Alpha-Skeletal Actin and Beta-actin

UnderGraduate Research TEAM

Arielle Adrian

Kendra Chichak

Mitch Demers

Andrew Wakabayashi

Keara Wong

Research Question

Do the extracellular matrix-derived proteins fibronectin or gelatin affect the morphology, ɑ-skeletal actin expression or ꞵ-actin expression in C2C12 cells during myogenesis?

Rational For this study

Using extracellular matrix (ECM)-derived protein coatings has been a popular method within literature to study myogenesis in vitro. However, many critical aspects are unknown regarding the specific effects of these protein coatings towards the morphology and overall gene expression of key myogenic markers. Regarding our target genes, α-skeletal actin is involved in myotube contractile function and is usually expressed late during myogenesis, while ꞵ-actin is highly expressed in early myogenesis during myoblast motility and fusion, before differentiation begins. This study aimed to compare how culturing of C2C12 murine myoblasts on plates coated with the ECM-derived proteins fibronectin and gelatin affect the mRNA gene expression of α-skeletal actin and ꞵ-actin. Additionally, we observed changes in morphology of the cells throughout myogenesis under the influence of these protein coatings. Such research provides foundational information about the effect of ECM-derived protein coatings on the morphology and gene expression of muscle cells during differentiation.

Target Genes

Alpha-Skeletal Actin

α-skeletal actin is an integral protein involved in the formation of Z-bodies, which develop in sarcomeres, the contractile units of myotubes. The formation of adhesion sites in myotubes relies on mechanical cues within the microenvironment. Actin polymerization is important in forming and maintaining these adhesion sites. Thus, quantification of this myogenic marker may provide insight into the development of myotubes, and whether such development represents that of in vivo myogenesis.

Because α-skeletal actin represents the development of mature myotubes with contractile function, it is beneficial to analyze its change in mRNA expression during the differentiation of C2C12 cells cultured on ECM-derived protein coatings. In comparison with traditional culture plates, the morphology and mRNA expression may be significantly different during late stage myogenesis. A model that utilizes ECM proteins found in the body may lead to the development of contractile muscle cultures, which would better simulate in vivo muscle.

Beta-Actin

β-actin is a key protein involved in cell fusion and motility. It is the most dominant cytoplasmic actin isoform in eukaryotes in almost all cell types. Localization of β-actin mRNA has been shown to directly influence cell motility during fusion of myoblasts throughout myogenesis. Studies of β-actin mRNA expression in C2C12 cells show significant expression before initial differentiation, and down-regulation after the development of myotubes. In this model we hypothesize similar observations of β-actin down-regulation.

There is evidence that β-actin mRNA expression is inversely related to α-actin mRNA expression. Therefore, β-actin is a suitable candidate for comparison to the α-skeletal actin aspect of this study. The model of ECM derived protein coating may promote the chemical environment necessary for C2C12 cell movement and fusion compared to standard cell culture methods.

workflow

Morphological Analysis

Figure 1. Morphology and growth of C2C12 cells are influenced by interactions with fibronectin and gelatin culture plate coatings. C2C12 murine myoblasts were seeded at 80% confluency and cultured in DMEM with 10% FBS during the proliferation stage (myoblast to day 0), followed by 2% horse serum after 100% confluency was reached to induce differentiation (day 0 to day 12). Images taken with confocal microscope at 100X magnification. Control cells seeded on standard culture dishes with no coatings. Experimental culture dishes were coated with either 500 μL of 50 μg/μL fibronectin or 0.2% gelatin and seeded. Fibronectin treated cells showed more branching and directional growth of myotubes at a faster rate compared to control and gelatin treated samples. Gelatin treated cells exhibited more limited patterns of growth, with shorter, thicker myotubes that were not developed as the fibronectin treated samples.

Relative mRNA Gene Expression

Figure 2. α-Skeletal actin and β-actin mRNA expression levels change in culture of C2C12 with exposure to different ECM-derived protein coatings. Data is expressed as a ratio of target mRNA expression levels normalized to GAPDH reference mRNA expression levels. Experiments of each sample were performed in triplicate and data are shown as mean +/- SD (n = 2). mRNA expression of α-skeletal actin (left) and β-actin (right) normalized to the GAPDH reference gene and plotted at the various stages of sampling (myoblast, day 0, day 4, day 7, and day 12). RNA was extracted from the aforementioned time-course samples with 100 ng of RNA used for reverse transcription to cDNA. Relative gene expression was performed using qPCR and calculated using the Pfaffl method, with each series calibrated to its own myoblast sample (n = 1). α-skeletal actin mRNA expression saw a stable level of expression in days 4 to 7 of differentiation, with a sudden decrease of expression on day 12 in the control and gelatin samples. β-actin expression remained low from the myoblast phase onwards, though it was increased in both experimental treatments from day 4 to day 12.

conclusions

Fibronectin treatment led to an altered cell morphology at day 7 of cell differentiation. Slender and long myotubes (~700µm) were formed as compared to the control and gelatin samples of short and thick myotubes (~200µm), which is consistent with published findings.5

Fibronectin coating may promote alignment and increase growth rate of myotubes in culture, which matches findings in the literature.5 Myotubes cultured on fibronectin had parallel alignment in all cultures, their altered morphology. When compared to control and gelatin treatments, they reached confluency at a faster rate with more advanced growth throughout all days of sampling.

In uncoated control cells, α-skeletal actin mRNA expression increased in early-mid myogenesis, between days 4 and 7, followed by a decrease at day 12. Alternatively, fibronectin treatment saw mRNA expression remain stable from days 4-12, which may be an effect of increased growth rate and a need for a steadier mRNA and protein production. Future research should involve protein analysis to verify these findings.

In control cells, β-actin mRNA expression increased at the start of myoblast differentiation (day 0), remained steady, then decreased at day 12. In contrast, fibronectin treatment led to largely decreased expression, though expression remained higher in fibronectin treated cells from days 4-12. β-actin is expressed in myoblasts,3 and the slower growth rate of the control cultures compared to the fibronectin treated cultures may account for this. Further investigation into the effect on growth rate of the different treatments may be beneficial.

Future Directions

Having more technical or biological replicates to reduce the variation in data and provide statistical significance towards the relation of myogenic specific marker with extracellular matrix-derived coatings.
Follow up studies should compare the effects of other adhesive treatment models on the physiological relevance of in vitro myogenesis, such as with the use of laminin or a combination of ECM proteins.
Introduce 2-D micropatterned surfaces, such as decellularized plant-derived scaffolds, in conjunction with the adhesion coating. This would aid in promoting a physiological relevant model.

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