Fig. 3: Effects of Speg deficiencies on T-tubule structure and SR Ca²⁺ leak (sparks).

FDB fibers were isolated from male mice as described in Methods. We used dual imaging of t-tubules (FM4-64) and Ca²⁺ (Fluo-4) to find areas of T-tubule disruption and SR Ca²⁺ leak. a, b T-tubule organization (raw and skeletonized) and spark localization (red squares) in FDB fibers from control (a) and HA-Speg (b) mice. c Spontaneous Ca²⁺ spark frequency in control (N_fibers=21, N_animals = 5) and HA-Speg fibers (N_fibers=28, N_animals = 6). d Analysis of T-tubule organization in control and HA-Speg fibers. e, f T-tubule organization (raw and skeletonized) and spark localization (red squares) in FDB fibers from control (e) and Speg-KO (f) mice. g Spontaneous Ca²⁺ spark frequency in control (N_fibers=20, N_animals = 5) and Speg-KO fibers (N_fibers=15, N_animals = 3). h Analysis of T-tubule organization in control and Speg-KO fibers. TE is the density of transverse elements; LE is the density of longitudinal elements. Regularity is the organization or spacing and is the magnitude of the major frequency derived from the FFT of the image. TTi is the t-tubule integrity, which considers both the regularity and density of the t-tubules. Data are plotted as the mean ± SD, ***P < 0.001, **P < 0.01, and *P < 0.05.