Morphological changes throughout C2C12 skeletal cell myogenesis. Images of fixed unstained and uncoated C2C12 cells were captured using an Olympus CKX31 phase-contrast microscope at 200x magnification.

Here you can see clear morphological changes from star-shaped myoblasts to elongated myotubes. The myoblasts show clear filopodia which is evidence of the cell migration in early stages of myogenesis. Day 0 at 100% confluency, C2C12 cells have turned off cell division and started the recruitment of transcription factor to allow for cell differentiation. Day 4 and 7 myotubes are formed which confirms the C2C12 myogenesis model.

Phase-contrast images showed that gelatin-coated plates allowed for a more organized and longer myotubes development compared to the control.

Localization of α-actin and Myosin During C2C12 Skeletal Muscle Cell Myogenesis. All cells were fixed, permeabilized, and viewed using an OLYMPUS IX51. Images were captured and combined using cellSense 1.16 software at 400x magnification and with α-actin and myosin exposure of 100x the exposure for DAPI. Cells were treated with Sarcomeric ɑ-Actin Monoclonal primary antibody (red), Myosin 4 polyclonal Rabbit primary antibody (green), and DAPI fluorescent stain (blue).

The first image shows undifferentiated myoblasts with very little myosin visible, and ɑ-Actin localized in cytoskeletal areas. Day 0 cells show an increase of myosin visible diffusely throughout the cell. ɑ-Actin remains in cytoskeletal areas. Cells show signs of division and migration. Day 5 cells show early myotube formation with ɑ-Actin and myosin colocalized within the myotube. Day 7 cells show intermediate myotube development. Low levels of α-Actin with myosin aligned in a parallel fashion and colocalized within myotubes.

Localization of α-actin and Myosin During C2C12 Skeletal Muscle Cell Myogenesis. All cells were fixed, permeabilized, and viewed using an OLYMPUS IX51. Images were captured and combined using cellSense 1.16 software at 400x magnification and with α-actin and myosin exposure of 100x the exposure for DAPI. A-E cells were treated with Sarcomeric ɑ-Actin Monoclonal primary antibody (red), Myosin 4 polyclonal Rabbit primary antibody (green), and DAPI fluorescent stain (blue).

Undifferentiated star-shaped myoblast where α-actin is localized outside the nucleus and myosin is diffuse along the cell periphery. Day 0 cells were taken at 100% confluency and individual myoblast is starting to anchor their filopodia and initiate fusion. Day 5 cells, Intermediate myotube formation with alternating bands of actin and myosin bands. Day 7 cells Intermediate and mature myotube with a clear parallel arrangement. Day 12 mature myotubes that are arranged linearly. there was no clear difference between the localization of α-actin and myosin between the control and gelatin-coated plates.

Gelatin's role in the EMC is to provide tensile strength, promote cell development, and regulate cell adhesion and chemotaxis (Rozario & DeSimone, 2010). The results shown in this research demonstrate that gelatin provides an environment that enhances cell development and growth due to higher confluency (faster growth, proliferation, and migration) at earlier stages of myogenesis. These findings are consistent with previous research stating that collagens have been found to control the release of certain growth factors during myogenesis, therefore regulating cell differentiation and behaviour (Nishimura, 2015).

By observing the changes in the sarcomeric proteins, α-actin and myosin, through the stages of myogenesis, it is possible to infer whether the extracellular protein gelatin impacts the process of myogenesis. Here we see inconsistencies in the observable expression of both α-actin and myosin which both are expressed in the early undifferentiated stages. This is unexpected because α-sarcomeric actin and sarcomeric-myosin are the molecules that make up sarcomeres which do not develop until the later stages of myogenesis. However, previous research has shown that sarcomeric actin and myosin are present in undifferentiated myoblasts which progressively develop a structured (co-localized) pattern as arranged in sarcomeres and myofibrils about 5 days after differentiation (Burattini et al., 2004). It is also discussed that sarcomeric actin and filamentous actin are expressed in undifferentiated C2C12 myoblasts mainly in the cell periphery and focal contacts (Burattini et al., 2004). This could account for the presence of the α-actin and myosin expressed in C2C12 myoblasts but does not account for the lack of sarcomeric protein levels in the later stages of growth. Perhaps gelatin can impact the expression of sarcomeric proteins in later stages of myogenesis, however, further research is needed to explore the impact of ECMs on myogenesis.

• Finding more specific antibodies that are able to differentiate between α-actin and myosin isoforms

• Performing an experiment the uses all three ECM proteins to compare the relative gene expression

• Identify which specific myosin and alpha-actin isoforms are expressed in the early stages. It's possibly cardiac-skeletal actin but our group's preliminary testing suggests that cardiac-actin is highly expressed in the later stages