AAC1, alpha-skeletal actin & beta-actin

Current Students

Ami Yan

Christian Donnelly

Alec Faustino

Jennie Vuong

Michael Makhoul

Tanis Beaver

Research Question:

How does myogenic differentiation induced by cell-to-cell contact or serum starvation affect the mRNA expression of AAC1, ɑ-skeletal actin and β-actin in C2C12 muscle cells throughout myogenesis?

Rationale:

Previous studies have used C2C12 myoblasts in various growth serum conditions to induce differentiation. However, those studies did not conclude which condition would be favourable to yield the most accurate results. It is still unclear of which growth serum methodology would be best for obtaining data for mRNA expression studies. By studying the various growth serum conditions such as myogenic differentiation induced by cell-to-cell contact or serum starvation, we can further understand how culture conditions can impact certain mRNA expressions during myogenic differentiation.

Target Genes

ADP/ATP carrier protein 1 (AAC1)

Figure is from Stepien et al. (1992) p. 14594

*ANT = Adenine Nucleotide Translocator = AAC.

AAC1 is an isoform of ADP/ATP Carriers (AACs) that are responsible for exchanging cytosolic ADP with ATP produced in the mitochondria. Since there is a direct correlation between mitochondrial biogenesis and myogenesis, the levels of AAC1 are anticipated to be elevated in myotubes when compared to myoblasts. The goal of this target gene is to prove if AAC1 is a suitable biomarker of myogenesis. In a study by Lunardi et al. (1997), they concluded "it would appear that expression of the [human AAC1] gene is a marker of a late stage in myogenesis" (p. 15269).

We expect that, as in Fig. 3 to the left, AAC1 ("ANT1") mRNA will spike during late myogenesis.

AAC shown in green to exchange cytosolic ADP with mitochondrial generated ATP.

𝞪-skeletal Actin

A cytoskeletal protein essential for providing contractibility in sarcomeres after myoblast fusion. 𝞪-Skeletal actin, much like other actin isoforms is globular protein that combines to forms actin filaments. Within skeletal muscle cells, 𝞪-skeletal actin along with myosin form myofilaments which compose sarcomeres, and allow for contractile function. Within proliferating myoblast 𝞪-skeletal actin is transcriptionally silent. Playing a role in later stage myogenesis, 𝞪-skeletal actin mRNA expression increases during skeletal muscle maturation.

We hypothesize that the mRNA expression of alpha-skeletal actin would increase upon the initiation of fusion in both cell-to-cell contact and cell starvation. However with cell starvation increasing the rate of differentiation, we hypothesize that the mRNA expression would begin increasing earlier compared to that of cell-to-cell contact cell culturing.

Northern blot results, tracking mRNA expression of 𝞪-skeletal actin and 𝞪-cardiac actin in differentiating C2C12 myoblasts. Day 0 corresponds to the day the proliferating myoblasts reached 90% confluence and were switched from growth medium (20% FBS) to differentiation medium (2% HS). The significant spike in 𝞪-cardiac actin mRNA expression seen on day 1 was an unexpected result from this paper.

Figure is from Bains, Ponte, Blau, and Kedes. (1984).

Expression of 𝞪-skeletal actin, 𝞪-cardiac actin, MLC1emb, and MLC1F/3f mRNA in differentiating C2/7 myoblasts. Expression was measured using northern blotting. They used two different methods for inducing differentiation: serum starvation (differentiation in 2% serum), or addition of bovine insulin and human transferrin.

Figure is from Cox, Garner, and Buckingham. (1990).

𝜷-actin

𝜷-actin is a cytoskeletal actin that plays an important role in cell migration, cell-to-cell adhesion and cell proliferation. 𝜷-actin has been commonly used as a housekeeping gene for numerous molecular biology studies involving techniques such as qPCR and western blot. It has been used to normalize quantitative data since previous researchers thought that was constantly expressed throughout differentiation. However, a previous study conducted by Bains et al. (1984) have demonstrated that during myogenic differentiation of C2C12 cells, 𝜷-actin was differentially expressed. During the myoblast stage, 𝜷-actin demonstrated high mRNA expression. At the onset of differentiation, a decline in mRNA expression was observed. This suggests that 𝜷-actin has an important role during early myogenesis but does not have a significant role during differentiation. Therefore, manipulation of variables contributing to later myogenic differentiation may not have a huge impact on 𝜷-actin mRNA expression.

Autoradiogram of northern blot showing mRNA expression for beta actin throughout differentiation. Well (a) is from proliferating C2C12 cells, (b) is from 90% confluent C2C12 cells before culturing in differentiation media and (c-j) is from cultured C2C12 cells in DMEM and 2% horse serum differentiation media in after 6, 12, 24, 36, 48, 72, 96 and 169 hours respectively.

Figure from Bains et al. (1984).

Figure from Hildyard & Wells (2014).

Both figures demonstrate mRNA expression levels of 𝜷-actin. Figure from Hildyard & Wells (2014) demonstrated that 𝜷-actin had the highest mRNA expression level at the myoblast stage. This suggests that 𝜷-actin has an important role in early myogenesis, especially during cell proliferation, cell-to-cell contact and cell migration. The figure from Bains et al. (1984) demonstrated that 𝜷-actin mRNA expression levels decreased throughout myogenic differentiation. This confirms that the role of 𝜷-actin is not as essential in later stages of myogenic differentiation but is necessary for early myogenesis.

𝜷-actin Hypothesis:

For this study, due to the role and function of 𝜷-actin in early myogenesis, we hypothesize that 𝜷-actin mRNA expression is not dependent on the differentiation media due to the reducing role it plays throughout myogenic differentiation.

Methodology and Workflow

Workflow of the Winter 2021 semester.

Results

Figure 1: Phase contrast microscopy images captured at 200x magnification to identify morphological changes throughout myogenic differentiation. C2C12 cells were grown on 60mm dishes and were imaged during the Myoblast (MB) stage, Day 0 (D0), Day 1 (D1), Day 6 (D6) and Day 7 (D7). Control plates were kept in 10% Fetal Bovine Serum (FBS) throughout differentiation. Experimental plates were kept in 10% FBS until Day 0 (D0) and switched to 2% Adult Horse Serum (HS). In both conditions, MB cells were round mononucleated cells and have not yet reached 100% confluency. D6 cells were elongated multinucleated cells that demonstrated a longitudinal arrangement. D7 control showed a greater amount of cells covering the surface of the dish when compared to D7 experimental. Images were captured using a mobile device with an ocular lens on a microscope lens.

Figure 2: qPCR gene expression analysis in C2C12 cells throughout myogenic differentiation between control and experimental serum conditions. Control samples (blue) remained in 10% Fetal Bovine Serum (FBS) throughout differentiation. Experimental samples (orange) remained in 10% FBS until Day 0 (D0) and switched to 2% Adult Horse Serum (HS). Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) normalized expression of both control and experimental serum conditions. All samples were normalized to myoblast (MB) samples. Relative gene expressions of A) AAC1, B) ɑ-skeletal actin, and C) β-actin were calculated using the Pfaffl method.

Conclusions

Morphology of the C2C12 cells underwent differentiation as expected going from mononucleated myoblasts to multinucleated myotubes. Both differentiation conditions were sufficient to complete myogenesis (Fig. 1). However, those differentiated with 2% HS (experimental) appeared to have undifferentiated myoblasts and less myotube confluency.
AAC1 mRNA expression surprisingly peaked at Day 1 and proceeded to decrease. This contrasts Stepien et al.’s (1992) findings (p. 14594), and is inconsistent with the findings in human muscle cells by Lunardi et al. (1992), who saw almost no AAC1 expression in early myogenesis (p. 15269). (Fig. 2A).
ɑ-skeletal actin reveals an increasing trend of gene expression in both conditions which is supported by Bains et al. (1984) and Cox et al. (1990). More biological replicates would be needed to confirm the trend of days 5/6, 7, and 12. (Fig. 2B)
β-actin demonstrated an increase in gene expression at day 0 when cells were at 100% confluency and declined rapidly after. This matches the literature in Bains et al. (1984) when their data demonstrated the rapid decrease of expression as cells reach confluency and begin to differentiate. (Fig. 2C)
ɑ-skeletal actin and β-actin demonstrated a switch between the actins. When β-actin rapidly decreased in gene expression, ɑ-skeletal actin had a rapid increase in gene expression. (Fig. 2)
With no significant difference in gene expression of our genes of interest, we conclude both conditions are suitable for C2C12 cell growth and culturing.

Future Directions

We suggest future studies could have more biological replicates and ensuring that each sample day is gathered at a specific time point in the day to confirm whether or not serum conditions impact gene expression data and cell growth.