Expression Of MAFbx/Atrogin-1 And MuRF1 In Rat Gracilis Muscle Following Muscle Free Transfer And Its Significance

【Background】Free muscle transplantation by microneurovascular anastomosis is a valuable clinical option in the reconstruction of functional deficits in the extremities and the face. The diminution in strength of the transferred muscle that invariably follows the procedure is problematic. Despite advances in nerve repair by microsurgical techniques, careful restoration of muscle tension, less period of ischemia during the transfer, clinical observations and experimental studies still document residual force deficits in transferred muscle. Recent studies have found that the weakness of the muscle after free transfer may be associated with the muscular atrophy. Muscle atrophy occurs when there is an imbalance between protein synthesis and degradation and mainly by acceleration of proteolysis. Among the various systems involved in muscle protein degradation, it is now well-established that the ubiquitin proteasome system is of primary importance in the enhanced protein degradation that occurs in muscle as it atrophies. Moreover, among the system or pathway, ubiquitin protein ligases (E3) play a critical role in polyubiquitination, as they are responsible for the selective recognition of protein substrates. Recently, two E3s havebeen identified and shown to be markedly induced in skeletal muscles atrophying in response to a wide array of catabolic conditions, which are MuRF1 and MAFbx/Atrogin-1. And importantly, mice lacking either gene show a blunted response to muscle wasting in response to denervation. This key finding represents the first solid evidence for a critical role of the ubiquitin pathway in mediating the atrophic process. But levels of mRNA for these E3s in free muscle transfer and levels of protein for these E3s in muscle wasting have not been reported to date.【Objective】The purpose of this study was to establish an animal model of free muscle transfer with nerve anastomosis using rat gracilis muscle and evaluate some related indexes for muscle atropht; to study the quantitative changes of mRNA and Protein expression of MuRF1 and MAFbx/Atrogin-1 following free transfer; to analyze the relationship of muscle atrophy and expression of these two E3s, and to explore the possible mechanism of E3s'expression change in muscle following free transfer.【Methods】(1) In part I: The gracilis muscle was orthotopic transferred in adult rat to establish the animal model. According the different time after free muscle transfer, muscle weight was measured and compared to that of the control side which had no operation, so did the transaction areas of muscular fiber using NIH Image J software following HE staining. Then muscular function including maximum strength of single contraction and tetanus was recorded using a multiple function physiological recording device. (2) In part II: Total RNA extraction and RT procedures were done in gracilis muscle specimens at different time following muscle transfer. Forwards and reverse primers of MAFbx/Atrogin-1 and MuRF1 for PCR were designed using Primer Premier software. A relative quantitative SYBR? Green realtime-PCR method (2–??CT Method), using GAPDH as an internal standard, was applied to study the expression of specific mRNAs for MAFbx/Atrogin-1 and MuRF1. (3) In partⅢ: Semiquantitative protein expression results of MAFbx/Atrogin-1 were obtained using Western blot method taking GAPDH as an internal standard. (4) In partⅣ: The expression of IGF1 mRNA was detected using the same method in Part II. Possible roles of IGF1/PI3K pathway in the activation of MAFbx/Atrogin-1 and MuRF1 for mucle atrophy following free transfer were discussed.【Results】(1) In part I: Fifty-four SD rats with an average weight of 250±25 g were operated successfully, and animal model of of free muscle transfer with nerve anastomosis was available. Muscle wet weight reservation rate (muscle weight of operative side / muscle weight of control side×100%) had a dramatically decrease in the first 4 weeks following the operation and increased gradually from then on, but remained lower compared with the control (P<0.05). Transaction areas of muscular fiber at different stage postoperatively were calculated and exhibitd a similar change with that of muscle wet weight. Then muscular function including maximum strength of single contraction and tetanus was successfully recorded using a multiple function physiological recording device. The recovery ratio was consistent with these index for muscular atrophy mentioned above. (2) In part II: Total RNA extraction and RT procedures were done successfully in gracilis muscle specimens at different time following muscle transfer. At 3 weeks after transfer ,the copies of mRNA expression of MAFbx/Atrogin-1 and MuRF1 reached the highest level, as much as 4-7 times of control, and then decreased gradually. At 30 weeks the mRNA expression of these two genes was still higher than the control but much lower than its highest in the reinnervated muscle (P<0.05). (3) In partⅢ:Western blot results documented increased expression of MAFbx/Atrogin-1 protein at 4 weeks following the operation but decreased remarkably at 15 weeks and remained lower-level high expression until 30 weeks after transfer. (4) In partⅣ: The expression of IGF1 mRNA decreased in the first 4 weeks after transfer and increased gradually from then on , which indicated that IGF1/PI3K pathway play a critical role in the activation of MAFbx/Atrogin-1 and MuRF1.【Conclusions】(1) Animal model of of free muscle transfer with nerve anastomosis using rat gracilis muscle was easy and stable. Muscle wet weight reaservation rate and transection area of muscle fiber are better indexes for muscle atrophy following free transfer, according the results of muscle function. (2) The detection of mRNA expression of MAFbx/Atrogin-1 and MuRF1 by SYBR? Green real-time fluorescent quantitative RT-PCR is more credible, accurate, sensitive, specific and time-saved. (3) The quantitative changes of mRNA of MAFbx/Atrogin-1 is consistent with that of protein, which reached the highest level, as much as 7-9 times of control, at 4 weeks after transfer and then decreased gradually. The muscle atrophy and persistent weakness of muscle function following free transfer may be attributed to these changes. (4) The expression of IGF1 mRNA indicated that IGF1/PI3K pathway play a critical role in the activation of MAFbx/Atrogin-1 and MuRF1.Perhaps the most direct strategy for identifying useful anti-atrophy targets is to determine the signalling pathways that are required for atrophy. In conclusion, our study elucidates that MAFbx/Atrogin-1 or MuRF1 maybe a potential target for drug discovery. First, their enzymatic activity seems to be required for muscle atrophy; second: they are expressed specifically by muscle cells; third, they do not seem to be required for normal muscle growth or function. Ongoing work is aimed at determining whether the muscle hat is preserved in the knockout animals maintains a normal functional capacity.