Altogether, 103 mice (average age, 2 mo) were used in the study. Genotype was determined based on tailtip PCR. Nebulin expression was assessed with Western blot analysis using anti-nebulin N-terminal antibody (SI Appendix, Fig. S6). Protein levels of tropomyosin (Tm), troponin-C (TnC), troponin-T (TnT), and troponin-I (TnI) as well as MHC isoform expression were also determined. Routine picrosirius red, Gömöri-trichrome stains (SI Appendix, Fig. S8) and electron microscopy were performed on fixed soleus cross-sections. All animal experiments were approved by the University of Arizona and the Illinois Institute of Technology Institutional Animal Care and Use Committees and followed the Guide for the Care and Use of Laboratory Animals (39 ).
Intact soleus muscles were electrically stimulated at optimal muscle length in a custom-built test system (Fig. 1A). Blebbistatin was used to inhibit muscle contraction and lower tetanic force. Alternatively, the tetanic force was rapidly lowered by adjusting the muscle length (Fig. 2C). Sarcomere length was measured on fixed soleus fiber bundles using a CCD camera (SI Appendix, Fig. S7B). X-ray diffraction images were recorded using a high-flux 12 keV X-ray beam provided by Beamline 18 at the Advanced Photon Source (Argonne National Laboratory). Individual image frames were summed to be equivalent to at least 0.375 s exposure. The MUSICO computational platform (27 (link)) was used to simulate isometric force development and instantaneous muscle stiffness.
Descriptive statistical results are shown as mean ± SD unless stated otherwise. Differences between groups were considered to be statistically significant at a probability value of P < 0.05. Symbols used in statistical tests and on figures include ns, P ≥ 0.05; *P < 0.05; **P < 0.01; ***P < 0.001; and ****P < 0.0001. Detailed statistical evaluation is provided in SI Appendix, Table S1. Further details are in SI Appendix, Supplemental Methods.