Biomechanische Charakterisierung von Skelettmuskelzellen aus R155C VCP knock-in und W2710X filamin C knock-in Mäusen: Ein neuer Ansatz zum Verständnis von myofibrillären Myopathien
Biophysik
Zusammenfassung der Projektergebnisse
The aim of this project was to assess and understand at the cellular level the biomechanical failure of striated muscle with pathogenic VCP and filamin C mutations. Specifically, we measured the influence of these mutations on the biomechanical function of cultured myoblasts and myotubes using immortalized cells obtained from knock-in mouse models (R155C VCP and W2710X filamin C). For the biomechanical characterization, we used traction force microscopy, magnetic tweezer microrheology, and uniaxial cell stretching in combination with high resolution confocal microscopy. With these methods, we tested the hypothesis that muscle cells carrying pathogenic VCP and filamin C mutations showed an increased stiffness compared to control cells, which in turn is expected to contribute to higher intracellular stress at physiologic stretch and shear deformation, causing progressive muscle fiber degeneration. First observations demonstrated that filamin C mutant myoblast cells do not detach to a higher percentage compared to wildtype cells after external stress application. Immunofluorescent imaging, however, showed that filamin C was not highly expressed in myoblasts (wild-type and mutant alike) and had not yet taken its role as "scaffolding protein" of the sarcomere. Therefore, we attempted to differentiate the myoblasts to myotubes with clearly developed sarcomeres in order to obtain more meaningful results. Our insight after large-scale trial-and-error experiments was that myoblasts with heterozygous and in particular homozygous mutations differentiate poorly into myotubes under 2-D culture conditions, which makes a direct comparison of biomechanical stress responses with wildtype cells questionable. Our experiments and the refinement of various differentiation protocols were performed in close collaboration with the laboratories of Prof. Schröder (Erlangen), Prof. Clemen (Cologne), and Prof. Fürst (Bonn), who had developed the cell lines. We also tested several mouse myoblast cell lines with a mutant desmin knock-in (R350P) developed by Prof. Clemen, but likewise we were unable to differentiate the mutant cells with the same efficiency as wildtype cells. Of note were also the marked differences in the proliferation rate of wildtype versus mutated cells. We attributed our failure to efficiently culture and differentiate mutated cells to non-physiologic 2-D cell culture conditions. We therefore developed methods to perform the planned measurements (i.e. stiffness measurements, contractility measurements, and response to cyclic stretch) under 3-D culture conditions. To this end, we mix a collagen/Matrigel matrix with myoblasts in 2x2x4 mm elastic silicone wells with two elastic vertical pillars (0.5 mm diameter) that are 2 mm apart. Cells and matrix components spontaneously form functional 3-D muscle tissue between the two pillars. These pillars bend visibly (under the microscope) in response to contractile force of the muscle tissue. Since we know the pillars’ bending stiffness, we can directly measure forces generated by the muscle tissue. Moreover, the wells can be mounted to a uniaxial stretching device for measuring the active and passive mechanical properties the tissue. Finally, we can apply cyclic stretch and synchronized electric pulses, all under continuous observation on an inverted epi-fluorescent or upright confocal microscope. We have successfully grown 3-D contractile skeletal muscle tissues from an immortalized mouse myoblast cell line (C2C12), from primary myoblasts isolated from adult wt mice, from satellite cells isolated from adult wt mice, and from neonatal mouse myoblasts. Further, we also cultured contractile heart muscle tissue from neonatal rat and wt mouse cardiomyocytes and measured the contractility of all of these bioengineered micro-muscle tissues in differently stiff environments and determined the force-length-relationship to extract the active and passive mechanical properties, and we have imposed cyclic stretch to investigate the stretch vulnerability of those tissues. We found that our immortalized cells obtained from knock-in mouse models do not form 3-D contractile muscle tissues, despite extensive efforts to adjust culture conditions, differentiation protocols, etc. The reasons for the unsuccessful experiments are currently unknown. Therefore, we have transferred desmin mutant R350P knock-in mice to Erlangen, from which we isolate satellite cells and grow them into 3-D micro-muscle tissues for the experiments as outlined in our grant proposal. Although this work is no longer funded by the grant, we are now in an excellent position to approach the question of mechanical stress-related muscle failure in human myopathies. With the institutional support of the University of Erlangen-Nuremberg, we will carry on with this line of work.
Projektbezogene Publikationen (Auswahl)
- (2015) Determining the mechanical properties of plectin in mouse myoblasts and keratinocytes, Experimental cell research, 331, 331-337
Bonakdar, N., Schilling, A., Spörrer, M., Lennert, P., Mainka, A., Winter, L., Walko, G., Wiche, G., Fabry, B., and Goldmann, W. H.
(Siehe online unter https://doi.org/10.1016/j.yexcr.2014.10.001) - Biomechanical behavior of myotubes with a desmin R350P mutation. 6th European Cell Mechanics Meeting, Barcelona, 2015
Spörrer M., Winter L., Schröder R., Fabry B., Goldmann W.H.
- (2016) Mechanical plasticity of cells, Nature materials 15, 1090-1094
Bonakdar, N., Gerum, R., Kuhn, M., Spörrer, M., Lippert, A., Schneider, W., Aifantis, K. E., and Fabry, B.
(Siehe online unter https://doi.org/10.1038/NMAT4689) - Biomechanical and cell biological analysis of a Desmin R350P mutation in myoblasts. International Meeting of the German Society for Cell Biology, Martinsried, 2016
Spörrer M., Gerum R., Winter L., Haug M., Schröder R., Fabry B., Goldmann W.H.
- The human Des R350P mutations results in early disruption of subcellular cytoarchitecture Altered biomechanical properties in skeletal muscle from young Des R350P knock-in mice. 60th Annual Meeting of Biophysical Society, Los Angeles, 2016
Diermeier S., Iberl J., Vetter K., Haug M., Reischl B., Buttgereit A., Schürmann S., Spörrer M., Goldmann W.H., Fabry B., Elhimine F., Stehle R., Pfitzer G., Winter L., Schröder R., Friedrich O.
- (2017) Early signs of architectural and biomechanical failure in isolated myofibers and immortalized myoblasts from desminmutant knock-in mice, Scientific reports 7, 1391
Diermeier, S., Iberl, J., Vetter, K., Haug, M., Pollmann, C., Reischl, B., Buttgereit, A., Schurmann, S., Spörrer, M., Goldmann, W. H., Fabry, B., Elhamine, F., Stehle, R., Pfitzer, G., Winter, L., Clemen, C. S., Herrmann, H., Schröder, R., and Friedrich, O.
(Siehe online unter https://doi.org/10.1038/s41598-017-01485-x) - (2017) The role of focal adhesion anchoring domains of CAS in mechanotransduction, Scientific reports 7, 46233
Branis, J., Pataki, C., Spörrer, M., Gerum, R. C., Mainka, A., Cermak, V., Goldmann, W. H., Fabry, B., Brabek, J., and Rosel, D.
(Siehe online unter https://doi.org/10.1038/srep46233) - A 3-D system for mechanical characterization of artificial skeletal muscle microtissue. 15th International Congress on Neuromuscular Diseases, Vienna, 2018
Spörrer M., Kah D., Wiedenmann S., Thievessen I., Schröder R., Goldmann W.H., Fabry B.
