(A) Time-resolved passive tension decreases with or without TEV-protease treatment, at 3.3 µm SL; experimental protocol (left) and representative tension-time traces (middle). Raw data was normalized to the averaged time-matched tension without TEV protease treatment. Following mechanical measurements, a subset of fibers was analyzed by titin protein-gel electrophoresis to confirm expected cleavage (Coomassie-stained example, right). (B) Averaged values at 0, 1, 2, 3, 4, 5, 10, 15, 20, 25, and 30 min after TEV protease treatment or no treatment for control (n = 6 WT, 6 Het, 6 Hom animals). (C) Representative electron micrographs of relaxed Wt, Het, and Hom fibers after treatment with TEV protease while being held passively for 30 min at the stretched length. Scale bars, 1 µm. (D) Z-disk order of TEV protease treated fibers after hold protocol, quantified by fracture area (see Materials and methods; Wt+TEV: n = 60, Het+TEV: n = 239, Hom+TEV: n = 104). The images below the graph show representative electron micrographs and processed images used to calculate fracture area, for each genotype, after TEV treatment. Scale bars, 1.5 µm. Average length of sarcomeres on images, 3.3 μm (range, 3.0–3.6 µm). Stats: ANOVA with Tukey’s HSD post hoc procedure.