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Monolayer cell cultures as model systems for studying paroxysmal atrial fibrillation
Journal of Electrocardiology Vol. 37 Supplement 2004
Monolayer Cell Cultures as Model Systems for Studying Paroxysmal Atrial Fibrillation
Gil Bub, PhD, and Nabil El-Sherif, MD
Atrial fibrillation is often paroxysmal in nature, characterized by periods of rapid irregular activity inter-dispersed with periods of normal sinus rhythm. Paroxysmal atrial fibrillation (PAF) is often associated with serious symptoms, such as dizziness, increased chance of thrombosis, and may also lead to sustained atrial fibrillation via remodeling of the tissue over time(1). The mechanisms that lead to PAF are thought to be similar to sustained AF, in that they include reentrant mechanisms, rapid pulmonary vein ectopy, or a combination of the two (2,3). Mechanisms that lead to the initiation and termination of AF are difficult to elucidate due to the complex geometry of the atria, which displays significant intra and inter species differences. Perhaps the greatest difficulty in understanding PAF is due to the heterogeneous nature of the substrate, which consists of pacemaker cells embedded in excitable and fibrotic tissue. A first step to understanding PAF is to develop an experimental and theoretical framework for studying the dynamics such heterogeneous substrates. This paper outlines tissue culture models that are potential systems for studying fundamental processes related to the sudden onset and offset of complex rhythms.
tal cardiac cells. Cardiac cells from very young animals have the capacity to easily form gap junctional connections with neighboring cells in culture. After a few days in culture, embryonic cardiac cells are capable of supporting propagating waves of excitation over long distances. Cardiac monolayers were popular 30 years ago as model systems of two-dimensional conduction (4). More recently, the availability of potential mapping techniques have renewed interest in cultured monolayers, as they allow controlled environments for studying conduction on microscopic and macroscopic scales. Optical mapping experiments on cultured cardiac monolayers have several advantages when compared to mapping conduction in intact tissue. Signals from individual cells can be identified and mapped with subcellular resolution in cell culture while optical signals from intact tissue are compound signals from cells at the surface and deeper intramural layers. The geometry of cell cultures can be precisely controlled, allowing conduction patterns to be observed independent of the influence of micro and macro anatomy. In addition, cardiac monolayers can be grown in conditions that cause cells to have similar anisotropy, connectivity and longitudinal to transverse cell diameter ratios to cells in intact tissue. As a result of these advantages, several fundamental experiments have been performed on cell culture systems that could not easily be addressed in whole tissue studies.
Mapping Conduction in Cardiac Cell Culture Heart cell monolayers are thin layers of tissue grown in culture dishes from embryonic or neona-
Measuring Microscopic Conduction Patterns: Factors that Effect Conduction
From the VA Medical Center, Brooklyn NY. Reprint requests: Gil Bub, PhD, SUNY Downstate Medical Center, VA Medical Center, 800 Poly Place, room 7-100, Brooklyn, NY 11203; e-mail: [email protected] © 2004 Elsevier Inc. All rights reserved. 0022-0736/04/370S-0013$30.00/0 doi:10.1016/j.jelectrocard.2004.08.014
Propagation in cardiac muscle depends on both excitable and passive electrical properties of the 44
RRH: Monolayer Cell Cultures •
coupled cell network. The passive properties of the tissue are influenced by factors such as discontinuities introduced by capillaries and connective tissue, variations in cell connectivity, and tissue anisotropy. The local structure dictates whether waves propagate, block, or form reentrant circuits and is fundamental to understanding arrhythmogenesis. The development of techniques for controlling cell orientation by brushing the collagen substrate, and cell position using photolithographic techniques have spurred investigations on the effects of geometry on propagation. The stability of propagating waves is altered at abrupt changes in the geometry of the substrate. In the case of a sudden expansion, current generated by the propagating wavefront may not be sufficient to stimulate cells immediately upstream. Optical mapping of propagation in monolayers plated as strands of different widths connected to a large tissue area demonstrated that impulses propagating into the site of expansion are blocked if the strand diameter is less than 200 m (5). The instability at the expansion site may be modulated by electrophysiological characteristics: rapid pacing, which reduces excitability by lowering I(Na), results in conduction block in wider strands, while gap junction block, which lowers the number of cells directly driven by cells at the expansion, permits propagation through narrower strands. Tissue geometry may also act to stabilize propagation. Propagation through a culture plated as a main strand with many side branches is slowed due to the current load imposed by the branches. As the wave propagates past each side branch, current is sourced back to the main branch, improving wave front stability. In preparations with reduced excitability conduction blocks at wave speeds of ⬃15 cm/s but is stable at speeds of ⬃1 cm/s in a highly branched structure (5). These results may explain how diseased myocardium, which is often a complex patchwork of healthy and dead tissue, supports stable reentry at small space scales.
Measuring Macroscopic Conduction: Cell Culture Models of Arrhythmia Reentry and ectopy are the underlying mechanism for many arrhythmias and have been mapped in whole tissue using a wide variety of methods. However, understanding basic mechanisms of these rhythms is complicated by the fact that activation patterns are only directly visible on the myocardial
Bub and El-Sherif 45
surface and the relative contribution of the underlying microanatomy of the tissue to the observed behavior is difficult to ascertain. Cardiac monolayers have been developed as relatively tractable models of reentrant and ectopic arrhythmias (6 – 9,11–16). Macroscopic reentrant activity has been observed in preparations with (8,11) and without (6, 14) obstacles, and in rapidly propagating excitable (13,15) and slowly propagating spontaneously active (6 –9,16) tissue. In two-dimensional sheets, reentry is typically in the form of a spiral wave, where spirals either are single armed or display a “figure of eight” morphology, but can also display a complex multi-armed morphology (15) and turbulent activity (16). Spiral wave stability has been experimentally investigated by changing cell coupling (16), mixing cell types (17), locally changing cell orientation (18), and by modulating ionic currents (19). The first macroscopic images (⬃1cm2) of cardiac conduction using optical methods showed that monolayer culture created from embryonic chick myocytes are capable of supporting reentrant circuits (6). In order to determine spatiotemporal dynamics from spontaneously active tissue, a technique was developed where images could be continuously acquired at low light levels for long time periods (also see ref 9 for a different method). Interestingly, reentrant waves in the form spirals were not necessarily stable, but would spontaneously initiate and terminate giving rise to a paroxysmal rhythm. Similar results were seen in cultured rings of cardiac tissue (8). Subsequent work on the two-dimensional monolayer preparation showed that the paroxysmal behavior depended on time in culture and plating density: at high plating densities and for old (⬎3 day) cultures, the monolayer displayed target patterns or stable spiral waves. For lower density preparations and younger preparations (⬃1.5 day), the monolayer displayed paroxysmal rhythms driven by the onset and offset of spiral waves. Theoretical modeling using generic excitable media formulations indicated that the transition between stable and paroxysmal reentrant rhythms can be accounted for by changes in cellcell connectivity and changes in the degree of heterogeneity (7). Interestingly, bursting rhythms which appeared to originate from focal sources are observed in the same preparation when older embryos are used. In this case, the monolayer displays ectopic beats from one location which may trigger a focus to rapidly fire from a second location. The mechanism of focal bursting in the chick preparation is not known, however, simple models show that pacemaker cells
46 Journal of Electrocardiology Vol. 37 Supplement 2004 can occasionally synchronize for prolonged periods giving rise to bursts of focal activity (7). This behavior has also been observed in rat ventricle cell cultures monitored using a noninvasive phase contrast technique (9). A similar mechanism has been suggested for coupled networks of neuronal cells (10). Bursting rhythms from foci have also been observed in cell culture models of ischemia reperfusion injury. In this case, macroscopic images were acquired from a monolayer of rat myocytes, which were treated in such a way as to create a transient ischemic zone (12,13). During washout, a portion of the monolayers (17%) display a burst of rapid focal activity originating from within the original ischemic zone. The authors speculate that this effect is caused by elevation of Ca⫹⫹ and Na⫹, as well as alterations in inward and outward currents which act to bring cells closer to threshold. It is interesting to speculate that local transient ischemia in the intact heart may generate conditions that lead to PAF. Additional work is required to determine which of these mechanisms, if any, contribute to paroxysmal rhythms in the intact atria. While some combination paroxysmal spiral waves, pacemaker synchronization, and localized changes in ionic concentrations may lead to paroxysmal rhythms in vivo, it is also possible that none of these effects plays a significant role. It is clear, however, that the behavior found in these simple biological preparations should be taken into account when studying complex paroxysmal arrhythmias in the diseased myocardium.
6.
7.
8.
9.
10.
11.
12.
13.
14.
15.
16.
References 1. Van Wagoner DR: Electrical remodeling in human atrial fibrillation. PACE 26:1572, 2003 2. Khairy P, Nattel S: New insights into the mechanisms and management of atrial fibrillation. JAMC 167: 1012, 2002 3. Jalife J: Rotors and spiral waves in atrial fibrillation. J Cardiovasc Electrophysiol 14:776, 2003 4. DeHaan RL, Gottlieb SH: The electrical activity of embryonic chick heart cells isolated in tissue culture singly or in interconnected cell sheets. J Gen Physiol 52:643, 1968 5. Rohr S, Kleber AG, Kucera J: Optical recording of
17.
18.
19.
impulse propagation in designer cultures: Cardiac tissue architectures inducing ultra-slow conduction. Trends Cardiovasc Med 9:173, 1999 Bub G, Glass L, Publicover NG, et al: Bursting calcium rotors in cultured cardiac myocytes monolayers. Proc Natl Acad Sci USA 95:10283, 1998 Bub G, Tateno K, Shrier A, et al: Spontaneous initiation and termination of complex cardiac rhythms. J Cardiovasc Electrophysiol 14:S229, 2003 Nagai Y, Gonzalez H, Shrier A, et al: Paroxysmal starting and stopping of circulating waves in an excitable media. Phys Rev Lett 84:4248, 2000 Hwang S, Yea K, Lee KJ: Regular and alternant spiral waves of contractile motion on rat ventricle cell cultures. Phys Rev Lett 92:198103.1, 2004 Lewis T, Rinzel J: Self-organized synchronous oscillations in a network of excitable cells coupled by gap junctions. Network: Comput Neural Syst 11:299, 2000 Entcheva E, Lu S N, Troppman RH, et al: Contact fluorescence imaging of reentry in monolayers of cultured neonatal rat ventricular myocytes. J Cardiovasc Electrophysiol 11:665, 2000 Arutunyan A, Webster DR, Swift LM, et al: Localized injury in cardiomyocyte network: A new experimental model of ischemia-reperfusion arrhythmias. 280: H1905, 2001 Arutunyan A, Swift L, Sarvazyan N: Initiation and propagation of ectopic waves: Insights from an in vitro model of ischemia-reperfusion injury. Am J Physiol Heart Circ Physiol 283:H741, 2002 Iravanian S, Nabutovsky Y, Kong CR, et al: Functional reentry in cultured monolayers of neonatal rat cardiac cells. Am J Physiol Heart Circ Physiol 285: H449, 2003 Bursac N, Aguel F, Tung L: Multi-arm spirals in two dimensional heart substrate. Proc Natl Acad Sci 2004 (in press) Bub G, Shrier A, Glass L: Spiral wave generation in heterogeneous excitable media. Phys Rev Lett 88: 058101, 2002 Abraham RM, Henrikson CA, Maskara B, et al: Sustained reentry in co-cultured monolayers of skeletal muscle myoblasts and neonatal rat cardiomyocytes. Heart Rhythm 1:1S, 2004 (abstr 595) Valderrabano M, Parker JM, Weiss JN: High-resolution optical calcium imaging of stable reentry in patterned monolayers of cultured neonatal rat cardiomyocytes. Heart Rhythm 1:1S, 2004 (abstr 492) Lucca E, Berenfeld O, Mironov SF, et al: Sodium channel blockade reduces wavefront curvature and terminates reentry in neonatal rat myocyte monolayers. Heart Rhythm 1:1S, 2004 (abstr 293)
Monolayer Cell Cultures as Model Systems for Studying Paroxysmal Atrial Fibrillation
Gil Bub, PhD, and Nabil El-Sherif, MD
Atrial fibrillation is often paroxysmal in nature, characterized by periods of rapid irregular activity inter-dispersed with periods of normal sinus rhythm. Paroxysmal atrial fibrillation (PAF) is often associated with serious symptoms, such as dizziness, increased chance of thrombosis, and may also lead to sustained atrial fibrillation via remodeling of the tissue over time(1). The mechanisms that lead to PAF are thought to be similar to sustained AF, in that they include reentrant mechanisms, rapid pulmonary vein ectopy, or a combination of the two (2,3). Mechanisms that lead to the initiation and termination of AF are difficult to elucidate due to the complex geometry of the atria, which displays significant intra and inter species differences. Perhaps the greatest difficulty in understanding PAF is due to the heterogeneous nature of the substrate, which consists of pacemaker cells embedded in excitable and fibrotic tissue. A first step to understanding PAF is to develop an experimental and theoretical framework for studying the dynamics such heterogeneous substrates. This paper outlines tissue culture models that are potential systems for studying fundamental processes related to the sudden onset and offset of complex rhythms.
tal cardiac cells. Cardiac cells from very young animals have the capacity to easily form gap junctional connections with neighboring cells in culture. After a few days in culture, embryonic cardiac cells are capable of supporting propagating waves of excitation over long distances. Cardiac monolayers were popular 30 years ago as model systems of two-dimensional conduction (4). More recently, the availability of potential mapping techniques have renewed interest in cultured monolayers, as they allow controlled environments for studying conduction on microscopic and macroscopic scales. Optical mapping experiments on cultured cardiac monolayers have several advantages when compared to mapping conduction in intact tissue. Signals from individual cells can be identified and mapped with subcellular resolution in cell culture while optical signals from intact tissue are compound signals from cells at the surface and deeper intramural layers. The geometry of cell cultures can be precisely controlled, allowing conduction patterns to be observed independent of the influence of micro and macro anatomy. In addition, cardiac monolayers can be grown in conditions that cause cells to have similar anisotropy, connectivity and longitudinal to transverse cell diameter ratios to cells in intact tissue. As a result of these advantages, several fundamental experiments have been performed on cell culture systems that could not easily be addressed in whole tissue studies.
Mapping Conduction in Cardiac Cell Culture Heart cell monolayers are thin layers of tissue grown in culture dishes from embryonic or neona-
Measuring Microscopic Conduction Patterns: Factors that Effect Conduction
From the VA Medical Center, Brooklyn NY. Reprint requests: Gil Bub, PhD, SUNY Downstate Medical Center, VA Medical Center, 800 Poly Place, room 7-100, Brooklyn, NY 11203; e-mail: [email protected] © 2004 Elsevier Inc. All rights reserved. 0022-0736/04/370S-0013$30.00/0 doi:10.1016/j.jelectrocard.2004.08.014
Propagation in cardiac muscle depends on both excitable and passive electrical properties of the 44
RRH: Monolayer Cell Cultures •
coupled cell network. The passive properties of the tissue are influenced by factors such as discontinuities introduced by capillaries and connective tissue, variations in cell connectivity, and tissue anisotropy. The local structure dictates whether waves propagate, block, or form reentrant circuits and is fundamental to understanding arrhythmogenesis. The development of techniques for controlling cell orientation by brushing the collagen substrate, and cell position using photolithographic techniques have spurred investigations on the effects of geometry on propagation. The stability of propagating waves is altered at abrupt changes in the geometry of the substrate. In the case of a sudden expansion, current generated by the propagating wavefront may not be sufficient to stimulate cells immediately upstream. Optical mapping of propagation in monolayers plated as strands of different widths connected to a large tissue area demonstrated that impulses propagating into the site of expansion are blocked if the strand diameter is less than 200 m (5). The instability at the expansion site may be modulated by electrophysiological characteristics: rapid pacing, which reduces excitability by lowering I(Na), results in conduction block in wider strands, while gap junction block, which lowers the number of cells directly driven by cells at the expansion, permits propagation through narrower strands. Tissue geometry may also act to stabilize propagation. Propagation through a culture plated as a main strand with many side branches is slowed due to the current load imposed by the branches. As the wave propagates past each side branch, current is sourced back to the main branch, improving wave front stability. In preparations with reduced excitability conduction blocks at wave speeds of ⬃15 cm/s but is stable at speeds of ⬃1 cm/s in a highly branched structure (5). These results may explain how diseased myocardium, which is often a complex patchwork of healthy and dead tissue, supports stable reentry at small space scales.
Measuring Macroscopic Conduction: Cell Culture Models of Arrhythmia Reentry and ectopy are the underlying mechanism for many arrhythmias and have been mapped in whole tissue using a wide variety of methods. However, understanding basic mechanisms of these rhythms is complicated by the fact that activation patterns are only directly visible on the myocardial
Bub and El-Sherif 45
surface and the relative contribution of the underlying microanatomy of the tissue to the observed behavior is difficult to ascertain. Cardiac monolayers have been developed as relatively tractable models of reentrant and ectopic arrhythmias (6 – 9,11–16). Macroscopic reentrant activity has been observed in preparations with (8,11) and without (6, 14) obstacles, and in rapidly propagating excitable (13,15) and slowly propagating spontaneously active (6 –9,16) tissue. In two-dimensional sheets, reentry is typically in the form of a spiral wave, where spirals either are single armed or display a “figure of eight” morphology, but can also display a complex multi-armed morphology (15) and turbulent activity (16). Spiral wave stability has been experimentally investigated by changing cell coupling (16), mixing cell types (17), locally changing cell orientation (18), and by modulating ionic currents (19). The first macroscopic images (⬃1cm2) of cardiac conduction using optical methods showed that monolayer culture created from embryonic chick myocytes are capable of supporting reentrant circuits (6). In order to determine spatiotemporal dynamics from spontaneously active tissue, a technique was developed where images could be continuously acquired at low light levels for long time periods (also see ref 9 for a different method). Interestingly, reentrant waves in the form spirals were not necessarily stable, but would spontaneously initiate and terminate giving rise to a paroxysmal rhythm. Similar results were seen in cultured rings of cardiac tissue (8). Subsequent work on the two-dimensional monolayer preparation showed that the paroxysmal behavior depended on time in culture and plating density: at high plating densities and for old (⬎3 day) cultures, the monolayer displayed target patterns or stable spiral waves. For lower density preparations and younger preparations (⬃1.5 day), the monolayer displayed paroxysmal rhythms driven by the onset and offset of spiral waves. Theoretical modeling using generic excitable media formulations indicated that the transition between stable and paroxysmal reentrant rhythms can be accounted for by changes in cellcell connectivity and changes in the degree of heterogeneity (7). Interestingly, bursting rhythms which appeared to originate from focal sources are observed in the same preparation when older embryos are used. In this case, the monolayer displays ectopic beats from one location which may trigger a focus to rapidly fire from a second location. The mechanism of focal bursting in the chick preparation is not known, however, simple models show that pacemaker cells
46 Journal of Electrocardiology Vol. 37 Supplement 2004 can occasionally synchronize for prolonged periods giving rise to bursts of focal activity (7). This behavior has also been observed in rat ventricle cell cultures monitored using a noninvasive phase contrast technique (9). A similar mechanism has been suggested for coupled networks of neuronal cells (10). Bursting rhythms from foci have also been observed in cell culture models of ischemia reperfusion injury. In this case, macroscopic images were acquired from a monolayer of rat myocytes, which were treated in such a way as to create a transient ischemic zone (12,13). During washout, a portion of the monolayers (17%) display a burst of rapid focal activity originating from within the original ischemic zone. The authors speculate that this effect is caused by elevation of Ca⫹⫹ and Na⫹, as well as alterations in inward and outward currents which act to bring cells closer to threshold. It is interesting to speculate that local transient ischemia in the intact heart may generate conditions that lead to PAF. Additional work is required to determine which of these mechanisms, if any, contribute to paroxysmal rhythms in the intact atria. While some combination paroxysmal spiral waves, pacemaker synchronization, and localized changes in ionic concentrations may lead to paroxysmal rhythms in vivo, it is also possible that none of these effects plays a significant role. It is clear, however, that the behavior found in these simple biological preparations should be taken into account when studying complex paroxysmal arrhythmias in the diseased myocardium.
6.
7.
8.
9.
10.
11.
12.
13.
14.
15.
16.
References 1. Van Wagoner DR: Electrical remodeling in human atrial fibrillation. PACE 26:1572, 2003 2. Khairy P, Nattel S: New insights into the mechanisms and management of atrial fibrillation. JAMC 167: 1012, 2002 3. Jalife J: Rotors and spiral waves in atrial fibrillation. J Cardiovasc Electrophysiol 14:776, 2003 4. DeHaan RL, Gottlieb SH: The electrical activity of embryonic chick heart cells isolated in tissue culture singly or in interconnected cell sheets. J Gen Physiol 52:643, 1968 5. Rohr S, Kleber AG, Kucera J: Optical recording of
17.
18.
19.
impulse propagation in designer cultures: Cardiac tissue architectures inducing ultra-slow conduction. Trends Cardiovasc Med 9:173, 1999 Bub G, Glass L, Publicover NG, et al: Bursting calcium rotors in cultured cardiac myocytes monolayers. Proc Natl Acad Sci USA 95:10283, 1998 Bub G, Tateno K, Shrier A, et al: Spontaneous initiation and termination of complex cardiac rhythms. J Cardiovasc Electrophysiol 14:S229, 2003 Nagai Y, Gonzalez H, Shrier A, et al: Paroxysmal starting and stopping of circulating waves in an excitable media. Phys Rev Lett 84:4248, 2000 Hwang S, Yea K, Lee KJ: Regular and alternant spiral waves of contractile motion on rat ventricle cell cultures. Phys Rev Lett 92:198103.1, 2004 Lewis T, Rinzel J: Self-organized synchronous oscillations in a network of excitable cells coupled by gap junctions. Network: Comput Neural Syst 11:299, 2000 Entcheva E, Lu S N, Troppman RH, et al: Contact fluorescence imaging of reentry in monolayers of cultured neonatal rat ventricular myocytes. J Cardiovasc Electrophysiol 11:665, 2000 Arutunyan A, Webster DR, Swift LM, et al: Localized injury in cardiomyocyte network: A new experimental model of ischemia-reperfusion arrhythmias. 280: H1905, 2001 Arutunyan A, Swift L, Sarvazyan N: Initiation and propagation of ectopic waves: Insights from an in vitro model of ischemia-reperfusion injury. Am J Physiol Heart Circ Physiol 283:H741, 2002 Iravanian S, Nabutovsky Y, Kong CR, et al: Functional reentry in cultured monolayers of neonatal rat cardiac cells. Am J Physiol Heart Circ Physiol 285: H449, 2003 Bursac N, Aguel F, Tung L: Multi-arm spirals in two dimensional heart substrate. Proc Natl Acad Sci 2004 (in press) Bub G, Shrier A, Glass L: Spiral wave generation in heterogeneous excitable media. Phys Rev Lett 88: 058101, 2002 Abraham RM, Henrikson CA, Maskara B, et al: Sustained reentry in co-cultured monolayers of skeletal muscle myoblasts and neonatal rat cardiomyocytes. Heart Rhythm 1:1S, 2004 (abstr 595) Valderrabano M, Parker JM, Weiss JN: High-resolution optical calcium imaging of stable reentry in patterned monolayers of cultured neonatal rat cardiomyocytes. Heart Rhythm 1:1S, 2004 (abstr 492) Lucca E, Berenfeld O, Mironov SF, et al: Sodium channel blockade reduces wavefront curvature and terminates reentry in neonatal rat myocyte monolayers. Heart Rhythm 1:1S, 2004 (abstr 293)