- Email: [email protected]
Bridging functional and structural cardiotoxicity assays using human stem cell-derived cardiomyocytes for a more comprehensive risk assessment
340
Abstracts
models form an integral part of future cardiac safety assessment guidelines, it is important that hERG channel kinetics are accurately represented at both temperatures. We have previously presented sine wave voltage protocols designed to rapidly explore hERG channel kinetics (Mirams et al. (2015), Biophys. J.; 108(2):121a). We use these novel protocols to explore temperature-dependence of hERG channel kinetics. On individual hERG-1a transfected HEK 293 cells we have performed both voltage-step and sine wave protocols at both room and physiological temperatures. We use currents recorded in response to the sine wave protocols to construct cell-specific mathematical models describing hERG channel kinetics at both room and physiological temperatures. We validate the models using voltagestep protocols performed on the same cell and explore the differences in the resulting kinetic models. We will extend this approach to determine compound-specific models describing kinetics of drug interactions with the hERG channel, which may enhance the predictive ability of in silico approaches for cardiac safety assessment.
doi:10.1016/j.vascn.2016.02.017
0017 Optimization of optical imaging of cardiac tissue slices as a tool in safety pharmacology Christian Bollensdorff a,c, Ken Wangb, Razik Mu-u-mina,c, Abbirami Sathappana, Derek Terrarc, Magdi Yacouba,d a
Qatar Foundation, QCRC, Doha, Qatar Oxford University, Department for Computer Science, Oxford, UK c Oxford University, Department of Pharmacology, Oxford, UK d Imperial College London, Harefield Heart Science Center, London, UK b
In addition to atrial tissue, cardiac tissue slices are gaining consideration as an upcoming model to test compounds. This method contributes to the principles of the 3 Rs to reduce the burden on animal usage in research due to the possibility of harvesting several slices from a single heart. However, cardiac tissue slices are a more challenging preparation in terms of handling and treatment as compared to the whole hearts and require special attention in order to reproduce reliable results. For cardiac tissue slices, we were able to systemize a detailed guideline to handle slices in order to maintain high reproducibility. We have established an improved methodology, along with the use of different voltage and calcium sensitive dyes individually, as well as in combination, to deduce the most appropriate methods to optically map the electrophysiological properties of the slices. Important considerations include speed and angle of slicing, a recovery period of about 1 h after slicing, keeping the point of stimulation constant as it directly defines the action potential duration, tight control of temperature, the need to keep the size of the slices comparable. Also, the APD changes throughout the ventricular wall and should therefore be considered if comparisons between slices are necessary.The concurrent measurement of action potential and calcium transients gives advanced opportunities for the characterization of compounds. The use of optical imaging, multi-parametric measurement in particular, increases the information obtained per slice and improves the statistical power.
doi:10.1016/j.vascn.2016.02.018
0018 LPS-induced cytokine storm produces QTc prolongation which can be prevented by an anti-inflammatory eutectic blend Annie Boucharda, Peter Sordillob, Lawrence Helsonb, George Shoppc, Sabrina Baillargeona, Walter Shawd, Dany Salvaila a
IPS Therapeutique Inc., Sherbrooke, QC, Canada SignPath Pharma Inc., Quakertown, PA, USA c Shopp NonClinical Consulting LLC., Boulder, CO, USA d Avanti Polar Lipids Inc., Alabaster, AL, USA b
There is increasing evidence that excess levels of pro-inflammatory cytokines play a major role in the pathogenesis of the prolonged QT syndrome. Inversely, blockers such as tocilizumab (IL-6), or anticytokine antibodies (TNFα) contribute to a shortening of the previously-prolonged QT interval. In this study, LPS and Kdo2Lipid-A were used to induce cytokine release in guinea-pigs with concomitant ECG monitoring and blood draws, followed by Q-ELISA measurement of cytokine production. The guinea pig was selected because it yields reliable QTc prolongation as a result of proarrhythmic challenge, with consistently visible T-waves on the ECG. Male adult guinea pigs received 300 μg/kg LPS at time 0, and had ECGs analyzed at 1 h, 2 h, and 4 hours post-LPS, with simultaneous blood draw. Animals receiving LPS only exhibited a 8-msec increase in QTc after 1 h post-LPS, when TNFα levels were maximal at 5.5-fold the pre-LPS values. A 29-msec QTc prolongation 2 h post-LPS correlated with 7- and 9- fold increases in IL-1β and IL-6, respectively. The QTc prolongation remained (27 msec) after 4 hours post-LPS, when the animals were euthanized. When 9 mg/kg SP2020 (a lipid blend shown to prevent IKr-channel block by a variety of hERG blockers) was given 1 h prior to LPS-induction, QTc prolongation was limited to 5 ms after 2 h, and completely prevented at 1 and 4 hours post-LPS. Plasma levels of TNFα, IL1β, and IL-6 were significantly lower in EU8120-administered animals. It is suggested that SP2020 reduces QT prolongation through suppression of proinflammatory cytokines. doi:10.1016/j.vascn.2016.02.019
0019 Bridging functional and structural cardiotoxicity assays using human stem cell-derived cardiomyocytes for a more comprehensive risk assessment Mike Clements, Val Millar GE Healthcare, Buckinghamshire, UK Cardiotoxicity is a major cause of compound attrition in preclinical drug development. More relevant and reliable tests are required to improve drug safety and reduce the cost of drug development. One fundamental limitation of current in vitro cardiotoxicity tests is that none of the cell line and ex vivo models can be utilized as a single testing model across multiple analytical platforms to generate a cohesive data set to access drug cardiac liabilities. The introduction of assays utilizing human stem cellderived cardiomyocytes (hSC-CMs) could potentially address the shortcomings of these existing models. Here we have profiled 4 compounds with well characterized cardiac liabilities (Aspirin, Astemizole, Doxorubicin, and Sunitinib) in converging functional (multi-electrode array, MEA) and structural (high content image analysis, HCA) assays. Using multi-parameter phenotypic profiling techniques we reveal the dynamic relationship
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
Abstracts
models form an integral part of future cardiac safety assessment guidelines, it is important that hERG channel kinetics are accurately represented at both temperatures. We have previously presented sine wave voltage protocols designed to rapidly explore hERG channel kinetics (Mirams et al. (2015), Biophys. J.; 108(2):121a). We use these novel protocols to explore temperature-dependence of hERG channel kinetics. On individual hERG-1a transfected HEK 293 cells we have performed both voltage-step and sine wave protocols at both room and physiological temperatures. We use currents recorded in response to the sine wave protocols to construct cell-specific mathematical models describing hERG channel kinetics at both room and physiological temperatures. We validate the models using voltagestep protocols performed on the same cell and explore the differences in the resulting kinetic models. We will extend this approach to determine compound-specific models describing kinetics of drug interactions with the hERG channel, which may enhance the predictive ability of in silico approaches for cardiac safety assessment.
doi:10.1016/j.vascn.2016.02.017
0017 Optimization of optical imaging of cardiac tissue slices as a tool in safety pharmacology Christian Bollensdorff a,c, Ken Wangb, Razik Mu-u-mina,c, Abbirami Sathappana, Derek Terrarc, Magdi Yacouba,d a
Qatar Foundation, QCRC, Doha, Qatar Oxford University, Department for Computer Science, Oxford, UK c Oxford University, Department of Pharmacology, Oxford, UK d Imperial College London, Harefield Heart Science Center, London, UK b
In addition to atrial tissue, cardiac tissue slices are gaining consideration as an upcoming model to test compounds. This method contributes to the principles of the 3 Rs to reduce the burden on animal usage in research due to the possibility of harvesting several slices from a single heart. However, cardiac tissue slices are a more challenging preparation in terms of handling and treatment as compared to the whole hearts and require special attention in order to reproduce reliable results. For cardiac tissue slices, we were able to systemize a detailed guideline to handle slices in order to maintain high reproducibility. We have established an improved methodology, along with the use of different voltage and calcium sensitive dyes individually, as well as in combination, to deduce the most appropriate methods to optically map the electrophysiological properties of the slices. Important considerations include speed and angle of slicing, a recovery period of about 1 h after slicing, keeping the point of stimulation constant as it directly defines the action potential duration, tight control of temperature, the need to keep the size of the slices comparable. Also, the APD changes throughout the ventricular wall and should therefore be considered if comparisons between slices are necessary.The concurrent measurement of action potential and calcium transients gives advanced opportunities for the characterization of compounds. The use of optical imaging, multi-parametric measurement in particular, increases the information obtained per slice and improves the statistical power.
doi:10.1016/j.vascn.2016.02.018
0018 LPS-induced cytokine storm produces QTc prolongation which can be prevented by an anti-inflammatory eutectic blend Annie Boucharda, Peter Sordillob, Lawrence Helsonb, George Shoppc, Sabrina Baillargeona, Walter Shawd, Dany Salvaila a
IPS Therapeutique Inc., Sherbrooke, QC, Canada SignPath Pharma Inc., Quakertown, PA, USA c Shopp NonClinical Consulting LLC., Boulder, CO, USA d Avanti Polar Lipids Inc., Alabaster, AL, USA b
There is increasing evidence that excess levels of pro-inflammatory cytokines play a major role in the pathogenesis of the prolonged QT syndrome. Inversely, blockers such as tocilizumab (IL-6), or anticytokine antibodies (TNFα) contribute to a shortening of the previously-prolonged QT interval. In this study, LPS and Kdo2Lipid-A were used to induce cytokine release in guinea-pigs with concomitant ECG monitoring and blood draws, followed by Q-ELISA measurement of cytokine production. The guinea pig was selected because it yields reliable QTc prolongation as a result of proarrhythmic challenge, with consistently visible T-waves on the ECG. Male adult guinea pigs received 300 μg/kg LPS at time 0, and had ECGs analyzed at 1 h, 2 h, and 4 hours post-LPS, with simultaneous blood draw. Animals receiving LPS only exhibited a 8-msec increase in QTc after 1 h post-LPS, when TNFα levels were maximal at 5.5-fold the pre-LPS values. A 29-msec QTc prolongation 2 h post-LPS correlated with 7- and 9- fold increases in IL-1β and IL-6, respectively. The QTc prolongation remained (27 msec) after 4 hours post-LPS, when the animals were euthanized. When 9 mg/kg SP2020 (a lipid blend shown to prevent IKr-channel block by a variety of hERG blockers) was given 1 h prior to LPS-induction, QTc prolongation was limited to 5 ms after 2 h, and completely prevented at 1 and 4 hours post-LPS. Plasma levels of TNFα, IL1β, and IL-6 were significantly lower in EU8120-administered animals. It is suggested that SP2020 reduces QT prolongation through suppression of proinflammatory cytokines. doi:10.1016/j.vascn.2016.02.019
0019 Bridging functional and structural cardiotoxicity assays using human stem cell-derived cardiomyocytes for a more comprehensive risk assessment Mike Clements, Val Millar GE Healthcare, Buckinghamshire, UK Cardiotoxicity is a major cause of compound attrition in preclinical drug development. More relevant and reliable tests are required to improve drug safety and reduce the cost of drug development. One fundamental limitation of current in vitro cardiotoxicity tests is that none of the cell line and ex vivo models can be utilized as a single testing model across multiple analytical platforms to generate a cohesive data set to access drug cardiac liabilities. The introduction of assays utilizing human stem cellderived cardiomyocytes (hSC-CMs) could potentially address the shortcomings of these existing models. Here we have profiled 4 compounds with well characterized cardiac liabilities (Aspirin, Astemizole, Doxorubicin, and Sunitinib) in converging functional (multi-electrode array, MEA) and structural (high content image analysis, HCA) assays. Using multi-parameter phenotypic profiling techniques we reveal the dynamic relationship
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