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Inducible CaV1.2 cell line optimized for automated patch clamp assays
Abstracts
that exists between functional and structural toxicity, and the benefits of this more holistic approach to risk assessment. We demonstrate for the first time how the advent of transparent MEA plate technology enables functional and structural cardiotoxic responses to be recorded from the same cell population. This approach more directly links changes in morphology of the hSC-CMs with recorded electrophysiology signatures, offering even greater insight into the wide range of potential drug impacts on cardiac physiology, with a throughput that is more amenable to early drug discovery. doi:10.1016/j.vascn.2016.02.020
0020 Mitogen activated kinase kinase 7 is involved in Sunitinib induced myocardial injury Samantha Cooper, Helen Maddock, Afthab Hussain, Hardip Sandhu Coventry University, Coventry, UK Introduction: Sunitinib is highly effective in the treatment of cancer. However, adverse cardiovascular events have been reported. MAP kinase signalling cascades play significant roles in the development of cardiac hypertrophy, in response to stress. ASK-1 is a partial activator of MKK7, which in turn activates c-Jun-terminal kinase (JNK). MKK7 expression was investigated with co-administration of Sunitinib +/- ASK-1 specific inhibitor NQDI-1; to identify the involvement of MKK7 in sunitinib induced cardiotoxicity and the potential cardioprotective role of NQDI-1. Methods: Langendorff heart models were perfused with vehicle (Krebs buffer) or Sunitinib +/- NQDI-1 for 120 minutes. Infarct to risk size was analysed with triphenyltetrazolium chloride (n = 6). MKK7 mRNA was detected by real time PCR (n = 6) and phosphorylated levels of MKK7 (pMKK7) was analysed by western blot (n = 4). Results: Haemodynamic analysis showed a reduction in heart rate (30%), plus an increase in coronary flow (15%) in Sunitinib treatment alone. Sunitinib causes a significant increase in infarct size compared to vehicle (vehicle = 5.4 ± 2%; Sunitinib = 46 ± 4.5%, P b 0.0001). NQDI-1 co-administration reduced the level of Sunitinib induced myocardial injury. MKK7 mRNA levels were significantly decreased in Sunitinib treatment alone. Plus, a decrease in pMKK7 levels were detected with Sunitinib treatment alone, however NQDI1 co-administration did not cause a significant change in MKK7 mRNA or pMKK7. Conclusion: Sunitinib induced cardiotoxicity is associated with a reduction in MKK7 mRNA expression and phosphorylation. NQDI-1 coadministration with Sunitinib is cardioprotective. This is the first study to highlight the potential involvement of mitogen-activated protein (MAP) kinase kinase family in Sunitinib induced cardiotoxicity. doi:10.1016/j.vascn.2016.02.021
0021 Use of high throughput electric field stimulation (EFS) coupled with intracellular Ca2 + kinetics measurements on iPSC-derived cardiomyocytes Natsumi Katoa, Satoshi Yamamotoa, Taira Itoa, Masanori Matsubaraa, Shouming Dub, Takuji Kataokaa, Fumio Iwasea, Ralf Kettenhofenc, Felix von Hanielc, Jean Marc d’Angelod, a
Systems Division, Hamamatsu Photonics K.K., Hamamatsu, Japan Hamamatsu Corporation, Bridgewater, NJ, USA
b
341
c
Axiogenesis AG, Cologne, Germany Hamamatsu Photonics, Massy, France
d
Hamamatsu has developed a 96-channel electrode array system that is mounted on the FDSS/μCELL. It adds electric field stimulations (EFSs) to all 96 wells in a microplate simultaneously while fluorescence/ luminescence signals are monitored. We measured oscillations of intracellular Ca2 + concentration, which occurs along with the beating of the cells, with a calcium sensitive fluorescent dye in human iPSCderived cardiomyocytes (Cor.4U, Axiogenesis AG). We observed that the Ca2 + oscillation in cardiomyocytes was synchronized to the electric stimulation, which indicates that the EFS system is able to pace the beating of cardiomyocytes. We also observed that the Ca2 + oscillations in the presence of some ion channel blockers were modulated by adding EFS. Use of EFS coupled with intracellular Ca2 + kinetics measurements, in the high-throughput manner, should be useful in in vitro assessment of cardiac toxicity of pharmacological compounds using cardiomyocytes, in particular in toxicity screening at early stages of drug development, as well as in cardiomyocyte research. doi:10.1016/j.vascn.2016.02.022
0022 Inducible CaV1.2 cell line optimized for automated patch clamp assays Timm Dankera, Manfred Freyb, Elke Guenthera a
NMI TT GmbH, Reutlingen, Germany Steinbeis-Transferzentrum für Angewandte Biologische Chemie, Mannheim, Germany
b
The Cav1.2 ion channel is expressed in heart, smooth muscle and neurons, and is a therapeutic target in cardiac arrhythmia and hypertension. Because of its key role in shaping the cardiac action potentials plateau phase, testing of drug candidates against CaV1.2 on automated patch clamp platforms has become an important part of cardiac safety assessments, and will be included in the upcoming CIPA proposal. This requires the availability of a cell line which is stably expressing all three subunits of the CaV1.2 channel and shows high performance in automated patch clamp assays. Since high levels of CaV1.2 expression negatively affect the growth rate of cultured cells, it is particular difficult to develop a stable CaV1.2 cell line having optimal characteristics for automated patch clamp. We addressed these issues by developing a CHO cell line with inducible expression of CaV1.2, based on our proprietary IGAMI expression system. The resulting cell line is easy to maintain in cell culture and shows excellent performance on the Qpatch automated patch clamp system. After induction of ion channel expression, CaV1.2 currents are reliably recorded with the Qpatch system. The expression level, and therefore the current amplitudes can be optimized for the automated patch clamp platform by varying the duration of the induction phase. This results in high success rates and excellent data quality of the automated assay. doi:10.1016/j.vascn.2016.02.023
0023 Variability in cardiac ion channel assays: A major challenge for CiPA paradigm implementation Annie Delaunois, Aurore Colomar, Jean-Pierre Valentin UCB Biopharma sprl, Braine-l'Alleud, Belgium
that exists between functional and structural toxicity, and the benefits of this more holistic approach to risk assessment. We demonstrate for the first time how the advent of transparent MEA plate technology enables functional and structural cardiotoxic responses to be recorded from the same cell population. This approach more directly links changes in morphology of the hSC-CMs with recorded electrophysiology signatures, offering even greater insight into the wide range of potential drug impacts on cardiac physiology, with a throughput that is more amenable to early drug discovery. doi:10.1016/j.vascn.2016.02.020
0020 Mitogen activated kinase kinase 7 is involved in Sunitinib induced myocardial injury Samantha Cooper, Helen Maddock, Afthab Hussain, Hardip Sandhu Coventry University, Coventry, UK Introduction: Sunitinib is highly effective in the treatment of cancer. However, adverse cardiovascular events have been reported. MAP kinase signalling cascades play significant roles in the development of cardiac hypertrophy, in response to stress. ASK-1 is a partial activator of MKK7, which in turn activates c-Jun-terminal kinase (JNK). MKK7 expression was investigated with co-administration of Sunitinib +/- ASK-1 specific inhibitor NQDI-1; to identify the involvement of MKK7 in sunitinib induced cardiotoxicity and the potential cardioprotective role of NQDI-1. Methods: Langendorff heart models were perfused with vehicle (Krebs buffer) or Sunitinib +/- NQDI-1 for 120 minutes. Infarct to risk size was analysed with triphenyltetrazolium chloride (n = 6). MKK7 mRNA was detected by real time PCR (n = 6) and phosphorylated levels of MKK7 (pMKK7) was analysed by western blot (n = 4). Results: Haemodynamic analysis showed a reduction in heart rate (30%), plus an increase in coronary flow (15%) in Sunitinib treatment alone. Sunitinib causes a significant increase in infarct size compared to vehicle (vehicle = 5.4 ± 2%; Sunitinib = 46 ± 4.5%, P b 0.0001). NQDI-1 co-administration reduced the level of Sunitinib induced myocardial injury. MKK7 mRNA levels were significantly decreased in Sunitinib treatment alone. Plus, a decrease in pMKK7 levels were detected with Sunitinib treatment alone, however NQDI1 co-administration did not cause a significant change in MKK7 mRNA or pMKK7. Conclusion: Sunitinib induced cardiotoxicity is associated with a reduction in MKK7 mRNA expression and phosphorylation. NQDI-1 coadministration with Sunitinib is cardioprotective. This is the first study to highlight the potential involvement of mitogen-activated protein (MAP) kinase kinase family in Sunitinib induced cardiotoxicity. doi:10.1016/j.vascn.2016.02.021
0021 Use of high throughput electric field stimulation (EFS) coupled with intracellular Ca2 + kinetics measurements on iPSC-derived cardiomyocytes Natsumi Katoa, Satoshi Yamamotoa, Taira Itoa, Masanori Matsubaraa, Shouming Dub, Takuji Kataokaa, Fumio Iwasea, Ralf Kettenhofenc, Felix von Hanielc, Jean Marc d’Angelod, a
Systems Division, Hamamatsu Photonics K.K., Hamamatsu, Japan Hamamatsu Corporation, Bridgewater, NJ, USA
b
341
c
Axiogenesis AG, Cologne, Germany Hamamatsu Photonics, Massy, France
d
Hamamatsu has developed a 96-channel electrode array system that is mounted on the FDSS/μCELL. It adds electric field stimulations (EFSs) to all 96 wells in a microplate simultaneously while fluorescence/ luminescence signals are monitored. We measured oscillations of intracellular Ca2 + concentration, which occurs along with the beating of the cells, with a calcium sensitive fluorescent dye in human iPSCderived cardiomyocytes (Cor.4U, Axiogenesis AG). We observed that the Ca2 + oscillation in cardiomyocytes was synchronized to the electric stimulation, which indicates that the EFS system is able to pace the beating of cardiomyocytes. We also observed that the Ca2 + oscillations in the presence of some ion channel blockers were modulated by adding EFS. Use of EFS coupled with intracellular Ca2 + kinetics measurements, in the high-throughput manner, should be useful in in vitro assessment of cardiac toxicity of pharmacological compounds using cardiomyocytes, in particular in toxicity screening at early stages of drug development, as well as in cardiomyocyte research. doi:10.1016/j.vascn.2016.02.022
0022 Inducible CaV1.2 cell line optimized for automated patch clamp assays Timm Dankera, Manfred Freyb, Elke Guenthera a
NMI TT GmbH, Reutlingen, Germany Steinbeis-Transferzentrum für Angewandte Biologische Chemie, Mannheim, Germany
b
The Cav1.2 ion channel is expressed in heart, smooth muscle and neurons, and is a therapeutic target in cardiac arrhythmia and hypertension. Because of its key role in shaping the cardiac action potentials plateau phase, testing of drug candidates against CaV1.2 on automated patch clamp platforms has become an important part of cardiac safety assessments, and will be included in the upcoming CIPA proposal. This requires the availability of a cell line which is stably expressing all three subunits of the CaV1.2 channel and shows high performance in automated patch clamp assays. Since high levels of CaV1.2 expression negatively affect the growth rate of cultured cells, it is particular difficult to develop a stable CaV1.2 cell line having optimal characteristics for automated patch clamp. We addressed these issues by developing a CHO cell line with inducible expression of CaV1.2, based on our proprietary IGAMI expression system. The resulting cell line is easy to maintain in cell culture and shows excellent performance on the Qpatch automated patch clamp system. After induction of ion channel expression, CaV1.2 currents are reliably recorded with the Qpatch system. The expression level, and therefore the current amplitudes can be optimized for the automated patch clamp platform by varying the duration of the induction phase. This results in high success rates and excellent data quality of the automated assay. doi:10.1016/j.vascn.2016.02.023
0023 Variability in cardiac ion channel assays: A major challenge for CiPA paradigm implementation Annie Delaunois, Aurore Colomar, Jean-Pierre Valentin UCB Biopharma sprl, Braine-l'Alleud, Belgium