Bridging integrator-1 (BIN1) is a family of banana-shaped molecules implicated in cell membrane tubulation. To understand the curvature sensitivity and functional roles of BIN1 splicing isoforms, we engineered vertical nanobars on a cell culture substrate to create high and low curvatures. When expressed individually, BIN1 isoforms with phosphoinositide-binding motifs (pBIN1) appeared preferentially at high-curvature nanobar ends, agreeing well with their membrane tubulation in cardiomyocytes. In contrast, the ubiquitous BIN1 isoform without phosphoinositide-binding motif (uBIN1) exhibited no affinity to membranes around nanobars but accumulated along Z-lines in cardiomyocytes. Importantly, in pBIN1-uBIN1 coexpression, pBIN1 recruited uBIN1 to high-curvature membranes at nanobar ends, and uBIN1 attached the otherwise messy pBIN1 tubules to Z-lines. The complementary cooperation of BIN1 isoforms (comboBIN1) represents a novel mechanism of T-tubule formation along Z-lines in cardiomyocytes. Dysregulation of BIN1 splicing, e.g., during myocardial infarction, underlied T-tubule disorganization, and correction of uBIN1/pBIN1 stoichiometry rescued T-tubule morphology in heart disease.