BIN1 Induces the Formation of T-Tubules and Adult-Like Ca²⁺ Release Units in Developing Cardiomyocytes.

Abstract

Human embryonic stem cell-derived cardiomyocytes (hESC-CMs) are at the center of new cell-based therapies for cardiac disease, but may also serve as a useful in vitro model for cardiac cell development. An intriguing feature of hESC-CMs is that although they express contractile proteins and have sarcomeres, they do not develop transverse-tubules (T-tubules) with adult-like Ca²⁺ release units (CRUs). We tested the hypothesis that expression of the protein BIN1 in hESC-CMs promotes T-tubules formation, facilitates Ca_V 1.2 channel clustering along the tubules, and results in the development of stable CRUs. Using electrophysiology, [Ca²⁺ ]_i imaging, and super resolution microscopy, we found that BIN1 expression induced T-tubule development in hESC-CMs, while increasing differentiation toward a more ventricular-like phenotype. Voltage-gated Ca_V 1.2 channels clustered along the surface sarcolemma and T-tubules of hESC-CM. The length and width of the T-tubules as well as the expression and size of Ca_V 1.2 clusters grew, as BIN1 expression increased and cells matured. BIN1 expression increased Ca_V 1.2 channel activity and the probability of coupled gating within channel clusters. Interestingly, BIN1 clusters also served as sites for sarcoplasmic reticulum (SR) anchoring and stabilization. Accordingly, BIN1-expressing cells had more Ca_V 1.2-ryanodine receptor junctions than control cells. This was associated with larger [Ca²⁺ ]_i transients during excitation-contraction coupling. Our data support the view that BIN1 is a key regulator of T-tubule formation and Ca_V 1.2 channel delivery. By studying the role of BIN1 during the differentiation of hESC-CMs, we show that BIN1 is also important for Ca_V 1.2 channel clustering, junctional SR organization, and the establishment of excitation-contraction coupling. Stem Cells 2019;37:54-64.