Rashida Aamir
Jake Hedrick
Reverse Transcription Expert
Neldimar Khamvongsa
Petra Lushai
Nicholas Pannell
Johnny Tran
Is the mRNA expression level of myoD, m-cadherin, as well as mitochondrial biogenesis (ATP synthase ß-subunit and Drp-1) affected throughout myogenesis in muscle cells cultured on extracellular matrix (ECM) coated proteins for C2C12 cells?
The murine myoblast cell line, C2C12, is frequently used to study myogenesis where mononucleated myoblasts grow in standard cell culture and undergo differentiation into multinucleated myotubes. However, these plates lack cell-to-extracellular matrix (ECM) interaction within this microenvironment which is required for the complex process of skeletal muscle formation in vivo. Studies have shown that culture plates coated with ECM proteins, such as fibronectin and collagen, promote adhesion and proliferation of myoblasts in vitro. Furthermore, how these coatings could impact the gene expression of myogenic regulatory factors (MRFs) and other biomarkers involved in myogenesis in vitro still requires consideration. Here, this study investigated the effect of either fibronectin or gelatin-coated plates on the gene expression of four myogenic markers: m-cadherin, MyoD, DRP-1 (gene DNM1L), and the 𝛽-subunit of ATP synthase (gene ATP5F1B) as compared to cells cultured on uncoated plates at myoblast, days 0, 4, and 7.
Figure 1. The interaction of ECM to its surrounding cell. Fibronectin protein mediates between the plasma membrane’s embedded integrin and other ECM proteins such as cross-linked collagen.
Khan Academy. (2021). The Extracellular Matrix and Cell. https://www.khanacademy.org/science/biology/structure-of-a-cell/cytoskeleton-junctions-and-extracellular-structures/a/the-extracellular-matrix-and-cell-wall.
M-cadherin is a specific type of cellular adhesion molecule from the cadherin superfamily, which like the other members of this family, are transmembrane proteins that partially constitute adherens junctions. Cadherins have an intracellular domain, that attaches to the cytoskeleton as an anchor, and an extracellular domain that will homodimerize with other cadherin molecules to form calcium-dependent cell-to-cell linkages.
Specifically, m-cadherin is expressed during skeletal muscle differentiation and is important in cell-cell adhesion, which is vital for allowing the fusion of neighbouring myoblast to form myotubes during myogenesis. Therefore, m-cadherin exists as a marker of midpoint myogenesis, which provides ample opportunity to observe whether its expression will be affected as myoblasts are cultured on different ECM coated proteins, fibronectin or gelatin.
MyoD belongs to a family of transcription factors, including Myf5, myogenin, and MRF4 that strictly regulate skeletal muscle cell differentiation. In myoblasts, myogenic differentiation is initiated by high confluency or by inhibiting the growth factors, MyoD gene expression is increased, and it plays potential roles:
It activates muscle-specific genes by interacting with E protein and MEF2 transcription factors,
withdraws the cells from cell cycles,
abolishes cell proliferating genes expressions,
and finally leads to matured myotubes formation after myoblasts alignment and fusion.
Additionally, functional MyoD (or Myf5) is crucial for muscle regeneration in injured cells and has a specific role in lost muscle replenishment.
Due to its roles as a master regulator in the myogenic differentiation of C2C12 cells, myoD is chosen as one of the markers to observe whether or not its mRNA expression is affected during myogenesis, induced on ECM coated proteins, fibronectin or gelatin.
Figure 2. MyoD upregulation is required for satellite cells' differentiation during myogenesis. Extrinsic signalling triggers satellite cells' activation, downregulating Pax7, while upregulating MyoD. MyoD promotes cell proliferation, upregulates myogenin and activates muscle-specific genes, which consequently leads to terminally differentiated muscle cell formation.
The C2C12 cells undergo many changes during myogenesis, including a shift from mainly glycolysis to a heavier reliance on aerobic respiration and oxidative phosphorylation. During cellular differentiation, the mitochondria undergo biogenesis in order to keep up with the cells changing energy requirements. It is believed that the mitochondria increase in volume (fusion) followed by an increase in number (fission) throughout the process, although this experiment on fibronectin and gelatin has not been previously published. We have thus chosen these two different mitochondrial enzymes for molecular markers to investigate and follow the biogenesis of mitochondria during myogenesis, a process in which we expect to observe an upregulation of relative gene expression for both markers under ECM conditions.
ATP synthase is the enzyme within the inner-membrane of each mitochondria that facilitates the production of the energy storage molecule adenosine triphosphate (ATP) from adenosine diphosphate (ADP). Muscle cells require more ATP production than almost any other cell due to their contractile abilities amongst all the other cellular functions. The 𝛽-subunit of this enzyme is responsible for the catalytic activity in the ATP → ADP reaction, and was therefore chosen as one of the mitochondrial markers.
Dynamin-related protein 1 (Drp-1) consists of a GTPase enzyme and a GTPase effector domain, with amino acid helical segments separating the two. Three different isoforms of Drp-1 exist in mice, and six different isoforms exist in humans.
Functions carried out by dynamin, a protein which cleaves vesicles from a membrane via physical torsion of the membrane, resulting in delocalization from the constituent membrane, include:
Mitochondrial Fission, the process by which mitochondria undergo physical separation during differentiation
Mitophagy, the degradation/recycling of damaged mitochondria
Fission processes, affiliated with intracellular calcium flux, and are thus related to apoptotic and oncogenic signal transduction initiation
Intracellular [ROS], the concentration of reactive oxygen species arising from incompletely-fused/fragmented mitochondria, is controlled (indirectly) by the precise fission of mitochondria via Drp-1