Introductory remarks

Myogenesis is triggered by multiple factors, including the expression of muscle regulatory factors as well as external cell signaling mediated by the ECM (Grzelkowska-Kowalczyk, 2016). This signaling enhances proliferation and differentiation of precursor muscle cells (Grzelkowska-Kowalczyk, 2016). Cells grown in vitro often lack an ECM, meaning that certain cell signaling processes essential for proper development are absent. Including ECM proteins in cell culture processes has the potential to enhance myoblast growth and proliferation, as well as aid in the development and the formation of mature myotubes.

ECM proteins are known to contribute to both the proliferation and migration of myoblasts, and the alignment and elongation of myotubes (Vaz et al., 2012). In the ECM, fibronectin is present in a mesh-like pattern and provides a substrate for cell growth (Grzelkowska-Kowalczyk, 2016). Fibronectin is known to speed up cell proliferation while simultaneously aiding in migration (Vaz et al., 2012). This may promote the linear alignment of myotubes during myogenesis. Lastly, fully developed muscle cells have regenerative capabilities that are enhanced by the ECM (Grezelkowska-Kowalczyk, 2016). Down-regulated or mutant ECM proteins contribute to a vast number of connective tissue disorders, such as osteogenesis, Ehlers-Danlos syndrome, and chondrodysplasias (Shireen & Bateman, 2020). The overall health and functionality of muscle tissue appears highly dependent on ECM proteins. Any deficiencies or abnormalities can lead to dysfunction and improper formation (Zhang et al., 2012). Therefore, the addition of ECM proteins such as fibronectin to cell cultures has the potential to produce a more physiologically accurate model of myogenesis, and may provide implications for studying muscle-related diseases and therapies.

cell ORganization & proliferation

Phase contrast images show enhanced organization of myotubes which can be seen growing in parallel across the entirety of the plates surface. Uncoated plates are in disarray and grow in random clusters. Fibronectin also seemed to enhance myoblast growth rate as well. More myoblasts can be seen during myoblast stage in experimental than control groups even though both samples were taken on the same day. It seems that myoblasts grow faster when placed on an ECM coating.

Figure 1. C2C12 mouse cells were grown in 10% FBS and 1% PENSTREP in DMEM, and fixed during the myoblast stage (MB), at 100% confluency (D0), Day 4 (D4), Day 7 (D7), and Day 11 (D11). The samples were observed using an Olympus CKX31 phase contrast microscope at 200x magnification. Fibronectin appears to promote the linear organization of myotubes during myogenesis.

Cell Morphology

Cell growth and organization was enhanced on fibronectin-coated plates, and this caused the production of thicker myotubes with higher levels of multinucleation.

Relative gene expression

Figure 5. RNA was extracted and purified from C2C12 control cells, and subject to purification and concentration testing. The sample was run on a 1% agarose gel to confirm the presence of the large (28S) and small (18S) ribosomal subunits. Qubit IQ was used to confirm the percentages of small and large RNA present within the samples. Lastly, Qubit BR was used to determine the concentration of RNA in each sample.

Figure 7. qPCR cardiac alpha-actin primer efficiency curve and amplicon length verification. 100 ng of C2C12 Day 0 cDNA was diluted in a 1:3 series using primers for the target gene cardiac α-actin. (A) Efficiency curve of cardiac α-actin primers obtained from qPCR shows a slope of -3.37 and an R2 value of 0.998. Primer efficiency was calculated to be 98% using slope of regression. (B) Relative amplicon length was verified using a 1% agarose gel; amplicons were expected to be 144 bps for cardiac α-actin.

Figure 8. The effect of fibronectin, gelatin, and laminin on relative myosin mRNA expression throughout myogenesis. Relative mRNA expression of myosin was steadily low from myoblast to day 5 of differentiation on all protein coatings. Laminin seems to have the most drastic effect on myosin mRNA expression espeacially in day 7 and 12 samples with peak expression on day 7. All data points are normalized with GAPDH as a reference gene and MB control samples as calibrator (n=9).

Figure 9. The effect of fibronectin and gelatin on relative myosin mRNA expression throughout myogenesis. Laminin results were removed. Relative mRNA expression of myosin was steadily low from myoblast to day 5 of differentiation on all protein coatings. Fibronectin drastically decreased myosin mRNA expression on D5, D7, and D12. Gelatin caused higher expression of myosin mRNA in early stages, from MB-D5, but caused a slight decrease in expression on D7-D12 compared to controls.

Figure 10. The effect of fibronectin, gelatin, and laminin on relative skeletal α-actin mRNA expression throughout myogenesis. Relative mRNA expression increased on gelatin and fibronectin coated plates. Fibronectin increased actin mRNA expression from days 4 to 12, gelatin increased expression from day 4-7 which then drastically decreased on day 12 and laminin did not show significant changes. All data points are normalized with GAPDH as a reference gene and MB control samples as calibrator (n=9).

Figure 11. The effect of fibronectin on relative cardiac α-actin mRNA expression throughout myogenesis. Relative mRNA expression was relatively low on fibronectin coated plates during myoblast and day 0 of differentiation. Fibronectin significantly decreased cardiac actin mRNA expression from days 5 to 12 compared to control. Uncoated plates show a continuous increase in cardiac α-actin mRNA expression throughout differentiation. All data points are normalized with GAPDH as a reference gene and MB control samples as calibrator (n=9).

Interestingly, the relative mRNA expression of alpha-cardiac actin, when grown on the fibronectin ECM, decreases significantly from D7 to D12, contrary to initial hypotheses.

Immunocytochemistry (ICC) images revealed the early expression of alpha-actin and myosin. Since we expected to see these two proteins at later stages of myogenesis, as sarcomeres developed, we believed that our fluorescent antibodies were labeling alternate isoforms of these proteins. We theorized that our alpha-actin antibodies were labeling both sarcomeric alpha-actin, which is seen on later days, and cardiac alpha-actin, which is expressed in early myogenesis. Surprisingly, cardiac alpha-actin mRNA expression was relatively low in early myoblasts and then increased over the course of myogenesis. This indicates that the alpha-actin labeled in early days during ICC is representative of an alternate isoform. The presence of cardiac alpha-actin within mature myotubes, especially in higher amounts, has been reported to be an indicator of muscle disease (Vandekerckhove et al, 1986; Tondeleir, Vandamme, Vandekerckhove, Ampe, & Lambrechts 2009). Specifically, Nemaline Myopathy (NM), which is characterized by muscle weakness throughout the body (Tondeleir et al, 2009). In patients with NM, an increased amount of cardiac alpha-actin is often reported, especially in severe cases (Tondeleir et al, 2009). Cardiac alpha-actin started increasing on day 0 and peaked at day 12 in uncoated control samples. Cardiac alpha-actin levels were 5-fold higher than skeletal alpha-alpha on day 12 within control groups. Fibronectin appears to reduce the presence of cardiac actin within culture and therefore could be more accurate of mature and healthy muscle cells. We also theorized that the early observance of myosin was in fact alternate isoforms of myosin, outside of the expected myosin IIb. These could include myosin X, which has previously been found in mouse myoblast filopodia (Hammers et al., 2021) and myosin XVIIIb, which has been found in human myoblasts (Salamon et al, 2003) Myosin XVIIIb relocates to the nucleus after the differentiation process has been initiated, which is consistent with our ICC images, particularly on D4 and D7. To reiterate, we believe that the fluorescent antibodies used here are not specific to skeletal alpha-actin and myosin IIb as previously thought.

While fibronectin is commonly known to be beneficial for myoblast proliferation, fusion, and migration, Vaz et al. have reported that fibronectin may inhibit the differentiation process if present at later stages of myogenesis. This might, in part, explain the relatively low myosin and alpha-actin expression seen in our D7 and D12 samples. In contrast, laminin is thought to be important in later stages of myogenesis for promoting myotube formation and adhesion (Grzelkowska-Kowalczyk, 2016). This might explain the drastic increase in myosin mRNA expression seen with laminin samples.

While fibronectin appears beneficial during early stages of myogenesis, particularly in relation to promoting myoblast proliferation, fusion, and linear migration, it appears to have some negative effects on the differentiation process. Myotube branching was observed more frequently in cells grown on fibronectin. Branching of myotubes is considered abnormal, and is often related to muscle dystrophy, or muscle tissue that has been damaged and subsequently regenerated. Branching has also been found to occur more frequently on smooth, flat surfaces, compared to patterned or grooved surfaces. In this study, fibronectin was applied evenly across the coverslips, which were laid flat to dry, resulting in a smooth growth surface. Therefore, it is not clear whether the branching seen here was a result of fibronectin hindering the differentiation process, or a result of the flat growth surface that the cells were grown on. It would be insightful to explore the effect of a combination of ECM proteins on myogenesis. Since gelatin is known to stimulate myogenesis and help in muscle regeneration, it may prove useful in the prevention of branching.

In a physiologically relevant sense, the magnitude of cardiac alpha-actin mRNA expression was indeed more consistent with previous publications, especially those which use a fibronectin ECM coating (Chaturvedi, V., et al., 2015). That is, a relatively consistent pattern emerges in recent literature pertaining to the turning off and turning on of genetic transcription in C2C12 cells. Specifically, mRNA expression of cardiac/skeletal alpha-actin by C2C12 cells have been correlated to be inversely proportional to one another, depending on the relative extracellular/intracellular concentration balances immediately surrounding the cell. For this reason, it can certainly be theorized that the implementation of a fibronectin scaffold during developmental stages of C2C12 myogenesis increases the applicability of the findings, in a physiologically-relevant sense. Of particular importance, the notion of physiological relevance, when used throughout any of the discussion points, refers to the increased relevance of our findings to those of previously published literature. That is, physiological relevance is used interchangeably with accurate data acquisition, specifically pertaining to in-Vivo experimental assays of similar nature.

Ultimately, we saw that fibronectin enhanced the linear, parallel growth of myotubes during myogenesis. This is a better representation of myogenesis in vivo. At later time points (D5-D12), fibronectin decreased myosin, cardiac alpha-actin, and skeletal alpha-actin mRNA expression. We theorize that significantly less mRNA transcript is produced because sarcomeres have formed at earlier time points, although this would need to be confirmed.

• Why do we see myosin and alpha-actin fluorescence as early as myoblast stages in skeletal muscle cells in control and experimental groups? What isoforms are being labeled here?

• Why does fibronectin decrease the relative mRNA expression of myosin, cardiac, and skeletal alpha-actin at all time points compared to controls?

• Myosin and skeletal alpha actin antibodies need to be more specific to the isoform we are interested in looking at. Ordering better antibodies for these proteins can truly determine if other isoforms of myosin and actin are present within cell cultures during myogenesis.

• It would be insightful to explore the effect of a combination of ECM proteins on myogenesis. Since gelatin is known to stimulate myogenesis and help in muscle regeneration, it may prove useful in the prevention of branching, since branching is often a sign of weak or damaged muscle tissue. Fibronectin and laminin are crucial at opposite time points during myogenesis: fibronectin is important for myoblast proliferation and migration, while laminin is conversely important for myotube formation and adhesion.

• A proteomics study would be extremely beneficial to deepen our understanding of myosin and alpha-actin expression during myogenesis.

• Implementation of a fluorescence in-Situ hybridization (FISH) assay, whereby two separate fluorophores are spliced into the genetic transcripts of alpha-cardiac and alpha-skeletal actin, respectively, may lead to a more conclusive understanding of the actin isoforms which are present in various stages throughout myogenesis.

• Third-generation genomic assays, such as Oxford Nanopore, may also provide additional insight into the exact genetic codon sequences/distinctions of C2C12 myocytes

Additional photos