Thyroid and Glucocorticoid Hormones Promote Functional T-Tubule Development in Human-Induced Pluripotent Stem Cell-Derived Cardiomyocytes

Abstract

Rationale: Human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CM) are increasingly being used for modeling heart disease and are under development for regeneration of the injured heart. However, incomplete structural and functional maturation of hiPSC-CM, including lack of T-tubules, immature excitation-contraction coupling, and inefficient Ca-induced Ca release remain major limitations.

Objective: Thyroid and glucocorticoid hormones are critical for heart maturation. We hypothesized that their addition to standard protocols would promote T-tubule development and mature excitation-contraction coupling of hiPSC-CM when cultured on extracellular matrix with physiological stiffness (Matrigel mattress).

Methods and results: hiPSC-CM were generated using a standard chemical differentiation method supplemented with T3 (triiodothyronine) and/or Dex (dexamethasone) during days 16 to 30 followed by single-cell culture for 5 days on Matrigel mattress. hiPSC-CM treated with T3+Dex, but not with either T3 or Dex alone, developed an extensive T-tubule network. Notably, Matrigel mattress was necessary for T-tubule formation. Compared with adult human ventricular cardiomyocytes, T-tubules in T3+Dex-treated hiPSC-CM were less organized and had more longitudinal elements. Confocal line scans demonstrated spatially and temporally uniform Ca release that is characteristic of excitation-contraction coupling in the heart ventricle. T3+Dex enhanced elementary Ca release measured by Ca sparks and promoted RyR2 (ryanodine receptor) structural organization. Simultaneous measurements of L-type Ca current and intracellular Ca release confirmed enhanced functional coupling between L-type Ca channels and RyR2 in T3+Dex-treated cells.

Conclusions: Our results suggest a permissive role of combined thyroid and glucocorticoid hormones during the cardiac differentiation process, which when coupled with further maturation on Matrigel mattress, is sufficient for T-tubule development, enhanced Ca-induced Ca release, and more ventricular-like excitation-contraction coupling. This new hormone maturation method could advance the use of hiPSC-CM for disease modeling and cell-based therapy.

Keywords: calcium; cardiac electrophysiology; extracellular matrix; stem cells.

Figure 1. T3+Dex promotes t-tubule formation and synchronizes intracellular Ca release

A) Hormone-based cardiac differentiation protocol (D=day, see online methods for details). B) Representative examples of t-tubule staining in hiPSC-CM (upper panels) and human adult myocardium (lower panels) with quantitation of t-tubule index (% cell area) (n=19–59 cells/group, *p<0.05 and ***p<0.001 vs vehicle). Images are planar projections of 3D reconstructions. C) Representative transverse line scans of Fluo-4AM loaded hiPSC-CM electrically-stimulated at 0.2 Hz (left panels) and summary data (right panel) comparing Ca transient time to peak between periphery (P) and center (C) of cell. Scan line was positioned across the middle of each cell. n=18–21 cells/group; *p<0.05, **p<0.01, ***p<0.001, vs vehicle, ns – non significant. All data are reported as mean±SEM.

Figure 2. T3+Dex treated hiPSC-CM demonstrate greater dependence on SR Ca release for EC coupling

A) Representative examples of intracellular Ca transients recorded from Fura-2AM loaded hiPSC-CM with an overlay of the Ca transient from a T3+Dex treated cell (red trace) and a vehicle-treated cell (black trace). Cells were paced at 0.2 Hz followed by rapid caffeine (Caff) application to measure SR Ca content. Note the significantly faster rate of Ca rise and rate of Ca decay in the T3+Dex treated cells. B-D) Summary data for Ca transient amplitude (B), time to peak (C), and transient decay tau (D); (n=68–73 cells/group). E) Representative paced Ca transients before (black) and after (grey) block of SR Ca release with thapsigargin and ryanodine. Note the larger contribution of SR Ca release to the Ca transient in T3+Dex treated cells. F–H) Summary data following SR blockade for Ca transient amplitude (F), time to peak (G), and decay tau (H). Data are expressed as percent change from baseline before SR block. Data reported as mean±SEM (n=16–21 cells/group); *p<0.05, **p<0.01, and ***p<0.001 vs vehicle.

Figure 3. T3+Dex increases cell capacitance and EC-coupling gain

A) Representative examples of simultaneously-recorded intracellular Ca fluorescence traces (top) and L-type Ca currents (bottom) from vehicle-treated (black) and T3+Dex treated (red) hiPSC-CM. Records were obtained from voltage-clamped hiPSC-CM loaded with the fluorescent Ca indicator Fluo-4 and measured using the indicated voltage protocol. B) Average current-voltage relationship with associated intracellular Ca fluorescence at indicated membrane potentials in T3+Dex versus vehicle treated hiPSC-CM. C) EC coupling gain calculated as a ratio of normalized Ca fluorescence to normalized Ca current. D) SR Ca content. E) Cell capacitance. All data reported as mean±SEM (n=6–7); *p<0.05, **p<0.01, ***p<0.001, vs vehicle, ns – non significant.

Figure 4. T3+Dex enhances elementary Ca release (Ca sparks) and RyR2 organization

A) Representative line-scans of Ca sparks recorded from saponin permeabilized hiPSC-CM. B) Overlay of representative Ca spark. C–E) Summary data for spark parameters for the different treatment groups (n=15–37 cells/group). F) Representative cell images and fast Fourier transform of RyR2 immunostaining in T3+Dex and vehicle treated cells. Summary data of peak power of RyR2 immunostaining (n=8–10 cells/group). All data reported as mean±SEM; *p<0.05 and ***p<0.001 vs vehicle.