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.

Keywords: BIN1; CaV1.2; Calcium release units; Cardiac myocytes; T-tubules; hESC.

Figure 1

BIN1 expression promotes the formation of T‐tubules in human embryonic stem cell‐derived cardiomyocyte (hESC‐CM). (A): Schematic representation of the formation and maturation of T‐tubules at differentiation days (DD) 10, DD20, and DD30 in hESC‐CM transduced with BIN1‐EGFP. (B): Representative confocal images of Di‐8‐ANEPPS labeled membrane (red) in control (left) and BIN1 hESC‐CM (right). Additional right panels show the expression pattern of BIN1‐EGFP (green). Colocalization of Di‐8‐ANEPPS and BIN1 appears as yellow in the merged images. Zoom in right panels show with more detail the organization of the tubular structures. (C): Front orthogonal view of a Αiryscan 3D reconstruction of a hESC‐CM BIN1 cell. Zoom‐in at the right shows the projection of T‐tubules from the top to the bottom of the cell. (D): T‐tubules density, length and diameter at DD10, DD20, and DD30 in hESC‐CM BIN1 cells, n = 4 cells/group. Bars are averages ± SEM. *, p < .05; **, p < .01; ***, p < .001.

Figure 2

BIN1 expression increases ventricular‐like phenotype in human embryonic stem cell‐derived cardiomyocyte (hESC‐CM). (A): Representative ventricular‐, atrial‐ and nodal‐like action potentials (APs) in control and BIN1 hESC‐CM. (B): Proportion of each electrical phenotype in control (n = 39) and BIN1 hESC‐CM (n = 32). (C): Comparison of the action potential duration at 90% of repolarization (ADP90) in control and BIN1 ventricular‐like hESC‐CM (*, p < .05). (D): Summary of AP parameters in control and BIN1 hESC‐CM. Abbreviations: MDP, maximum diastolic potential; ADP, action potential duration at the specified percentage of repolarization. Data in the table in panel D are presented as mean ± SEM.

Figure 3

BIN1 increases Ca_V1.2 channel clustering in human embryonic stem cell‐derived cardiomyocyte (hESC‐CM). (A): 3D reconstruction of GSD super‐resolution images from a representative differentiation days (DD) 30 BIN1 hESC‐CM immunostained against Ca_V1.2 (green) and BIN1 (red). ROI outlined in yellow and enlarged at the bottom show juxtapositioning of Ca_V1.2 and BIN1 clusters. (B): Representative GSD 2D super‐resolution images of typical Ca_V1.2 and BIN1 cluster organizations observed in BIN1 hESC‐CMs at DD10, DD20 and DD30. (C): Ca_V1.2 and BIN1 cluster mean area plots (n = 4 cells/group). Bars are averages ± SEM. ***, p < .001.

Figure 4

BIN1 increases whole‐cell Ca²⁺ currents in human embryonic stem cell‐derived cardiomyocyte (hESC‐CM). Representative control (black) and BIN1 (red) hESC‐CMs calcium currents normalized to the cell's capacitance and elicited by a 300 ms depolarizing pulse from −70 mV to +0 mV at specific differentiation days (DD) 10 (A), DD20 (B), and DD30 (C). Right: Mean current density–voltage relationships obtained in control (n = 10 cells/DD) and BIN1 hESC‐CMs (n = 10 cells/DD) from three different cell batches.

Figure 5

BIN1 increases Ca_V1.2 channel coupling. (A): TIRF images control and BIN1 human embryonic stem cell‐derived cardiomyocyte (hESC‐CM) at differentiation days (DD) 30, green circles show active Ca_V1.2 sparklets sites. Time courses of [Ca²⁺]_i in the outlined sites are shown on the right. (B): Event amplitude histograms of Ca_V1.2 sparklets recorded control (gray) and BIN1 hESC‐CMs (red). The quantal amplitude of a sparklet was calculated by fitting histograms with multicomponent Gaussian functions. (C): Scatter plot of coupling coefficients (κ) in control (black), BIN1 high (red) and BIN1 low (light red) hESC‐CMs (n = 7 cells/group). Solid lines and error bars superimposed over the individual points indicate mean ± SEM. ***, p < .001.

Figure 6

BIN1 expression promotes Ca_V1.2‐RyR juxtaposition. (A): representative 2D GSD images of Ca_V1.2 (green) and RyR (red) cluster arrangements at differentiation days (DD) 10, DD20 and DD30 in control and BIN1 human embryonic stem cell‐derived cardiomyocytes (hESC‐CMs). (B): Density of RyR localized within a distance of 50 nm from Ca_V1.2 clusters in control (gray) and BIN1 (red) hESC‐CMs (n = 4 cells/group). Data is displayed as mean ± SEM. ***, p < .001.

Figure 7

BIN1 increases and coordinates intracellular calcium release. (A): Representative spontaneous calcium transients from control (black) and BIN1 (red) human embryonic stem cell‐derived cardiomyocyte (hESC‐CM) cells at differentiation days (DD) 10, DD20, and DD30. (B): Analysis of spontaneous calcium transient peak amplitudes (n = 5–11 cells for each group; *, p < .05. (C): Ratio of the time constant of activation for the calcium transients at the periphery and center of the cells. (D): Above, representative confocal line‐scan images of spontaneous calcium transients from control (left) and BIN1 (right) hESC‐CM cells. Below, normalized calcium transients from line scan images recorded at center (red) and periphery (black) of each cell. Center time course of the control calcium transients was delayed 50 ms compared with periphery time course. Time courses at periphery and center are synchronized in BIN1 cells.