Experimental Studies on the Differentiation of Fibroblasts into Myoblasts induced by MyoD Genes in vitro

Abstract

To evaluate the biological functions of myogenic regulatory factors, we have examined the effects of ectopic expression of MyoD and Cx43 genes in the fibroblasts on the differentiation of myoblast in vitro. The expression of MyoD and Cx43 in the transfectants was confirmed by RT-PCR and Western blot. More than 50% of fibroblasts transfected with MyoD or both MyoD and Cx43 genes displayed typical morphological features of myoblast-like cells at 20 days following gene transfection, including cell elongation, cytoplasm enrichment and granule manifold. Moreover, these myoblast-like cells also expressed both desmin and α-actin. These results demonstrate that direct exogenous expression of the myogenic regulatory factors is sufficient to induce transdifferentiation of fibroblasts into a myoblast-like lineage and provide new insights into the trauma repair after myocardial infraction.

Keywords: Cx43; MyoD; fibroblast; lentivirus; myogenesis.

Figure 1

Culture of packaging cells 293FT and production of lentivirus. (A) Morphology of 293FT cells before viral packaging. The packaging cells were shown in good condition; (B) Morphology of 293FT cells during ingathering. The pLenti-based expression vector containing gene of rat Cx43 or MyoD and the ViraPower packing were transfected into 293FT cell line to produce lentivirus. The viruses were ingathered after culturing for 72h.

Figure 2

Expression of rat Cx43 and MyoD after lentivirus transfection. MyoD and Cx43 expression levels in four groups of fibroblasts: non-treated fibroblast (N), MyoD gene-transfected fibroblast (M), Cx43 gene-transfected fibroblast (C) and MyoD-Cx43-transfected fibroblast (MC) were analyzed. (A) MyoD mRNA expression was determined by RT-PCR. The 254bp band is MyoD gene amplifying product. The figure shows that MyoD mRNA was highly expressed in the MyoD- and MyoD plus Cx43- transfected fibroblasts; (B) Cx43 mRNA expression was determined by RT-PCR. The 580 bp band indicates Cx43 gene PCR product. Panel B reveals that Cx43 mRNA was only detected highly in the Cx43- transfected fibroblasts. In addition, low level expression of Cx43 was found in MyoD gene-transfected group. Non-treated fibroblasts did not express mRNA of these two genes; (C) MyoD and (D) Cx43 protein level was analyzed by western blotting. GAPDH was used as a sample loading control. MyoD or Cx43 protein was expressed in the MyoD- or Cx43-transfected fibroblasts. Note low level expression of Cx43 was found in MyoD gene-transfected fibroblasts.

Figure 3

Transfected-Myo D and Cx43 genes enable morphology conversion of fibroblast to myoblast like cells. Positive clones of four groups of fibroblasts: (A) non-treated fibroblast (N), (B) MyoD gene-transfected fibroblast (M), (C) Cx43 gene-transfected fibroblast (C) and (D) MyoD-Cx43-transfected fibroblast (MC) were screened out after lentivirus with Cx43 or MyoD gene transfection into rat fibroblast cell line RFL-6. The figures show that RFL-6 cells in M and MC group had changed with cell elongation, cytoplasm enrichment and granule manifold and become myoblast like cells. Cell morphology of C group was similar with N group but proliferation ability decreased obviously (data not shown).

Figure 4

Expression of Desmin and α-actin in different transfected group were analyzed by western blotting. The antibodies of α-actin and Desmin were muscle cell specific. GAPDH was also detected as a sample loading control. The figure shows that MyoD downstream molecules, Desmin and α-actin, were all detected in cells transfected with MyoD or MyoD plus Cx43 genes but not in the cells transfected with Cx43 gene alone or control.