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but decreased by day 8 in myoblasts on muscle-matrix and fibronectin. Brown et al found that Myf5 decreased in C2C12 cells during differentiation, in contrast MYOG levels increased initially but decreased as the cells fully differentiated. This is similar to our findings with C2C12 cells on fibronectin. Another study measured mRNA levels of Myf5 and MYOG in primary porcine muscle stem cells cultured on collagen I, laminin, fibronectin, gelatin and Matrigel. In this case the expression of Myf5 decreased with cell differentiation regardless of substrate; but MYOG expression in cells on Matrigel increased in a time dependent manner and was higher than in cells cultured on the other substrates at day 5 of differentiation. On muscle matrix we found MYOG expression PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19666601 in C2C12 myoblasts increased as differentiation progressed. Like solubilised muscle matrix, Matrigel is a complex mix of ECM proteins. Stern et al measured the levels of myogenin protein at day 1 and 3 of differentiation on plastic and 21 / 27 An Acellular Muscle Matrix Supports Myoblast Differentiation ECM coated surfaces and found that myogenin levels decreased while MyHC levels increased as the cells differentiated, and myogenin levels were higher in C2C12 cells cultured on skeletal muscle matrix surfaces than on uncoated surfaces. This mirrors our mRNA data. Interestingly MYOG null myoblasts, that cannot differentiate in vivo, form myotubes as efficiently as wild-type myoblasts when cultured on gelatin. Possibly, the in vitro requirements for myogenin are less stringent than in vivo, and factors in complex matrices maintain expression of this protein further into the differentiation pathway than is usually seen in vitro. Myosin, the muscle contractile protein, consists of four light chains and two heavy chains. Myosin heavy chain has different isoforms: MYH3 is an embryonic form whereas MYH1 and MYH7 are adult isoforms; MYH1 is expressed in fast-twitch fibres and MYH7 in slow-twitch muscle fibres. Cardiac actin is expressed early during differentiation of skeletal muscle, but is down regulated as skeletal muscle alpha actin is expressed. ACTA1 is the major actin isoform in muscle and is a late differentiation marker. The mRNA levels for MYH3 and ACTC1 increased at day 4 of differentiation on all three surfaces, PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19666849 but decreased by day 8; with the drop in expression more pronounced on muscle matrix and fibronectin than on collagen I. It is thought Myf5 initiates the expression of MYH3 and MyoD enhances MYH3 expression. MyoD is expressed slightly later than Myf5, appearing in myoblasts and myocytes, but decreasing markedly in myotubes. It might be expected that a decrease in Myf5 expression will be followed a day or two later by a corresponding decrease in MYH3 expression. This was observed in a study of C2C12 cells. Our data do not allow such a detailed interpretation, but the expression pattern of Myf5 and MYH3 in cells grown on fibronectin and muscle matrix are consistent with this report. An early peak in MYH3 expression was followed by a decline in human muscle myoblasts cultured on gelatin, which resembles our data for cells on collagen I. The decrease in ACTC1 expression levels by day 8 on all three surfaces is probably associated with an increased expression of other actin isoforms, a conclusion that is supported by the expression pattern observed for ACTA1 on all substrates. Myotube maturation was indicated by the time dependent Sunset Yellow FCF site increase in expression of adult myosin and sk