Sarcomere length measurement permits high resolution normalization of muscle fiber length in architectural studies

The use of sarcomere length to normalize fiber length in architectural studies is commonly practiced but has not been explicitly validated. Using mouse hindlimb muscles as a model system, ankle joints were intentionally set to angles ranging from 30 degrees to 150 degrees and their muscles fixed. Tibialis anterior (TA), extensor digitorum longus (EDL) and soleus muscles were removed and their raw fiber length measured. Sarcomere length was then measured for each fiber length sample and fiber length was normalized to a standard sarcomere length. As expected, raw fiber length was dependent on tibiotarsal angle (P < 0.0005 for all muscles, r2 range 0.22-0.61), while sarcomere length normalization eliminated the joint-angle dependent variation in fiber length (P > 0.24, r2 range 0.001-0.028). Similarly, one-way ANOVA revealed no significant differences in normalized fiber length among ankle angles for any of the three muscles (P > 0.1), regardless of animal size. To determine the resolution of the method, power calculations were performed. For all muscles studied, there was >90% chance of detecting a 15% fiber length difference among muscles and >60% chance of detecting fiber length differences as small as 10%. We thus conclude that the use of sarcomere length normalization in architectural studies permits resolution of fiber length variations of 15% and may even be effective at resolving 10% fiber length variations.