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Microelectrode arrays: A new tool to measure embryonic heart activity
Journal of Electrocardiology Vol. 37 Supplement 2004
Microelectrode Arrays: A New Tool to Measure Embryonic Heart Activity Michael Reppel, MD,* Frank Pillekamp, MD,† Zhong Ju Lu,* Marcel Halbach,* Konrad Brockmeier, MD,† Bernd K. Fleischmann, MD,* and Juergen Hescheler, MD*
Abstract: The analysis of the sequential excitation of cardiac tissue is of high relevance, both for clinical pathophysiological purposes, eg, detection of sustained ventricular arrhythmias, as well as for experimental electrophysiology. Clinically, different technical approaches such as single electrode measurements and bipolar mapping electrode catheters have been used. In experimental setups several techniques to record cardiac activity have been proposed. Beside the well-established intracellular current-clamp recordings of action potentials, recent studies have performed extracellularly activation sequence mapping or simultaneous multichannel action potential electrode array measurements. Measurement of extracellularly recorded field potentials (FPs) hereby especially provides detailed information about the origin and spread of excitation in the heart. A similar analytical approach for cardiac FPs advanced the analysis of excitation spread and arrhythmic activity in multicellular preparations like developmental differentiation tissue of mouse embryonic stem cells, multicellular preparations of isolated native embryonic cardiomyocytes or the embryonic heart in toto. The use of substrate-integrated Microelectrode Arrays (MEAs, Multi Channel Systems, Reutlingen, Germany) with 60 electrodes of 10 –30 m diameters on a 100 –200 m grid, coated with porous titanium nitride to minimize the impedance allows recording of FPs at a high signal to noise ratio. The possibility to electrically stimulate the tissue further expands the range of applications and bioassays. It may thus facilitate the evaluation of drug research providing detailed information about the interplay of the complex cardiac network, and might improve the predictability of physiological and pathophysiological conditions or drug effects in embryonic heart tissue. Key words: Electrophysiology, embryonic heart, MEA, cardiac mapping.
Abnormalities of the heartbeat belong to the most frequent causes of mortality and morbidity in hu-
mans, often occurring in a setting of end-stage heart failure (1). The understanding of the basic mechanisms underlying pathophysiological conditions has grown rapidly in the course of the last century. In 1952, Hodgkin and Huxley were the first who discovered the ionic basis of electrical activity in nerve and muscle (2). The introduction of the patch-clamp method by Neher and Sakmann was an additional step towards the understanding of the molecular mechanism of cardiac action potential
*From the Institute of Neurophysiology and †Pedriatic Cardiology of the University of Cologne, Germany. Reprint requests: J. Hescheler, MD, Institute of Neurophysiology, University of Cologne, Robert-Kochstr. 39, D-50931-Cologne; e-mail: [email protected] © 2004 Elsevier Inc. All rights reserved. 0022-0736/04/370S-0033$30.00/0 doi:10.1016/j.jelectrocard.2004.08.033
104
MEAs and Embryonic Cardiac Mapping •
Reppel et al. 105
Fig. 1. MEA system, comparison of extra- and intracellular recordings, nomenclature. (A) 8x8 layout electrode grid (interelectrode distance: 200 m, electrode diameter: 30 m) embedded in a (B) substrate-integrated MEA culture dish. (C) MEA amplifier (B and C were kindly provided by Multichannelsystems, Reutlingen, Germany). (D) Simultaneous measurements of FPs (upper panel) and APs recorded from multicellular preparations of spontaneously active isolated embryonic mouse cardiomyocytes. A close correlation of the depolarization and repolarizing phase of the APs with FPMin and FPMax was characteristic for all recordings. (E) Standardarized FP nomenclature according to Halbach et al. (6). Interspike intervals (ISI, time between individual FPMin.) are calculated to determine beating frequency.
(AP) generation and its propagation in heart (3). Since then it was possible to separate ion channel and transporter related contribution to the AP. However, three major disadvantages of AP measurements exist in parallel: 1) Intracellular measurement of APs is limited to single-cell activities, 2) Manipulation of the cytosolic ionic compound mea-
surements may not reflect in-vivo conditions, and 3) Long-term measurements are difficult to handle although they are essentially needed for drug response testing. The complex interactions among the many molecular events that determine the functional and structural cardiac phenotype exclude a simple and linear interpretation of phenomena ob-
106 Journal of Electrocardiology Vol. 37 Supplement 2004
Fig. 2.
MEAs and Embryonic Cardiac Mapping •
served at the tissue and whole heart level in terms of transsarcolemmal processes. Since relatively small perturbations in elementary processes can have a major effect on the overall system behavior, it is particularly important to integrate results obtained from single cell systems into more complex models that take the integrated-system complexities into account. For example, mapping of extracellular FPs in cardiac tissue is used to localize the pacemaker region and characterize the excitation spread out of this area (4,5). Although these studies provide a well defined basis for the fundamental understanding of cardiac physiology, they were performed mainly using adult heart tissue. In experimental setups MEAs with 60 electrodes integrated two-dimensionally in a culture dish (Figs 1A and B) allow to perform long-term observations also in embryonic and neonatal cardiac preparations by multi-site extracellular recordings of FPs (6 – 8). This overview article focuses on the technical properties of extracellular recordings of cardiac preparations using microelectrode recordings. It attempts to review recent technical applications and experimental observations, in an effort to compare field potential measurements to transmembrane potentials and to describe basic characteristics of field potentials in working myocardium and pacemaker areas.
Comparison Between Intracellular and Extracellular Current Recordings, Characterization, and Nomenclature of FP Parameters in Embryonic Heart Tissue
Reppel et al. 107
isolated hearts of dogs and rabbits (9). They found using a simultaneously approach of intra- and extracellular measurements a strong and consistent correlation between the time of the upstroke of the intracellular AP and the fast downstroke of the extracellular wave form. In 1977, Cramer et al. identified low-frequency extracellular potential changes associated with electrical activity of the sinoatrial pacemaker of the rabbit (4). These potential changes consist of a slow diastolic and a more rapid upstroke slope that correspond to phase 4 and phase 0 depolarization of dominant pacemaker fibres of the sinus node. Based on these observations, the technique for recording extracellular field potential electrocardiograms was applied in human beings during cardiac surgery and cardiac catheterization to allow direct assessment of the sinus node function (10). In a study by Haberl et al. extracellular and intracellular potentials were recorded simultaneously from multiple sites of the sinus node in rabbits to determine whether extracellular recordings can accurately determine sinoatrial conduction time under clinical conditions (11). A study by Halbach et al. using MEAs demonstrated not only that the FPs recorded from multicellular preparations of spontaneously active isolated embryonic mouse cardiomyocytes contain information about the AP rise time, AP duration and individual transmembrane currents, but also provided detailed information about a standardarized nomenclature of typical extracellular field potential recordings (Fig. 1E) (6). Although the interpretation of the FP components does not reveal absolute values for AP properties, it can assist in the analysis of the contribution of the different electrophysiological parameters and their modulation in association with physiological and pathophysiological changes of excitation generation and excitation spread in multicellular preparations of cardiac myocytes.
In 1971, Spach et al. investigated extracellular potentials in the atrial septum and AV nodes in
Š Fig. 2. Measurement of excitation in embryonic cardiac tissue, pacemaker localization. Plating embryonic stem cell (ES)-derived cardiomyocytes or embryonic hearts in toto on MEAs, the system allows determining spontaneous electrical activity (A, C, respectively). (B) FPs of ES cell-derived cardiomyocytes or (D) embryonic hearts at higher resolution. At ⫽ atrial FP, V ⫽ ventricular FP. (E) Early onset of typical slow descending primary negativation (2) indicating pacemaking area. Delay comparing FPMin of individual electrodes, expressed as FP delay, characterizes conduction velocities. # indicates individual electrodes.
Determination of the Pacemaking Area An example of one of the first extracellular excitation maps of the cardiac pacemaker was provided by Sir Thomas Lewis in 1915 (12). He noted that at the site of the earliest activation the unipolar electrocardiogram showed a characteristic morphology termed “primary negativity.” This was defined as the electrocardiogram that showed an initial negative deflection before the onset of the P wave. Using a bipolar electrode Rijlant observed similar prepotentials prior to the onset of the P
108 Journal of Electrocardiology Vol. 37 Supplement 2004 wave, in the region of the sinus node (13). Van der Kooi et al. noted that the region of primary negativity was located in a very small region within the sinus node (14). A study by Yamamoto et al. provided information about extracellular potentials measured by the use of bipolar electrodes (15). Near the leading pacemaker a slow primary negative deflection (25–30 ms in duration) was preceded by a gradual increase of the negativity. In our experiments using embryonic mouse heart tissue, prepotentials around the leading pacemaker site showed a slow primary negative deflection of comparable duration (⬇30ms) which was followed by a substantial subsequent increase of negativity (Fig. 2E). As has also been reviewed by Schuessler and coworkers for experimental setups using adult heart preparations (16), the early onset of the primary negative deflection correlated in our MEA setup with the early onset of secondary negative deflection pointing to the pacemaking area in the embryonic heart. Thus, the concept of primary negativity can generally be used to define the point of impulse initiation.
niques. Even though biophysical properties and ionic channel regulation are dramatically changing during development and differentiation and arrhythmias are of major importance also in the neonatal and embryonic heart (8), due to the small size of the embryonic heart and the lack of appropriate techniques, the mechanisms of excitation, properties of conduction, involvement of different ion channels and cell-cell interplay of the early heart remained elusive so far. ES cell-derived cardiomyocytes, cellular monolayers of native embryonic cardiomyocytes (6) and whole embryonic hearts in toto serve as an ideal model of early heart development at different stages of development. For example, whole heart measurements improve electrophysiological characterisation of transgenic mice as has been shown by Lu et al. (8). The knowledge of electrical cardiac activity in the embryonic heart could be used in several fields that are in the forefront of science such as developmental biology, functional genomics, pharmacological testing, gene and cell-based therapies. Summarizing, MEAs serve as an ideal standardarized tool to measure excitation and propagation in excitable tissue, eg, the embryonic heart.
Potential of Micro-Electrode Recordings in Heart References Electrode recordings in heart tissue were mainly performed in adult isolated hearts or cardiac cell monolayers. Using multiple micro-electrodes Sano and Yamagishi were the first to map systematically the spread of excitation within the sinus node and into the surrounding atrial myocardium (17). They showed asymmetric and slow velocity in the sinus node and a clearly increased propagation velocity after reaching atrial tissue. Bleeker et al. performed the most comprehensive mapping study of the rabbit sinus node (18). They suggested that the dominant pacemaker area was composed of approximately 5,000 cells covering an area of 0.1mm2 without clear indication of specific pathways out of the node. However, long-term measurements were impossible to perform. Substrate integrated MEAs serve as a novel tool to circumvent these challenges. High-density extracellular mapping using culture dishes with integrated TiN/SiN electrodes (Figs. 1A and B) and electrical stimulation are possible to perform simultaneously. The ability to record long-term electrophysiological information with extremely high spatio-temporal resolution and the correlation of this information with single cell and morphological data represents a paradigm shift from conventional tech-
1. Murray CJ, Lopez AD: Alternative projections of mortality and disability by cause 1990 –2020: Global Burden of Disease Study. Lancet 349:1498, 1997 2. Hodgkin AL, Huxley AF: A quantitative description of membrane current and its application to conduction and excitation in nerve. J Physiol 117:500, 1952 3. Neher E, Sakmann B: Single-channel currents recorded from membrane of denervated frog muscle fibres. Nature 260:799, 1976 4. Cramer M, Siegal M, Bigger JT Jr, et al: Characteristics of extracellular potentials recorded from the sinoatrial pacemaker of the rabbit. Circ Res 41:292, 1977 5. Tweddell JS, Branham BH, Harada A, et al: Potential mapping in septal tachycardia: Evaluation of a new intraoperative mapping technique. Circulation 80: I97, 1989 6. Halbach M, Egert U, Hescheler J, et al: Estimation of action potential changes from field potential recordings in multicellular mouse cardiac myocyte cultures. Cell Physiol Biochem 13:271, 2003 7. Banach K, Halbach MD, Hu P, et al: Development of electrical activity in cardiac myocyte aggregates derived from mouse embryonic stem cells. Am J Physiol Heart Circ Physiol 284:H2114, 2003 8. Lu ZJ, et al: Arrhythmia in isolated prenatal hearts after ablation of the Cav2.3 (alpha1E) subunit of
MEAs and Embryonic Cardiac Mapping •
9.
10.
11.
12.
voltage-gated Ca2⫹ channels. Cell Physiol Biochem 14:11, 2004 Spach MS, Barr RC, Serwer GS, et al: Collision of excitation waves in the dog Purkinje system: Extracellular identification. Circ Res 29:499, 1971 Hariman RJ, Krongrad E, Boxer RA, et al: Methods for recording electrograms of the sinoatrial node during cardiac surgery in man. Circulation 61:1024, 1980 Haberl R, Steinbeck G, Luderitz B: Comparison between intracellular and extracellular direct current recordings of sinus node activity for evaluation of sinoatrial conduction time. Circulation 70: 760, 1984 Lewis T: Lectures on the heart, New York, NY. Paul B. Hoeber, 1915, p 1
Reppel et al. 109
13. Rijlant R: La conduction dans le coeur du mammife`re. Arch Internat Physiol 1931; 179 14. Van Der Kooi MW, Durrer D, Van Dam RT, et al: Electrical activity in sinus node and atrioventricular node. Am Heart J 51:684, 1956 15. Yamamoto M, Honjo H, Niwa R, et al: Low-frequency extracellular potentials recorded from the sinoatrial node. Cardiovasc Res 39:360, 1998 16. Schuessler RB, Boineau JP, Bromberg BI: Origin of the sinus impulse. J Cardiovasc Electrophysiol 7:263, 1996 17. Sano T, Yamagishi S: Spread of excitation from the sinus node. Circ Res 16:423, 1965 18. Bleeker WK, Mackaay AJ, Masson-Pevet M, et al: Functional and morphological organization of the rabbit sinus node. Circ Res 46:11, 1980
Microelectrode Arrays: A New Tool to Measure Embryonic Heart Activity Michael Reppel, MD,* Frank Pillekamp, MD,† Zhong Ju Lu,* Marcel Halbach,* Konrad Brockmeier, MD,† Bernd K. Fleischmann, MD,* and Juergen Hescheler, MD*
Abstract: The analysis of the sequential excitation of cardiac tissue is of high relevance, both for clinical pathophysiological purposes, eg, detection of sustained ventricular arrhythmias, as well as for experimental electrophysiology. Clinically, different technical approaches such as single electrode measurements and bipolar mapping electrode catheters have been used. In experimental setups several techniques to record cardiac activity have been proposed. Beside the well-established intracellular current-clamp recordings of action potentials, recent studies have performed extracellularly activation sequence mapping or simultaneous multichannel action potential electrode array measurements. Measurement of extracellularly recorded field potentials (FPs) hereby especially provides detailed information about the origin and spread of excitation in the heart. A similar analytical approach for cardiac FPs advanced the analysis of excitation spread and arrhythmic activity in multicellular preparations like developmental differentiation tissue of mouse embryonic stem cells, multicellular preparations of isolated native embryonic cardiomyocytes or the embryonic heart in toto. The use of substrate-integrated Microelectrode Arrays (MEAs, Multi Channel Systems, Reutlingen, Germany) with 60 electrodes of 10 –30 m diameters on a 100 –200 m grid, coated with porous titanium nitride to minimize the impedance allows recording of FPs at a high signal to noise ratio. The possibility to electrically stimulate the tissue further expands the range of applications and bioassays. It may thus facilitate the evaluation of drug research providing detailed information about the interplay of the complex cardiac network, and might improve the predictability of physiological and pathophysiological conditions or drug effects in embryonic heart tissue. Key words: Electrophysiology, embryonic heart, MEA, cardiac mapping.
Abnormalities of the heartbeat belong to the most frequent causes of mortality and morbidity in hu-
mans, often occurring in a setting of end-stage heart failure (1). The understanding of the basic mechanisms underlying pathophysiological conditions has grown rapidly in the course of the last century. In 1952, Hodgkin and Huxley were the first who discovered the ionic basis of electrical activity in nerve and muscle (2). The introduction of the patch-clamp method by Neher and Sakmann was an additional step towards the understanding of the molecular mechanism of cardiac action potential
*From the Institute of Neurophysiology and †Pedriatic Cardiology of the University of Cologne, Germany. Reprint requests: J. Hescheler, MD, Institute of Neurophysiology, University of Cologne, Robert-Kochstr. 39, D-50931-Cologne; e-mail: [email protected] © 2004 Elsevier Inc. All rights reserved. 0022-0736/04/370S-0033$30.00/0 doi:10.1016/j.jelectrocard.2004.08.033
104
MEAs and Embryonic Cardiac Mapping •
Reppel et al. 105
Fig. 1. MEA system, comparison of extra- and intracellular recordings, nomenclature. (A) 8x8 layout electrode grid (interelectrode distance: 200 m, electrode diameter: 30 m) embedded in a (B) substrate-integrated MEA culture dish. (C) MEA amplifier (B and C were kindly provided by Multichannelsystems, Reutlingen, Germany). (D) Simultaneous measurements of FPs (upper panel) and APs recorded from multicellular preparations of spontaneously active isolated embryonic mouse cardiomyocytes. A close correlation of the depolarization and repolarizing phase of the APs with FPMin and FPMax was characteristic for all recordings. (E) Standardarized FP nomenclature according to Halbach et al. (6). Interspike intervals (ISI, time between individual FPMin.) are calculated to determine beating frequency.
(AP) generation and its propagation in heart (3). Since then it was possible to separate ion channel and transporter related contribution to the AP. However, three major disadvantages of AP measurements exist in parallel: 1) Intracellular measurement of APs is limited to single-cell activities, 2) Manipulation of the cytosolic ionic compound mea-
surements may not reflect in-vivo conditions, and 3) Long-term measurements are difficult to handle although they are essentially needed for drug response testing. The complex interactions among the many molecular events that determine the functional and structural cardiac phenotype exclude a simple and linear interpretation of phenomena ob-
106 Journal of Electrocardiology Vol. 37 Supplement 2004
Fig. 2.
MEAs and Embryonic Cardiac Mapping •
served at the tissue and whole heart level in terms of transsarcolemmal processes. Since relatively small perturbations in elementary processes can have a major effect on the overall system behavior, it is particularly important to integrate results obtained from single cell systems into more complex models that take the integrated-system complexities into account. For example, mapping of extracellular FPs in cardiac tissue is used to localize the pacemaker region and characterize the excitation spread out of this area (4,5). Although these studies provide a well defined basis for the fundamental understanding of cardiac physiology, they were performed mainly using adult heart tissue. In experimental setups MEAs with 60 electrodes integrated two-dimensionally in a culture dish (Figs 1A and B) allow to perform long-term observations also in embryonic and neonatal cardiac preparations by multi-site extracellular recordings of FPs (6 – 8). This overview article focuses on the technical properties of extracellular recordings of cardiac preparations using microelectrode recordings. It attempts to review recent technical applications and experimental observations, in an effort to compare field potential measurements to transmembrane potentials and to describe basic characteristics of field potentials in working myocardium and pacemaker areas.
Comparison Between Intracellular and Extracellular Current Recordings, Characterization, and Nomenclature of FP Parameters in Embryonic Heart Tissue
Reppel et al. 107
isolated hearts of dogs and rabbits (9). They found using a simultaneously approach of intra- and extracellular measurements a strong and consistent correlation between the time of the upstroke of the intracellular AP and the fast downstroke of the extracellular wave form. In 1977, Cramer et al. identified low-frequency extracellular potential changes associated with electrical activity of the sinoatrial pacemaker of the rabbit (4). These potential changes consist of a slow diastolic and a more rapid upstroke slope that correspond to phase 4 and phase 0 depolarization of dominant pacemaker fibres of the sinus node. Based on these observations, the technique for recording extracellular field potential electrocardiograms was applied in human beings during cardiac surgery and cardiac catheterization to allow direct assessment of the sinus node function (10). In a study by Haberl et al. extracellular and intracellular potentials were recorded simultaneously from multiple sites of the sinus node in rabbits to determine whether extracellular recordings can accurately determine sinoatrial conduction time under clinical conditions (11). A study by Halbach et al. using MEAs demonstrated not only that the FPs recorded from multicellular preparations of spontaneously active isolated embryonic mouse cardiomyocytes contain information about the AP rise time, AP duration and individual transmembrane currents, but also provided detailed information about a standardarized nomenclature of typical extracellular field potential recordings (Fig. 1E) (6). Although the interpretation of the FP components does not reveal absolute values for AP properties, it can assist in the analysis of the contribution of the different electrophysiological parameters and their modulation in association with physiological and pathophysiological changes of excitation generation and excitation spread in multicellular preparations of cardiac myocytes.
In 1971, Spach et al. investigated extracellular potentials in the atrial septum and AV nodes in
Š Fig. 2. Measurement of excitation in embryonic cardiac tissue, pacemaker localization. Plating embryonic stem cell (ES)-derived cardiomyocytes or embryonic hearts in toto on MEAs, the system allows determining spontaneous electrical activity (A, C, respectively). (B) FPs of ES cell-derived cardiomyocytes or (D) embryonic hearts at higher resolution. At ⫽ atrial FP, V ⫽ ventricular FP. (E) Early onset of typical slow descending primary negativation (2) indicating pacemaking area. Delay comparing FPMin of individual electrodes, expressed as FP delay, characterizes conduction velocities. # indicates individual electrodes.
Determination of the Pacemaking Area An example of one of the first extracellular excitation maps of the cardiac pacemaker was provided by Sir Thomas Lewis in 1915 (12). He noted that at the site of the earliest activation the unipolar electrocardiogram showed a characteristic morphology termed “primary negativity.” This was defined as the electrocardiogram that showed an initial negative deflection before the onset of the P wave. Using a bipolar electrode Rijlant observed similar prepotentials prior to the onset of the P
108 Journal of Electrocardiology Vol. 37 Supplement 2004 wave, in the region of the sinus node (13). Van der Kooi et al. noted that the region of primary negativity was located in a very small region within the sinus node (14). A study by Yamamoto et al. provided information about extracellular potentials measured by the use of bipolar electrodes (15). Near the leading pacemaker a slow primary negative deflection (25–30 ms in duration) was preceded by a gradual increase of the negativity. In our experiments using embryonic mouse heart tissue, prepotentials around the leading pacemaker site showed a slow primary negative deflection of comparable duration (⬇30ms) which was followed by a substantial subsequent increase of negativity (Fig. 2E). As has also been reviewed by Schuessler and coworkers for experimental setups using adult heart preparations (16), the early onset of the primary negative deflection correlated in our MEA setup with the early onset of secondary negative deflection pointing to the pacemaking area in the embryonic heart. Thus, the concept of primary negativity can generally be used to define the point of impulse initiation.
niques. Even though biophysical properties and ionic channel regulation are dramatically changing during development and differentiation and arrhythmias are of major importance also in the neonatal and embryonic heart (8), due to the small size of the embryonic heart and the lack of appropriate techniques, the mechanisms of excitation, properties of conduction, involvement of different ion channels and cell-cell interplay of the early heart remained elusive so far. ES cell-derived cardiomyocytes, cellular monolayers of native embryonic cardiomyocytes (6) and whole embryonic hearts in toto serve as an ideal model of early heart development at different stages of development. For example, whole heart measurements improve electrophysiological characterisation of transgenic mice as has been shown by Lu et al. (8). The knowledge of electrical cardiac activity in the embryonic heart could be used in several fields that are in the forefront of science such as developmental biology, functional genomics, pharmacological testing, gene and cell-based therapies. Summarizing, MEAs serve as an ideal standardarized tool to measure excitation and propagation in excitable tissue, eg, the embryonic heart.
Potential of Micro-Electrode Recordings in Heart References Electrode recordings in heart tissue were mainly performed in adult isolated hearts or cardiac cell monolayers. Using multiple micro-electrodes Sano and Yamagishi were the first to map systematically the spread of excitation within the sinus node and into the surrounding atrial myocardium (17). They showed asymmetric and slow velocity in the sinus node and a clearly increased propagation velocity after reaching atrial tissue. Bleeker et al. performed the most comprehensive mapping study of the rabbit sinus node (18). They suggested that the dominant pacemaker area was composed of approximately 5,000 cells covering an area of 0.1mm2 without clear indication of specific pathways out of the node. However, long-term measurements were impossible to perform. Substrate integrated MEAs serve as a novel tool to circumvent these challenges. High-density extracellular mapping using culture dishes with integrated TiN/SiN electrodes (Figs. 1A and B) and electrical stimulation are possible to perform simultaneously. The ability to record long-term electrophysiological information with extremely high spatio-temporal resolution and the correlation of this information with single cell and morphological data represents a paradigm shift from conventional tech-
1. Murray CJ, Lopez AD: Alternative projections of mortality and disability by cause 1990 –2020: Global Burden of Disease Study. Lancet 349:1498, 1997 2. Hodgkin AL, Huxley AF: A quantitative description of membrane current and its application to conduction and excitation in nerve. J Physiol 117:500, 1952 3. Neher E, Sakmann B: Single-channel currents recorded from membrane of denervated frog muscle fibres. Nature 260:799, 1976 4. Cramer M, Siegal M, Bigger JT Jr, et al: Characteristics of extracellular potentials recorded from the sinoatrial pacemaker of the rabbit. Circ Res 41:292, 1977 5. Tweddell JS, Branham BH, Harada A, et al: Potential mapping in septal tachycardia: Evaluation of a new intraoperative mapping technique. Circulation 80: I97, 1989 6. Halbach M, Egert U, Hescheler J, et al: Estimation of action potential changes from field potential recordings in multicellular mouse cardiac myocyte cultures. Cell Physiol Biochem 13:271, 2003 7. Banach K, Halbach MD, Hu P, et al: Development of electrical activity in cardiac myocyte aggregates derived from mouse embryonic stem cells. Am J Physiol Heart Circ Physiol 284:H2114, 2003 8. Lu ZJ, et al: Arrhythmia in isolated prenatal hearts after ablation of the Cav2.3 (alpha1E) subunit of
MEAs and Embryonic Cardiac Mapping •
9.
10.
11.
12.
voltage-gated Ca2⫹ channels. Cell Physiol Biochem 14:11, 2004 Spach MS, Barr RC, Serwer GS, et al: Collision of excitation waves in the dog Purkinje system: Extracellular identification. Circ Res 29:499, 1971 Hariman RJ, Krongrad E, Boxer RA, et al: Methods for recording electrograms of the sinoatrial node during cardiac surgery in man. Circulation 61:1024, 1980 Haberl R, Steinbeck G, Luderitz B: Comparison between intracellular and extracellular direct current recordings of sinus node activity for evaluation of sinoatrial conduction time. Circulation 70: 760, 1984 Lewis T: Lectures on the heart, New York, NY. Paul B. Hoeber, 1915, p 1
Reppel et al. 109
13. Rijlant R: La conduction dans le coeur du mammife`re. Arch Internat Physiol 1931; 179 14. Van Der Kooi MW, Durrer D, Van Dam RT, et al: Electrical activity in sinus node and atrioventricular node. Am Heart J 51:684, 1956 15. Yamamoto M, Honjo H, Niwa R, et al: Low-frequency extracellular potentials recorded from the sinoatrial node. Cardiovasc Res 39:360, 1998 16. Schuessler RB, Boineau JP, Bromberg BI: Origin of the sinus impulse. J Cardiovasc Electrophysiol 7:263, 1996 17. Sano T, Yamagishi S: Spread of excitation from the sinus node. Circ Res 16:423, 1965 18. Bleeker WK, Mackaay AJ, Masson-Pevet M, et al: Functional and morphological organization of the rabbit sinus node. Circ Res 46:11, 1980