MATHEMATICAL MODELLING OF ACTION POTENTIAL IN EMBRYONIC STEM CELL-DERIVED CARDIOMYOCYTES INTRODUCTION Human embryonic stem cell-derived cardiomyocytes (hES-CM) represent a promising tool for cell therapy and drug screening. Identification of their in vitro functional properties is mandatory to envisage appropriate cardiac cell-based therapies. To this purpose an appropriate experimental approach is represented by different electrophysiological and molecular techniques. A challenging, innovative approach is to use suitable modelling to simulate electrophysiological properties of hES-CM and their response to pharmacological tools. METHODS We characterized hES-CM action potentials (AP) at two developmental stages (Early 15-40 days, Late, 50-110 days) with a combination of single cell patch-clamp for currents characterization, multicellular AP recordings, RT-PCR to assess the expression of different currents [1] and modelling tools. These experiments led to the characterization of transient outward current Ito, delayed rectifier current IKr, f-current If, inward rectifier IK1 and L-type current ICaL. Experimental data were integrated into a modified version of Ten Tusscher model of human adult cardiomyocyte [2], tuning current parameters to match the experimentally assessed stage-dependent current modifications and integrating the hyperpolarization-activated If, not present in adult ventricular cardiomyocytes. If was incorporated following a Hodking-Huxley formulation with a single activation gate. Data about the other ionic currents were derived from [3-6]. This led to a model showing a spontaneous beating activity. Effects of 3 current blockers were assessed, in particular E4031 for IKr, Zatebradine (Zat) for If and Barium (Ba++) for IK1. These simulations were performed by reducing to 25% the maximal conductance for IKr, If and IK1 respectively. AP registrations were not performed on single cells but on embryoid bodies, aggregates containing different cell phenotypes among which hES-CM and fibroblasts. Therefore, an additional human fibroblast model, to couple resistively to the hES-CM, was developed in order to evaluate its effects on hES-CM AP. RESULTS AND DISCUSSION The simulated APs mimic satisfactorily the recorded ones at both developmental stages. Several AP features were calculated. Changes in AP occur during in-vitro maturation (Early vs. Late): AP duration (APD) and amplitude increase, diastolic depolarization rate (DDR) and rate of spontaneous beating (F) decrease. Our model well reproduces the experimentally observed changes in AP profile in terms of APD, DDR and F. Simulations of current blockers show that our model well reproduces the effects of E4031 (reduced F and increased APD), of Zat (reduced F and DDR). Ba++ shows a small DDR increase but F increases as well as side effect. The most important effects of coupling with one fibroblast are an increase of DDR (+4 mV/s) and F (+3 bpm) and a small reduction of the AP peak (-0.4 mV). These results suggest that our novel mathematical model can serve as a predictive approach to interpret and refine in vitro experiments on hES-CM. REFERENCES [1] Sartiani L et al.,Stem Cells, 2007, 25(5): 1136-1144. [2] Grandi E et al., J Mol Cell Cardiol, 2009, 46(3): 332-342. [3] Satin J et al., J Physiol, 2004, 559: 479-496. [4] Itoh H et al., Syst Synth Biol, 2007, 1: 11-23. [5] Qu Y et al., Cardiovasc Res, 2000, 45(4): 866-73. [6] Otsu K et al., Cell Calcium, 2005, 37(2): 137-51.

Mathematical modelling of action potential in embryonic stem cell-derived cardiomyocytes.

SEVERI, STEFANO;PACI, MICHELANGELO;
2010

Abstract

MATHEMATICAL MODELLING OF ACTION POTENTIAL IN EMBRYONIC STEM CELL-DERIVED CARDIOMYOCYTES INTRODUCTION Human embryonic stem cell-derived cardiomyocytes (hES-CM) represent a promising tool for cell therapy and drug screening. Identification of their in vitro functional properties is mandatory to envisage appropriate cardiac cell-based therapies. To this purpose an appropriate experimental approach is represented by different electrophysiological and molecular techniques. A challenging, innovative approach is to use suitable modelling to simulate electrophysiological properties of hES-CM and their response to pharmacological tools. METHODS We characterized hES-CM action potentials (AP) at two developmental stages (Early 15-40 days, Late, 50-110 days) with a combination of single cell patch-clamp for currents characterization, multicellular AP recordings, RT-PCR to assess the expression of different currents [1] and modelling tools. These experiments led to the characterization of transient outward current Ito, delayed rectifier current IKr, f-current If, inward rectifier IK1 and L-type current ICaL. Experimental data were integrated into a modified version of Ten Tusscher model of human adult cardiomyocyte [2], tuning current parameters to match the experimentally assessed stage-dependent current modifications and integrating the hyperpolarization-activated If, not present in adult ventricular cardiomyocytes. If was incorporated following a Hodking-Huxley formulation with a single activation gate. Data about the other ionic currents were derived from [3-6]. This led to a model showing a spontaneous beating activity. Effects of 3 current blockers were assessed, in particular E4031 for IKr, Zatebradine (Zat) for If and Barium (Ba++) for IK1. These simulations were performed by reducing to 25% the maximal conductance for IKr, If and IK1 respectively. AP registrations were not performed on single cells but on embryoid bodies, aggregates containing different cell phenotypes among which hES-CM and fibroblasts. Therefore, an additional human fibroblast model, to couple resistively to the hES-CM, was developed in order to evaluate its effects on hES-CM AP. RESULTS AND DISCUSSION The simulated APs mimic satisfactorily the recorded ones at both developmental stages. Several AP features were calculated. Changes in AP occur during in-vitro maturation (Early vs. Late): AP duration (APD) and amplitude increase, diastolic depolarization rate (DDR) and rate of spontaneous beating (F) decrease. Our model well reproduces the experimentally observed changes in AP profile in terms of APD, DDR and F. Simulations of current blockers show that our model well reproduces the effects of E4031 (reduced F and increased APD), of Zat (reduced F and DDR). Ba++ shows a small DDR increase but F increases as well as side effect. The most important effects of coupling with one fibroblast are an increase of DDR (+4 mV/s) and F (+3 bpm) and a small reduction of the AP peak (-0.4 mV). These results suggest that our novel mathematical model can serve as a predictive approach to interpret and refine in vitro experiments on hES-CM. REFERENCES [1] Sartiani L et al.,Stem Cells, 2007, 25(5): 1136-1144. [2] Grandi E et al., J Mol Cell Cardiol, 2009, 46(3): 332-342. [3] Satin J et al., J Physiol, 2004, 559: 479-496. [4] Itoh H et al., Syst Synth Biol, 2007, 1: 11-23. [5] Qu Y et al., Cardiovasc Res, 2000, 45(4): 866-73. [6] Otsu K et al., Cell Calcium, 2005, 37(2): 137-51.
2010
A. CAPPELLO, T. D’ALESSIO, M. KNAFLITZ E F.M. MONTEVECCHI
151
152
S. Severi; M. Paci; L. Sartiani; M.E. Jaconi; A. Mugelli ; E. Cerbai
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/11585/100574
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