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Evaluation of ion channel trafficking in human stem cell-derived cardiomyocytes for cardiotoxicity screening
Abstracts
chronic treatment, probucol acts via the KCNE1 molecule to exert IKs inhibition. Since probucol has cholesterol-lowering effect in the cells; we are currently testing effects of another type of cholesterollowering drugs, simvastatin and triparanol, on the IKs and KCNQ1 current using KCNQ1/KCNE1-CHO and KCNQ1-CHO cells, respectively to determine detailed inhibition mechanisms of the probucol on the IKs current. doi:10.1016/j.vascn.2013.01.033
022 Evaluation of ion channel trafficking in human stem cell-derived cardiomyocytes for cardiotoxicity screening Yasuyuki Abea, Tomoko Sakakuraa, Kiyoshi Takasunaa, Atsushi Sanbuisshoa, Fumimasa Nomurab, Tomoyo Hamadab, Tomoyuki Kanekob, Kenji Yasudab a
e7
purification and selection of the in vitro differentiated cardiomyocytes. The cardiomyocytes produced from these hiPS cells showed spontaneously beating and positive immuostaining for cardiac markers like alpha-actinin. We have then implemented these Cor.4U cardiomyocytes in three different assays widely used for cardiac safety assessment: Patch Clamp (voltage and current clamp), Microelectrode Array (MEA), and the RTCA Cardio xCELLigence system. A set of compounds with known effects on cardiac physiology (e.g. ion channel modulation, hERG traficking, contractility, adrenergic and muscarinic regulation) were tested; and the results obtained were compared to data available in the literature. All compounds tested so far (Patch clamp: 4; MEA: 8; RTCACardio: 17) induced the expected effects on cardiomyocyte function, suggesting that all major mechanisms underlying cardiac physiology can be addressed using these hiPS derived Cor.4U cardiomyocytes. Moreover, our results demonstrate that the combination of standardized, stable and physiologically relevant stem cell-derived cardiomyocytes in combination with advanced assay technologies provide a reliable and convenient in vitro model to screen for cardiac safety as well as efficacy.
Daiichi Sankyo Co., Ltd, Tokyo, Japan Tokyo Medical and Dental University, Tokyo, Japan
b
doi:10.1016/j.vascn.2013.01.035 We have investigated the applicability of human stem cell-derived cardiomyocytes for the evaluation of sub-chronic effects such as ion channel trafficking. Human embryonic stem (ES) cell-derived cardiomyocytes clusters were cultivated on multi electrode array (MEA) chips under 37 °C, 5% CO2 condition. Pentamidine (10 and 30 μM) and 17-AAG (1 and 3 μM) were applied to the incubation medium, and the field potential duration (FPD) was periodically evaluated before (control) and 2, 4, 8, and 24 h after drug application. FPD prolongation by pentamidine was observed from 4 h onwards and finally reached 179% at 10 μM and 429% at 30 μM, after 24 h application. In contrast, 17AAG at 3 μM shortened FPD by 28% after 24 h application. Immunohistochemical staining showed reduced expression of hERG, Nav1.5, and Cav1.2 channels in 17-AAG treated cardiomyocytes, indicating that the shortened FPD in 17-AAG might result from the reduction of inward currents via Nav1.5 and Cav1.2 overcoming the reduction of outward current via hERG. The results suggest that the FPD analysis in human stem cell-derived cardiomyocytes with sub-chronic drug exposure has a potential to be an integrated ion channel function assay to predict the cardio-toxicity risks such as ion channel trafficking inhibitions induced by long term drug exposure. doi:10.1016/j.vascn.2013.01.034
023 In vitro pharmacology and safety testing using human iPS-derived Cor.4U cardiomyocytes Ralf Kettenhofena, Kristina Tressata, Eugen Kolossova, Simon Hebeisenb, Evelyn Jaehneb, Andreas Ehlicha, Silke Schwengberga, Heribert Bohlena a
Axiogenesis AG, Koeln, Germany bSYS GmbH, Witterswill, Switzerland
b
The lack of a predictive and standardized in vitro cardiomyocyte cell models hampers the development of new cardiac drugs and leads to costly late stage failures of potential drug candidates due to safety issues. Human induced-pluripotent stem cell-derived cardiomyocytes (hiPS-CM) have the capacity to bridge this gap. We have created a human iPS cell line harboring a puromycin resistance cassette under control of the cardiac alph-MHC promotor. This construct allows for
024 Pluripotent stem cell-derived cardiomyocytes: Modeling hypertrophy in a dish Daniella Steel, Kerstin Dahlenborg, Sofie Andersson, Peter Sartipy Cellectis Stem Cells/Cellartis AB, Gothenburg, Sweden Cardiac hypertrophy occurs as a natural reaction following exercise but also in response to stress stimulation or injury, where individual cardiomyocytes increase in size as compensation for dysfunctional heart tissue. Pathological cardiac hypertrophy can provoke heart failure and is an independent risk for torsades de pointes. Progress in safety pharmacology has been hindered by the lack of readily available human cardiac tissue and interspecies differences in animal hypertrophy models. Human pluripotent stem cell derived cardiomyocytes clusters (hPSC-CMC) represent a renewable source of relevant cells to substitute for ex vivo material. hPSC-CMC have been characterized at the genetic, molecular and functional level, and demonstrate a clear cardiac phenotype. Furthermore, the tissue-like format of hPSC-CMC offers a complex system for studying the multiple signal transduction pathways involved in the hypertrophic response. Here, we have tested inducing hypertrophy in hPSC-CMC through the inactivation of GSK-3 which removes the negative constraint of GSK-3 on hypertrophy. Automated imaging during 90 h treatment showed significant increase in hPSCCMC area (mean 33.0 ± 1.1% St. Dev). Testing was repeated in monolayers of dissociated hPSC-CMC, which demonstrated no increase in cellular proliferation but rather an increase in cardiomyocyte size. Molecular profiling showed changes in mRNA MHY7: MHY6 ratio, suggesting induction of a fetal gene program typically associated with a hypertrophic response. Interestingly, induction of hypertrophy also predisposed hPSC-CMC to increased sensitivity to doxorubicin exposure, as measured by release of cardiac troponin. Taken together, these results suggest that hPSC-CMC is a suitable model system to study pathologic and non-pathologic cardiac hypertrophy in vitro.
doi:10.1016/j.vascn.2013.01.036
chronic treatment, probucol acts via the KCNE1 molecule to exert IKs inhibition. Since probucol has cholesterol-lowering effect in the cells; we are currently testing effects of another type of cholesterollowering drugs, simvastatin and triparanol, on the IKs and KCNQ1 current using KCNQ1/KCNE1-CHO and KCNQ1-CHO cells, respectively to determine detailed inhibition mechanisms of the probucol on the IKs current. doi:10.1016/j.vascn.2013.01.033
022 Evaluation of ion channel trafficking in human stem cell-derived cardiomyocytes for cardiotoxicity screening Yasuyuki Abea, Tomoko Sakakuraa, Kiyoshi Takasunaa, Atsushi Sanbuisshoa, Fumimasa Nomurab, Tomoyo Hamadab, Tomoyuki Kanekob, Kenji Yasudab a
e7
purification and selection of the in vitro differentiated cardiomyocytes. The cardiomyocytes produced from these hiPS cells showed spontaneously beating and positive immuostaining for cardiac markers like alpha-actinin. We have then implemented these Cor.4U cardiomyocytes in three different assays widely used for cardiac safety assessment: Patch Clamp (voltage and current clamp), Microelectrode Array (MEA), and the RTCA Cardio xCELLigence system. A set of compounds with known effects on cardiac physiology (e.g. ion channel modulation, hERG traficking, contractility, adrenergic and muscarinic regulation) were tested; and the results obtained were compared to data available in the literature. All compounds tested so far (Patch clamp: 4; MEA: 8; RTCACardio: 17) induced the expected effects on cardiomyocyte function, suggesting that all major mechanisms underlying cardiac physiology can be addressed using these hiPS derived Cor.4U cardiomyocytes. Moreover, our results demonstrate that the combination of standardized, stable and physiologically relevant stem cell-derived cardiomyocytes in combination with advanced assay technologies provide a reliable and convenient in vitro model to screen for cardiac safety as well as efficacy.
Daiichi Sankyo Co., Ltd, Tokyo, Japan Tokyo Medical and Dental University, Tokyo, Japan
b
doi:10.1016/j.vascn.2013.01.035 We have investigated the applicability of human stem cell-derived cardiomyocytes for the evaluation of sub-chronic effects such as ion channel trafficking. Human embryonic stem (ES) cell-derived cardiomyocytes clusters were cultivated on multi electrode array (MEA) chips under 37 °C, 5% CO2 condition. Pentamidine (10 and 30 μM) and 17-AAG (1 and 3 μM) were applied to the incubation medium, and the field potential duration (FPD) was periodically evaluated before (control) and 2, 4, 8, and 24 h after drug application. FPD prolongation by pentamidine was observed from 4 h onwards and finally reached 179% at 10 μM and 429% at 30 μM, after 24 h application. In contrast, 17AAG at 3 μM shortened FPD by 28% after 24 h application. Immunohistochemical staining showed reduced expression of hERG, Nav1.5, and Cav1.2 channels in 17-AAG treated cardiomyocytes, indicating that the shortened FPD in 17-AAG might result from the reduction of inward currents via Nav1.5 and Cav1.2 overcoming the reduction of outward current via hERG. The results suggest that the FPD analysis in human stem cell-derived cardiomyocytes with sub-chronic drug exposure has a potential to be an integrated ion channel function assay to predict the cardio-toxicity risks such as ion channel trafficking inhibitions induced by long term drug exposure. doi:10.1016/j.vascn.2013.01.034
023 In vitro pharmacology and safety testing using human iPS-derived Cor.4U cardiomyocytes Ralf Kettenhofena, Kristina Tressata, Eugen Kolossova, Simon Hebeisenb, Evelyn Jaehneb, Andreas Ehlicha, Silke Schwengberga, Heribert Bohlena a
Axiogenesis AG, Koeln, Germany bSYS GmbH, Witterswill, Switzerland
b
The lack of a predictive and standardized in vitro cardiomyocyte cell models hampers the development of new cardiac drugs and leads to costly late stage failures of potential drug candidates due to safety issues. Human induced-pluripotent stem cell-derived cardiomyocytes (hiPS-CM) have the capacity to bridge this gap. We have created a human iPS cell line harboring a puromycin resistance cassette under control of the cardiac alph-MHC promotor. This construct allows for
024 Pluripotent stem cell-derived cardiomyocytes: Modeling hypertrophy in a dish Daniella Steel, Kerstin Dahlenborg, Sofie Andersson, Peter Sartipy Cellectis Stem Cells/Cellartis AB, Gothenburg, Sweden Cardiac hypertrophy occurs as a natural reaction following exercise but also in response to stress stimulation or injury, where individual cardiomyocytes increase in size as compensation for dysfunctional heart tissue. Pathological cardiac hypertrophy can provoke heart failure and is an independent risk for torsades de pointes. Progress in safety pharmacology has been hindered by the lack of readily available human cardiac tissue and interspecies differences in animal hypertrophy models. Human pluripotent stem cell derived cardiomyocytes clusters (hPSC-CMC) represent a renewable source of relevant cells to substitute for ex vivo material. hPSC-CMC have been characterized at the genetic, molecular and functional level, and demonstrate a clear cardiac phenotype. Furthermore, the tissue-like format of hPSC-CMC offers a complex system for studying the multiple signal transduction pathways involved in the hypertrophic response. Here, we have tested inducing hypertrophy in hPSC-CMC through the inactivation of GSK-3 which removes the negative constraint of GSK-3 on hypertrophy. Automated imaging during 90 h treatment showed significant increase in hPSCCMC area (mean 33.0 ± 1.1% St. Dev). Testing was repeated in monolayers of dissociated hPSC-CMC, which demonstrated no increase in cellular proliferation but rather an increase in cardiomyocyte size. Molecular profiling showed changes in mRNA MHY7: MHY6 ratio, suggesting induction of a fetal gene program typically associated with a hypertrophic response. Interestingly, induction of hypertrophy also predisposed hPSC-CMC to increased sensitivity to doxorubicin exposure, as measured by release of cardiac troponin. Taken together, these results suggest that hPSC-CMC is a suitable model system to study pathologic and non-pathologic cardiac hypertrophy in vitro.
doi:10.1016/j.vascn.2013.01.036