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Title

ELASTIC ELEMENTS IN THE SARCOMERES OF STRIATED MUSCLE

Creator

Ma, Weikang

Date

2016, 2016-07

Description

The flight muscle of the Hawkmoth, Manduca sexta, is an emerging model system for structure and function studies. M. sexta flight muscle shows...
Show moreThe flight muscle of the Hawkmoth, Manduca sexta, is an emerging model system for structure and function studies. M. sexta flight muscle shows several interesting properties such as its length tension curve is similar with cardiac muscle, but the detailed protein compositions of M. sexta flight muscle is not known. Here we identified proteins that might be responsible for the elastic properties of M. sexta flight muscle. 1% vertical SDS-agarose gel electrophoresis combined with western blot analysis was used to separate and identify high molecular weight proteins in M. sexta flight muscle. Two projectin isoforms as well as two kettin isoforms were found in M. sexta flight muscle. In addition, two high molecular weight proteins were seen in agarose gels which turned out to be Sallimus (Sls) proteins isoforms based on the sallimus (sls) gene map. The localization and orientation of projectin and Sallimus proteins were determined by immuno-localization using confocal microscopy. The thin and thick filament lengths were also determined, and shown to be consistent with the length tension curve data. Knowledge of myofilament compliance is critical in interpreting cross bridges kinetics interpretation and modeling. Here we used small angle X-ray diffraction to study thick filament compliance in intact mouse soleus muscle. The thick filament compliance was estimated by plotting the spacing changes of myosin based meridional reflections against tension generated by the muscle during contraction. A non-linear relationship of thick filament compliance was seen for the first time. Nebulin is a giant thin filament protein and has been proposed to play significant roles in muscle physiology, but the underlying mechanisms are largely unknown. A conditional nebulin gene knockout mouse model was used here to study any structural and functional changes caused by nebulin deficiency using small angle X-ray diffraction. The thin filament compliance was estimated, and the results showed that the thin filament compliance in nebulin deficient muscle was larger than muscle from control animals, whereas no difference was seen in thick filament compliance between knockout and control muscles. The inter-filament spacing was larger in knock out muscle than in control muscle, and less force was generated by each cross bridge. The larger interfilament spacing and less force per cross bridge might explain the muscle weakness seen in both the knock out mouse model, as well as in nemaline myopathy patients. Other structural changes caused by nebulin deficiency was also characterized by small angle X-ray diffraction.
Ph.D. in Molecular Biochemistry and Biophysics, July 2016
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