(A–C) Representative STED images and corresponding intensity plot profiles of DCM^TTNtv–, negative control (human papillary muscle), and DCM^TTNtv+ cardiac samples, respectively. Arrows and arrowheads point to the MIR and A170 titin epitopes, respectively. The plot profiles represent the fluorescence intensity distribution along the long axis of the sarcomere; the x axis therefore represents the distance (in nm), and the y axis indicates the fluorescence intensity (in AU). The sarcomere images are coaligned with respect to their left-side Z-lines, indicated by the vertical dotted line. Note the decreased A170 intensity in DCM^TTNtv+ sarcomeres relative to that of the MIR epitope. The bottom panel in C is the respective confocal microscopic image, shown here to indicate the critical power of STED in resolving the separate A170 epitopes. Scale bar: 500 nm. (D) A-band titin length (measured as the distance between 2 consecutive MIR epitopes bounding an A170 epitope doublet) increased in both DCM^TTNtv– and DCM^TTNtv+ cardiac fibers when the sarcomeres were passively stretched. (E) Linear regression analysis of the A-band titin length in the 1.8–2.6 μm sarcomere length range. There was a significant difference in the slopes of the linear fit (for statistics, see Supplemental Table 7). (F) Comparison of A-band titin lengths between the negative control and DCM^TTNtv– samples by linear regression. There was no significant difference in the slopes of the linear fit, but there was a significant, 44 nm difference in the y axis intercepts (for statistics, see Supplemental Table 8). (G) M-line–to–TK distance, calculated as the half value of the distance of 2 consecutive A170 epitopes, as a function of sarcomere length. (H) Linear regression analysis of the sarcomere length–dependent M-line–to–TK distance in the 1.8–2.6 μm sarcomere length range (for statistics, see Supplemental Table 9). (I) M-line–to–A170 epitope distance as a function of sarcomere length, for negative control and DCM^TTNtv– samples, compared with linear regression. The slope of the control data was not significantly different from zero (for statistics, see Supplemental Table 10). Three quasi-control (TTNtv^–) and 3 TTNtv⁺ cardiac muscle samples (septum) were processed, and 3 skinned fiber bundles (technical replicates) used as a negative control were prepared from each papillary muscle sample. The fiber bundles were dissected from different locations of the cardiac muscle samples. At least 15 frozen sections were processed for STED imaging per technical replicate.