Muscle cell differentiation in vitro and effects of antisense oligodeoxyribonucleotides on gene expression of contractile proteins

Abstract

Coordinated and stoichiometric expression of contractile apparatus genes during the differentiation of L6 and C2C12 cells was studied. Expression of the genes initiated prior to fusion, and coordination in-between amply expressed actin and tropomyosin (TM), and among fast members of a troponin complex, was apparent. Pronounced synthesis of members of this complex initiated slightly later than that of actin and TM. Expression seemed to start with troponins I (TnI) and C (TnC), whereas troponin T (TnT) showed variation in the cell lines. The order and abundance of expression is supported by the regulatory sequences that the genes have. The results suggest that assembly of thin filament starts with the interaction of α-actin and TM. Initial basic TnI may interact with slightly later accumulating acidic TnC, and the complex may be then joined through a basic TnT to TM-actin structure. α-actin and TM were shown to be more stable proteins than the members of the troponin complex, especially TnC. The shorter half-life member of a TM dimer may determine the renewal of the TnT-TM-actin structure, whereas the most labile TnC may have the same impact on the troponin complex. The other goal of the study was to disturb the normal expression of TnT in L6 cells and TnC in C2Cl2 cells by using antisense oligonucleotides (ODNs) and/or antisense RNA. Treatment of L6 cells with 25 μM antisense AUG-TnT 3'end phosphorothioated ODNs (3'SODN), and C2C12 cells with 50 μM antisense TnC unmodified ODNs (umODNs) for 12 h or antisense TnC RNA, reduced the synthesis of the respective proteins by 20-50%. Antisense capTnC umODNs were more effective than antisense AUG-TnC umODNs or any other site along the mRNA-binding ODNs. Treatment with antisense TnT 3'5-0DN also altered cellular mRNA levels of actin, TM, and TnC, and its effects on contraction apparatus protein synthesis were greater than by treatment with antisense TnC. However, phosphorothioated antisense S-0DNs against the internal region of c/sTnC mRNA (int-c/sTnC) unexpectedly increased the synthesis of both TnC proteins in C2C12 cells, when treated for 48-72 h with 20-100 μM concentration of S-ODNs. The reason for the increase may be due to the induction of Spl by S-ODNs. An Increase was apparent also in around 120 h differentiated myotubes treated with antisensec/ sTnC umODNs together with Actinomycin D for 3 h. C/sTnC synthesis also increased, when in vitro transcribed c/sTnC mRNA was translated in the presence of either antisense cape/ sTnC or AUG-c/sTnC umODNs, or sense int-c/sTnC umODN in rabbit reticulocyte lysate. Translation with antisense AUG-c/sTnC umODN in wheat germ extract, however, reduced the synthesis. Antisense cap-c/sTnC umODN had no effect. The results suggest that ODNs may open the folding of c/sTnC mRNA to a more translatable form. The unusual folding of the mRNA is also supported by its long half-life. The differentiation study also suggested, that TnC is post-transcriptionally regulated. RNAse H activity in C2C12 myocytes withdrawn from the division cycle may also decline during differentiation, which may be one reason for the differing results of the antisense cap-c/sTnC umODN treatments.