanism of CNR1on muscle fiber types, Quantitative PCR, immunohistochemistry, Western Blot and other technologies were used to establish the in vitro and in vivo overexpression and gene silencing in cells or animal models. The findings are as follows:1. Expression patterns of the cannabinoid receptor1gene in pig skeletal muscle and skeletal satellite muscle cellsUp to now, detailed analysis of CNR1gene expression in pig muscles and muscle cells has not been reported. Here, quantitative PCR were used to characterize the CNR1in different muscles of pig and isolation of porcine myogenic satellite cells. Results showed that the expression of CNR1mRNA was greatest in longissimus dorsi muscle (p<0.01)among that in the gastrocnemius (mix), soleus muscle (slow-type) and longissimus doris muscle (fast-type). As to age-related changes of CNR1gene in different pig muscles, the higher expression was found at the30th day, then declined (p<0.05) in all three kinds of muscles to the90th day. At the150th day, the expression of CNR1steadily decreased (p<0.05) in the gastrocnemius compared to the90th day, but not in longissimus doris muscle and soleus. Meanwhile the breed differences in the expression of CNR1mRNA between Jinhua pig and Landrace were also identified. The mRNA abundance of CNR1in Landrace was higher compared with that of Jinhua pigs and strong differences were observed in longissimus doris muscle (p<0.05) and soleus (p<0.01). In vitro, with the differentiation of the satellite cells, the mRNA expression of the CNR1was down-regulated (p<0.05). The lowest mRNA expression was observed at the9th day. These data provide a framework of CNR1gene expression patterns and implicate a novel gene for regulating muscle fiber types.2. Effects and molecular mechanism of CNR1agonist and antagonist on muscle fiber typeswe analyzed the expression patterns of CNR1and MyHC isforms types includingⅠ,Ⅱa,Ⅱx Ⅱb with the cell differentiate in L6cells.After that,L6cell were coped with CNR1selective agonist MAEA and antagonist AM251.The changes of CNR1expression, MyHC isforms(MyHCⅠ, MyHCⅡa, MyHCⅡx and MyHCⅡb) and the key enzyme gene (LDH,SDH and MDH)as well as the key gene of muscle fiber types transition(AMPKal, PGC-1, ERK1and ERK2) were detected using quantitative PCR.The results show that:CNR1gene expression was higher at the3th and7th day. The levels of MyHCI, and MyHCⅡa expression peaked at the3thday, then dramatically decreased. MyHCⅡx gene expression showed the increased trend before the7th day (P<0.05). MyHClIb gene was significantly increased at the first two2days, at the3th day,reached the higest level and then significant decreased.Treated L6cells with different concentrations of MAEA(0,5,10,15μM/L), we found that CNR1expression was significantly increased at15μM/L. Further treated with different time at15μM/L.Under the both conditions, the MyHCⅡb gene expression was reduced (p<0.01). but MyHCIIa gene expression was increased The gege expression of MyHCl and MyHCⅡx had no significant difference. Meanwhile gene expression of glycolytic key enzyme LDH were decreased (p<0.05). Key enzyme genes of oxidation SDH and MDH had no significant change.The key gene of muscle fiber types transition ERK1expression was increased (P<0.05).On the contrary, treated L6cells with different concentrations of AM251(0,0.1,1,10μM/L), the results showed that CNR1expression was significantly down regulated at10μM/L. Further treated with different time at10μM/L.Under the both conditions, the gene expression MyHCⅡa, and MyHCⅡx was significantly decreased (P<0.05), MyHCⅡb gene expression significantly increased (P<0.01). The expression level of LDH was elevated (P>0.05), MDH and SDH gene expression were significantly decreased (P<0.05). AMPKal and ERK1expression was significantly down regulated (P<0.01).3. Effects and molecular mechanism of interference CNR1on muscle fiber types in vitroThis study was conducted to construct and identify CNRl gene siRNA expression vectors and screen the stable CNR1-interference positive L6cell clones. Three pairs of CNR1-specific double-strand siRNAs were designed, synthesized, annealed and inserted into the pYr-1.1vector.The CNR1gene siRNA expression vectors were identified by restriction enzyme digestion and sequencing. After that siRNAs were transfected with L6cells by LipofectamineTM (Lip)2000. Then, the transfection efficiency was detected by EGFP and FCM.CNRI gene expression was determined by real-time PCR and the stable transgenic L6cell clones were screened by G418. The results revealed that the CNR1gene siRNA expression vectors have been constructed successfully. The transient transfection efficiency to L6cells were10.45%(P<0.01)、8.57%(P<0.01) and8.71%(P<0.01),and the silencing efficacy of the transient transfected L6cells was39%(P<0.05)、64%(P<0.01) and68%(p<0.01),respectively. The optimal selection concentration of G418for stable transfected L6cell clones was800ug/ml. The silencing efficacy of CNR-1-positive transgenic cell clones were43%(P<0.05).78%(P<0.01) and91%(P<0.01). The results showed that CNR1-3expression vector is the optimal silencing vector and CNR1-3stable transgenic cell clones is best silencing cell line. This study provides a successful CNR1gene silencing method by siRNA and the screening of CNR1-interference positive L6cell clones renders basic tools for further studying the functions of CNR1gene.After effectively silence CNR1gene by RNA interference. The gene expression of MyHCⅡa was decreased (P<0.05); the levels of MyHCⅡb was increased significantly (P<0.05), stable interference cells inducted by1,3,5days; gene expression of MyHCⅡb was significantly increased (P<0.05); Meanwhile,MyHCⅡa gene expression showed a decline trend. At the3th day, MyHCIIa gene expression decreased significantly (P<0.05). Expression of the glycolytic key enzyme gene LDH was significantly increased (P<0.05); the key enzyme genes of oxidation MDH and SDH expression was decreased, but no significant difference. ERK2gene expression was significantly increased (P<0.05). These results were consistent with treated with AM251.4. Effects and molecular mechanism of overexpression CNR1on muscle fiber types in vitro4.1. Effects and molecular mechanism of overexpression CNR1on muscle fiber types in vitroStable CNR1-overexpression positive L6cell clones were screened by800ug/ml G418.The level of CNR1gene and protein both was increased(P<0.05),it means that stable CNR1overexpression cell model was eatablished successfully. In stable overexpression CNR1cells, gene expression of MyHCⅡa was increased (P<0.01), MyHCIIb gene expression was decreased (P<0.01), The expression of MyHCI and MyHCIIX had no significant changes.While differentiated stable overexpression cells differentiated, gene expression of MyHCIIa was increased (P<0.05). In addition, the expression of glycolytic key enzyme gene LDH was decreased (P<0.01), key enzyme gene of oxidative metabolism including MDH (P<0.01) and SDH (P<0.05) were significantly increased. Key genes involved in Muscle fiber transformation, gene expression of ERK1significantly increased (P<0.05), AMPKa1, PGC-1and ERK2expression had no significant difference. These results were consistent with treated with MAEA in L6cells.4.2. Effects and molecular mechanism of overexpression CNR1on muscle fiber types in vivoIn vivo, gene overexpression system of rat gastrocnemius and soleus electroporation was established successfully, the expression of both gene and protein of CNR1were increased(P<0.05).Using the CNR1gene force expression technique to explore the effect and the molecular mechanism of CNR1on the muscle fiber types and energy metabolism.The result showed that the expression of gene and protein of MyHCⅡa significantly higher (P<0.01) than that of control group (empty vector group and saline injected group) in gastrocnemius.In selous.the gene expression of MyHCⅡa and MyHCⅡx were also significantly increased (P<0.05), the protein expression of MyHCⅡa was increased,but had no significantly differences. The Glycolysis key enzyme LDH activity was significantly decresed both in gastrocnemius and soleus (p<0.05);The gene and enzyme activity of MDH in gastrocnemius and soleus were significantly increased (P<0.05); SDH activity increased but had no significandifference in gastrocnemius; But in soleus,the RNA level and activity of SDH were significantly increased (P<0.01). The key gene which involved inmuscle fiber type transition, gene expression of ERK1(p<0.01) and PGC-1(p-<0.05) gene expression were significantly increased in both gastrocnemius and soleus.Based on the data of vitro and vivo experimental results, we can conclude that CNR1has the function of increasing the expression of MyHCⅡa, promoting the conversion of muscle fibers to fast oxidative type. It also can effect energy metabolism with increasing the oxidation enzyme MDH and SDH and decreasing glycolysis key enzyme LDH. ERK1/2may play an important role in this process. In addition, the regulation and mechanism of CNR1in muscle fiber types exist differences between vivo and vitro.