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Efficient in vitro model of myotubes is a principle platform to study developmental processes occurring in vivo, investigation of drug activity and designing new forms of therapy. Myotubes grown in vitro do not mature efficiently and have low insulin sensitivity. There is published evidence suggesting "co-culture of myotube and spinal cord explant improves myotube maturity". In this project the hypothesis will be tested using rat C2C12 and N2a neuroblastoma cell line. Real time PCR will be used to amplify and quantify mRNA transcripts of fully matured myotube markers and a non-radioactive glucose uptake assay will be performed to test for insulin sensitivity of myotubes. It is expected that this attempt at co-culturing cell line will improve insulin sensitivity and in turn will allow development of a better experimental model for variety of investigations.
Background to investigation
The human body consists of three types of muscle tissues, namely skeletal muscles, smooth muscles and cardiac muscles. Smooth muscles and cardiac muscles are involuntary muscles while skeletal muscles are voluntary muscles. Skeletal muscles contribute to 30% and 38% of the body weight of women and men respectively (Janssen et al., 200). Skeletal muscles are also called striated muscles and they are parallel in orientation. Skeletal muscles contain myofibres which are made up of compact bundle of myofibryls packed together. Myofibryls have sarcomere which is responsible for contraction of the skeletal muscles and hence contributes to movements of the body. The regenerative property of skeletal muscles is imparted by presence of satellite cells, which differentiate into myoblasts and travel to the damaged location and get incorporated (Goldring et al., 2002 and Anderson, 2006). Myogenesis is a phenomenon of formation of multinucleated myotubes by fusion of single nucleated myoblasts (Lawson and Purslaw, 2000). Myotubes undergo further differentiation to form fully functional, contracting muscle cells (Burattini et. al., 2009).
Differentiation of skeletal muscles
The skeletal muscle differentiation is subject to expression of transcriptional factors. Myogenic regulatory factors (MRFs) are expressed in the early stages of differentiation. Paired box (Pax) proteins are the first one to be expressed which has a DNA binding domain of 128 amino acid in length (Chi et. al., 2002). Pax 3 and Pax 7 proteins are expressed in embryonic stage in muscle progenitor cell; and are homologous (Godinho, 2006). The cells failing to express Pax 3/Pax 7 are not differentiated into muscle cells (Relaix et. al., 2005). Pax 7 is also expressed in the satellite cell of a mature muscle fibre. At the myoblast stage of differentiation minimum four MRF's are needed to be expressed in the cell. These belong to the basic helix-loop-helix nuclear phosphoproteins family (Berkes and Tapscott, 2005). The most important and well-studied MRF's are MyoD (Pinney et. al., 1988), Myf5 (Braun et. al., 1990), Myogenin (Wright et. al., 1989) and MRF4 (Rhodes et. al., 1989; Braun et. al., 1990).
MRF's can be categorized in two sets, the first being MyoD and Myf5; and the second Myogenin and MRF4. MyoD and Myf5 are indicators of the cell undergoing myogenisis hence expressed initially, whereas Myogenin and MRF4 are markers for differentiation of myoblasts (Sabourin and Rudnicki, 2000). Rudnicki et. al. showed that by knocking out MyoD or Myf5 gene in a mouse did not hinder the formation of skeletal muscles, suggesting that MyoD and Myf5 can compensate for each other (Rudnicki et. al., 1992). Myogenin is only expressed in differentiated myoblasts (Braun et. al., 1989) and knocking out of myogenin gene results in perinatal death (Rudnicki et. al., 1993), this depicts the importance of myogenin in formation of myotube. MRF4 has a very limited role in differentiation and it is expressed early embryonic stage imparting myogenicity to the embryonic cells (Kassar-Duchossoy et. al., 2004). At the end of cell cycle the myoblasts start to extend themselves and interact with other myoblasts. This eventually leads to fusion of a number of myoblasts to form a multinucleated myotube. Keven Watt and colleagues showed Yap, a transcriptional cofactor (member of Hippo pathway) that acts as a regulator for skeletal muscle differentiation. They showed that for skeletal muscles to differentiate Yap must be phosphorylated at serine 127 residue (Watt et. al., 2010). Another interesting fact about skeletal muscle differentiation is that caspase3; a key apoptotic enzyme is required for skeletal muscle differentiation. Caspase3 activates MST1, a member of MAPK which is essential for skeletal muscle differentiation (Fernando et. al., 2002).
The formation of myotube triggers transcription of gene specific to mature muscle fibres like muscle creatine kinase and myosin heavy chain. Creatine kinase mRNA is found in large quantities in striated muscles (Tai et. al., 2011) and hence is reliable a muscle differentiation marker. It is involved in energy transport pathway necessary in muscle contraction and hence expressed in large quantity in muscles. Myosin heavy chain has four isoforms in skeletal muscles MyHC-Î², MyHC-IIa, MyHC-IId/x and MyHC-IIb, only MyHC-Î² is present in cardiac as well as skeletal muscles but the other three are skeletal muscle specific (Harrison et. al., 2011). Prenatal MyHC and embryonic MyHC are differentiation isoforms. Prenatal MHC in cell lines can be detected after 7 days of growth in differentiation medium (Miller, 1990). The fact that MyHC-IIb is expressed in mouse cell line but is not expressed in human skeletal muscles is intriguing and in humans MyHC-IIb is only expressed in dystrophic skeletal muscles (Harrison et. al., 2011).
Skeletal Neuromuscular Junction
The muscle fibre and motor neuron form the most well understood synapsis known as skeletal neuromuscular junction (Grinnell, 1995 and Sanger et. al., 2002). At the later stage of myogenesis, the nicotinic acetylcholine receptors (nAChR's) are expressed on the myotube surface and are concentrated around each nuclei of myotube (Godinho, 2006). The nAChR's are trans-membrane channels, made up of five subunits (two Î±, Î², Î³ and Î´) and Charbonnier et. al. demonstrated their expression is influenced by MRF's in Xenopus laevis embryo (Godinho, 2006. see page 176). Acetylcholine esterase (AChE) is a vital part of neuromuscular junction. The contact between nerve and myotube triggers the accumulation of AChE beneath the nerve terminal. AChE exists as dimers or tetramers and is attached to collagen like tail via covalent interaction to form AChE-Col Q, which is secreted into synaptic cleft at adult synapsis (Rotundo et. al., 2005). The presence of AChE-Col Q at mature synapsis enables its use as a valid marker for mature neuromuscular junction. Neuregulin Î² and argin activate MuSK (tyrosine kinase receptor) and Col Q binds to MuSK, this inturn triggers the clustering of AChR's (Cartaud et. al., 2004 and Lin et. al., 2001).
Innervation of skeletal muscles influences differentiation, organization and maintenance of adult muscle fibres. Acetylcholine receptors have an important role in fusion of myoblasts shown by in vitro experiments (Entwistle et. al., 1988 and Krause et. al., 1995). In vivo experiment performed on mice by knocking out gene for choline acetyl transferase production, showed poorly developed muscle tissue (Misgeld et. al., 2002). The work of Defez and Brachet; and Ecob-Prince et. al. suggests that in vitro co-culture with spinal cord explant improves myotube maturity (Harper and Buttery, 1992). These evidences suggest that neuromuscular junction formed at skeletal muscle improves the differentiation and maturity of skeletal muscles in vivo and in vitro. It would be a worthwhile question to ask, "Can this be replicated using established cell line of mouse and neuronal cell line?"
Insulin Dependent Glucose uptake
Blood glucose level is regulated mainly by skeletal muscles and this uptake is mediated by insulin. About 25% of body glucose is utilized by insulin dependent tissue, mainly skeletal and cardiac muscles and all muscles do not take the same amount of glucose (James et. al., 1985). The glucose that is taken up by skeletal muscles is either utilised by glycolysis to produce ATP or stored in form of glycogen (Nielsen and Richter, 2003). Insulin mediated glucose uptake by skeletal muscles occurs insulin signalling pathway, which is further split up into two different pathways namely, phostidylinisitol 3 kinase (PI3K) dependent and TC10 pathway (not discussed) or PI3K independent pathway (Whiteman et. al., 2002). In PI3K dependent pathway binding of insulin to insulin receptors causes insulin receptor substrate 1 (IRS1) mediated activation of PI3K and PI3K phosphorylates Akt/protein kinase B, which in turn results in translocation of GLUT4 to the plasma membrane (Whiteman et. al., 2002) and extracellular glucose is ingested into the skeletal muscle. The experiments of Stenbit et. al. on knockout mice heterozygous of GLUT 4 showed insulin resistance and shows symptoms for diabetes mellitus (Stenbit et. al., 1997). In humans SLC2A4 gene code for GLUT 4 mRNA (Yamamoto et. al., 2011). GLUT 4 could be used as a marker for matured myotube.
Muscle Cell Culture
In vitro culture system of myotubes is widely used as model of developmental processes occurring in vivo. Animal derived tissue culture system are used since a long time for conceiving biological systems, understanding human diseases and developing novel strategies for treatment and therapy. Development of fully functional muscle based system is vital for muscle based diseases such as Muscular dystrophy Amyotrophic lateral sclerosis (ALS) and Spinal muscular atrophy. The major advantage of in vitro system is that it allows study of isolated cell types. The most widely used cell line as in vitro skeletal muscle system is rat C2C12. Rat C2 cell line was developed by Yaffe and Saxel (Yaffe and Saxel, 1977). C2C12 cell line is a sub-clone of C2 cell line (Blau et. al., 1983). C2C12 cell lines display many of the differentiation markers of various stages of muscle development and can form mature myotubes (Portièr et. al., 1998). C2C12 have also been used in various studies. Myotubes grown in vitro despite displaying markers for fully differentiated myotube, fail to mature fully, which shows very poor insulin sensitivity. The work of Tortorella and Pilch, showed that mouse C2C12 cells expressing GLUT 4 show no significant change in insulin dependent glucose transport (Tortorella and Pilch, 2002). This suggests that there are other factors involved in glucose uptake by cultured cells. Mackrell James showed that MHC isoforms have different insulin dependent glucose uptake and hybrid muscles (muscles that have more than one isoforms) have higher insulin mediated glucose uptake than individual isoforms (MacKrell and Cartee, 2012).
Myotubes cultured in vitro are commonly used as a model system to study processes occurring in vivo. It has been observed that these in vitro culture systems do not mature completely, for instance they fail to show insulin sensitivity (Tortorella and Pilch, 2002). In this project we will be testing effects of co culturing myotube and neuronal cells on the maturation of myotubes and also study insulin sensitivity of the co culture system.
To develop skeletal neuromuscular junction using rat C2C12 muscle cell line and N2a neurobastoma neuronal cell line.
To test whether coculturing of cell lines enables complete maturation of skeletal myotubes by
Looking for expression marker of mature myotubes and NMJ.
Checking insulin sensitivity by measuring glucose uptake.
Looking for expression of GLUT4 in response to insulin.
C2C12 cell line will be cultured and differentiated in Dulbecco's modified Eagle's medium (DMEM). DMEM was formulated in 1969 by Hary Egal. It is one of the most widely used medium for cell culture experiments. C2C12 cell will be maintained in DMEM and will be supplemented with 10% fetal bovine serum, 2mM glutamine, 1% antibiotics, 0.5% anti-mycoplasm and 25mM Hepes, pH 7.5. Cell will be cultivated with humidified 5% CO2 at 37Â°C (Burattini et. al., 2004). When cells reach 80% confluence, they will be transferred into differentiation medium with 1% fetal bovine serum (Burattini et. al., 2004). Dye exclusion method will be used to check viability at various stages of cell growth.
Neuronal cell line N2a neurobalstoma (from mouse) will be used for coculturing. These cells will be cultured and differentiated in DMEM medium with 10% fetal bovine serum and for differentiation of N2a, serum free DMEM will be used, with other constituents of the medium same as that of C2C12 cell line (De Girolamo et. al., 2001).
To obtain a Nerve-muscle co-culture the N2a neuroblastoma cells will be plated along with mature myotubes and incubated for 2 days (Vianney et. al., 2011)
Protein content by Coomassie dye binding assay (Bardfords assay)
Total protein content in the cell culture is a good indicator of cell growth and viability. Bardfords assay will be used to check total protein content as it is comparatively simple to perform and it is also sensitive to a wide range of proteins. This method is widely used for cell culture experiments. It is based on the principle that binding of coomassie blue dye to the protein cause shift in absorption maximum of the dye from 465 nm to 595 nm, which can be detected spectrophotometric analysis (Bradford, 1976).
Creatine kinase assay
As mentioned in the background creatine kinase is a marker for muscle differentiation and hence measuring creatine kinase production in serum is very vital. This will be performed using creatine kinase enzymatic assay kit, this assay is based on coupled reaction of two enzymes, it is a plate based colorimetric enzymatic assay and detection is based on amount of NADH produced. The absorbance will be read 340 nm at 37Â°C for 30 minutes and rate of production of creatine kinase will be determined.
RNA extraction and quantitative real time PCR
The total RNA will be extracted from the cell culture using RNA extraction kit (Eg. RNeasy plus mini kit) and stored at -80oÂ C. The extracted RNA will be subjected to reverse transcription by reverse transcriptase enzyme to synthesis cDNA.
Bioinformatics techniques will be used to design forward and reverse primers for genes of interest for instance prenatal MHC gene, AChE gene, GLUT 4 gene etc. It is very important to consider number positions to be matched while designing a primer.
Quantitative real time PCR will be performed using the newly synthesized primers and target gene sequence will be amplified and quantified. The transcript abundance will be determined by plotting a standard curve. Real time PCR is a fast and easy method that enables quantification of sample during each amplification cycle for multiple DNA sequences at the same time.
Non-radiolabelled Glucose uptake assay (Yamamoto et. al., 2011)
One of the end points of this project will be to test the hypothesis that co-culturing myotubes with neuronal cell line improves maturity of myotubes. For this purpose insulin sensitivity of the cultured cells will be tested by quantifying glucose uptake by cells in response to insulin. The non-radiolabelled 2-deoxyglucose uptake assay will be performed which has been described by Yamamoto et. al. in their work. This assay works on the following principle: 2-deoxyglucose 6-phosphate (DG6P) is converted to 6-phospho2-deoxyglucuronic acid with the help of enzyme glucose 6-phosphate dehydrogenase (G6PDH) with simultaneous conversion of NADP+ to NADPH. At the same time enzyme diaphorase converts resazurin to resorufin with simultaneous conversion of NADPH back to NADP+. This chain reaction continues until DG6P is completely utilized. Amount of glucose uptake can be measured by quantifying amount of resorufin fluorophore (Yamamoto et. al., 2011). This assay was chosen as it is sensitive and non-radioactive hence cost effective.