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Relative importance of funny current in human versus rabbit sinoatrial node
Journal of Molecular and Cellular Cardiology 48 (2010) 799–801
Contents lists available at ScienceDirect
Journal of Molecular and Cellular Cardiology j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / y j m c c
Letter to the Editor Relative importance of funny current in human versus rabbit sinoatrial node To the editor,
We read with great interest the recent Point/Counterpoint article by Lakatta and DiFrancesco on the relative role of the hyperpolarization-activated “funny current” If vs. that of intracellular Ca2+ cycling in controlling the normal pacemaker cell automaticity [1]. In this article, it is argued by Dr. Lakatta, referring to experimental data from our laboratory [2,3], that “the extent to which If becomes activated during diastolic depolarization in primary sinoatrial node cells must be low, in general, but especially in humans.” We would like to comment on the suggestion that the funny current is less important in the human sinoatrial (SA) node than in rabbit. In our patch-clamp study of If in human SA nodal cells [2], we found that the half-maximal activation voltage was ≈20 mV more negative and the fully-activated conductance 3–4 times smaller than in rabbit SA nodal cells [3,4]. But does this imply that If is less important in human than in rabbit? Intuitively, as in the Point/ Counterpoint article [1] and also in a recent editorial in another journal [5], one might conclude that with the above biophysical properties If can only play a minor role in human pacemaker activity. Yet, as already noted by Dobrev [5], there is a considerable bradycardic effect of the If blocker ivabradine. Furthermore, sinus bradycardia is also observed in families with sinus node dysfunction linked to loss-of-function mutations in HCN4, i.e. the gene encoding one of the four isoforms of the HCN gene family (HCN proteins of various isoforms self-assemble or co-assemble in tetramers to form the central pore of functional If pacemaker channels [4]). At the mRNA level, HCN4 is the most abundant HCN isoform in the rabbit SA node. The relative abundance of the four isoforms is HCN4 N HCN1 N HCN2 N HCN3. The amount of HCN3 is negligible [6], whereas values of 81.2%, 18.2%, and 0.6% (RNase protection assay) [6] and 86.4%, 11.7%, and 1.9% (quantitative PCR) [7] have been reported for the relative abundance of HCN4, HCN1, and HCN2 mRNA, respectively. Recently, highly similar results have been reported for the human SA node, with a relative abundance of 84%, 13%, 2.5%, and 0.5% for HCN4, HCN1, HCN2, and HCN3 mRNA, respectively (quantitative PCR) [8]. Immunofluorescence clearly shows the presence of the HCN4 protein in the sarcolemma of both rabbit and human SA nodal myocytes [7,8], whereas immunolabeling of HCN1 is close to detection threshold [7]. Despite the similar HCN isoform expression in rabbit and human SA node, there is a striking difference in the biophysical properties of If, which may be due to the strong “context dependence” of If channel properties [7]. As mentioned above, the half-maximal activation voltage of human If is ≈20 mV more negative than that of rabbit If and the fully-activated conductance 3–4 times smaller. Furthermore, the typical bell-shaped curve compiled from the (de)activation time constants is also shifted to more negative voltages [3]. To assess the functional implications of these differences in If properties 0022-2828/$ – see front matter © 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.yjmcc.2009.09.020
between rabbit and human, we carried out a numerical reconstruction of If during rabbit and human SA nodal pacemaker activity based on the experimentally obtained voltage clamp data on If, and compared this reconstructed If to the net membrane current (Inet) computed from Inet = − Cm × dVm/dt, where Cm and Vm denote membrane capacitance and membrane potential, respectively (Fig. 1). The full set of equations used in our numerical reconstruction of rabbit and human If is detailed and explained in the Online Supplement. For rabbit, we used a 1-s train of typical rabbit SA nodal action potentials (Fig. 1A, top; maximum diastolic potential of −63 mV, cycle length of ≈320 ms) as the repeatedly applied “command potential” in an “action potential clamp” of rabbit If. For this rabbit If, we used the If equations that van Ginneken and Giles derived from their comprehensive voltage clamp data on rabbit If [9], with a slight correction for temperature as in the If equations of the SA nodal cell models by Kurata et al. [10] and Maltsev and Lakatta [11], which are also based on the van Ginneken and Giles data. Our rabbit If has a halfmaximal activation voltage of − 76 mV and results in a fully-activated current density of 23 pA/pF at −130 mV, which is three times larger than the fully-activated human If at this voltage (8 pA/pF) [2] and in line with the values of 21 pA/pF (at −130 mV) [12], 19 pA/pF (at −130 mV) [13] and 17 pA/pF (at − 125 mV and room temperature) [7] reported for rabbit If. For human, we used the trains of action potentials obtained from three different human SA nodal cells (Figs. 1B–D, top panels; maximum diastolic potential between −65 and −55 mV, cycle length between 786 and 846 ms) [2] in an “action potential clamp” of human If. The pacemaker activity of human SA nodal cells is characterized by a slow and long diastolic depolarization (Figs. 1B– D, top panels), allowing sufficient time for If activation. The human If equations were those derived from our voltage clamp data from human SA nodal cells [3]. Notably, this human If has a half-maximal activation voltage of − 97 mV and a fully-activated conductance that is one third of rabbit. Its reversal potential (−22 mV) is similar to that of our rabbit If (− 24 mV). As expected from the biophysical properties, the numerically reconstructed human If (Figs. 1B–D, bottom panels) is considerably smaller than the typical rabbit If of Fig. 1A (bottom). However, Fig. 1 also shows a striking similarity between human and rabbit If if compared to the net membrane current: in either case, If is of about the same magnitude as Inet during the diastolic depolarization phase of the action potential (but see the "Note on rabbit If" in the Online Supplement). Similar results were obtained with first-order instead of second-order kinetic schemes or vice versa (not shown). In line with the apparently similar relative contribution of If to Inet in human and rabbit, we observed a similar increase in cycle length upon blockade of If by Cs+ (2 mmol/L), as illustrated in Fig. 2. The arrows in Fig. 2 indicate the ≈25% increase in cycle length in both rabbit (25 ± 3% (mean ± SEM, n = 5) [13]) and human (26% [2]). As an important caveat, it should be noted that our data on human If originate from only three SA nodal pacemaker cells that were isolated from a single patient with inappropriate tachycardias
800
Letter to the Editor
Fig. 1. Numerical reconstruction of If during rabbit (A) or human (B–D) SA nodal pacemaker activity. (A) Experimentally recorded action potentials of a single rabbit SA nodal pacemaker cell (top), associated time derivative (dVm/dt, middle), and associated net membrane current density derived from dVm/dt (Inet, bottom). The time course of If (red solid line, bottom) was reconstructed using the membrane potential (Vm) values of the recorded action potentials and the second-order Hodgkin-Huxley type kinetic scheme put forward by van Ginneken and Giles based on their voltage clamp data from rabbit SA nodal pacemaker cells [9], with the correction for temperature as used in the If equations of the SA nodal cell models by Kurata et al. [10] and Maltsev and Lakatta [11]. (B–D) Experimentally recorded action potentials of three different single human SA nodal pacemaker cells (top), associated time derivative (dVm/dt, middle), and associated net membrane current density derived from dVm/dt (Inet, bottom), together with the time course of If (red solid line, bottom) as reconstructed using the recorded Vm values and a first-order Hodgkin-Huxley type kinetic scheme [3] based on the voltage clamp data from human SA nodal pacemaker cells [2], as used previously [3,4] and detailed in the Online Supplement.
Letter to the Editor
801
References
Fig. 2. Effect of If blockade by Cs+ (2 mmol/L) on the action potential of a single rabbit (A) or human (B) SA nodal pacemaker cell. Arrows indicate the increase in cycle length. Note the difference in time scale. See Refs. [2] and [13] for recording conditions.
originating from the SA node region. Although their sudden onset and termination suggested that these tachycardias were based on reentrant excitation, we cannot exclude If remodeling in response to these tachycardias, e.g. a reduction in If current density without changes in voltage dependence or kinetics [14]. Furthermore, although our data on the increase in cycle length of rabbit SA nodal pacemaker cells by Cs+ are consistent with data from other laboratories, it should be noted that there are also groups that have reported much smaller pacemaker slowing effects of Cs+ [15]. In conclusion, the available data on human SA nodal If are consistent with an equally important role of the funny current in human vs. rabbit. Whether the funny current is more important than the “calcium clock” remains a matter of debate [1,15]. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.yjmcc.2009.09.020.
[1] Lakatta EG, DiFrancesco D. What keeps us ticking: a funny current, a calcium clock, or both? J Mol Cell Cardiol 2009;47:157–70. [2] Verkerk AO, Wilders R, van Borren MMGJ, Peters RJG, Broekhuis E, Lam K, et al. Pacemaker current (If) in the human sinoatrial node. Eur Heart J 2007;28:2472–8. [3] Verkerk AO, van Borren MMGJ, Peters RJG, Broekhuis E, Lam KY, Coronel R, et al. Single cells isolated from human sinoatrial node: action potentials and numerical reconstruction of pacemaker current. Conf Proc IEEE Eng Med Biol Soc 2007;1:904–7. [4] Verkerk AO, van Ginneken ACG, Wilders R. Pacemaker activity of the human sinoatrial node: role of the hyperpolarization-activated current If. If Int J Cardiol 2009;132:318–36. [5] Dobrev D. Ion channel portrait of the human sinus node: useful for a better understanding of sinus node function and dysfunction in humans? Circulation 2009;119:1556–8. [6] Shi W, Wymore R, Yu H, Wu J, Wymore RT, Pan Z, et al. Distribution and prevalence of hyperpolarization-activated cation channel (HCN) mRNA expression in cardiac tissues. Circ Res 1999;85:e1–6. [7] Brioschi C, Micheloni S, Tellez JO, Pisoni G, Longhi R, Moroni P, et al. Distribution of the pacemaker HCN4 channel mRNA and protein in the rabbit sinoatrial node. J Mol Cell Cardiol 2009;47:221–7. [8] Chandler NJ, Greener ID, Tellez JO, Inada S, Musa H, Molenaar P, et al. Molecular architecture of the human sinus node: insights into the function of the cardiac pacemaker. Circulation 2009;119:1562–75. [9] van Ginneken ACG, Giles W. Voltage clamp measurements of the hyperpolarization-activated inward current If in single cells from rabbit sino-atrial node. J Physiol 1991;434:57–83. [10] Kurata Y, Hisatome I, Imanishi S, Shibamoto T. Dynamical description of sinoatrial node pacemaking: improved mathematical model for primary pacemaker cell. Am J Physiol Heart Circ Physiol 2002;283:H2074–101. [11] Maltsev VA, Lakatta EG. Synergism of coupled subsarcolemmal Ca2+ clocks and sarcolemmal voltage clocks confers robust and flexible pacemaker function in a novel pacemaker cell model. Am J Physiol Heart Circ Physiol 2009;296:H594–615. [12] Verkerk AO, Wilders R, Coronel R, Ravesloot JH, Verheijck EE. Ionic remodeling of sinoatrial node cells by heart failure. Circulation 2003;108:760–6. [13] Verkerk AO, den Ruijter HM, Bourier J, Boukens BJ, Brouwer IA, Wilders R, et al. Dietary fish oil reduces pacemaker current and heart rate in rabbit. Heart Rhythm 2009;6:1485–92. [14] Yeh YH, Burstein B, Qi XY, Sakabe M, Chartier D, Comtois P, et al. Funny current downregulation and sinus node dysfunction associated with atrial tachyarrhythmia: a molecular basis for tachycardia-bradycardia syndrome. Circulation 2009;119:1576–85. [15] Eisner DA, Cerbai E. Beating to time: calcium clocks, voltage clocks, and cardiac pacemaker activity. Am J Physiol Heart Circ Physiol 2009;296:H561–2.
Arie O. Verkerk Ronald Wilders⁎,1 Heart Failure Research Center, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands E-mail address: [email protected] (R. Wilders). ⁎Corresponding author. Department of Anatomy, Embryology and Physiology, Academic Medical Center, University of Amsterdam, Meibergdreef 15, 1105 AZ Amsterdam, The Netherlands. Fax: +31 20 6976177. 1 Postal address: PO Box 22700, 1100 DE Amsterdam, The Netherlands. 31 July 2009
Contents lists available at ScienceDirect
Journal of Molecular and Cellular Cardiology j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / y j m c c
Letter to the Editor Relative importance of funny current in human versus rabbit sinoatrial node To the editor,
We read with great interest the recent Point/Counterpoint article by Lakatta and DiFrancesco on the relative role of the hyperpolarization-activated “funny current” If vs. that of intracellular Ca2+ cycling in controlling the normal pacemaker cell automaticity [1]. In this article, it is argued by Dr. Lakatta, referring to experimental data from our laboratory [2,3], that “the extent to which If becomes activated during diastolic depolarization in primary sinoatrial node cells must be low, in general, but especially in humans.” We would like to comment on the suggestion that the funny current is less important in the human sinoatrial (SA) node than in rabbit. In our patch-clamp study of If in human SA nodal cells [2], we found that the half-maximal activation voltage was ≈20 mV more negative and the fully-activated conductance 3–4 times smaller than in rabbit SA nodal cells [3,4]. But does this imply that If is less important in human than in rabbit? Intuitively, as in the Point/ Counterpoint article [1] and also in a recent editorial in another journal [5], one might conclude that with the above biophysical properties If can only play a minor role in human pacemaker activity. Yet, as already noted by Dobrev [5], there is a considerable bradycardic effect of the If blocker ivabradine. Furthermore, sinus bradycardia is also observed in families with sinus node dysfunction linked to loss-of-function mutations in HCN4, i.e. the gene encoding one of the four isoforms of the HCN gene family (HCN proteins of various isoforms self-assemble or co-assemble in tetramers to form the central pore of functional If pacemaker channels [4]). At the mRNA level, HCN4 is the most abundant HCN isoform in the rabbit SA node. The relative abundance of the four isoforms is HCN4 N HCN1 N HCN2 N HCN3. The amount of HCN3 is negligible [6], whereas values of 81.2%, 18.2%, and 0.6% (RNase protection assay) [6] and 86.4%, 11.7%, and 1.9% (quantitative PCR) [7] have been reported for the relative abundance of HCN4, HCN1, and HCN2 mRNA, respectively. Recently, highly similar results have been reported for the human SA node, with a relative abundance of 84%, 13%, 2.5%, and 0.5% for HCN4, HCN1, HCN2, and HCN3 mRNA, respectively (quantitative PCR) [8]. Immunofluorescence clearly shows the presence of the HCN4 protein in the sarcolemma of both rabbit and human SA nodal myocytes [7,8], whereas immunolabeling of HCN1 is close to detection threshold [7]. Despite the similar HCN isoform expression in rabbit and human SA node, there is a striking difference in the biophysical properties of If, which may be due to the strong “context dependence” of If channel properties [7]. As mentioned above, the half-maximal activation voltage of human If is ≈20 mV more negative than that of rabbit If and the fully-activated conductance 3–4 times smaller. Furthermore, the typical bell-shaped curve compiled from the (de)activation time constants is also shifted to more negative voltages [3]. To assess the functional implications of these differences in If properties 0022-2828/$ – see front matter © 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.yjmcc.2009.09.020
between rabbit and human, we carried out a numerical reconstruction of If during rabbit and human SA nodal pacemaker activity based on the experimentally obtained voltage clamp data on If, and compared this reconstructed If to the net membrane current (Inet) computed from Inet = − Cm × dVm/dt, where Cm and Vm denote membrane capacitance and membrane potential, respectively (Fig. 1). The full set of equations used in our numerical reconstruction of rabbit and human If is detailed and explained in the Online Supplement. For rabbit, we used a 1-s train of typical rabbit SA nodal action potentials (Fig. 1A, top; maximum diastolic potential of −63 mV, cycle length of ≈320 ms) as the repeatedly applied “command potential” in an “action potential clamp” of rabbit If. For this rabbit If, we used the If equations that van Ginneken and Giles derived from their comprehensive voltage clamp data on rabbit If [9], with a slight correction for temperature as in the If equations of the SA nodal cell models by Kurata et al. [10] and Maltsev and Lakatta [11], which are also based on the van Ginneken and Giles data. Our rabbit If has a halfmaximal activation voltage of − 76 mV and results in a fully-activated current density of 23 pA/pF at −130 mV, which is three times larger than the fully-activated human If at this voltage (8 pA/pF) [2] and in line with the values of 21 pA/pF (at −130 mV) [12], 19 pA/pF (at −130 mV) [13] and 17 pA/pF (at − 125 mV and room temperature) [7] reported for rabbit If. For human, we used the trains of action potentials obtained from three different human SA nodal cells (Figs. 1B–D, top panels; maximum diastolic potential between −65 and −55 mV, cycle length between 786 and 846 ms) [2] in an “action potential clamp” of human If. The pacemaker activity of human SA nodal cells is characterized by a slow and long diastolic depolarization (Figs. 1B– D, top panels), allowing sufficient time for If activation. The human If equations were those derived from our voltage clamp data from human SA nodal cells [3]. Notably, this human If has a half-maximal activation voltage of − 97 mV and a fully-activated conductance that is one third of rabbit. Its reversal potential (−22 mV) is similar to that of our rabbit If (− 24 mV). As expected from the biophysical properties, the numerically reconstructed human If (Figs. 1B–D, bottom panels) is considerably smaller than the typical rabbit If of Fig. 1A (bottom). However, Fig. 1 also shows a striking similarity between human and rabbit If if compared to the net membrane current: in either case, If is of about the same magnitude as Inet during the diastolic depolarization phase of the action potential (but see the "Note on rabbit If" in the Online Supplement). Similar results were obtained with first-order instead of second-order kinetic schemes or vice versa (not shown). In line with the apparently similar relative contribution of If to Inet in human and rabbit, we observed a similar increase in cycle length upon blockade of If by Cs+ (2 mmol/L), as illustrated in Fig. 2. The arrows in Fig. 2 indicate the ≈25% increase in cycle length in both rabbit (25 ± 3% (mean ± SEM, n = 5) [13]) and human (26% [2]). As an important caveat, it should be noted that our data on human If originate from only three SA nodal pacemaker cells that were isolated from a single patient with inappropriate tachycardias
800
Letter to the Editor
Fig. 1. Numerical reconstruction of If during rabbit (A) or human (B–D) SA nodal pacemaker activity. (A) Experimentally recorded action potentials of a single rabbit SA nodal pacemaker cell (top), associated time derivative (dVm/dt, middle), and associated net membrane current density derived from dVm/dt (Inet, bottom). The time course of If (red solid line, bottom) was reconstructed using the membrane potential (Vm) values of the recorded action potentials and the second-order Hodgkin-Huxley type kinetic scheme put forward by van Ginneken and Giles based on their voltage clamp data from rabbit SA nodal pacemaker cells [9], with the correction for temperature as used in the If equations of the SA nodal cell models by Kurata et al. [10] and Maltsev and Lakatta [11]. (B–D) Experimentally recorded action potentials of three different single human SA nodal pacemaker cells (top), associated time derivative (dVm/dt, middle), and associated net membrane current density derived from dVm/dt (Inet, bottom), together with the time course of If (red solid line, bottom) as reconstructed using the recorded Vm values and a first-order Hodgkin-Huxley type kinetic scheme [3] based on the voltage clamp data from human SA nodal pacemaker cells [2], as used previously [3,4] and detailed in the Online Supplement.
Letter to the Editor
801
References
Fig. 2. Effect of If blockade by Cs+ (2 mmol/L) on the action potential of a single rabbit (A) or human (B) SA nodal pacemaker cell. Arrows indicate the increase in cycle length. Note the difference in time scale. See Refs. [2] and [13] for recording conditions.
originating from the SA node region. Although their sudden onset and termination suggested that these tachycardias were based on reentrant excitation, we cannot exclude If remodeling in response to these tachycardias, e.g. a reduction in If current density without changes in voltage dependence or kinetics [14]. Furthermore, although our data on the increase in cycle length of rabbit SA nodal pacemaker cells by Cs+ are consistent with data from other laboratories, it should be noted that there are also groups that have reported much smaller pacemaker slowing effects of Cs+ [15]. In conclusion, the available data on human SA nodal If are consistent with an equally important role of the funny current in human vs. rabbit. Whether the funny current is more important than the “calcium clock” remains a matter of debate [1,15]. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.yjmcc.2009.09.020.
[1] Lakatta EG, DiFrancesco D. What keeps us ticking: a funny current, a calcium clock, or both? J Mol Cell Cardiol 2009;47:157–70. [2] Verkerk AO, Wilders R, van Borren MMGJ, Peters RJG, Broekhuis E, Lam K, et al. Pacemaker current (If) in the human sinoatrial node. Eur Heart J 2007;28:2472–8. [3] Verkerk AO, van Borren MMGJ, Peters RJG, Broekhuis E, Lam KY, Coronel R, et al. Single cells isolated from human sinoatrial node: action potentials and numerical reconstruction of pacemaker current. Conf Proc IEEE Eng Med Biol Soc 2007;1:904–7. [4] Verkerk AO, van Ginneken ACG, Wilders R. Pacemaker activity of the human sinoatrial node: role of the hyperpolarization-activated current If. If Int J Cardiol 2009;132:318–36. [5] Dobrev D. Ion channel portrait of the human sinus node: useful for a better understanding of sinus node function and dysfunction in humans? Circulation 2009;119:1556–8. [6] Shi W, Wymore R, Yu H, Wu J, Wymore RT, Pan Z, et al. Distribution and prevalence of hyperpolarization-activated cation channel (HCN) mRNA expression in cardiac tissues. Circ Res 1999;85:e1–6. [7] Brioschi C, Micheloni S, Tellez JO, Pisoni G, Longhi R, Moroni P, et al. Distribution of the pacemaker HCN4 channel mRNA and protein in the rabbit sinoatrial node. J Mol Cell Cardiol 2009;47:221–7. [8] Chandler NJ, Greener ID, Tellez JO, Inada S, Musa H, Molenaar P, et al. Molecular architecture of the human sinus node: insights into the function of the cardiac pacemaker. Circulation 2009;119:1562–75. [9] van Ginneken ACG, Giles W. Voltage clamp measurements of the hyperpolarization-activated inward current If in single cells from rabbit sino-atrial node. J Physiol 1991;434:57–83. [10] Kurata Y, Hisatome I, Imanishi S, Shibamoto T. Dynamical description of sinoatrial node pacemaking: improved mathematical model for primary pacemaker cell. Am J Physiol Heart Circ Physiol 2002;283:H2074–101. [11] Maltsev VA, Lakatta EG. Synergism of coupled subsarcolemmal Ca2+ clocks and sarcolemmal voltage clocks confers robust and flexible pacemaker function in a novel pacemaker cell model. Am J Physiol Heart Circ Physiol 2009;296:H594–615. [12] Verkerk AO, Wilders R, Coronel R, Ravesloot JH, Verheijck EE. Ionic remodeling of sinoatrial node cells by heart failure. Circulation 2003;108:760–6. [13] Verkerk AO, den Ruijter HM, Bourier J, Boukens BJ, Brouwer IA, Wilders R, et al. Dietary fish oil reduces pacemaker current and heart rate in rabbit. Heart Rhythm 2009;6:1485–92. [14] Yeh YH, Burstein B, Qi XY, Sakabe M, Chartier D, Comtois P, et al. Funny current downregulation and sinus node dysfunction associated with atrial tachyarrhythmia: a molecular basis for tachycardia-bradycardia syndrome. Circulation 2009;119:1576–85. [15] Eisner DA, Cerbai E. Beating to time: calcium clocks, voltage clocks, and cardiac pacemaker activity. Am J Physiol Heart Circ Physiol 2009;296:H561–2.
Arie O. Verkerk Ronald Wilders⁎,1 Heart Failure Research Center, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands E-mail address: [email protected] (R. Wilders). ⁎Corresponding author. Department of Anatomy, Embryology and Physiology, Academic Medical Center, University of Amsterdam, Meibergdreef 15, 1105 AZ Amsterdam, The Netherlands. Fax: +31 20 6976177. 1 Postal address: PO Box 22700, 1100 DE Amsterdam, The Netherlands. 31 July 2009