Bogaards, S., Yuen, M., Onderwater, Y., Clara, C., Galli, R., Vizoso, M., Conijn, S., Peters, E., Nahidi, L., Jalink, K., van Rheenen, J., Granzier, H., and Ottenheijm, C.
To date, it is unknown how giant muscle proteins are integrated and replaced in continuously contracting muscles. A prime example is the thin filament-associated, sarcomeric protein nebulin (800 kDa), mutations in which cause nemaline myopathy. To establish the replacement kinetics of nebulin in sarcomeres, we designed a mouse model with fluorescent Dendra2 inserted in nebulin's N-terminus (NebN-D2) and used intra-vital (imaging window on m. gluteus maximus) or ex vivo FRAP microscopy on isolated FDB fibers. Dendra2 was converted from green to red fluorescent state and fluorescence was followed over time. Both intra-vital and ex vivo data indicate that nebulin's replacement kinetics are very slow with respectively 38% and 28% of converted (red) Dendra2 replaced after 9 days. During intra-vital imaging, after 19 days, ∼half of the converted Dendra2 was still present in sarcomeres. Other novel mouse models with Dendra2 attached to sarcomeric proteins were engineered. Data showed much faster replacement kinetics of tropomodulin-4 (69% in 15h) and leiomodin-3 (>90% in 2 min). Recovery of titin, another giant sarcomere protein (∼3.7 MDa) showed similar replacement kinetics as nebulin (∼29% in 9 days). Next, NebN-D2 mice were crossed with NebΔEx55 mice (NebD2/ΔEx55) to mimic patients heterozyous for the NEBΔEx55 mutation. The NebΔEx55 allele does not produce nebulin protein, and, therefore, all nebulin molecules carry Dendra2. Viability of isolated fibers from the NebD2/ΔEx55 mice was reduced and the sarcomere structure was affected. Interestingly, experiments using photo-activated localization microscopy revealed a lower fraction of unincorporated nebulin molecules in NebD2/ΔEx55 mice and the replacement kinetics of nebulin were reduced compared to NebN-D2 mice. Summarizing, our data indicate that the replacement kinetics of the giant proteins nebulin and titin in mature muscle are very slow. In NebD2/ΔEx55 mice, replacement kinetics of nebulin are slower due to a lower fraction of unincorporated nebulin protein. [ABSTRACT FROM AUTHOR]