Fusion and proliferation
The ability of the cultured myoblasts to fuse strongly depended on cell type (Fig. 2). C1F myoblasts showed the greatest ability to fuse, while human skeletal myoblasts (HuSK) and C2C12 myoblasts were less efficient at fusing. The ability of the myoblasts to fuse is demonstrated by confocal images of the myotubes formed following fusion of the myoblasts on the different surfaces, after 5 days under differentiation conditions (Fig. 2). These cells have been fixed and stained for skeletal muscle myosin, which is only expressed once the cells have differentiated. For the mouse myoblast line C1F, the surfaces are almost completely covered by long, skeletal myosin positive, myotubes. For the mouse myoblast line C2C12, while myotubes are present, there are fewer of them, although all the cells were plated at the same density in each experiment. HuSK myoblasts were similar to C2C12 myoblasts in terms of fusion.
Quantification of fusion, which measures the numbers of nuclei present in a field of view, and how many of these nuclei are present in the myotubes, showed that C1F cells had the highest fusion index (Fig. 3), in that the majority of the nuclei were in multinucleated myotubes (~60–80 % on most surfaces). The fusion index obtained for human skeletal muscle cells (HuSK) and C2C12 cells was similar, and lower than that obtained for C1F cells at ~40 % on average (Fig. 3).
Tests of the single LAMA2 peptides for all three cell types, showed that Orla C (with the sequence GLLFYMARINHA, Table 1) was the best in promoting fusion for all three cell types, as quantified by the fusion indices obtained. While the differences in the fusion indices for the different single peptides were generally small, fusion on OrlaC was significantly higher for C2C12 myoblasts than fusion on gelatin, which is commonly used as a substrate for fusion and differentiation of myoblasts. In contrast, fusion of C1F and C2C12 cells on the c-myc Orla peptide, which is not expected to support fusion, was significantly reduced compared to fusion on gelatin, and to fusion on Orla C, for C2C12 and C1F cells. Thus, the c-myc peptide is unable to support fusion, while the LAMA2 peptides promote fusion for all the cell lines tested.
Tests for the mixture of pepides showed that an equimolar mixture of all three LAMA2 peptides, together with FGF1 (Figs. 2, 3) increased fusion significantly for both C2C12 and C1F cells, compared to differentiation on a gelatin only surface, or on c-myc peptide surfaces. In contrast, mixtures of FGF1 and one of the LAMA2 peptides (A&D, B&D or C&D) did not improve fusion compared to the single LAMA2 peptides alone. In addition, the mixture of all three LAMA2 peptides (A&B&C) was not markedly different from the results obtained for each peptide in isolation. A similar trend was observed for HuSK cells, although this increase in fusion was not significant. These results suggest that each peptide is reasonably good in promoting fusion in isolation, and they do not work synergistically, unless all 3 LAMA2 peptides and the FGF1 peptide is present. This effect was most marked for the two mouse muscle cell lines, C1F and C2C12.
To test if this effect on fusion was due to differences in proliferation of the cells prior to fusion, we analysed rates of proliferation on three of the surfaces: FGF1 alone, the mixture of all three LAMA2 peptides together with FGF1 (A&B&C&D), and the c-myc peptide (Fig. 4). None of these surfaces affected proliferation of the C2C12 myoblasts, confirming that any differences in fusion observed were not due to changes in proliferation. For the C1F myoblasts, FGF1 did increase proliferation significantly above that observed for the mixture of all 4 peptides (A&B&C&D), and c-myc. However, the rate of proliferation for C1F cells on the c-myc peptide, and the mixture of all 4 peptides was not significantly different. As fusion on the mixture of all 4 peptides was significantly higher than in the other two conditions, we can conclude any effects on proliferation are not the underlying cause of improvements in fusion.
Sarcomere organisation also varied markedly between the three different cell types used (Fig. 5). Sarcomere organisation was best for the differentiated HuSK myotubes, intermediate for C1F myotubes, and worst for C2C12 myotubes (Fig. 5c). This was quantified by measuring the number of peaks along a line of fixed length across multiple myotubes in multiple fields (see methods). The higher the average number of peaks, the more likely that the majority of myotubes contain a more highly striated organisation of skeletal myosin, and thus good sarcomere organisation. These results show that sarcomere organisation does not necessarily correlate with the ability of the cells to fuse, as HuSK myoblasts had a similar fusion index to C2C12 cells, but showed better sarcomere organisation, whereas C1F cells fused well, but had intermediate levels of sarcomere organisation.
Tests of the Orla peptides showed that, as with fusion, the best sarcomere organisation for C1F cells was obtained for the mixture of all 4 peptides (Fig. 5) and the improvement in sarcomere organisation was significantly increased compared to that obtained on gelatin. This suggests that surfaces that promote fusion for individual cell types, can also support better levels of sarcomere organisation, as C1F cells fused best on the mixture of all 4 peptides, and sarcomere organisation was also best on that surface. However, for single peptides, Orla B (GLLFYMARINHA), rather than C showed the best results for sarcomere organisation. Adding in FGF1 to single LAMA2 peptides slightly improved results for IKVSV (A&D), but not the other two LAMA2 peptides. Generally, sarcomere organisation was improved on all of the Orla surfaces, compared to gelatin alone. C-myc peptides were very poor at promoting sarcomere organisation, and almost no myotubes were observed with recognisable banded structures (Fig. 5a).
For C2C12 cells, the best sarcomere organisation was again obtained for the mixture of all 4 peptides (Fig. 5), again linking an improvement in fusion with that of differentiation for an individual cell type. The improvement in sarcomere organisation was significantly increased compared to gelatin. The best single peptide was IKVSV (A). Adding in FGF1 to the single LAMA2 peptides did not improve sarcomere organisation beyond that of the LAMA2 peptides alone. In general, all the peptides improved sarcomere organisation compared to gelatin alone. As with CIF cells, C-myc peptides were very poor at promoting sarcomere organisation, and almost no myotubes were observed with recognisable banded structures (Fig. 5a).
HuSK cells appeared to have well organised sarcomeres on most surfaces, with the exception of Orla C. As their sarcomeric organisation is already the highest compared to the other cell lines, it is possible that Orla peptides have less effect on their sarcomere organisation compared to C1F and C2C12 cells.
These data show that the extent to which each of the different types of cultured cells (C1F, C2C12 and HuSk) can fuse and differentiate is largely determined by the inherent properties of these cells. However, use of the Orla surfaces is able to improve fusion and differentiation above that of using gelatin, and is significantly better than using an unrelated peptide such as c-myc, which is not expected to promote fusion. Moreover, using the combination of all 3 LAMA2 peptides and FGF1 significantly improved fusion and differentiation for the two mouse myogenic cell lines (C1F and C2C12), and was the best surface for the human myogenic (HuSK) cells. It is also noticeable that there is less variation in the fusion index and sarcomere organisation on this particular surface compared to the other surfaces used. For fusion, GLLFYMARINHA appears to be the most effective of the three LAMA2 peptides we tested, when used as a single peptide.
Overall, the combination of all three of the LAMA2 peptides with FGF1 was best for both fusion and differentiation for the two mouse myogenic cell lines (C2C12 and C1F). This combination was also better for HuSK cells. It is interesting that fusion and differentiation can be improved by adding FGF1 to the mixture of all three LAMA2 peptides, although the combination of FGF1 with any of the LAMA2 peptides in isolation is less effective. Exogenous FGF1 might be expected to promote proliferation rather than differentiation (Uruno et al. 1999). However, using the mixture of all 4 peptides did not show significant effects on proliferation for C2C12 cells, and only a small increase for C1F cells, although this was significant. Although the FGF1 is covalently coupled to the glass surface via the Orla peptide, other studies have shown that these growth factors remain biologically active when immobilized onto surfaces (Feito et al. 2011). Thus, we suspect that when used in combination with the three LAMA2 peptides, FGF1 is able to promote myotube survival, and the slightly lower relative concentration of FGF1 in the mixture of all 4 peptides, where FGF is at 25 % of the concentration compared to a surface that only contains the FGF1 peptide, is better optimised for promotion of proliferation and myoblast survival prior to and during differentiation, than the higher levels of FGF1 present in the mixtures with a single peptide. Alternatively, there is a synergistic effect between FGF1 and the 3 LAMA2 peptides that promotes myotube differentiation.
In conclusion, using the three LAMA2 peptides in combination with FGF1 promotes fusion and differentiation, and gives more consistent results for each of the cell lines tested here, and should be useful for studies that require well-differentiated cultured muscle cells in the future.