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  • Prolonged APs and EADs are

    2018-11-02

    Prolonged APs and EADs are typical of pharmacologically induced LQTS modeled in nonhuman hearts (Shimizu and Antzelevitch, 2000; Studenik et al., 2001; Terentyev et al., 2014), yet few such studies examined [Ca2+]i transients. The lengthening of APs and [Ca2+]i transients after pharmacologically blocking IKr in control iPS-CM, in direct parallel to animal studies, well corroborates our evidence from LQTS myocytes. Insights such as this suggest that benefits may accrue from routinely deriving iPS-CM from LQTS patients, both for risk assessment, and to guide treatments including whether to employ Ca2+ channel blockers (Terrenoire et al., 2013). In conclusion, our results contend that existing or novel Ca2+ antagonists (perhaps acting to shift the level of cytosolic Mg2+) may help us to reduce the risk of cardiac events in the LQTS population.
    Experimental Procedures
    Author Contributions C.I.S. designed and performed patch clamp, fluorescence, and pharmacological studies in iPS-CM and wrote the manuscript; S. Baba designed and performed fibroblast reprogramming, molecular confirmation of DNA, and protein muscarinic receptor antagonists in iPSCs and iPS-CM and assisted with the manuscript; K.N. recruited patients, assisted with reprogramming, advised on molecular confirmation of DNA and protein expression, and assisted with the manuscript; E.A.H. prepared and maintained iPSCs and iPS-CM cultures; M.A.F.S. referred patients, collected patient information, and prepared and maintained iPSCs and iPS-CM cultures; C.F. programmed analysis software in the LabView environment; J.Z. advised on methods for preparing iPS-CM cultures and assisted with the manuscript; S. Balijepalli performed IKr voltage clamp electrophysiology and assisted with the manuscript; K.T. advised on reprogramming and molecular confirmation of DNA and protein expression and advised on manuscript; Y.H. advised on reprogramming and molecular confirmation of DNA and protein expression and advised on manuscript; P.L. prepared and maintained iPSCs and iPS-CM cultures; J.W. and M.M.S. referred patients, collected patient information, and assisted with the manuscript; K.A.-S. helped conceive project design, assisted with recruiting of patients, and assisted with the manuscript; C.T.J. managed iPS-CM production and voltage-clamp experiments in Wisconsin and assisted with the manuscript; J.C.M. and K.E.H. assisted with the manuscript; T.J.K. advised on iPS-CM production, electrophysiology, and all aspects of the manuscript; S.Y. advised on iPS-CM production and assisted with the manuscript; B.R.C. was overall project leader and designer, recruited patients, supervised iPS-CM production, collaborations, and assisted with the manuscript.
    Acknowledgments
    Introduction Transcriptional programs are believed to maintain cellular identities and are stabilized through various mechanisms, including chromatin modifications and lineage-determining transcription factors (Vierbuchen and Wernig, 2012). However, under several experimental approaches, imposed changes in the intrinsic and extrinsic cues have been shown to overcome these epigenetic barriers, driving the cells to pluripotency or completely unrelated somatic lineages (Jaenisch and Young, 2008; Ladewig et al., 2013; Vierbuchen and Wernig, 2011). Lineage conversion of embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) or already differentiated somatic cells into other cell types, such as neuronal cells, has recently attracted immense interest due to its possible application in the therapy of developmental diseases and in regenerative medicine (Blanpain et al., 2012; Han et al., 2011; Marchetto and Gage, 2012). We initially reported that forced expression of the three transcription factors ASCL1, BRN2, and MYT1L (BAM factors) successfully converts mesodermal fibroblasts into induced neuronal (iN) cells (Vierbuchen et al., 2010). In subsequent studies, we and others generated functional iN cells from human fibroblasts based on the same three BAM factors but adding additional transcription factors, microRNAs, or small molecules (Caiazzo et al., 2011; Ladewig et al., 2012; Pang et al., 2011; Pfisterer et al., 2011; Yoo et al., 2011). Thus, just like the critical breakthrough for generating iPSCs, a combination of factors was thought to be required for iN cell reprogramming from fibroblasts, and use of single transcription factors was considered insufficient.