Thyroid and Glucocorticoid Hormones Promote Functional T-Tubule Development in Human-Induced Pluripotent Stem Cell–Derived Cardiomyocytes
Authors: Theis Tønnessen
Christen P Dahl
Dmytro O Kryshtal
William E Louch
Michael Frisk
Bjorn C Knollmann
Kyungsoo Kim
Shan S Parikh
Daniel J Blackwell
Nieves Gomez-Hurtado
Arnt Fiane
Lili Wang
Journal: Circulation Research (2017)
Discipline: CLINICAL MEDICINE
Keywords: calcium; cardiac electrophysiology; extracellular matrix; stem cells.
Affiliations: From the Vanderbilt Center for Arrhythmia Research and Therapeutics, Department of Medicine (S.S.P., D.J.B., N.G.-H., L.W., K.K., D.O.K., B.C.K.), Department of Pharmacology (S.S.P., B.C.K.), Vanderbilt University Medical School, Nashville, TN; Institute for Experimental Medical Research, Oslo University Hospital, Norway (M.F., T.T., W.E.L.); University of Oslo, Norway (M.F., T.T., W.E.L.); Department of Cardiology (C.P.D.), and Department of Cardiothoracic Surgery (A.F.), Oslo University Hospital Rikshospitalet, Norway; and Oslo University Hospital Ullevål, Norway (T.T.)., From the Vanderbilt Center for Arrhythmia Research and Therapeutics, Department of Medicine (S.S.P., D.J.B., N.G.-H., L.W., K.K., D.O.K., B.C.K.), Department of Pharmacology (S.S.P., B.C.K.), Vanderbilt University Medical School, Nashville, TN; Institute for Experimental Medical Research, Oslo University Hospital, Norway (M.F., T.T., W.E.L.); University of Oslo, Norway (M.F., T.T., W.E.L.); Department of Cardiology (C.P.D.), and Department of Cardiothoracic Surgery (A.F.), Oslo University Hospital Rikshospitalet, Norway; and Oslo University Hospital Ullevål, Norway (T.T.). .

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. 

Retracted: No

PubPeer comments:
 Dear authors, I came across article, who are first one to show the presence of t-tubules in the iPSC derived cardiomyocytes. Congratulations on getting more mature cardiomyocytes derived from iPSCs. Is it possible for authors to upload the raw image of Figure 1B and online figure 2A,B. You have only showed processed images of t-tubule staining, however, it would be wise for readers to see how the raw t-tubule staining will look on iPSC derived cardiomyocytes. Another question, in Figure 1B and online Figure 3, you show that hormones (T3+D) with the mattresses (red bar graph) enhance the morphological parameters of the cells (tubule density, cell size, volume, length, width). However, with the hormones (T3+D) without mattress, you don t see this improved morphology which remain as non treated cells. Do you have data of cells without hormones with the mattress. It looks like your enhanced maturity can only come from the mattress and not really from the hormones. In your previous publication (Matrigel Mattress: A Method for the Generation of Single Contracting Human-Induced Pluripotent Stem Cell-Derived Cardiomyocytes) from 2015, where you only use matrigel mattress you unfortunately didn t measure the same cell morphology parameters. ,