Cardiomyocyte T-tubules are densely folded by BIN1. (α–b) Representative confocal images (a, scale bars: 5 µm) and the fluorescent profiles (b) of live WT and Bin1 HT cardiomyocytes labeled with Di-8-ANNEPS. (c) Quantification of T-tubules peak intensity. (n = 40 from 4–5 cells, P < 0.0001). (d) Cell size normalized membrane capacitance in WT (n = 14) and Bin1 HT (n = 12) cardiomyocytes (P = 0.0181). WC indicates reported whole cell capacitance without T-tubules. (e) 2D transmission electron microscope (TEM) images (Left to right: gross morphology, transverse cross section, and axial cross section) and 3D electron tomography images (right) of WT and Bin1 HT heart sections. Scale bars (left to right): 1 µm, 250 nm, 100 nm, and 100 nm. (f) Electron density profiles (middle) across individual T-tubules marked by the lines in the images above, with average T-tubule electron density in the bottom (n = 75, P < 0.0001). (g) T-tubule lumen area of axial cross sections (n = 80, P < 0.0001). (h) Cardiomyocyte T-tubule contour score (1, circular shape and no folds and spatial complexity; 2, non-circular shape and no folds and spatial complexity; or 3, multiple folds with spatial complexity) distribution (n = 196, P < 0.0001). Data are presented as mean ± SEM, cardiomyocytes are from three mice per genotype, and six left ventricular sections from three hearts per genotype were used for TEM analysis. Student’s t-test and one way-ANOVA were used for statistical analysis.