Enhanced growth and myogenic differentiation of spheroid-derived C2C12 cells

Among many factors of controlling stem cell differentiation, the key transcription factor upregulation via physical force is a good strategy on the lineage-specific differentiation of stem cells. The study aimed to compare growth and myogenic potentials between the parental cells (PCs) and the 1-day-old C2C12 spheroid-derived cells (SDCs) in two-dimensional (2D) and three-dimensional (3D) culture conditions through examination of the cell proliferation and the expression of myogenic genes. The data showed that 1-day-old spheroids had more intense expression of MyoD gene with respect to the PCs. The proliferation of the SDCs significantly higher than the PCs in a time dependent manner. The SDCs had also significantly higher myogenic potential than the PCs in 2D and 3D culture conditions. The results suggest that MyoD gene upregulation through cell-cell contacts is the good approach for preparation of seed cells in muscle tissue engineering.

Lactate Promotes Myoblast Differentiation and Myotube Hypertrophy via a Pathway Involving MyoD In Vitro and Enhances Muscle Regeneration In Vivo

Lactate is a metabolic substrate mainly produced in muscles, especially during exercise. Recently, it was reported that lactate affects myoblast differentiation; however, the obtained results are inconsistent and the in vivo effect of lactate remains unclear. Our study thus aimed to evaluate the effects of lactate on myogenic differentiation and its underlying mechanism. The differentiation of C2C12 murine myogenic cells was accelerated in the presence of lactate and, consequently, myotube hypertrophy was achieved. Gene expression analysis of myogenic regulatory factors showed significantly increased myogenic determination protein (MyoD) gene expression in lactate-treated cells compared with that in untreated ones. Moreover, lactate enhanced gene and protein expression of myosin heavy chain (MHC). In particular, lactate increased gene expression of specific MHC isotypes, MHCIIb and IId/x, in a dose-dependent manner. Using a reporter assay, we showed that lactate increased promoter activity of the MHCIIb gene and that a MyoD binding site in the promoter region was necessary for the lactate-induced increase in activity. Finally, peritoneal injection of lactate in mice resulted in enhanced regeneration and fiber hypertrophy in glycerol-induced regenerating muscles. In conclusion, physiologically high lactate concentrations modulated muscle differentiation by regulating MyoD-associated networks, thereby enhancing MHC expression and myotube hypertrophy in vitro and, potentially, in vivo.

Evolutionary aspects of a new MyoD gene in amphioxus (Branchiostoma belcheri) and its promoter specificity in skeletal and cardiac muscles

Vertebrate MyoD family of transcription factors contains four members including MyoD, Myf5, Myogenin and MRF4. These myogenic regulatory factors (MRFs) play key roles in regulating skeletal muscle development and growth. Evolutionary analysis suggests that the four vertebrate MRF genes were derived by gene duplications from a single ancestral gene during chordate evolution. Better understanding of the structure and regulation of MyoD expression in amphioxus Branchiostoma belcheri may provide insight into the evolutionary history of myogenic gene duplications because of the unique position of amphioxus in evolution. We report here that isolation and characterization of a new MyoD gene, AmphiMyoD, in B. belcheri. Sequence analysis revealed that the AmphiMyoD is more closely related to myogenic transcription factors in invertebrates and vertebrates compared with the previously identified three MyoD like genes in amphioxus, suggesting that the AmphiMyoD might be the closest relative of the ancestral myogenic gene. To determine if the AmphiMyoD gene promoter controls muscle-specific expression, the AmphiMyoD promoter was linked with the green fluorescence protein (GFP) reporter and the construct was microinjected into zebrafish embryos for transient expression assay. AmphiMyoD promoter directed skeletal muscle-specific GFP expression in zebrafish embryos. In addition, it also drove GFP expression in cardiac muscles of the injected embryos, but not in other non-muscle tissues. These data demonstrated that the AmphiMyoD promoter contained regulatory elements for skeletal and cardiac muscle-specific expression. Moreover, the regulatory element(s) could function across species.