Abstract 342: The Absence of S-nitrosoglutathione Reductase (GSNOR -/- ) Reduces Maturation of iPSC-derived Cardiomyocytes

Abstract

Introduction: Induced pluripotent stem cells (iPSCs) are a potential source of cardiac regenerative therapy. However, incomplete structural and functional maturation of iPSC-derived cardiomyocytes (iPSC-CMs), including lack of T-tubules, immature sarcoplasmic reticulum (SR), and inefficient Ca 2+ -induced Ca 2+ release remain major limitations. We assessed the influence of nitric oxide (NO) on differentiation and maturation of iPSC-CMs. Objective: We hypothesized that enhanced S-nitrosylation impairs SR calcium (Ca 2+ ) handling (storage and uptake) in iPSC-CMs. Methods: iPSCs were derived from fibroblasts from wildtype mice and mice lacking S-nitrosoglutathione reductase (GSNOR -/- ), a denitrosylase that regulates protein S-nitrosylation. iPSCs were differentiated into CMs from embryoid bodies via the hanging-drop method. Analyses of cell morphology and structural changes, gene expression (cardiac markers), and functional tests (intracellular calcium transients) were performed at an early stage of cardiac development (day 21 of cell culture). The involvement of the SR Ca 2+ ATPase (SERCA2) in iPSC-CM contraction was tested by blocking Ca 2+ re-uptake with 1 μM Thapsigargin and SR Ca 2+ stores were estimated by addition of 20 mM caffeine. The role of the cardiac ryanodine receptor (RyR2) was assessed by blocking the channel with 10 μM ryanodine. The dependence of Ca 2+ transients on L-type Ca 2+ channels (LTCC) was demonstrated using 10 μM nifedipine, which eliminated [Ca 2+ ] i transients and cell contraction. Length of sarcomeres within the contractile apparatus was determined by staining cells with α-actinin. Results: Sarcomeres were more organized in WT iPSC-CMs than in GSNOR -/- iPSC-CMs after 21 days in culture. The loss of GSNOR activity reduced SR Ca 2+ content and sarcomere organization in iPSC-CMs. While iPSC-CMs are relatively immature in terms of ultrastructure and Ca 2+ handling; GSNOR –/– iPSC-CMs were less able to recycle intracellular Ca 2+ . Conclusions: Our results provide novel insights into NO-mediated gene regulation and cell proliferation and suggest that the absence of GSNOR affects CM differentiation. These findings have important implications for regenerative medicine and provide new targets for iPSC-based therapy.