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Simultaneous recording of action potentials and calcium transients from stem cell-derived cardiomyocytes: Applications for cardiotoxicity testing
Abstracts
e47
Effects of selective versus nonselective alpha adrenergic receptor blockade on cardiovascular parameters, body temperature and sympathetic tone in rats Siddhartha Bhatt, Laura Ringer, Todd Wisialowski, Jill Steidl-Nichols, Declan Flynn
studied. Compound A, at a dose of 80 mpk, induced a mild, delayed increase in BP and HR, and a mild decrease in locomotor activity in both WT and KO mice. These results indicate that compound A-induced cardiovascular liability is not target related and may be avoided with a different chemical structure. The mechanism responsible for the hemodynamic changes induced by compound A remains unclear.
Global Safety Pharmacology, Pfizer Worldwide R&D, Groton, CT, USA
doi:10.1016/j.vascn.2013.01.169
162
Doxazosin (selective alpha1) and phentolamine (nonselective alpha) cause hypotension and reflex tachycardia through alpha adrenergic receptor antagonism. However, the effects of these agents in vivo, including changes in body temperature (BT) and circulating catecholamine levels, may be relatively different. In this study, the effects of orally administered doxazosin (10 mg/kg) and phentolamine (20 and 100 mg/kg) on blood pressure (BP), heart rate (HR), and BT were assessed for 24 h post dose (hpd) in telemetered male Wistar rats. Norepinephrine levels were evaluated as an indicator of sympathetic tone, and plasma drug concentrations were measured. Doxazosin 10 mg/kg caused a sustained decrease in BP from 0 to 20 hpd; the maximum change in mean BP (−12.4 mm Hg) occurred 16–20 hpd and was accompanied by reflex tachycardia (65 bpm). Doxazosin induced ~3-fold increase in plasma norepinephrine levels and had no significant effects on BT. Phentolamine also decreased BP: maximum changes of −9.2 (20 mg/kg) and −29.1 (100 mg/kg) occurred 0–2 hpd with reflex tachycardia (71 and 111 bpm, respectively). BP reduction with phentolamine was of shorter duration, and tachycardia was of greater magnitude compared to doxazosin. Phentolamine 100 mg/kg also reduced BT (−1.26 °C, 2–4 hpd) and caused larger increases in plasma norepinephrine (~10 fold) compared to doxazosin. The increase in norepinephrine is suggestive of enhanced sympathetic tone. In conclusion, doxazosin and phentolamine produce relatively different effects on cardiovascular endpoints, BT, and plasma catecholamine levels; which may be due to blockade of presynaptic alpha2 receptors by phentolamine but not doxazosin. doi:10.1016/j.vascn.2013.01.168
163 Target related or off-target cardiovascular liabilities of receptor × agonists in telemetrized rats and knockout mice Julia Li, George Thalody, Oliver Flint, Paul Levesque, James Hennan Bristol-Myers Squibb, Pennington, NJ, USA Compound A, a selective receptor × agonist, has demonstrated efficacy in animals with type-2 diabetes; however, in vitro data shows that compound A induces an increase in glucagon-like peptide 1 (GLP-1) release that may be associated with increases in blood pressure (BP) and heart rate (HR) in rats. These studies were designed to determine 1) whether compound A induces hemodynamic changes following single oral doses administered in conscious telemetrized rats; 2) if compound A will induce changes in BP and HR after a single oral dose in conscious male receptor × wild type (WT) and knockout (KO) mice. Four groups were studied: vehicle, compound A at 80, 160, and 240 mg/kg. Compound A induced increases in SBP, HR and body temperature in rats. There were no compound related changes in DBP or locomotor activity. To further understand the cardiovascular effects and the role of the target and GLP-1, four groups of telemetrized receptor × wild type (WT) and knockout (KO) (vehicle and compound A 80 mg·kg) mice were
164 Simultaneous recording of action potentials and calcium transients from stem cell-derived cardiomyocytes: Applications for cardiotoxicity testing Ross Whittakera, Fabio Cerignolia, Randall Ingermansona, Rob Towartc, David J. Gallacherc, Mark Mercolab, Jeffrey Pricea,d a Vala Sciences Inc., San Diego, CA, USA b Muscle Development and Regeneration Program, Sanford-Burnham Medical Research Institute, La Jolla, CA, USA c Center of Excellence for Cardiovascular Safety Research and Mechanistic Pharmacology, Jassen Pharmaceutica NV, Beerse, Belgium d Quantitative Microscopy Laboratory, Sanford-Burnham Medical Research Institute, La Jolla, CA, USA Current methods for preclinical cardio-safety testing generally examine the effects of candidate compounds on the activity of single ion channels using manual or planar patch clamp methods. Limitations in these assays require that the tests be performed with cell lines which stably express the ion channel of interest. This reductionist approach grossly underestimates the complexity of cardiomyocyte excitability and physiology. We have developed a new automated image cytometer and associated software which facilitates a more physiologically relevant test of compound effects on cardiomyocyte excitation-contraction coupling. This new approach utilizes a dual channel automated Image cytometer that allows for simultaneous measurement of the cardiomyocyte action potential and calcium transient using voltage and calcium sensitive dyes. By using these advanced imaging techniques this system avoids the need for the assay apparatus to interact physically with the cells, allowing the use of a wide array of cell types including more clinically relevant models such as cardiomyocytes derived from human induced pluripotent stem cells (hIPSC). Here we demonstrate the application of this system to a small panel of known cardioactive compounds in hIPSC derived cardiomyocytes. Our results suggest that the ability to identify perturbations in the cardiomyocyte action potential and/or calcium transient due to exposure to cardioactive compounds is similar to existing technologies. However, this system offers a much higher throughput than existing systems and provides a more complete analysis of compound effects on excitation-contraction coupling in the cardiomyocyte. doi:10.1016/j.vascn.2013.01.170
165 Detection of complex mechanisms of drug action for vardenafil using stem cell-derived human cardiomyocytes Carlos Obejero-Paz, Andrew Bruening-Wright, Shengde Peng, Alina O'Connell, Zhixiong Lu, Weimin Pei, Tianen Yang, James Kramer, Yuri Kuryshev, Arthur Brown ChanTest Corporation, Cleveland, OH, USA
e47
Effects of selective versus nonselective alpha adrenergic receptor blockade on cardiovascular parameters, body temperature and sympathetic tone in rats Siddhartha Bhatt, Laura Ringer, Todd Wisialowski, Jill Steidl-Nichols, Declan Flynn
studied. Compound A, at a dose of 80 mpk, induced a mild, delayed increase in BP and HR, and a mild decrease in locomotor activity in both WT and KO mice. These results indicate that compound A-induced cardiovascular liability is not target related and may be avoided with a different chemical structure. The mechanism responsible for the hemodynamic changes induced by compound A remains unclear.
Global Safety Pharmacology, Pfizer Worldwide R&D, Groton, CT, USA
doi:10.1016/j.vascn.2013.01.169
162
Doxazosin (selective alpha1) and phentolamine (nonselective alpha) cause hypotension and reflex tachycardia through alpha adrenergic receptor antagonism. However, the effects of these agents in vivo, including changes in body temperature (BT) and circulating catecholamine levels, may be relatively different. In this study, the effects of orally administered doxazosin (10 mg/kg) and phentolamine (20 and 100 mg/kg) on blood pressure (BP), heart rate (HR), and BT were assessed for 24 h post dose (hpd) in telemetered male Wistar rats. Norepinephrine levels were evaluated as an indicator of sympathetic tone, and plasma drug concentrations were measured. Doxazosin 10 mg/kg caused a sustained decrease in BP from 0 to 20 hpd; the maximum change in mean BP (−12.4 mm Hg) occurred 16–20 hpd and was accompanied by reflex tachycardia (65 bpm). Doxazosin induced ~3-fold increase in plasma norepinephrine levels and had no significant effects on BT. Phentolamine also decreased BP: maximum changes of −9.2 (20 mg/kg) and −29.1 (100 mg/kg) occurred 0–2 hpd with reflex tachycardia (71 and 111 bpm, respectively). BP reduction with phentolamine was of shorter duration, and tachycardia was of greater magnitude compared to doxazosin. Phentolamine 100 mg/kg also reduced BT (−1.26 °C, 2–4 hpd) and caused larger increases in plasma norepinephrine (~10 fold) compared to doxazosin. The increase in norepinephrine is suggestive of enhanced sympathetic tone. In conclusion, doxazosin and phentolamine produce relatively different effects on cardiovascular endpoints, BT, and plasma catecholamine levels; which may be due to blockade of presynaptic alpha2 receptors by phentolamine but not doxazosin. doi:10.1016/j.vascn.2013.01.168
163 Target related or off-target cardiovascular liabilities of receptor × agonists in telemetrized rats and knockout mice Julia Li, George Thalody, Oliver Flint, Paul Levesque, James Hennan Bristol-Myers Squibb, Pennington, NJ, USA Compound A, a selective receptor × agonist, has demonstrated efficacy in animals with type-2 diabetes; however, in vitro data shows that compound A induces an increase in glucagon-like peptide 1 (GLP-1) release that may be associated with increases in blood pressure (BP) and heart rate (HR) in rats. These studies were designed to determine 1) whether compound A induces hemodynamic changes following single oral doses administered in conscious telemetrized rats; 2) if compound A will induce changes in BP and HR after a single oral dose in conscious male receptor × wild type (WT) and knockout (KO) mice. Four groups were studied: vehicle, compound A at 80, 160, and 240 mg/kg. Compound A induced increases in SBP, HR and body temperature in rats. There were no compound related changes in DBP or locomotor activity. To further understand the cardiovascular effects and the role of the target and GLP-1, four groups of telemetrized receptor × wild type (WT) and knockout (KO) (vehicle and compound A 80 mg·kg) mice were
164 Simultaneous recording of action potentials and calcium transients from stem cell-derived cardiomyocytes: Applications for cardiotoxicity testing Ross Whittakera, Fabio Cerignolia, Randall Ingermansona, Rob Towartc, David J. Gallacherc, Mark Mercolab, Jeffrey Pricea,d a Vala Sciences Inc., San Diego, CA, USA b Muscle Development and Regeneration Program, Sanford-Burnham Medical Research Institute, La Jolla, CA, USA c Center of Excellence for Cardiovascular Safety Research and Mechanistic Pharmacology, Jassen Pharmaceutica NV, Beerse, Belgium d Quantitative Microscopy Laboratory, Sanford-Burnham Medical Research Institute, La Jolla, CA, USA Current methods for preclinical cardio-safety testing generally examine the effects of candidate compounds on the activity of single ion channels using manual or planar patch clamp methods. Limitations in these assays require that the tests be performed with cell lines which stably express the ion channel of interest. This reductionist approach grossly underestimates the complexity of cardiomyocyte excitability and physiology. We have developed a new automated image cytometer and associated software which facilitates a more physiologically relevant test of compound effects on cardiomyocyte excitation-contraction coupling. This new approach utilizes a dual channel automated Image cytometer that allows for simultaneous measurement of the cardiomyocyte action potential and calcium transient using voltage and calcium sensitive dyes. By using these advanced imaging techniques this system avoids the need for the assay apparatus to interact physically with the cells, allowing the use of a wide array of cell types including more clinically relevant models such as cardiomyocytes derived from human induced pluripotent stem cells (hIPSC). Here we demonstrate the application of this system to a small panel of known cardioactive compounds in hIPSC derived cardiomyocytes. Our results suggest that the ability to identify perturbations in the cardiomyocyte action potential and/or calcium transient due to exposure to cardioactive compounds is similar to existing technologies. However, this system offers a much higher throughput than existing systems and provides a more complete analysis of compound effects on excitation-contraction coupling in the cardiomyocyte. doi:10.1016/j.vascn.2013.01.170
165 Detection of complex mechanisms of drug action for vardenafil using stem cell-derived human cardiomyocytes Carlos Obejero-Paz, Andrew Bruening-Wright, Shengde Peng, Alina O'Connell, Zhixiong Lu, Weimin Pei, Tianen Yang, James Kramer, Yuri Kuryshev, Arthur Brown ChanTest Corporation, Cleveland, OH, USA