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  • In proof of principle experiments we examined the response

    2018-11-09

    In proof-of-principle experiments, we examined the response of hESC-derived CMs to verapamil and isoproterenol, two drugs commonly used in cardiovascular research. Single CMs exhibited a significantly reduced calcium transient amplitude associated with a negative inotropic response to treatment with the L-type Ca2+ channel blocker verapamil. These results are in accordance with previous reports from hPSC-CM-based tissue-engineered heart tissues (Turnbull et al., 2014), embryoid bodies (EBs; Dolnikov et al., 2006), and hPSC-CM monolayers (Himmel, 2013). Likewise, we observed a dose-dependent positive inotropic response to isoproterenol. Prior studies using hPSC-CMs have found a variable inotropic response to isoproterenol. Although some studies show a significant inotropic response in EBs and single hPSC-CMs (Hayakawa et al., 2014; Reppel et al., 2004), other studies show a transient or no response in EBs and engineered heart tissue (Brito-Martins et al., 2008; Turnbull et al., 2014). Over the past decades, numerous drug development projects have been halted by concerns over cardiotoxicity. Because significant differences exist between humans and rodents, hPSC-CMs have been suggested as a promising alternative to rodent adult CMs. Several studies using hPSC-CMs for cardiotoxicity screening have been performed with different readouts of toxicity, such as increased raf inhibitors duration and the occurrence of early afterdepolarizations (Braam et al., 2010; Liang et al., 2013). We demonstrate our ability to rapidly detect drug-induced cardiac toxicity by assessing contractility and action potential simultaneously. Our finding that excitation-contraction coupling may be perturbed under toxic conditions suggests that simultaneously assessing action potential characteristics and contractile behavior may improve the sensitivity of drug-induced cardiac toxicity screening.
    Experimental Procedures
    Author Contributions J.D.K. and D.H. designed and performed the experiments reported in Figures 1, 2, 3, 4, 5, 6, and 7 and wrote the manuscript. N.M. and E.K. contributed the mechanical model for force generation calculations. P.V.D.M. mentored colleagues and edited the manuscript. A.G. wrote the Visible software and mentored colleagues. I.J.D. designed experiments, mentored colleagues, and wrote the manuscript.
    Acknowledgments We thank the Massachusetts General Hospital Program in the Membrane Biology Microscopy Core Facility for contributions to the setup and image acquisition on the Nikon A1R. We thank Reify Corporation for the use of the Visible software. We thank C. Cowan from the Harvard Stem Cell Institute for the generous gift of the HUES-9 cell line and D. Eliot from the Murdoch Children’s Research Institute for the generous gift of the HES-3 NKX2-5eGFP/w cell line. We thank Nikita Shah for assistance with the data analysis and isolation of adult murine cardiomyocytes. This work was supported by grants from the NIH/National Heart, Lung, and Blood Institute (U01HL100408-01 and 1K08 HL091209) and a grant from the Dutch Heart Foundation (2013SB013). A.G. is a founder of Reify.
    Introduction Heart failure is the leading cause of death worldwide; however, current therapies such as surgical interventions are capable only of delaying the progression of this devastating disease (Go et al., 2013). In particular, patients suffering from myocardial infarction (MI), a major cause of heart failure, have cardiac dysfunction due to significant loss of cardiomyocytes (CMs) (Laflamme and Murry, 2011). The adult mammalian heart has very limited ability to regenerate after such a loss. Due to their self-renewal and multi-lineage differentiation capacity, embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs), collectively called pluripotent stem cells (PSCs), have emerged as a highly promising and renewable source for generating CMs (Kehat et al., 2001; Laflamme et al., 2007; Yang et al., 2008; Zwi et al., 2009). Studies have shown that cell cultures directed toward differentiation into CMs include three types of CMs, nodal, atrial, and ventricular CMs, in varying ratios as well as other lineage cells (Huber et al., 2007; Lian et al., 2012; Shiba et al., 2012; Tohyama et al., 2013). Each type of cardiac-chamber-specific CM has unique functional, structural, and electrophysiological characteristics (Ng et al., 2010). Thus, transplantation of cardiomyogenically differentiated cells, which include heterogeneous CMs and other lineage cells, into injured myocardium might induce dysrhythmia, asynchronous cardiac contraction, or aberrant tissue formation (Liao et al., 2010). Since ventricular CMs are the most extensively affected cell type in MI and the major source for generating cardiac contractile forces, there has been great interest in producing ventricular CMs from stem cells for treatment of MI (Bizy et al., 2013; Lee et al., 2012; Müller et al., 2000; Zhang et al., 2011). It would therefore be ideal to generate a pure population of ventricular CMs from PSCs for cardiac-cell-based therapies.