Efficient in vitro theoretical account of myotubes is a principle platform to analyze developmental procedures happening in vivo, probe of drug activity and planing new signifiers of therapy. Myotubes grown in vitro do non maturate expeditiously and have low insulin sensitiveness. There is published grounds proposing “ co-culture of myotube and spinal cord explant improves myotube adulthood ” . In this undertaking the hypothesis will be tested utilizing rat C2C12 and N2a neuroblastoma cell line. Real clip PCR will be used to magnify and quantify mRNA transcripts of to the full matured myotube markers and a non-radioactive glucose uptake check will be performed to prove for insulin sensitiveness of myotubes. It is expected that this effort at co-culturing cell line will better insulin sensitiveness and in bend will let development of a better experimental theoretical account for assortment of probes.
Background to probe
The human organic structure consists of three types of musculus tissues, viz.
skeletal musculuss, smooth musculuss and cardiac musculuss. Smooth musculuss and cardiac musculuss are nonvoluntary musculuss while skeletal musculuss are voluntary musculuss. Skeletal musculuss contribute to 30 % and 38 % of the organic structure weight of adult females and work forces severally ( Janssen et al. , 200 ) .
Skeletal musculuss are besides called striated musculuss and they are parallel in orientation. Skeletal musculuss contain myofibres which are made up of compact package of myofibryls packed together. Myofibryls have sarcomere which is responsible for contraction of the skeletal musculuss and hence contributes to motions of the organic structure. The regenerative belongings of skeletal musculuss is imparted by presence of orbiter cells, which differentiate into myoblasts and travel to the damaged location and acquire incorporated ( Goldring et al. , 2002 and Anderson, 2006 ) .
Myogenesis is a phenomenon of formation of multinucleated myotubes by merger of individual nucleated myoblasts ( Lawson and Purslaw, 2000 ) . Myotubes undergo farther distinction to organize to the full functional, undertaking musculus cells ( Burattini et. al. , 2009 ) .
Differentiation of skeletal musculuss
The skeletal musculus distinction is capable to look of transcriptional factors. Myogenic regulative factors ( MRFs ) are expressed in the early phases of distinction.
Paired box ( Pax ) proteins are the first one to be expressed which has a Deoxyribonucleic acid adhering sphere of 128 amino acid in length ( Chi et. al. , 2002 ) . Pax 3 and Pax 7 proteins are expressed in embryologic phase in musculus primogenitor cell ; and are homologous ( Godinho, 2006 ) .
The cells neglecting to show Pax 3/Pax 7 are non differentiated into musculus cells ( Relaix et. al. , 2005 ) . Pax 7 is besides expressed in the orbiter cell of a mature musculus fiber. At the myoblast phase of distinction minimum four MRF ‘s are needed to be expressed in the cell. These belong to the basic helix-loop-helix atomic phosphoproteins household ( Berkes and Tapscott, 2005 ) .
The most of import 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 2nd Myogenin and MRF4.
MyoD and Myf5 are indexs of the cell undergoing myogenisis hence expressed ab initio, whereas Myogenin and MRF4 are markers for distinction of myoblasts ( Sabourin and Rudnicki, 2000 ) . Rudnicki et. Al. showed that by strike harding out MyoD or Myf5 cistron in a mouse did non impede the formation of skeletal musculuss, proposing that MyoD and Myf5 can counterbalance for each other ( Rudnicki et. al. , 1992 ) .
Myogenin is merely expressed in differentiated myoblasts ( Braun et. al. , 1989 ) and strike harding out of myogenin cistron consequences in perinatal decease ( Rudnicki et.
al. , 1993 ) , this depicts the importance of myogenin in formation of myotube. MRF4 has a really limited function in distinction and it is expressed early embryologic phase leaving myogenicity to the embryologic cells ( Kassar-Duchossoy et. al. , 2004 ) .
At the terminal of cell rhythm the myoblasts start to widen themselves and interact with other myoblasts. This finally leads to merger of a figure of myoblasts to organize a multinucleated myotube. Keven Watt and co-workers showed Yap, a transcriptional cofactor ( member of Hippo tract ) that acts as a regulator for skeletal musculus distinction. They showed that for skeletal musculuss to distinguish Yap must be phosphorylated at serine 127 residue ( Watt et.
al. , 2010 ) . Another interesting fact about skeletal musculus distinction is that caspase3 ; a cardinal apoptotic enzyme is required for skeletal musculus distinction. Caspase3 activates MST1, a member of MAPK which is indispensable for skeletal musculus distinction ( Fernando et. al. , 2002 ) .
The formation of myotube triggers written text of cistron specific to maturate musculus fibers like musculus creatine kinase and myosin heavy concatenation. Creatine kinase messenger RNA is found in big measures in striated musculuss ( Tai et. al. , 2011 ) and hence is dependable a musculus distinction marker.
It is involved in energy conveyance pathway necessary in musculus contraction and hence expressed in big measure in musculuss. Myosin heavy concatenation has four isoforms in skeletal musculuss MyHC-I? , MyHC-IIa, MyHC-IId/x and MyHC-IIb, merely MyHC-I? is present in cardiac every bit good as skeletal musculuss but the other three are skeletal musculus particular ( Harrison et. al.
, 2011 ) . Prenatal MyHC and embryologic MyHC are distinction isoforms. Prenatal MHC in cell lines can be detected after 7 yearss of growing in distinction medium ( Miller, 1990 ) . The fact that MyHC-IIb is expressed in mouse cell line but is non expressed in human skeletal musculuss is fascinating and in worlds MyHC-IIb is merely expressed in dystrophic skeletal musculuss ( Harrison et. al.
, 2011 ) .
Skeletal Neuromuscular Junction
The musculus fiber and motor nerve cell form the most good understood synapsis known as skeletal neuromuscular junction ( Grinnell, 1995 and Sanger et. al.
, 2002 ) . At the ulterior phase of myogenesis, the nicotinic acetylcholine receptors ( nAChR ‘s ) are expressed on the myotube surface and are concentrated around each karyon of myotube ( Godinho, 2006 ) . The nAChR ‘s are trans-membrane channels, made up of five fractional monetary units ( two I± , I? , I? and I? ) and Charbonnier et. Al. demonstrated their look is influenced by MRF ‘s in Xenopus laevis embryo ( Godinho, 2006. see page 176 ) .
Acetylcholine esterase ( AChE ) is a critical portion of neuromuscular junction. The contact between nervus and myotube triggers the accretion of AChE beneath the nervus terminus. AChE exists as dimers or tetramers and is attached to collagen like tail via covalent interaction to organize AChE-Col Q, which is secreted into synaptic cleft at grownup synapsis ( Rotundo et. al. , 2005 ) . The presence of AChE-Col Q at mature synapsis enables its usage as a valid marker for mature neuromuscular junction. Neuregulin I? and argin activate MuSK ( tyrosine kinase receptor ) and Col Q binds to MuSK, this inturn triggers the bunch of AChR ‘s ( Cartaud et.
al. , 2004 and Lin et. al. , 2001 ) .Excitation of skeletal musculuss influences distinction, organisation and care of grownup musculus fibers. Acetylcholine receptors have an of import function in merger of myoblasts shown by in vitro experiments ( Entwistle et. al.
, 1988 and Krause et. al. , 1995 ) . In vivo experiment performed on mice by strike harding out cistron for choline acetyl transferase production, showed ill developed musculus 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 adulthood ( Harper and Buttery, 1992 ) . These groundss suggest that neuromuscular junction formed at skeletal musculus improves the distinction and adulthood of skeletal musculuss in vivo and in vitro. It would be a worthwhile inquiry to inquire, “ Can this be replicated utilizing established cell line of mouse and neural cell line? ”
Insulin Dependent Glucose consumption
Blood glucose degree is regulated chiefly by skeletal musculuss and this consumption is mediated by insulin.
About 25 % of organic structure glucose is utilized by insulin dependent tissue, chiefly skeletal and cardiac musculuss and all musculuss do non take the same sum of glucose ( James et. al. , 1985 ) . The glucose that is taken up by skeletal musculuss is either utilized by glycolysis to bring forth ATP or stored in signifier of animal starch ( Nielsen and Richter, 2003 ) . Insulin mediated glucose uptake by skeletal musculuss occurs insulin signalling tract, which is further split up into two different tracts viz. , phostidylinisitol 3 kinase ( PI3K ) dependant and TC10 tract ( non discussed ) or PI3K independent tract ( 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 bend consequences in translocation of GLUT4 to the plasma membrane ( Whiteman et. al. , 2002 ) and extracellular glucose is ingested into the skeletal musculus. The experiments of Stenbit et. Al. on smasher mice heterozygous of GLUT 4 showed insulin opposition and shows symptoms for diabetes mellitus ( Stenbit et. al.
, 1997 ) . In worlds SLC2A4 cistron codification for GLUT 4 messenger RNA ( Yamamoto et. al. , 2011 ) . GLUT 4 could be used as a marker for full-blown myotube.
Muscle Cell Culture
In vitro civilization system of myotubes is widely used as theoretical account of developmental procedures happening in vivo. Animal derived tissue civilization system are used since a long clip for gestating biological systems, understanding human diseases and developing fresh schemes for intervention and therapy. Development of to the full functional musculus based system is critical for musculus based diseases such as Muscular dystrophy Amyotrophic sidelong induration ( ALS ) and Spinal muscular wasting.
The major advantage of in vitro system is that it allows survey of stray cell types. The most widely used cell line as in vitro skeletal musculus 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 distinction markers of assorted phases of musculus development and can organize mature myotubes ( Portier et. al. , 1998 ) . C2C12 have besides been used in assorted surveies. Myotubes grown in vitro despite exposing markers for to the full differentiated myotube, fail to maturate to the full, which shows really hapless insulin sensitiveness. The work of Tortorella and Pilch, showed that mouse C2C12 cells showing GLUT 4 show no important alteration in insulin dependant glucose conveyance ( Tortorella and Pilch, 2002 ) . This suggests that there are other factors involved in glucose uptake by civilized cells.
Mackrell James showed that MHC isoforms have different insulin dependant glucose consumption and intercrossed musculuss ( musculuss that have more than one isoforms ) have higher insulin mediated glucose consumption than single isoforms ( MacKrell and Cartee, 2012 ) .Myotubes cultured in vitro are normally used as a theoretical account system to analyze procedures happening in vivo. It has been observed that these in vitro civilization systems do non maturate wholly, for case they fail to demo insulin sensitiveness ( Tortorella and Pilch, 2002 ) . In this undertaking we will be proving effects of carbon monoxide culturing myotube and neural cells on the ripening of myotubes and besides study insulin sensitiveness of the co civilization system.
To develop skeletal neuromuscular junction utilizing rat C2C12 musculus cell line and N2a neurobastoma neural cell line.To prove whether coculturing of cell lines enables complete ripening of skeletal myotubes byLooking for look marker of mature myotubes and NMJ.Checking insulin sensitiveness by mensurating glucose consumption.
Looking for look 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 civilization experiments. C2C12 cell will be maintained in DMEM and will be supplemented with 10 % foetal 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 37A°C ( Burattini et. al. , 2004 ) . When cells reach 80 % meeting, they will be transferred into distinction medium with 1 % foetal bovine serum ( Burattini et.
al. , 2004 ) . Dye exclusion method will be used to look into viability at assorted phases of cell growing.
Neuronal cell line N2a neurobalstoma ( from mouse ) will be used for coculturing. These cells will be cultured and differentiated in DMEM medium with 10 % foetal bovine serum and for distinction of N2a, serum free DMEM will be used, with other components 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 yearss ( Vianney et. al. , 2011 )
Protein content by Coomassie dye adhering check ( Bardfords assay )
Entire protein content in the cell civilization is a good index of cell growing and viability.
Bardfords assay will be used to look into entire protein content as it is relatively simple to execute and it is besides sensitive to a broad scope of proteins. This method is widely used for cell civilization experiments. It is based on the rule that binding of coomassie blue dye to the protein cause displacement in soaking up upper limit of the dye from 465 nanometers to 595 nanometers, which can be detected spectrophotometric analysis ( Bradford, 1976 ) .
Creatine kinase check
As mentioned in the background creatine kinase is a marker for musculus distinction and hence measurement creatine kinase production in serum is really critical. This will be performed utilizing creatine kinase enzymatic check kit, this check is based on conjugate reaction of two enzymes, it is a home base based colorimetric enzymatic check and sensing is based on sum of NADH produced. The optical density will be read 340 nanometers at 37A°C for 30 proceedingss and rate of production of creatine kinase will be determined.
RNA extraction and quantitative existent clip PCR
The entire RNA will be extracted from the cell civilization utilizing RNA extraction kit ( Eg. RNeasy plus mini kit ) and stored at -80oA C. The extracted RNA will be subjected to change by reversal written text by rearward RNA polymerase enzyme to synthesis complementary DNA.Bioinformatics techniques will be used to plan frontward and change by reversal primers for cistrons of involvement for case antenatal MHC cistron, AChE cistron, GLUT 4 cistron etc.
It is really of import to see figure places to be matched while planing a primer.Quantitative existent clip PCR will be performed utilizing the freshly synthesized primers and aim cistron sequence will be amplified and quantified. The transcript copiousness will be determined by plotting a standard curve. Real clip PCR is a fast and easy method that enables quantification of sample during each elaboration rhythm for multiple DNA sequences at the same clip.Non-radiolabelled Glucose consumption check ( Yamamoto et. al.
, 2011 )One of the terminal points of this undertaking will be to prove the hypothesis that co-culturing myotubes with neural cell line improves adulthood of myotubes. For this purpose insulin sensitiveness of the civilized cells will be tested by quantifying glucose uptake by cells in response to insulin. The non-radiolabelled 2-deoxyglucose consumption check will be performed which has been described by Yamamoto et. Al. in their work. This check works on the undermentioned rule: 2-deoxyglucose 6-phosphate ( DG6P ) is converted to 6-phospho2-deoxyglucuronic acid with the aid of enzyme glucose 6-phosphate dehydrogenase ( G6PDH ) with coincident transition of NADP+ to NADPH.
At the same clip enzyme diaphorase converts resazurin to resorufin with coincident transition of NADPH back to NADP+ . This concatenation reaction continues until DG6P is wholly utilised. Sum of glucose consumption can be measured by quantifying sum of resorufin fluorophore ( Yamamoto et. al. , 2011 ) .
This check was chosen as it is sensitive and non-radioactive hence cost effectual.