Einfluss von externer Belastung auf die myogene Differenzierung
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Beschreibung
The aim of this study was to investigate different external stresses with regard to their influence on myoblasts and their myogenic differentiation in vitro. The influence of mechanical stress in the form of fluid shear stress (FSS) and atmospheric stress in the form of hypoxia (3% O2) was investigated. In the context of the mechanical stress, a comparison was also performed between the influence of a periodic FSS (pFSS, 1 h per day) and a continuous FSS (kFSS).
The experiments were carried out with the C2C12 mouse myoblast cell line. First, the myoblasts were characterised with regard to their morphology, the expression of stem cell markers and myogenic markers as well as myogenic differentiation. Subsequently, the myoblasts were cultured for 3 d or differentiated for 7 d under the respective stress. Further analyses of the cells were performed at different time points (3 h, 24 h and 3 d after the start of cultivation and 3 h, 3 d and 7 d after the start of differentiation). Myoblasts cultivated and differentiated under standard conditions (static, 21% O2) served as controls.
The following results were achieved:
FSS
Based on phalloidin staining, an altered morphology of proliferating myoblasts under FSS was observed. The myoblasts showed more small, round or large, flat cell shapes and formed more cell processes and cell contacts in order to maximise their adhesion and minimise the shear forces on their surface. Improved cell growth was observed under FSS, as shown in the significantly increased total cell number after 24 h in the SRB staining. Also with regard to the viability of the myoblasts, no negative influences of FSS could be demonstrated in the MTT test.
In contrast, the colony forming potential and thus the self-renewal under FSS was significantly reduced with the formation of significantly fewer colonies in the CFU assay. Furthermore, gene expression of the early myogenic differentiation marker MyoD was investigated by RT-qPCR. The proliferating myoblasts showed reduced expression under FSS after 24 h and 3 d, although the differences were not significant. Immunofluorescence staining also showed a slight reduction in the number of MyoD-positive myoblasts after 24 h under FSS, with no significant difference. However, the results indicate that FSS inhibits the determination of proliferating myoblasts to the myogenic lineage and promotes the undifferentiated state.
Under both static and dynamic culture conditions, successful myogenic differentiation was demonstrated by phase contrast microscopy and phalloidin staining. However, under FSS, the differentiating myoblasts showed marked clumping as a possible indication that the myoblasts were increasingly seeking contact with each other in search of adhesion. Nevertheless, FSS resulted in improved myotube formation with the formation of significantly wider and with a tendency to the formation of larger myotubes. The fusion index and the number of myonuclei per myotube were also significantly increased under FSS, indicating that more myoblasts fused to form a myotube.
By RT-qPCR, the gene expression of the myogenic differentiation markers MyoD, Myogenin, MYH1 and MYH7 as well as the protein synthesis indicator mTOR was investigated. For all genes, a stronger increase in expression could be observed under FSS, although the differences did not show any significance. Immunofluorescence staining also showed no difference in the number of Myogenin-positive cells on day 3 of differentiation between the two culture conditions.
When comparing pFSS versus kFSS, the same morphological changes for the proliferating myoblasts as described above were observed under both FSS culture conditions. The most significant difference between cultivation under pFSS and kFSS was observed in the colony forming potential. In contrast to kFSS, cultivation under pFSS resulted in an improved colony forming potential with the formation of significantly more colonies, whereas smaller colonies were formed under both FSS conditions. Neither the MTT test, the SRB staining, the immunofluorescence staining, the RT-qPCR nor the myogenic differentiation showed any further significant differences between the culture conditions.
Hypoxia
The myoblasts showed an improved viability under hypoxia recognisable by the significantly increased number of living cells after 24 h and 3 d in the MTT test. The SRB staining also showed a significantly increased total cell number and thus improved cell growth after 3 h under hypoxia. Furthermore, a slightly increased colony number under hypoxia was observed in the CFU assay, although the differences were not significant.
In the immunofluorescence staining and RT-qPCR, a slightly reduced MyoD expression and a slightly reduced number of MyoD-positive cells were detected under hypoxia as an indication of an inhibition of myogenic determination, although no significant differences could be determined here either. In addition, the gene expression of the hypoxia markers HIF1α, VEGF and NOS1 was investigated. In response to hypoxia, the myoblasts showed significantly lower gene expression for HIF1α after 3 h. For VEGF, a significantly higher gene expression was detected after 24 h and 3 d under hypoxia, indicating an activation of the HIF-1 signalling pathway. NOS1 gene expression also tended to increase more under hypoxia. In line with this, proliferating myoblasts under hypoxia showed a tendency towards higher NO production at all time points in the DAF-FM staining.
In contrast, myoblasts showed inhibited myogenic differentiation under hypoxia. In phase contrast microscopy as well as in phalloidin staining, a reduced myotube formation could be observed under hypoxia with the formation of clearly longer but thinner myotubes. This could be confirmed in the morphometric analysis. While the length of the myotubes and the cytoplasm-to-myonucleus ratio were significantly increased under hypoxia, the width, the number of myonuclei per myotube and the fusion index were significantly reduced and the area of the myotubes tended to be reduced, indicating an impaired fusion process under hypoxia.
RT-qPCR showed reduced gene expression under hypoxia for MyoD, Myogenin and mTOR, especially on day 3 of differentiation, but no significant differences could be detected. In agreement, the number of Myogenin-positive myoblasts was significantly reduced on day 3 of differentiation under hypoxia. For MYH1 and MYH7, no significant differences in gene expression could be detected between the two culture conditions. Also for the hypoxia markers HIF1α and VEGF no significance could be determined, while for NOS1 a tendency towards lower expression on day 3 and 7 under hypoxia could be detected.
The results of this study show that mechanical stress in the form of FSS and atmospheric stress in the form of hypoxia have an influence on proliferating and differentiating myoblasts. For both factors, a positive effect on the function of proliferating myoblasts, especially with regard to viability and cell growth, could be demonstrated, possibly due to the presence of these factors in the physiological microenvironment of the cells. Specifically, pFSS also improved the self-renewal potential of myoblasts. The incorporation of these factors in the in vitro cultivation of muscle stem cells could effectively mimic their stem cell niche, potentially improving cell expansion prior to transplantation as well as the regeneration potential in the context of cell-based therapies. While hypoxia inhibited myogenic differentiation of C2C12 myoblasts, FSS promoted myogenic differentiation and may play a central role as a hypertrophic stimulus. The application of FSS should therefore be considered to optimise the myogenic differentiation protocol.
Autor*in
Paula Marie Hawlitschek
Themen in »Einfluss von externer Belastung auf die myogene Differenzierung«
Myoblasten-Differenzierung Flüssigkeitsscherspannung (FSS) Hypoxie (3 % O2) C2C12-Zelllinie Stammzellnische in vitro Zellbasierte Muskeltherapien / Regeneration