The developments in the field of genetically

The developments in the field of genetically encoded fluorescent indicators may provide a possible solution to the aforementioned challenges. Genetically encoded indicators are composed of a sensing element, which is usually fused to an autofluorescent protein (like circularly permuted enhanced GFP; cpEGFP) that alters its fluorescent intensity as a result of conformational changes in the sensing element. While utilized in numerous neuroscience-related experimental models (Akemann et al., 2010; Cao et al., 2013; Grienberger and Konnerth, 2012; Looger and Griesbeck, 2012; Tian et al., 2009), the use of similar indicators in non-neuronal tissues, such as the heart, has been more limited (Addis et al., 2013; Chong et al., 2014; Kaestner et al., 2014; Leyton-Mange et al., 2014). Here, we aimed to transfer these emerging technologies to the cardiac field, specifically focusing on genetically encoded calcium indicators (GECIs) (Grienberger and Konnerth, 2012; Kaestner et al., 2014; Tian et al., 2009) and genetically encoded voltage indicators (GEVIs) (Jin et al., 2012; Kralj et al., 2012; Leyton-Mange et al., 2014), in an attempt to establish experimental platforms to monitor the functional activity of hiPSC-CMs. To this end, we aimed to express GCaMP5G (Addis et al., 2013; Tian et al., 2009), a GECI that displays improved dynamic range, improved sensitivity, and maintains relatively stable nitric oxide booster levels, and ArcLight A242 (Cao et al., 2013; Jin et al., 2012; Leyton-Mange et al., 2014), a new variant of the Ciona intestinalis voltage-sensitive (CiVS)-based fluorescent protein voltage sensor (Barnett et al., 2012; Murata et al., 2005) super-family, in both healthy and diseased hiPSC-CMs.
Results
Discussion To fulfill the potential of the hiPSC technology for several cardiovascular applications, methods should be developed for efficient, long-term, and large-scale functional phenotyping of hiPSC-CMs. The successful utilization of genetically encoded fluorescent indicators in the neuroscience field (Akemann et al., 2010; Cao et al., 2013; Grienberger and Konnerth, 2012; Looger and Griesbeck, 2012; Tian et al., 2009) and initial reports using similar GEVIs (Leyton-Mange et al., 2014) and GECIs (Chong et al., 2014) in hESC-CMs suggest that similar approaches could also prove useful for studying different hiPSC-CMs. The aforementioned qualities allowed monitoring changes in AP and calcium-handling properties and the development of arrhythmias in hiPSC-CMs in response to application of various drugs and in the setting of different genetic disorders. First, we showed the potential of ArcLight-hiPSC-CMs for screening the effects of drugs known to prolong APD due to their hERG channel (IKr)-blocking activity (“QT screening”). Drug-induced AP prolongation, leading to life-threatening arrhythmias, represents the single most important reason for withdrawal of already approved drugs from the market (Roden, 2004). Our results revealed the ability of ArcLight-based analysis to robustly identify APD prolongation in the hiPSC-CMs, as well as markers of arrhythmogenicity (EADs and triggered beats), following application of a variety of QT-prolonging agents. Next, we provided evidence for the potential of nilotinib (a TKI used to treat multiple cancers) to prolong AP and induce arrhythmias in human cardiomyocytes. This finding may be important not only because of the clinical importance of TKIs but also because of the suggested mechanism underlying APD prolongation by these agents, as suggested in a recent canine study (Lu et al., 2012), which involves interaction with multiple currents and is not immediate. This highlights the potential advantages of using ArcLight-hiPSC-CMs over traditional heterologous expression systems used for QT screening (that evaluate only a single current) and even over intracellular recording of hiPSC-CMs, since ArcLight imaging allows us to follow AP properties in the same cell over time. Finally, we also showed the ability to use GCaMP5G (alone or combine with voltage-imaging) to detect drug-induced alterations in the calcium-handling properties of human cardiomyocytes leading to arrhythmias.