MyoD, Myogenin, and Myosin Heavy Chain

current students

Emma Bogner

"The cell culture queen"

Charleigha Cao-Gagnon

"The RNA extraction queen"

Jessica Lee

"The gel queen"

Daniel Major

"The qPCR queen"

Jaiden Peeace

"The quantification queen"

Research Question

Does mRNA expression of myogenic regulatory factors (myoD and myogenin (myoG)), or contractile apparatus protein myosin heavy chain II (myh4) change over the course of myogenesis when different serum conditions are used during differentiation?

(Image: Bogner & Major, BIOL 3102, 2020)

MyoD

MyoD is a myogenic regulatory factor (MRF) which plays an important role in myoblast determination and cell cycle regulation. MyoD, among other MRFs, contains a helix-loop-helix domain that allows for the heterodimerization with E proteins to efficiently bind to DNA. MyoD is able to activate the transcription program of muscle specific genes. It is able to induce differentiation in a variety of muscle cells by prompting cell-cycle withdrawal through the inhibition of cytokine signals and cross-talking with cell cycle regulators.

Myogenin (MYOG)

Myogenin (MyoG) is a myogenic regulatory factor that governs the latter portion of myogenesis. Together with MyoD, MyoG assists in cell cycle exit and terminal differentiation of myoblasts to form myocytes. Myogenin also plays an essential role in driving myocyte fusion to produce syncytial myotubes (in-vitro) or myofibers (in vivo). Despite functional redundancy between the MRFs, MyoG is a crucial factor in embryonic myogenesis as evidenced by lethality in MyoG-null neonatal mice. However, this indispensability does not appear to extend to postnatal (regenerative) myogenesis, where the role of Myogenin is less well-defined (Zammit, 2017).

Figure 1. Changes in expression of key proteins including MyoD, Myogenin (MyoG), and Myosin Heavy Chain (MHC) across myogenic differentiation. MyoD is most abundant during early myogenesis, where it prompts proliferative myoblasts to exit the cell cycle and become terminally differentiated myocytes. This stage of terminal differentiation is also marked by a drop in expression of stemness marker Pax7. MyoG also helps to stimulate terminal differentation, however it plays a more central role in regulating the subsequent fusion of myocytes to form myotubes (in vitro) or myofibers (in vivo) during later myogenesis. Expression of MHC begins to build near the end of myogenesis, as it is incorporated into sarcomeres that will make up the mature, contractile myofibers.

Myosin heavy Chain (MyHC)

Myosin is present within sarcomeres, which are the functional units of skeletal muscle. Myosin is essential for binding actin filaments and shortening sarcomeres - thereby causing contraction. Typically, myosin is composed of a motor head, neck and tail domain; MyHC makes up the motor head, which physically binds thin filamentous actin, while myosin light chain is present in the neck region.

Within MyHC there are 6 known isoforms: embryonic (emb), neonatal (neo), type I, type IIa, IIx, and IIb. Emb and neo isoforms are expressed during embryonic development as well as during states of muscle regeneration. In adult skeletal muscle type I is expressed in slow muscle while type II isoforms are expressed in fast muscle. In C2C12 muscle cells, there are two clusters of myosin differentiation. Myosin II are important genes to measure during myogenesis progression because they are expressed late in differentiation and are markers of contractile apparatus formation.

Specifically, we chose the MyHCIIb isoform (myh4 transcript) because C2C12 myoblasts express this isoform late in differentiation, which complements an investigation parallel to early expression of myogenic regulatory factors. In addition, previous studies have associated MyHCIIb expression with visual hypertrophy of myotubes in vitro and increased muscularity in vivo (Gunawan, 2007; Wimmers, 2008).

Rationale for Our Study

Serum starvation is used in the majority of studies exploring in vitro myogenesis, however the methods vary considerably and the impact of this intervention on gene expression is unclear. Our target genes MyoD, MyoG, and MyHCIIb were selected as they represent markers of early, middle, and late differentiation with previously defined expression trends found in literature. This study aims to determine whether varying serum conditions impact the mRNA expression of MyoD, MyoG, and MyHCIIb during myogenesis. This research provides important information about how culture conditions may impact differentiation and could inform future experiments investigating protein-level gene expression and optimization of muscle cell growth.

Results

mORPHOLOGY

Fig 1. Representative morphologies of C2C12 cells cultured in growth media (GM) or starvation media (SM) during myogenesis.

C2C12 myoblasts were cultured in GM until cells were ~100% confluent, at which point half were switched to SM and the other half maintained in GM for up to 12 days. (A) Images of the cells at each day that they were sampled. These sampling points were chosen based on the gene expression timing of target genes MyoD as an early marker, MyoG as a middle marker, and Myh4 as a late marker of myogenesis in the literature (Chal & Pourquié, 2017). In both conditions, the myoblasts began as fusiform, isolated cells which proliferated, made contact, and fused (day 0 and day 1) to form elongated myotubes (day 5, day 7 and day 12). Mature myotube formation can be seen in both GM and SM in the day 12 images. Plates were viewed using a phase contrast microscope at 200X total magnification. (B) DIC images (at 100X total magnification) of day 12 GM and SM. By day 12, substantially more developed myotubes (narrower, longer, and aligned) were visible using GM vs. SM and an abundance of myoblast cells were visible by day 12 in SM only.

c2c12_d12_10fbs_11032021_ccg_dm_2.avi

c2c12_d12_10fbs_11032021_ccg_dm.avi

Weird flex but okay... we have contraction! Video (at 100X total magnification) of spontaneously contracting cells observed on day 12 in growth media conditions exclusively.

Relative gene expression

"Don't quote me on this, but this is the best melt curve I've ever seen"

- Dr. Laura Atkinson after myh4 qPCR repeated trial due to mysterious amplicons appearing out of nowhere.

Fig 2. Representative time-course expression of myoD, myoG, and myh4 relative to gapdh in starvation media (SM; blue) and growth media (GM; yellow) culture conditions. C2C12 myoblasts were cultured in GM (10% FBS) and sampled at 60 - 90% confluency (MB) and ~100% confluency (D0). On day 0 (D0), the cultured cells were either switched to SM (2% HS) or maintained in GM up to day 12 (D12). RNA was extracted and 100 ng of RNA was input for reverse transcription to cDNA. N= 1 and qPCR was run in technical triplicate.

CONCLUSIONS

GM conditions yielded more mature (longer, narrower, and aligned) and functional myotubes in comparison to SM; this was indicated by the ability to contract exclusively in GM conditions. In addition, SM had an increasingly large population of myoblasts toward the day 12 timepoint.
myoD expression remains stable across myogenesis in GM conditions - aligning with the previously described role of MyoD as a post-transcriptionally regulated transcription factor. Expression appears more dynamic in SM.
myoG expression increased from day 0 and peaked by day 7 in SM conditions, while the trend was increasing in GM conditions. This pattern aligns with the morphological observation of myotube formation by day 5, and is consistent with the role of MyoG as a mediator of terminal differentiation.
myh4 expression was considerably upregulated by day 5, which was consistent with our observations of myotube formation; Myh4 is an essential component of the contractile apparatus in mature muscle. By day 12 we noticed divergence of myh4 expression under GM vs SM conditions. This could be due to the abundance of myoblast cells observed in day 12 SM.
The overall trends exhibited by each target support the pattern described in the literature: MyoD is responsible for activating downstream myogenic factors, including MyoG, which activates terminal differentiation and expression of muscle specific genes, including Myh4.
Preliminary data suggests there are differences in transcript expression of myoG and myh4 based on culture conditions, however, this could be due to the abundance of myoblast cells observed under starvation conditions
These results cast doubt on practice of using starvation media during myogenesis, especially at later time points. However, further data collection and analysis of protein expression is required to confirm this hypothesis

Future Directions

Investigate the patterns of protein expression for MyoD, MyoG, and MyHCII during myogenesis and compare to transcript expression to determine true gene expression and presence of post-transcriptional regulatory mechanisms.
Increase the validity of these preliminary results by increasing the number of trials by a future cohort of students. This would also demonstrate replicability of our approach. With additional trials, statistical tests could be used.

queens (2021-04-04 at 21:52 GMT-7)

2021 FST Research Days Poster Submission

Relevant Publications

References

Chal, J., & Pourquié, O. (2017). Making muscle: skeletal myogenesis in vivo and in vitro. Development (Cambridge), 144(12), 2104–2122. https://doi.org/10.1242/dev.151035

Ferri, P., Barbieri, E., Burattini, S., Guescini, M., D'Emilio, A., Biagiotti, L., ... & Falcieri, E. (2009). Expression and subcellular localization of myogenic regulatory factors during the differentiation of skeletal muscle C2C12 myoblasts. Journal of cellular biochemistry, 108(6), 1302-1317.

Gunawan, R. (2007). Ractopamine induces differential gene expression in porcine skeletal muscles. Journal of Animal Science, 85(9), 2115–2124. https://doi.org/10.2527/jas.2006-540

Wimmers, N. (2008). Relationship between myosin heavy chain isoform expression and muscling in several diverse pig breeds. Journal of Animal Science, 86(4), 795–803. https://doi.org/10.2527/jas.2006-521

Zammit, P. S. (2017, December). Function of the myogenic regulatory factors Myf5, MyoD, Myogenin and MRF4 in skeletal muscle, satellite cells and regenerative myogenesis. In Seminars in cell & developmental biology (Vol. 72, pp. 19-32). Academic Press.

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