Myogenin And M-cadherin

Student Researchers

Laura Reyes Palacios

4th year BSc. Cellular & Molecular Biology

Diana Carolina Ortiz Pérez

4th year BSc. Cellular & Molecular Biology

Noha Zeineldin

4th year BSc. Cellular & Molecular Biology

Meron Feleke

4th year BSc. Cellular & Molecular Biology

Background

The C2C12 cell line is used to study myogenesis as it can proliferate at the myoblast stage and will withdraw from the cell cycle, entering differentiation when certain conditions are met. These events can be followed through the mRNA expression of differentiation markers like m-cadherin and myogenin, which are known to have specific roles in the fusion and differentiation of myoblasts.

Myoblasts

Myoblast Fusion

Zeineldin, BIOL 3202, 2019

Matured Myotubes

Myogenin

(Myog) is a key developmental regulator for skeletal muscle formation because it facilitates the regeneration of adult myofibers during muscle damage. It plays a role in embryonic myofibers formation as well. Myogenin expression sharply increases during myoblast fusion and differentiation when the C2C12 cell line is cultured in the differentiation medium. The expression of myogenin in the late phase of myogenesis suggests that myogenin is a differentiation marker for the entry of myoblast into fusion and differentiation into myotubes.

Figure 1. Model showing the presence of Myogenin throughout the differentiation of C2C12 cells. This model shows the temporal relationship between the expression of myogenin and the different myogenic events - however it does not imply a causal relationship.

Image taken from: Andrés, W., Walsh, K. (1996). Myogenin expression, cell cycle withdrawal, and phenotypic differentiation are temporally separable events that precede cell fusion upon myogenesis. The Journal of Cell Biology, 132(4), 657–666. https://doi.org/10.1083/jcb.132.4.657

M-cadherin

M-cadherin is a transmembrane glycoprotein that mediates calcium dependent intercellular adhesion. It is important for the regulation of morphogenetic processes, such as myogenesis. As differentiation of C2C12 cells is induced by serum starvation or as they reach 100% confluency, this will result in the upregulation of M-cadherin, a myogenic marker of fusion, causing cells to withdraw from cell cycle to allow for myoblast fusion and trigger terminal cell differentiation.

Figure 2. M-cadherin interaction with β-catenin/plakoglobin and with α-catenin during differentiation to allow for myoblast fusion.

Image taken from: Kaufmann, U., Martin, B., Link, D., Witt, K., Zeitler, R., Reinhard, S., & Starzinski-Powitz, A. (1999). M-cadherin and its sisters in development of striated muscle. Cell and tissue research, 296(1), 191-198.

How is mRNA expression of Myogenin and M-cadherin affected during myogenesis of C2C12 cell line when differentiation takes place on 2% horse serum media in comparison 10% fetal Bovine serum?

Our Study Rationale

In this investigation the focus on two of the more common culturing & differentiating methods Fetal Bovine Serum (FBS) vs. Horse Serum (HS) comes due to their constant use in previous literature. According to the ATCC protocol, C2C12 cells are best grown in 10% fetal bovine serum (FBS). In this study, we aim to quantify any changes 10% FBS vs 2% HS may have on m-cadherin and myogenin mRNA expression during myogenesis. It is unclear if serum conditions can impact the mRNA expression levels of essential proteins in C2C12 cells. What we find through this study could prove to be significant at broadening our understanding of C2C12, and in the future allow us to choose the culturing method that will work best with this model and the study being conducted.

For this experiment, the control C2C12 were grown in 10% FBS throughout myogenesis, while the experimental C2C12 were grown in 10% FBS, and changed to 2%HS at D0, and maintained in 2%HS for the rest of myogenesis. The sampling days chosen were MB, D0, D4, D7 & D11, cells were incubated at 37°C, 5% CO₂_, and media were replaced every 3-4 days.

Validation experiments

RNA - Integrity and Purity

Figure 1. Validation of RNA integrity and purity. (A) Representative image of 1% agarose denaturing RNA gel electrophoresis of denatured RNA of a myoblast (MB) sample, displayed to check MB RNA quality after extraction.The 18S and 28S ribosomal RNA bands were clearly visible on the samples indicating that the extracted RNA is intact and has not been degraded. (B) A representative image of NanoDrop spectrophotometer analysis of a MB sample extracted RNA to determine RNA purity. A260/A280 ratio = 2.09 which indicates that there is no protein contamination and A260/A230 ratio = 2.18 which indicates no salt contamination. The RNA concentration was determined by Qubit broad range kit.

Dynamic Range of Reverse Transcriptase

Figure 2. Linear dynamic range and efficiency of reverse-transcription determination. Serial 10 fold dilutions were performed starting with a 1ug myoblast RNA quantity to determine the initial amount of RNA that will be used for reverse transcriptase reaction. This scatter plot was generated using Cq values (Y-axis) from quantitative PCR vs initial RNA concentration in ng/ul in each dilution (X-axis) using five different reference genes (GAPDH, Csnka2, Hprt, AP3D1, and Rer1).

Determination of Optimal Reference Gene

Figure 3. Optimal Reference primer efficiency (GAPDH). (A) Bar graph comparing the standard deviation for myoblast (MB) and myotube (MT) expression for optimal reference gene determination. Five different reference genes (either low, medium, or high expression) were tested to choose the best reference gene for future analysis. GAPDH was found to have the lowest standard deviation which is also less than 0.5. (B) GAPDH reference gene has a primer efficiency of 82%. qPCR was performed using 7 three-fold dilutions with starting cDNA quantity ranging from 1ug to 1pg. R²=0.996, which suggests a good amplification of the primers. The slope 3.82, was used to calculate the primer efficiency percentage.

Primer Efficiency and Amplification

Figure 4. Primer efficiency of M-cadherin and Myogenin. The experiment was performed using cDNA reverse transcribed from 100 ng starting RNA, then made 1:3 serial dilutions of the cDNA for the qPCR reactions. (A) Myogenin & M-cadherin. The x-axis indicates the logarithmic values of the dilution factors while the Y-axis indicates Cq value of each dilution sample. (Myog) R²=0.989, m=-3.5 & (M-cad) R²=0.994, m=-3.5. Calculated M-cad and Myog primer efficiency were 93% and 91% respectively. (B) M-cadherin (top) and Myogenin (bottom) primers melting curves were shown respectively. (C) 1% agarose DNA gel electrophoresis, showing the amplicon size of both the target gene (M-cadherin and Myogenin) and the reference gene (GAPDH)

C2C12 myoblast cells Morphology
in 10% FBS and 2% HS

Figure 1. Microscopy Imaging of Myoblast Cells Differentiating to Myotube under Two Different Serum Conditions. Images were taken using phase-contrast microscopy (at 100x and 200x magnification). Brightness and contrast were adjusted using ImageJ software. Images at the top lane represent the experimental group, in which growth media was switched to 2% Horse Serum (HS) at Day 4 – Day 11. Images at the bottom lane represent the control group, cells were grown in 10% Fetal bovine serum through the entire process. Cells were grown at 37°C and 5% CO₂. Culture media was changed for all dishes every 3-4 days. Days selection were based on the relevance to the target gene that is being analyzed (M-cadherin and Myogenin).

M-cadherin and myogenin Relative Gene expression

(A) myogenin relative gene expression

(B) m-Cadherin relative gene expression

Figure 2. Relative Gene Expression of M-cadherin and Myogenin. Pfaffl method was used to measure fold change in relative gene expression of M-cadherin and Myogenin, and GAPDH was used for normalization. (A) M-cadherin expression level was present at low levels at the MB stage and it got upregulated when differentiation (D0) is induced. There was a change in expression between cells grown under 10% FBS and 2% HS, where 2% HS seems to have higher expression of M-cadherin than cells grown in 10% FBS. (B) Myogenin expression showed an increase in D4/D5 and a decrease in D7 C2C12 cells grown in 10% FBS and in 2% HS. In both culture conditions, myog expression levels follow the expected trend as it started to show low myog expression at MB and D0, then increased in D4/D5 and decreased in D7 and D11 of cell differentiation. However, there is no a statistical significance difference ((M-cad p-value =0.328) and (myog P-value = 0.203)) between 10% FBS and 2 % HS according to two-way-ANOVA test. Bars represent the mean + SD of at least two independent experiments.

Conclusions

C2C12 cultures treated with 10% FBS during differentiation showed mononucleated star-like shaped myoblasts up to day 0. At day 4 cells started fusing and forming multinucleated myotubes later on. Myotubes numbers were increased on day 7 and 11 in both differentiating conditions however, myotube sizes were smaller on 10% FBS cultures in comparison to those grown in 2% HS. This indicated that HS had shown larger and more multinucleated myotubes (shown by the white-headed in figure 1, D11E).
In cells grown in 2% HS, we were expecting the mRNA expression levels of both M-cadherin and Myogenin to decrease. However, in our data the mRNA expression of M-cadherin and Myogenin did not decrease at D7 and D11 as expected. This can be explained by the fact that there was a large amount of myoblast population differentiating and fusing which was visibility seen at D11E (shown by the white arrow).
The results of relative gene expression of M-cadherin in C2C12 myoblast cells indicate there is a peak between days 0 and 4/5 when differentiation is induced, which follows the same results as previous literature.²
Our data show that there is a change in M-cadherin and Myogenin mRNA expression at days 7 and 11/12 of differentiation and even higher at day 11/12. However, statistical analysis showed no significant difference in gene expression between 10% FBS and 2% HS treated cells.

Future directions

Results could be improved if more trials for relative gene expression are performed, therefore future studies should aim in producing more trials to obtain more accurate data.
Future studies should focus also on exploring cellular contraction in myoblasts using 10% FBS and 2% HS growing media.
For future directions, more trials are required to investigate if there is a significant difference between 10% FBS and 2% HS, as well as investigating possible signaling pathways that might be involved in the activation of myoblast cells in later stages of myogenesis in cells grown in 2% HS.