Shan S Parikh1, Daniel J Blackwell1, Nieves Gomez-Hurtado1, Michael Frisk1, Lili Wang1, Kyungsoo Kim1, Christen P Dahl1, Arnt Fiane1, Theis Tønnessen1, Dmytro O Kryshtal1, William E Louch1, Bjorn C Knollmann2. 1. 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.). 2. 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.). bjorn.knollmann@vanderbilt.edu.
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.
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.
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