Bradycardia and Slowing of the Atrioventricular Conduction in Mice Lacking CaV3.1/1G T-Type Calcium Channels
http://www.100md.com
Matteo E. Mangoni, Achraf Traboulsie, An
参见附件。
the Institut de Génomique Fonctionnelle (M.E.M., A.T., B.C., L.M., E.K., A.C.-S., J.N., P.L.), CNRS UMR5203-INSERM U661-Université de Montpellier I–Université de Montpellier II, Département de Physiologie, Montpellier
Institut du Thorax (A.-L.L., K.L.Q., D.E., F.C.), INSERM U533, Faculté de Médecine, Nantes
Cardiovascular Research Center (J.V.), INSERM U689, Université Paris 7-Denis Diderot, Paris, France
Center for Calcium and Learning (H.-S.S.), Korea Institute of Science and Technology, Cheongryang, Seoul, Republic of Korea.
Abstract
The generation of the mammalian heartbeat is a complex and vital function requiring multiple and coordinated ionic channel activities. The functional role of low-voltage activated (LVA) T-type calcium channels in the pacemaker activity of the sinoatrial node (SAN) is, to date, unresolved. Here we show that disruption of the gene coding for Cav3.1/1G T-type calcium channels (cacna1g) abolishes T-type calcium current (ICa,T) in isolated cells from the SAN and the atrioventricular node without affecting the L-type Ca2+ current (ICa,L). By using telemetric electrocardiograms on unrestrained mice and intracardiac recordings, we find that cacna1g inactivation causes bradycardia and delays atrioventricular conduction without affecting the excitability of the right atrium. Consistently, no ICa,T was detected in right atrium myocytes in both wild-type and Cav3.1–/– mice. Furthermore, inactivation of cacna1g significantly slowed the intrinsic in vivo heart rate, prolonged the SAN recovery time, and slowed pacemaker activity of individual SAN cells through a reduction of the slope of the diastolic depolarization. Our results demonstrate that Cav3.1/T-type Ca2+ channels contribute to SAN pacemaker activity and atrioventricular conduction.
Key Words: pacemaker activity T-type calcium channel sinoatrial node conduction knockout mice
Introduction
The initiation of the heartbeat requires coordination between the automaticity of the sinoatrial node (SAN) and excitability of the atrioventricular (AV) conduction tissue: the AV node (AVN) and the His–Purkinje fiber network. SAN automaticity is caused by the presence of the diastolic depolarization.1 Multiple classes of ionic channels are expressed in the SAN,1,2 but the precise mechanism initiating the diastolic depolarization has not been entirely elucidated, and the relative contribution of different ionic channels in establishing the heart rate under specific physiological conditions is still a matter of debate.3–5 Strong functional, pharmacological, and genetic evidence show that the hyperpolarization-activated currents If6,7 and ICa,L8–10 play major roles in controlling the diastolic depolarization after decaying of the fast component of delayed-rectifier K+ currents (IKr). Diastolic release of Ca2+ mediated by ryanodine receptors (RyRs) has been indicated as an important mechanism for controlling SAN pacemaking under activation of the -adrenergic receptor.11 The SAN also expresses neuronal tetrodotoxin (TTX)-sensitive and cardiac TTX-resistant voltage-dependent Na+ currents (INa).12 TTX-sensitive INa is involved in SAN pacemaking in the newborn rabbit13 and in the adult mouse.12,14 The cardiac TTX-resistant SCN5A-mediated INa is important for conduction from the SAN to the atrium and in intranodal conduction.15 INa is also expressed in the rabbit16 and guinea pig17 AVN and contributes to the fast AV conduction pathway.18 Consistently, heterozygous mice lacking SCN5A channels have major AV conduction dysfunction.19
ICa,T has also been proposed to contribute to pacemaking in primary SAN20 as well as in latent pacemaker cells of the right atrium (RA).21 ICa,T is expressed in pacemaker cells of mammals, including the rabbit20 and the mouse.22 ICa,T is also expressed in the cardiac conduction system, the rabbit AVN,18 and in canine Purkinje cells.23,24 The functional role of ICa,T in the cardiac primary pacemaker and conduction tissue remains to be elucidated.
Molecular cloning of 3 T-type Ca2+ channel pore-forming subunits, namely the CaV3.1/1G, CaV3.2/1H, and CaV3.3/1I subunits, has fostered investigation of the role of native ICa,T.25 Here, we describe that CaV3.1 knockout (CaV3.1–/–) mice lack ICa,T in both SAN pacemaker and AVN cells and display slowed pacemaker activity and AV conduction. Our results constitute the first direct functional demonstration of the participation of Cav3.1 channels in pacemaking and cardiac conduction.
Materials and Methods
The generation of the CaV3.1–/– mouse line was originally described by Kim et al.26 The experimental procedure for electrophysiological recording of isolated cells from the SAN, the AVN, and the RA is described in the expanded Materials and Methods section in the online data supplement, available at http://circres.ahajournals.org. Detailed procedures for in vivo surface and intracardiac electrocardiograms (ECGs), measurement of blood pressure, RT-PCR, and quantitative RT-PCR experiments (supplemental Figure II) and numerical modeling of pacemaker activity are available in the expanded online Materials and Methods section. Results are presented as the mean±SEM. Statistical significance was assessed by the unpaired Student’s t test. A value of P<0.05 was considered as statistically significant. N indicates the number of mice used and n the number of cells considered.
Results
CaV3.1–/– SAN Pacemaker Cells Lack ICa,T
Electrophysiological recordings obtained on SAN cells of wild-type (WT) and CaV3.1–/– animals revealed that ICa,T related to CaV3.1 is a major component of the total Ca2+ current (ICa) in SAN cells (Figure 1). Figure 1A shows representative ICa traces recorded from a holding potential (HP) of –90 mV in WT (Figure 1Aa) and CaV3.1–/– (Figure 1Ab) SAN cells. No difference in the cell capacitance was detected (see supplementary Figure I). Corresponding current–voltage (I-V) curves are presented in Figure 1B (filled symbols). ICa,L was measured by applying depolarizing steps from a HP of –55 mV (Figure 1B, open symbols), revealing ICa,T by subtraction of records from a HP of –55 mV from those obtained from a HP of –90 mV (Figure 1Ba, dotted line). Consistently, switching the HP from –90 to –55 mV significantly shifted ICa voltage for half activation (V0.5act) to more positive values (–45±1 mV; k=5.7±0.6 mV from a HP of –90 mV and –28±1 mV; k=6.3±0.3 mV from a HP of –55 mV, N=9, n=17, P<0.05) in all tested cells (n=17), revealing further a V0.5act of –47±1 mV for the net ICa,T (Figure 1Ba, dotted line). In contrast to WT SAN cells, none of the tested Cav3.1–/– cells displayed detectable ICa,T (Figure 1Bb). Indeed, in CaV3.1–/– SAN cells, switching to a HP of –55 mV did not alter significantly voltage dependence of ICa activation (HP, –90 mV: V0.5act=–30±1 mV; k=8.0±1.3 mV, n=14; and HP –55 mV: –28±1 mV; k=6.3±0.3 mV, N=10, n=15, P=NS). Subtraction between traces from HP –90 mV and from HP –55 mV identified no residual ICa,T, because current waveforms displayed slow inactivation kinetics (Figure 1Bb, inset) and no criss-crossing of the current traces. A comparison of the current density values in WT and CaV3.1–/– SAN cells at HP –90 mV is shown in Figure 1C (black and white bars, respectively) to estimate the ICa,T component related to Cav3.1. At –40 mV, ICa,T density was 6.8±1.6 pA/pF (+/+, N=9, n=18) and ICa,L density was 2.6±0.7 pA/pF, (–/–, N=10, n=14). ICa,L density was not significantly affected by inactivation of Cav3.1 channels (Figure 1D). Indeed, ICa,L peak densities at –10 mV (HP, –55 mV) were 6.0±1.6 pA/pF, N=9, n=15; and 4.5±0.6 pA/pF, N=10, n=11 in CaV3.1–/– and WT SAN cells, respectively. Taken together, these data showed that inactivation of Cav3.1 channels abolished ICa,T and had no significant effect on ICa,L in SAN cells.
To determine whether the pharmacological sensitivity of the SAN ICa,T is consistent with the expression of the Cav3.1 subunit, we tested its sensitivity to Ni2+ ions (Figure 2A and 2B). Application of Ni2+ concentrations of 50 and 200 μmol/L inhibited 26±9% (N=2, n=4) and 61±10% (n=9) of ICa,T, respectively. Because the IC50 values for Ni2+ on recombinant CaV3.1 and CaV3.2 T-type channels are in the range of 100 to 200 μmol/L and 5 to 10 μmol/L, respectively,27 these results indicate that ICa,T in mouse adult SAN cells is generated by the CaV3.1 subunit. Consistent with these findings, real-time RT-PCR experiments showed that the Cav3.1 subunit is the predominant T-type Ca2+ channel isotype in the SAN of the adult mouse (supplemental Figure II). Furthermore, application of 2 μmol/L of the 1,4-dihydropyridine ICa,L blocker isradipine on Cav3.1–/– SAN cells blocked ICa by 93±4% (N=5, n=7, Figure 2C and 2E) from a HP of –90 mV and by 100% from a HP of –55 mV (N=5, n=8, Figure 2D and 2F). Such a sensitivity to isradipine further confirmed that the remaining ICa in SAN cells from CaV3.1–/– mice was ICa,L.
Sedated CaV3.1–/– Mice Have Intrinsic Slower Heart Rate and Prolonged AV Conduction
We next performed surface ECG recordings on sedated WT and CaV3.1–/– mice (Figure 3). Because the heart rate is highly regulated by the balance between the sympathetic and vagal input, which depends on the levels of stress and anesthesia, ECGs were also recorded after injection of propranolol and atropine to block the autonomic nervous system (ANS).28 Recording examples collected from WT (+/+) and CaV3.1–/– (–/–) mice before and after the ANS block are presented in Figure 3A. The loss of the CaV3.1 subunit induced a significant (9%) prolongation of the atrioventricular conduction (PQ interval) under baseline conditions (34±1 ms, N=11, and 37±1 ms, N=12 for WT and CaV3.1–/– mice, respectively; P<0.05). The heart beat (RR interval), P wave duration, and QRS interval (Figure 3B), as well as the QTc interval (data not shown; 62±1 ms and 61±1 ms for WT and CaV3.1–/– mice, respectively; P>0.05), were not significantly modified. In conditions of ANS block, the intrinsic heart rate was significantly slowed by &10%. Indeed, the RR interval was significantly longer in CaV3.1–/– mice than in WT animals (144±2 ms and 132±2 ms, respectively; P<0.05; Figure 3B). The PQ interval was also markedly prolonged (34±1 ms and 40±1 ms in WT and CaV3.1–/– animals, respectively; P<0.001; Figure 4B). The other ECG parameters were not significantly modified.
SAN Cells From CaV3.1–/– Mice Display Slowed Pacemaker Activity
The spontaneous activity of isolated SAN cells was studied to evaluate whether the slowing of the intrinsic heart rate observed in vivo in CaV3.1–/– mice was associated with dysfunction of SAN cell automaticity. Representative recordings obtained on WT and CaV3.1–/– SAN cells are presented in Figure 4A (upper and lower traces, respectively). Analysis of these recordings showed that inactivation of cacna1g gene resulted in a 37% slowing of the cellular beating rate (163±13 bpm, N=4, n=15 in CaV3.1–/–, and 234±19 bpm, N=5, n=14 in SAN cells from WT mice; P<0.01; Figure 4B). Slowing of cellular pacemaking was accompanied by a reduction of the diastolic depolarization slope of &44% (Figure 4C). No significant changes in the maximum diastolic potential (MDP) and the voltage threshold of the action potential upstroke (Eth) were observed (MDP: –56.9±1.3 mV in WT, n=15, and –58.2±1.4 mV for CaV3.1–/– SAN cells, n=14; Eth: –45±1.3 mV in WT and –43±1.4 mV in CaV3.1–/– SAN cells; Figure 4D). Also, the action potential duration (147±15 and 141±10 ms for CaV3.1–/– and WT SAN cells, respectively; Figure 4E) and the action potential amplitude (APA) (92±8 and 90±8 mV for CaV3.1–/– and WT SAN cells, respectively; not shown) were comparable in SAN cells from WT and Cav3.1–/– mice.
Intracardiac Electrophysiology Study of CaV3.1–/– Mice
The AV conduction delay in mice lacking Cav3.1 channels was measured under baseline intracardiac recording conditions. Eight of 10 WT and 7 of 9 CaV3.1–/– mice had a distinctly visible His-bundle electrogram (Figure 5A). In this group of animals, atrial-His (AH) conduction times were significantly longer in CaV3.1–/– mice (31±1 ms) than in WT animals (25±1 ms; P<0.001; Figure 5B). However, His-ventricle (HV) intervals were comparable (11±1 and 10±1 ms for WT and CaV3.1–/– mice, respectively; P>0.05). Effective refractory periods at the atrial (AERP), atrioventricular (AVERP), and ventricular (VERP) levels were investigated at a basic cycle length of 100 ms (Figure 5C). AERP and VERP were not significantly different between WT and Cav3.1–/– mice. In contrast, the AVERP was significantly longer in CaV3.1–/– mice (58±6 ms; N=8) than in WT animals (44±2 ms; N=10; P<0.05). Decremental atrial pacing revealed that Wenckebach cycle length was significantly longer in CaV3.1–/– mice than in WT mice (85±5 ms and 67±2 ms, respectively; P<0.01; Figure 5C). Atrial pacing confirmed SAN dysfunction. Indeed, the corrected SAN recovery time (cSNRT) was longer in CaV3.1–/– mice than in WT mice (31±5 in Cav3.1–/– and 17±2 ms in WT mice P<0.05; n=7 and 9, respectively).
AVN Cells From CaV3.1–/– Mice Lack ICa,T
We then tested whether dysfunction of AV conduction observed on inactivation of Cav3.1 channels was associated with downregulation of ICa,T in the AVN. ICa,T and ICa,L were recorded in isolated AVN cells using the same recording protocols as for SAN cells (Figure 6). ICa,T was found in all the WT AVN cells investigated (Figure 6A). Indeed, switching the HP from –90 to –60 mV significantly shifted the V0.5act of the total ICa from –34±0.7 to –23±0.8 mV (n=7, P<0.05). In contrast, no ICa,T could be recorded in Cav3.1–/– AVN cells (Figure 6Ab), and switching the HP from –90 to –60 mV did not shift the V0.5act value (–25±1.3 mV to –27±0.8 mV, respectively; P=NS; Figure 6Bb). The peak density of the residual ICa measured from a HP of –60 mV in AVN cells was not altered by inactivation of Cav3.1 channels (4.1±0.5 pA/pF, N=5, n=7 in WT and 4.6±0.7 pA/pF, N=4, n=7 in Cav3.1–/–; P=NS). In addition, no ICa,T could be detected in isolated cells from the RA of WT adult mice (Figure 6C and 6D), because switching the HP from –100 to –50 mV produced no change both in the V0.5act (–16±2 and –17±1 mV, N=3, n=8, P=NS; Figure 6D) and in the peak density (6.9±1.5 and 7±1 pA/pF, N=3, n=8, P=NS; Figure 6C).
Bradycardia in Conscious Unrestrained CaV3.1–/– Mice
Both the heart rate and the AV conduction were altered in freely moving CaV3.1–/– mice (Figure 7A and 7B and the Table). The mean heart rate was significantly slowed in CaV3.1–/– mice, when considering a 24-hour period or during the day and night periods (Figure 7B and the Table). The maximum cardiac frequency during short periods of activity (40 seconds) was unchanged (Table), suggesting that activation of other ionic channels involved in SAN automaticity can compensate for the lack of ICa,T during strong activation of the sympathetic nervous system. Spectral analysis of RR signals in the frequency domain revealed no differences between the 2 groups of mice. Total spectral power, ultralow (ULF), very-low (VLF), low (LF), and high (HF) frequencies were not significantly modified between WT and CaV3.1–/– mice (Figure 7C, data not shown for ULF and VLF), indicating that the degree of autonomic regulation of heart rate was not altered in CaV3.1–/– mice.
Telemetric recordings also confirmed the prolongation of the PQ interval in Cav3.1–/– mice (Table), demonstrating that the propagation of the heartbeat through the AV conduction system was delayed in CaV3.1–/– mice. This effect was independent from the heart rate because measurements were performed at comparable heart rates (600 bpm; cycle length of 100 ms). In contrast, the atrial conduction (as assessed by the P wave duration), the ventricular conduction (QRS interval), and ventricular repolarization (QT interval) were unchanged (Table). In addition, we did not find evidence for cardiac arrhythmias in CaV3.1–/– mice. Also, no significant difference in the systolic, diastolic, and mean arterial pressure (60±8 mm Hg, N=8 in WT and 72±5 mm Hg, N=8 in Cav3.1–/– mice; P=NS) was observed between the 2 mouse strains (Figure 7D).
Discussion
The major finding of this study is that genetic inactivation of the CaV3.1/1G T-type Ca2+ channels in mice results in a significant slowing of the heart rate and AV conduction. Downregulation of ICa,T in both SAN and AVN cells of CaV3.1–/– animals is documented, accounting for this in vivo phenotype. The lack of Cav3.1 channels in isolated SAN cells induces slowing of pacemaker activity through a reduction of the slope of the diastolic depolarization. Our study provides novel and compelling genetic evidence for a direct contribution of Cav3.1 channels in the setting of the mammalian cardiac impulse generation and propagation by contributing to both the diastolic depolarization in the SAN and impulse conduction through the AVN.
ICa,T in SAN and AVN Cells Following Inactivation of Cav3.1 Channels
A striking observation of this study is that ICa,T in the mouse SAN is predominantly generated by the CaV3.1 subunit because we found no evidence for a residual ICa,T in Cav3.1–/– mice in both SAN (Figure 1) and AVN cells (Figure 6). The low sensitivity of SAN ICa,T to Ni2+ is consistent with that observed on recombinant Cav3.1-mediated ICa,T.27 Also consistent with our general findings is the recent observation that CaV3.2–/– mice showed no ECG alterations.29 Inactivation of Cav3.1 channels did not significantly modify the expression of ICa,L in Cav3.1–/– in both SAN and AVN cells. Our data show, therefore, that expression of CaV3.2, Cav1.3, and Cav1.2 subunits do not compensate for the lack of Cav3.1 channels in Cav3.1–/– mice.
Bradycardia and Atrioventricular Dysfunction in Cav3.1–/– Mice
ECGs and intracardiac recordings document both bradycardia and slowing of the atrioventricular conduction in CaV3.1–/– mice. Several lines of evidence indicate that bradycardia is attributable to slowing of the pacemaker activity in the SAN. First, bradycardia was evident in sedated mice under pharmacological blockade of the ANS input for heart rates that were comparable with the mean heart rate observed in freely moving mice. Second, slowing of the heart rate was observed in freely moving mice in the absence of a significant change both in the heart rate variability profile and in the arterial blood pressure, thus arguing against the hypothesis that bradycardia would be caused by dysfunction in the autonomic regulation of the heart rate or could be secondary to a change in the control of the vascular tone. The prolongation of the SAN recovery time also indicates dysfunction in SAN automaticity. Finally, the slowing of pacemaker activity observed in isolated SAN pacemaker cells of Cav3.1–/– mice is consistent with bradycardia being attributable to the lack of ICa,T in the SAN. The reduction of the mean heart rate measured on freely moving Cav3.1–/– mice is moderate, compared with the slowing of the cycle length in isolated pacemaker cells (10% in freely moving mice and 37% in isolated cells). This difference can be caused by the compensatory adrenergic tone in vivo, which stimulates ionic mechanisms involved in pacemaking, such as Cav1.3-mediated ICa,L9,10 and If6 and RyR-dependent diastolic release of Ca2+.11 The observation that the maximal heart rate is comparable in WT and Cav3.1–/– mice is consistent with this hypothesis. Comparison of mice lacking Cav1.39 and Cav3.1 channels (this study) indicate that ICa,L and ICa,T play distinct roles in pacemaker activity in vivo. Indeed, knockout of Cav1.3 channels reduces the basal and intrinsic heart rate in sedated conditions by approximately 20% and 60%, respectively,9 compared with the 10% reduction observed in mice lacking Cav3.1 channels in both freely moving and block of ANS conditions. These observations suggest that Cav3.1 channels contribute to the setting of the basal heart rate but have less impact on the dynamic regulation of pacemaker activity by the ANS. The lack of a significant change in the variability of the RR intervals in Cav3.1 mice is consistent with this hypothesis.
We report that the slowing of the AV conduction in Cav3.1–/– mice is caused by dysfunction in the excitability of the AVN. Indeed, intracardiac recordings show a delay in the AH conduction time and AVERP, in the absence of a prolongation of the P wave, which indicates similar conduction velocities in atria from WT and Cav3.1–/– mice. Consistently with ECGs and intracardiac recordings, we found no ICa,T in atrial myocytes from WT mice. Also, AVN cells display ICa,T related to CaV3.1 channels because it was no longer detectable in CaV3.1–/– mice. We did not find significant changes in the HV, QRS, and QT intervals (see Figures 3 and 5 and supplemental Figure III), indicating that the lack of Cav3.1 channels does not affect conduction through the His–Purkinje fiber network or the ventricular muscle. This phenotype differs from that of SCN5A+/– mice,19 which shows prominent alteration of the P wave and the QRS complex duration,30 stressing the importance of the cardiac INa in intraatrial and His–Purkinje conduction. Also in contrast with SCN5A+/–,19 Cav3.1–/– mice do not present ventricular tachiarrhythmias. This SCN5A-mediated INa can possibly compensate for the lack of ICa,T in the His–Purkinje system.
Pacemaking in Cav3.1–/– Mice
We describe that inactivation of CaV3.1 channels significantly prolongs the pacemaker cycle length in isolated SAN cells by reducing the slope of the diastolic depolarization, demonstrating the involvement of Cav3.1-mediated ICa,T in the setting of the diastolic depolarization in mouse SAN cells. In an attempt to compare the physiological roles of Cav3.1-mediated ICa,T with that of Cav1.3-mediated ICa,L and INa, we have developed a numerical model of mouse SAN electrophysiology based on previous studies on mouse SAN pacemaking in normal and genetically modified mouse strains (see supplemental Figures IV and V). Our numerical simulations indicate that Cav3.1 channels contribute to pacemaking by activating during the diastolic depolarization for more negative voltages than TTX-sensitive INa and Cav1.3-mediated ICa,L (see supplemental Figure VB and VE). As a consequence, Cav3.1 channels can accelerate the diastolic depolarization rate in a voltage range in which INa is still not activated. This property can explain, at least in part, how Cav3.1 channels can contribute to pacemaking in spite of their relatively low availability at SAN diastolic potentials. An expanded discussion about the development, the features and limitations of our numerical model, and the physiological significance of Cav3.1-mediated ICa,T compared with that of ICa,L and INa is available in the online data supplement.
Conclusion
In conclusion, our study demonstrates that CaV3.1 channels contribute to the heartbeat by influencing pacemaking and the AV conduction. To date, the presence of T-type channels in human heart is poorly documented. Molecular analyses, such as dot blots and Northern blots of human heart mRNAs, have identified transcripts for CaV3.1 and CaV3.2.31,32 T-type channels may play a role in the human SAN and the conduction system. For instance, the causality between congenital heart block induced by maternal autoantibodies against T-type channels and children showing SAN bradycardia and atrioventricular block has recently been documented.33 There is growing evidence that T-type channels may constitute a promising pharmacological target for the treatment of human diseases, such as epilepsy and chronic pain.34 From our observations, T-type channel inhibition would have no deleterious consequences in cardiac physiology. It is, therefore, tempting to speculate that selective blockers of CaV3.1 channels may hold promise for the therapeutic management of the cardiac diseases that require moderate heart rate reduction, such as cardiac ischemia and coronary heart disease.
Acknowledgments
This work was supported by the Association Franaise Contre les Myopathies, the Action Concertée Incitative (Developmental Biology and Integrative Physiology) of the French Ministry for Education, the Fondation de France, and the Chemoinformatics Program of Korean Institute of Science and Technology, Korea. A.T. is supported by a fellowship from the CNRS Lebanon. We are grateful to Patrick Atger for excellent technical assistance.
Footnotes
Both authors contributed equally to this study.
Original received November 5, 2004; resubmission received December 5, 2005; revised resubmission received March 30, 2006; accepted April 27, 2006.
References
Irisawa H, Brown HF, Giles W. Cardiac pacemaking in the sinoatrial node. Physiol Rev. 1993; 73: 197–227.
Boyett MR, Honjo H, Kodama I. The sinoatrial node, a heterogeneous pacemaker structure. Cardiovasc Res. 2000; 47: 658–687. [Order article via Infotrieve]
Difrancesco D. Serious workings of the funny current. Prog Biophys Mol Biol. 2006; 90: 13–25. [Order article via Infotrieve]
Lakatta EG, Maltsev VA, Bogdanov KY, Stern MD, Vinogradova TM. Cyclic variation of intracellular calcium: a critical factor for cardiac pacemaker cell dominance. Circ Res. 2003; 92: e45–e50.
Honjo H, Inada S, Lancaster MK, Yamamoto M, Niwa R, Jones SA, Shibata N, Mitsui K, Horiuchi T, Kamiya K, Kodama I, Boyett MR. Sarcoplasmic reticulum Ca2+ release is not a dominating factor in sinoatrial node pacemaker activity. Circ Res. 2003; 92: e41–e44.
Baruscotti M, Bucchi A, Difrancesco D. Physiology and pharmacology of the cardiac pacemaker ("funny") current. Pharmacol Ther. 2005; 107: 59–79. [Order article via Infotrieve]
Stieber J, Herrmann S, Feil S, Loster J, Feil R, Biel M, Hofmann F, Ludwig A. The hyperpolarization-activated channel HCN4 is required for the generation of pacemaker action potentials in the embryonic heart. Proc Natl Acad Sci U S A. 2003; 100: 15235–15240.
Verheijck EE, van Ginneken AC, Wilders R, Bouman LN. Contribution of L-type Ca2+ current to electrical activity in sinoatrial nodal myocytes of rabbits. Am J Physiol. 1999; 276: H1064–H1077.
Zhang Z, Xu Y, Song H, Rodriguez J, Tuteja D, Namkung Y, Shin HS, Chiamvimonvat N. Functional Roles of Cav1.3 1D calcium channel in sinoatrial nodes: insight gained using gene-targeted null mutant mice. Circ Res. 2002; 90: 981–987.
Mangoni ME, Couette B, Bourinet E, Platzer J, Reimer D, Striessnig J, Nargeot J. Functional role of L-type Cav1.3 Ca2+ channels in cardiac pacemaker activity. Proc Natl Acad Sci U S A. 2003; 100: 5543–5548.
Vinogradova TM, Bogdanov KY, Lakatta EG. beta-Adrenergic stimulation modulates ryanodine receptor Ca2+ release during diastolic depolarization to accelerate pacemaker activity in rabbit sinoatrial nodal cells. Circ Res. 2002; 90: 73–79. [Order article via Infotrieve]
Lei M, Jones SA, Liu J, Lancaster MK, Fung SS, Dobrzynski H, Camelliti P, Maier SK, Noble D, Boyett MR. Requirement of neuronal- and cardiac-type sodium channels for murine sinoatrial node pacemaking. J Physiol. 2004; 559: 835–848.
Baruscotti M, DiFrancesco D, Robinson RB. A TTX-sensitive inward sodium current contributes to spontaneous activity in newborn rabbit sino-atrial node cells. J Physiol. 1996; 492: 21–30.
Maier SK, Westenbroek RE, Yamanushi TT, Dobrzynski H, Boyett MR, Catterall WA, Scheuer T. An unexpected requirement for brain-type sodium channels for control of heart rate in the mouse sinoatrial node. Proc Natl Acad Sci U S A. 2003; 100: 3507–3512.
Lei M, Goddard C, Liu J, Leoni AL, Royer A, Fung SS, Xiao G, Ma A, Zhang H, Charpentier F, Vandenberg JI, Colledge WH, Grace AA, Huang CL. Sinus node dysfunction following targeted disruption of the murine cardiac sodium channel gene Scn5a. J Physiol. 2005; 567: 387–400.
Munk AA, Adjemian RA, Zhao J, Ogbaghebriel A, Shrier A. Electrophysiological properties of morphologically distinct cells isolated from the rabbit atrioventricular node. J Physiol. 1996; 493: 801–818.
Yuill KH, Hancox JC. Characteristics of single cells isolated from the atrioventricular node of the adult guinea-pig heart. Pflugers Arch. 2002; 445: 311–320. [Order article via Infotrieve]
Efimov IR, Nikolski VP, Rothenberg F, Greener ID, Li J, Dobrzynski H, Boyett M. Structure-function relationship in the AV junction. Anat Rec A Discov Mol Cell Evol Biol. 2004; 280: 952–965. [Order article via Infotrieve]
Papadatos GA, Wallerstein PM, Head CE, Ratcliff R, Brady PA, Benndorf K, Saumarez RC, Trezise AE, Huang CL, Vandenberg JI, Colledge WH, Grace AA. Slowed conduction and ventricular tachycardia after targeted disruption of the cardiac sodium channel gene Scn5a. Proc Natl Acad Sci U S A. 2002; 99: 6210–6215.
Hagiwara N, Irisawa H, Kameyama M. Contribution of two types of calcium currents to the pacemaker potentials of rabbit sino-atrial node cells. J Physiol. 1988; 395: 233–253.
Huser J, Blatter LA, Lipsius SL. Intracellular Ca2+ release contributes to automaticity in cat atrial pacemaker cells. J Physiol. 2000; 524: 415–422.
Mangoni ME, Nargeot J. Properties of the hyperpolarization-activated current (If) in isolated mouse sino-atrial cells. Cardiovasc Res. 2001; 52: 51–64. [Order article via Infotrieve]
Hirano Y, Fozzard HA, January CT. Characteristics of L- and T-type Ca2+ currents in canine cardiac Purkinje cells. Am J Physiol. 1989; 256: H1478–H1492.
Tseng GN, Boyden PA. Multiple types of Ca2+ currents in single canine Purkinje cells. Circ Res. 1989; 65: 1735–1750.
Perez-Reyes E. Molecular physiology of low-voltage-activated T-type calcium channels. Physiol Rev. 2003; 83: 117–161.
Kim D, Song I, Keum S, Lee T, Jeong MJ, Kim SS, McEnery MW, Shin HS. Lack of the burst firing of thalamocortical relay neurons and resistance to absence seizures in mice lacking 1G T-type Ca2+ channels. Neuron. 2001; 31: 35–45. [Order article via Infotrieve]
Lee JH, Gomora JC, Cribbs LL, Perez-Reyes E. Nickel block of three cloned T-type calcium channels: low concentrations selectively block alpha1H. Biophys J. 1999; 77: 3034–3042.
Lande G, Demolombe S, Bammert A, Moorman A, Charpentier F, Escande D. Transgenic mice overexpressing human KvLQT1 dominant-negative isoform. Part II: pharmacological profile. Cardiovasc Res. 2001; 50: 328–334. [Order article via Infotrieve]
Chen CC, Lamping KG, Nuno DW, Barresi R, Prouty SJ, Lavoie JL, Cribbs LL, England SK, Sigmund CD, Weiss RM, Williamson RA, Hill JA, Campbell KP. Abnormal coronary function in mice deficient in 1H T-type Ca2+ channels. Science. 2003; 302: 1416–1418.
Royer A, van Veen TA, Le Bouter S, Marionneau C, Griol-Charhbili V, Leoni AL, Steenman M, van Rijen HV, Demolombe S, Goddard CA, Richer C, Escoubet B, Jarry-Guichard T, Colledge WH, Gros D, de Bakker JM, Grace AA, Escande D, Charpentier F. Mouse model of SCN5A-linked hereditary Lenegre’s disease: age-related conduction slowing and myocardial fibrosis. Circulation. 2005; 111: 1738–1746.
Cribbs LL, Lee JH, Yang J, Satin J, Zhang Y, Daud A, Barclay J, Williamson MP, Fox M, Rees M, Perez-Reyes E. Cloning and characterization of alpha1H from human heart, a member of the T-type Ca2+ channel gene family. Circ Res. 1998; 83: 103–109.
Monteil A, Chemin J, Bourinet E, Mennessier G, Lory P, Nargeot J. Molecular and functional properties of the human 1G subunit that forms T-type calcium channels. J Biol Chem. 2000; 275: 6090–6100.
Hu K, Qu Y, Yue Y, Boutjdir M. Functional basis of sinus bradycardia in congenital heart block. Circ Res. 2004; 94: e32–e38.
Birch PJ, Dekker LV, James IF, Southan A, Cronk D. Strategies to identify ion channel modulators: current and novel approaches to target neuropathic pain. Drug Discov Today. 2004; 9: 410–418. [Order article via Infotrieve]
the Institut de Génomique Fonctionnelle (M.E.M., A.T., B.C., L.M., E.K., A.C.-S., J.N., P.L.), CNRS UMR5203-INSERM U661-Université de Montpellier I–Université de Montpellier II, Département de Physiologie, Montpellier
Institut du Thorax (A.-L.L., K.L.Q., D.E., F.C.), INSERM U533, Faculté de Médecine, Nantes
Cardiovascular Research Center (J.V.), INSERM U689, Université Paris 7-Denis Diderot, Paris, France
Center for Calcium and Learning (H.-S.S.), Korea Institute of Science and Technology, Cheongryang, Seoul, Republic of Korea.
Abstract
The generation of the mammalian heartbeat is a complex and vital function requiring multiple and coordinated ionic channel activities. The functional role of low-voltage activated (LVA) T-type calcium channels in the pacemaker activity of the sinoatrial node (SAN) is, to date, unresolved. Here we show that disruption of the gene coding for Cav3.1/1G T-type calcium channels (cacna1g) abolishes T-type calcium current (ICa,T) in isolated cells from the SAN and the atrioventricular node without affecting the L-type Ca2+ current (ICa,L). By using telemetric electrocardiograms on unrestrained mice and intracardiac recordings, we find that cacna1g inactivation causes bradycardia and delays atrioventricular conduction without affecting the excitability of the right atrium. Consistently, no ICa,T was detected in right atrium myocytes in both wild-type and Cav3.1–/– mice. Furthermore, inactivation of cacna1g significantly slowed the intrinsic in vivo heart rate, prolonged the SAN recovery time, and slowed pacemaker activity of individual SAN cells through a reduction of the slope of the diastolic depolarization. Our results demonstrate that Cav3.1/T-type Ca2+ channels contribute to SAN pacemaker activity and atrioventricular conduction.
Key Words: pacemaker activity T-type calcium channel sinoatrial node conduction knockout mice
Introduction
The initiation of the heartbeat requires coordination between the automaticity of the sinoatrial node (SAN) and excitability of the atrioventricular (AV) conduction tissue: the AV node (AVN) and the His–Purkinje fiber network. SAN automaticity is caused by the presence of the diastolic depolarization.1 Multiple classes of ionic channels are expressed in the SAN,1,2 but the precise mechanism initiating the diastolic depolarization has not been entirely elucidated, and the relative contribution of different ionic channels in establishing the heart rate under specific physiological conditions is still a matter of debate.3–5 Strong functional, pharmacological, and genetic evidence show that the hyperpolarization-activated currents If6,7 and ICa,L8–10 play major roles in controlling the diastolic depolarization after decaying of the fast component of delayed-rectifier K+ currents (IKr). Diastolic release of Ca2+ mediated by ryanodine receptors (RyRs) has been indicated as an important mechanism for controlling SAN pacemaking under activation of the -adrenergic receptor.11 The SAN also expresses neuronal tetrodotoxin (TTX)-sensitive and cardiac TTX-resistant voltage-dependent Na+ currents (INa).12 TTX-sensitive INa is involved in SAN pacemaking in the newborn rabbit13 and in the adult mouse.12,14 The cardiac TTX-resistant SCN5A-mediated INa is important for conduction from the SAN to the atrium and in intranodal conduction.15 INa is also expressed in the rabbit16 and guinea pig17 AVN and contributes to the fast AV conduction pathway.18 Consistently, heterozygous mice lacking SCN5A channels have major AV conduction dysfunction.19
ICa,T has also been proposed to contribute to pacemaking in primary SAN20 as well as in latent pacemaker cells of the right atrium (RA).21 ICa,T is expressed in pacemaker cells of mammals, including the rabbit20 and the mouse.22 ICa,T is also expressed in the cardiac conduction system, the rabbit AVN,18 and in canine Purkinje cells.23,24 The functional role of ICa,T in the cardiac primary pacemaker and conduction tissue remains to be elucidated.
Molecular cloning of 3 T-type Ca2+ channel pore-forming subunits, namely the CaV3.1/1G, CaV3.2/1H, and CaV3.3/1I subunits, has fostered investigation of the role of native ICa,T.25 Here, we describe that CaV3.1 knockout (CaV3.1–/–) mice lack ICa,T in both SAN pacemaker and AVN cells and display slowed pacemaker activity and AV conduction. Our results constitute the first direct functional demonstration of the participation of Cav3.1 channels in pacemaking and cardiac conduction.
Materials and Methods
The generation of the CaV3.1–/– mouse line was originally described by Kim et al.26 The experimental procedure for electrophysiological recording of isolated cells from the SAN, the AVN, and the RA is described in the expanded Materials and Methods section in the online data supplement, available at http://circres.ahajournals.org. Detailed procedures for in vivo surface and intracardiac electrocardiograms (ECGs), measurement of blood pressure, RT-PCR, and quantitative RT-PCR experiments (supplemental Figure II) and numerical modeling of pacemaker activity are available in the expanded online Materials and Methods section. Results are presented as the mean±SEM. Statistical significance was assessed by the unpaired Student’s t test. A value of P<0.05 was considered as statistically significant. N indicates the number of mice used and n the number of cells considered.
Results
CaV3.1–/– SAN Pacemaker Cells Lack ICa,T
Electrophysiological recordings obtained on SAN cells of wild-type (WT) and CaV3.1–/– animals revealed that ICa,T related to CaV3.1 is a major component of the total Ca2+ current (ICa) in SAN cells (Figure 1). Figure 1A shows representative ICa traces recorded from a holding potential (HP) of –90 mV in WT (Figure 1Aa) and CaV3.1–/– (Figure 1Ab) SAN cells. No difference in the cell capacitance was detected (see supplementary Figure I). Corresponding current–voltage (I-V) curves are presented in Figure 1B (filled symbols). ICa,L was measured by applying depolarizing steps from a HP of –55 mV (Figure 1B, open symbols), revealing ICa,T by subtraction of records from a HP of –55 mV from those obtained from a HP of –90 mV (Figure 1Ba, dotted line). Consistently, switching the HP from –90 to –55 mV significantly shifted ICa voltage for half activation (V0.5act) to more positive values (–45±1 mV; k=5.7±0.6 mV from a HP of –90 mV and –28±1 mV; k=6.3±0.3 mV from a HP of –55 mV, N=9, n=17, P<0.05) in all tested cells (n=17), revealing further a V0.5act of –47±1 mV for the net ICa,T (Figure 1Ba, dotted line). In contrast to WT SAN cells, none of the tested Cav3.1–/– cells displayed detectable ICa,T (Figure 1Bb). Indeed, in CaV3.1–/– SAN cells, switching to a HP of –55 mV did not alter significantly voltage dependence of ICa activation (HP, –90 mV: V0.5act=–30±1 mV; k=8.0±1.3 mV, n=14; and HP –55 mV: –28±1 mV; k=6.3±0.3 mV, N=10, n=15, P=NS). Subtraction between traces from HP –90 mV and from HP –55 mV identified no residual ICa,T, because current waveforms displayed slow inactivation kinetics (Figure 1Bb, inset) and no criss-crossing of the current traces. A comparison of the current density values in WT and CaV3.1–/– SAN cells at HP –90 mV is shown in Figure 1C (black and white bars, respectively) to estimate the ICa,T component related to Cav3.1. At –40 mV, ICa,T density was 6.8±1.6 pA/pF (+/+, N=9, n=18) and ICa,L density was 2.6±0.7 pA/pF, (–/–, N=10, n=14). ICa,L density was not significantly affected by inactivation of Cav3.1 channels (Figure 1D). Indeed, ICa,L peak densities at –10 mV (HP, –55 mV) were 6.0±1.6 pA/pF, N=9, n=15; and 4.5±0.6 pA/pF, N=10, n=11 in CaV3.1–/– and WT SAN cells, respectively. Taken together, these data showed that inactivation of Cav3.1 channels abolished ICa,T and had no significant effect on ICa,L in SAN cells.
To determine whether the pharmacological sensitivity of the SAN ICa,T is consistent with the expression of the Cav3.1 subunit, we tested its sensitivity to Ni2+ ions (Figure 2A and 2B). Application of Ni2+ concentrations of 50 and 200 μmol/L inhibited 26±9% (N=2, n=4) and 61±10% (n=9) of ICa,T, respectively. Because the IC50 values for Ni2+ on recombinant CaV3.1 and CaV3.2 T-type channels are in the range of 100 to 200 μmol/L and 5 to 10 μmol/L, respectively,27 these results indicate that ICa,T in mouse adult SAN cells is generated by the CaV3.1 subunit. Consistent with these findings, real-time RT-PCR experiments showed that the Cav3.1 subunit is the predominant T-type Ca2+ channel isotype in the SAN of the adult mouse (supplemental Figure II). Furthermore, application of 2 μmol/L of the 1,4-dihydropyridine ICa,L blocker isradipine on Cav3.1–/– SAN cells blocked ICa by 93±4% (N=5, n=7, Figure 2C and 2E) from a HP of –90 mV and by 100% from a HP of –55 mV (N=5, n=8, Figure 2D and 2F). Such a sensitivity to isradipine further confirmed that the remaining ICa in SAN cells from CaV3.1–/– mice was ICa,L.
Sedated CaV3.1–/– Mice Have Intrinsic Slower Heart Rate and Prolonged AV Conduction
We next performed surface ECG recordings on sedated WT and CaV3.1–/– mice (Figure 3). Because the heart rate is highly regulated by the balance between the sympathetic and vagal input, which depends on the levels of stress and anesthesia, ECGs were also recorded after injection of propranolol and atropine to block the autonomic nervous system (ANS).28 Recording examples collected from WT (+/+) and CaV3.1–/– (–/–) mice before and after the ANS block are presented in Figure 3A. The loss of the CaV3.1 subunit induced a significant (9%) prolongation of the atrioventricular conduction (PQ interval) under baseline conditions (34±1 ms, N=11, and 37±1 ms, N=12 for WT and CaV3.1–/– mice, respectively; P<0.05). The heart beat (RR interval), P wave duration, and QRS interval (Figure 3B), as well as the QTc interval (data not shown; 62±1 ms and 61±1 ms for WT and CaV3.1–/– mice, respectively; P>0.05), were not significantly modified. In conditions of ANS block, the intrinsic heart rate was significantly slowed by &10%. Indeed, the RR interval was significantly longer in CaV3.1–/– mice than in WT animals (144±2 ms and 132±2 ms, respectively; P<0.05; Figure 3B). The PQ interval was also markedly prolonged (34±1 ms and 40±1 ms in WT and CaV3.1–/– animals, respectively; P<0.001; Figure 4B). The other ECG parameters were not significantly modified.
SAN Cells From CaV3.1–/– Mice Display Slowed Pacemaker Activity
The spontaneous activity of isolated SAN cells was studied to evaluate whether the slowing of the intrinsic heart rate observed in vivo in CaV3.1–/– mice was associated with dysfunction of SAN cell automaticity. Representative recordings obtained on WT and CaV3.1–/– SAN cells are presented in Figure 4A (upper and lower traces, respectively). Analysis of these recordings showed that inactivation of cacna1g gene resulted in a 37% slowing of the cellular beating rate (163±13 bpm, N=4, n=15 in CaV3.1–/–, and 234±19 bpm, N=5, n=14 in SAN cells from WT mice; P<0.01; Figure 4B). Slowing of cellular pacemaking was accompanied by a reduction of the diastolic depolarization slope of &44% (Figure 4C). No significant changes in the maximum diastolic potential (MDP) and the voltage threshold of the action potential upstroke (Eth) were observed (MDP: –56.9±1.3 mV in WT, n=15, and –58.2±1.4 mV for CaV3.1–/– SAN cells, n=14; Eth: –45±1.3 mV in WT and –43±1.4 mV in CaV3.1–/– SAN cells; Figure 4D). Also, the action potential duration (147±15 and 141±10 ms for CaV3.1–/– and WT SAN cells, respectively; Figure 4E) and the action potential amplitude (APA) (92±8 and 90±8 mV for CaV3.1–/– and WT SAN cells, respectively; not shown) were comparable in SAN cells from WT and Cav3.1–/– mice.
Intracardiac Electrophysiology Study of CaV3.1–/– Mice
The AV conduction delay in mice lacking Cav3.1 channels was measured under baseline intracardiac recording conditions. Eight of 10 WT and 7 of 9 CaV3.1–/– mice had a distinctly visible His-bundle electrogram (Figure 5A). In this group of animals, atrial-His (AH) conduction times were significantly longer in CaV3.1–/– mice (31±1 ms) than in WT animals (25±1 ms; P<0.001; Figure 5B). However, His-ventricle (HV) intervals were comparable (11±1 and 10±1 ms for WT and CaV3.1–/– mice, respectively; P>0.05). Effective refractory periods at the atrial (AERP), atrioventricular (AVERP), and ventricular (VERP) levels were investigated at a basic cycle length of 100 ms (Figure 5C). AERP and VERP were not significantly different between WT and Cav3.1–/– mice. In contrast, the AVERP was significantly longer in CaV3.1–/– mice (58±6 ms; N=8) than in WT animals (44±2 ms; N=10; P<0.05). Decremental atrial pacing revealed that Wenckebach cycle length was significantly longer in CaV3.1–/– mice than in WT mice (85±5 ms and 67±2 ms, respectively; P<0.01; Figure 5C). Atrial pacing confirmed SAN dysfunction. Indeed, the corrected SAN recovery time (cSNRT) was longer in CaV3.1–/– mice than in WT mice (31±5 in Cav3.1–/– and 17±2 ms in WT mice P<0.05; n=7 and 9, respectively).
AVN Cells From CaV3.1–/– Mice Lack ICa,T
We then tested whether dysfunction of AV conduction observed on inactivation of Cav3.1 channels was associated with downregulation of ICa,T in the AVN. ICa,T and ICa,L were recorded in isolated AVN cells using the same recording protocols as for SAN cells (Figure 6). ICa,T was found in all the WT AVN cells investigated (Figure 6A). Indeed, switching the HP from –90 to –60 mV significantly shifted the V0.5act of the total ICa from –34±0.7 to –23±0.8 mV (n=7, P<0.05). In contrast, no ICa,T could be recorded in Cav3.1–/– AVN cells (Figure 6Ab), and switching the HP from –90 to –60 mV did not shift the V0.5act value (–25±1.3 mV to –27±0.8 mV, respectively; P=NS; Figure 6Bb). The peak density of the residual ICa measured from a HP of –60 mV in AVN cells was not altered by inactivation of Cav3.1 channels (4.1±0.5 pA/pF, N=5, n=7 in WT and 4.6±0.7 pA/pF, N=4, n=7 in Cav3.1–/–; P=NS). In addition, no ICa,T could be detected in isolated cells from the RA of WT adult mice (Figure 6C and 6D), because switching the HP from –100 to –50 mV produced no change both in the V0.5act (–16±2 and –17±1 mV, N=3, n=8, P=NS; Figure 6D) and in the peak density (6.9±1.5 and 7±1 pA/pF, N=3, n=8, P=NS; Figure 6C).
Bradycardia in Conscious Unrestrained CaV3.1–/– Mice
Both the heart rate and the AV conduction were altered in freely moving CaV3.1–/– mice (Figure 7A and 7B and the Table). The mean heart rate was significantly slowed in CaV3.1–/– mice, when considering a 24-hour period or during the day and night periods (Figure 7B and the Table). The maximum cardiac frequency during short periods of activity (40 seconds) was unchanged (Table), suggesting that activation of other ionic channels involved in SAN automaticity can compensate for the lack of ICa,T during strong activation of the sympathetic nervous system. Spectral analysis of RR signals in the frequency domain revealed no differences between the 2 groups of mice. Total spectral power, ultralow (ULF), very-low (VLF), low (LF), and high (HF) frequencies were not significantly modified between WT and CaV3.1–/– mice (Figure 7C, data not shown for ULF and VLF), indicating that the degree of autonomic regulation of heart rate was not altered in CaV3.1–/– mice.
Telemetric recordings also confirmed the prolongation of the PQ interval in Cav3.1–/– mice (Table), demonstrating that the propagation of the heartbeat through the AV conduction system was delayed in CaV3.1–/– mice. This effect was independent from the heart rate because measurements were performed at comparable heart rates (600 bpm; cycle length of 100 ms). In contrast, the atrial conduction (as assessed by the P wave duration), the ventricular conduction (QRS interval), and ventricular repolarization (QT interval) were unchanged (Table). In addition, we did not find evidence for cardiac arrhythmias in CaV3.1–/– mice. Also, no significant difference in the systolic, diastolic, and mean arterial pressure (60±8 mm Hg, N=8 in WT and 72±5 mm Hg, N=8 in Cav3.1–/– mice; P=NS) was observed between the 2 mouse strains (Figure 7D).
Discussion
The major finding of this study is that genetic inactivation of the CaV3.1/1G T-type Ca2+ channels in mice results in a significant slowing of the heart rate and AV conduction. Downregulation of ICa,T in both SAN and AVN cells of CaV3.1–/– animals is documented, accounting for this in vivo phenotype. The lack of Cav3.1 channels in isolated SAN cells induces slowing of pacemaker activity through a reduction of the slope of the diastolic depolarization. Our study provides novel and compelling genetic evidence for a direct contribution of Cav3.1 channels in the setting of the mammalian cardiac impulse generation and propagation by contributing to both the diastolic depolarization in the SAN and impulse conduction through the AVN.
ICa,T in SAN and AVN Cells Following Inactivation of Cav3.1 Channels
A striking observation of this study is that ICa,T in the mouse SAN is predominantly generated by the CaV3.1 subunit because we found no evidence for a residual ICa,T in Cav3.1–/– mice in both SAN (Figure 1) and AVN cells (Figure 6). The low sensitivity of SAN ICa,T to Ni2+ is consistent with that observed on recombinant Cav3.1-mediated ICa,T.27 Also consistent with our general findings is the recent observation that CaV3.2–/– mice showed no ECG alterations.29 Inactivation of Cav3.1 channels did not significantly modify the expression of ICa,L in Cav3.1–/– in both SAN and AVN cells. Our data show, therefore, that expression of CaV3.2, Cav1.3, and Cav1.2 subunits do not compensate for the lack of Cav3.1 channels in Cav3.1–/– mice.
Bradycardia and Atrioventricular Dysfunction in Cav3.1–/– Mice
ECGs and intracardiac recordings document both bradycardia and slowing of the atrioventricular conduction in CaV3.1–/– mice. Several lines of evidence indicate that bradycardia is attributable to slowing of the pacemaker activity in the SAN. First, bradycardia was evident in sedated mice under pharmacological blockade of the ANS input for heart rates that were comparable with the mean heart rate observed in freely moving mice. Second, slowing of the heart rate was observed in freely moving mice in the absence of a significant change both in the heart rate variability profile and in the arterial blood pressure, thus arguing against the hypothesis that bradycardia would be caused by dysfunction in the autonomic regulation of the heart rate or could be secondary to a change in the control of the vascular tone. The prolongation of the SAN recovery time also indicates dysfunction in SAN automaticity. Finally, the slowing of pacemaker activity observed in isolated SAN pacemaker cells of Cav3.1–/– mice is consistent with bradycardia being attributable to the lack of ICa,T in the SAN. The reduction of the mean heart rate measured on freely moving Cav3.1–/– mice is moderate, compared with the slowing of the cycle length in isolated pacemaker cells (10% in freely moving mice and 37% in isolated cells). This difference can be caused by the compensatory adrenergic tone in vivo, which stimulates ionic mechanisms involved in pacemaking, such as Cav1.3-mediated ICa,L9,10 and If6 and RyR-dependent diastolic release of Ca2+.11 The observation that the maximal heart rate is comparable in WT and Cav3.1–/– mice is consistent with this hypothesis. Comparison of mice lacking Cav1.39 and Cav3.1 channels (this study) indicate that ICa,L and ICa,T play distinct roles in pacemaker activity in vivo. Indeed, knockout of Cav1.3 channels reduces the basal and intrinsic heart rate in sedated conditions by approximately 20% and 60%, respectively,9 compared with the 10% reduction observed in mice lacking Cav3.1 channels in both freely moving and block of ANS conditions. These observations suggest that Cav3.1 channels contribute to the setting of the basal heart rate but have less impact on the dynamic regulation of pacemaker activity by the ANS. The lack of a significant change in the variability of the RR intervals in Cav3.1 mice is consistent with this hypothesis.
We report that the slowing of the AV conduction in Cav3.1–/– mice is caused by dysfunction in the excitability of the AVN. Indeed, intracardiac recordings show a delay in the AH conduction time and AVERP, in the absence of a prolongation of the P wave, which indicates similar conduction velocities in atria from WT and Cav3.1–/– mice. Consistently with ECGs and intracardiac recordings, we found no ICa,T in atrial myocytes from WT mice. Also, AVN cells display ICa,T related to CaV3.1 channels because it was no longer detectable in CaV3.1–/– mice. We did not find significant changes in the HV, QRS, and QT intervals (see Figures 3 and 5 and supplemental Figure III), indicating that the lack of Cav3.1 channels does not affect conduction through the His–Purkinje fiber network or the ventricular muscle. This phenotype differs from that of SCN5A+/– mice,19 which shows prominent alteration of the P wave and the QRS complex duration,30 stressing the importance of the cardiac INa in intraatrial and His–Purkinje conduction. Also in contrast with SCN5A+/–,19 Cav3.1–/– mice do not present ventricular tachiarrhythmias. This SCN5A-mediated INa can possibly compensate for the lack of ICa,T in the His–Purkinje system.
Pacemaking in Cav3.1–/– Mice
We describe that inactivation of CaV3.1 channels significantly prolongs the pacemaker cycle length in isolated SAN cells by reducing the slope of the diastolic depolarization, demonstrating the involvement of Cav3.1-mediated ICa,T in the setting of the diastolic depolarization in mouse SAN cells. In an attempt to compare the physiological roles of Cav3.1-mediated ICa,T with that of Cav1.3-mediated ICa,L and INa, we have developed a numerical model of mouse SAN electrophysiology based on previous studies on mouse SAN pacemaking in normal and genetically modified mouse strains (see supplemental Figures IV and V). Our numerical simulations indicate that Cav3.1 channels contribute to pacemaking by activating during the diastolic depolarization for more negative voltages than TTX-sensitive INa and Cav1.3-mediated ICa,L (see supplemental Figure VB and VE). As a consequence, Cav3.1 channels can accelerate the diastolic depolarization rate in a voltage range in which INa is still not activated. This property can explain, at least in part, how Cav3.1 channels can contribute to pacemaking in spite of their relatively low availability at SAN diastolic potentials. An expanded discussion about the development, the features and limitations of our numerical model, and the physiological significance of Cav3.1-mediated ICa,T compared with that of ICa,L and INa is available in the online data supplement.
Conclusion
In conclusion, our study demonstrates that CaV3.1 channels contribute to the heartbeat by influencing pacemaking and the AV conduction. To date, the presence of T-type channels in human heart is poorly documented. Molecular analyses, such as dot blots and Northern blots of human heart mRNAs, have identified transcripts for CaV3.1 and CaV3.2.31,32 T-type channels may play a role in the human SAN and the conduction system. For instance, the causality between congenital heart block induced by maternal autoantibodies against T-type channels and children showing SAN bradycardia and atrioventricular block has recently been documented.33 There is growing evidence that T-type channels may constitute a promising pharmacological target for the treatment of human diseases, such as epilepsy and chronic pain.34 From our observations, T-type channel inhibition would have no deleterious consequences in cardiac physiology. It is, therefore, tempting to speculate that selective blockers of CaV3.1 channels may hold promise for the therapeutic management of the cardiac diseases that require moderate heart rate reduction, such as cardiac ischemia and coronary heart disease.
Acknowledgments
This work was supported by the Association Franaise Contre les Myopathies, the Action Concertée Incitative (Developmental Biology and Integrative Physiology) of the French Ministry for Education, the Fondation de France, and the Chemoinformatics Program of Korean Institute of Science and Technology, Korea. A.T. is supported by a fellowship from the CNRS Lebanon. We are grateful to Patrick Atger for excellent technical assistance.
Footnotes
Both authors contributed equally to this study.
Original received November 5, 2004; resubmission received December 5, 2005; revised resubmission received March 30, 2006; accepted April 27, 2006.
References
Irisawa H, Brown HF, Giles W. Cardiac pacemaking in the sinoatrial node. Physiol Rev. 1993; 73: 197–227.
Boyett MR, Honjo H, Kodama I. The sinoatrial node, a heterogeneous pacemaker structure. Cardiovasc Res. 2000; 47: 658–687. [Order article via Infotrieve]
Difrancesco D. Serious workings of the funny current. Prog Biophys Mol Biol. 2006; 90: 13–25. [Order article via Infotrieve]
Lakatta EG, Maltsev VA, Bogdanov KY, Stern MD, Vinogradova TM. Cyclic variation of intracellular calcium: a critical factor for cardiac pacemaker cell dominance. Circ Res. 2003; 92: e45–e50.
Honjo H, Inada S, Lancaster MK, Yamamoto M, Niwa R, Jones SA, Shibata N, Mitsui K, Horiuchi T, Kamiya K, Kodama I, Boyett MR. Sarcoplasmic reticulum Ca2+ release is not a dominating factor in sinoatrial node pacemaker activity. Circ Res. 2003; 92: e41–e44.
Baruscotti M, Bucchi A, Difrancesco D. Physiology and pharmacology of the cardiac pacemaker ("funny") current. Pharmacol Ther. 2005; 107: 59–79. [Order article via Infotrieve]
Stieber J, Herrmann S, Feil S, Loster J, Feil R, Biel M, Hofmann F, Ludwig A. The hyperpolarization-activated channel HCN4 is required for the generation of pacemaker action potentials in the embryonic heart. Proc Natl Acad Sci U S A. 2003; 100: 15235–15240.
Verheijck EE, van Ginneken AC, Wilders R, Bouman LN. Contribution of L-type Ca2+ current to electrical activity in sinoatrial nodal myocytes of rabbits. Am J Physiol. 1999; 276: H1064–H1077.
Zhang Z, Xu Y, Song H, Rodriguez J, Tuteja D, Namkung Y, Shin HS, Chiamvimonvat N. Functional Roles of Cav1.3 1D calcium channel in sinoatrial nodes: insight gained using gene-targeted null mutant mice. Circ Res. 2002; 90: 981–987.
Mangoni ME, Couette B, Bourinet E, Platzer J, Reimer D, Striessnig J, Nargeot J. Functional role of L-type Cav1.3 Ca2+ channels in cardiac pacemaker activity. Proc Natl Acad Sci U S A. 2003; 100: 5543–5548.
Vinogradova TM, Bogdanov KY, Lakatta EG. beta-Adrenergic stimulation modulates ryanodine receptor Ca2+ release during diastolic depolarization to accelerate pacemaker activity in rabbit sinoatrial nodal cells. Circ Res. 2002; 90: 73–79. [Order article via Infotrieve]
Lei M, Jones SA, Liu J, Lancaster MK, Fung SS, Dobrzynski H, Camelliti P, Maier SK, Noble D, Boyett MR. Requirement of neuronal- and cardiac-type sodium channels for murine sinoatrial node pacemaking. J Physiol. 2004; 559: 835–848.
Baruscotti M, DiFrancesco D, Robinson RB. A TTX-sensitive inward sodium current contributes to spontaneous activity in newborn rabbit sino-atrial node cells. J Physiol. 1996; 492: 21–30.
Maier SK, Westenbroek RE, Yamanushi TT, Dobrzynski H, Boyett MR, Catterall WA, Scheuer T. An unexpected requirement for brain-type sodium channels for control of heart rate in the mouse sinoatrial node. Proc Natl Acad Sci U S A. 2003; 100: 3507–3512.
Lei M, Goddard C, Liu J, Leoni AL, Royer A, Fung SS, Xiao G, Ma A, Zhang H, Charpentier F, Vandenberg JI, Colledge WH, Grace AA, Huang CL. Sinus node dysfunction following targeted disruption of the murine cardiac sodium channel gene Scn5a. J Physiol. 2005; 567: 387–400.
Munk AA, Adjemian RA, Zhao J, Ogbaghebriel A, Shrier A. Electrophysiological properties of morphologically distinct cells isolated from the rabbit atrioventricular node. J Physiol. 1996; 493: 801–818.
Yuill KH, Hancox JC. Characteristics of single cells isolated from the atrioventricular node of the adult guinea-pig heart. Pflugers Arch. 2002; 445: 311–320. [Order article via Infotrieve]
Efimov IR, Nikolski VP, Rothenberg F, Greener ID, Li J, Dobrzynski H, Boyett M. Structure-function relationship in the AV junction. Anat Rec A Discov Mol Cell Evol Biol. 2004; 280: 952–965. [Order article via Infotrieve]
Papadatos GA, Wallerstein PM, Head CE, Ratcliff R, Brady PA, Benndorf K, Saumarez RC, Trezise AE, Huang CL, Vandenberg JI, Colledge WH, Grace AA. Slowed conduction and ventricular tachycardia after targeted disruption of the cardiac sodium channel gene Scn5a. Proc Natl Acad Sci U S A. 2002; 99: 6210–6215.
Hagiwara N, Irisawa H, Kameyama M. Contribution of two types of calcium currents to the pacemaker potentials of rabbit sino-atrial node cells. J Physiol. 1988; 395: 233–253.
Huser J, Blatter LA, Lipsius SL. Intracellular Ca2+ release contributes to automaticity in cat atrial pacemaker cells. J Physiol. 2000; 524: 415–422.
Mangoni ME, Nargeot J. Properties of the hyperpolarization-activated current (If) in isolated mouse sino-atrial cells. Cardiovasc Res. 2001; 52: 51–64. [Order article via Infotrieve]
Hirano Y, Fozzard HA, January CT. Characteristics of L- and T-type Ca2+ currents in canine cardiac Purkinje cells. Am J Physiol. 1989; 256: H1478–H1492.
Tseng GN, Boyden PA. Multiple types of Ca2+ currents in single canine Purkinje cells. Circ Res. 1989; 65: 1735–1750.
Perez-Reyes E. Molecular physiology of low-voltage-activated T-type calcium channels. Physiol Rev. 2003; 83: 117–161.
Kim D, Song I, Keum S, Lee T, Jeong MJ, Kim SS, McEnery MW, Shin HS. Lack of the burst firing of thalamocortical relay neurons and resistance to absence seizures in mice lacking 1G T-type Ca2+ channels. Neuron. 2001; 31: 35–45. [Order article via Infotrieve]
Lee JH, Gomora JC, Cribbs LL, Perez-Reyes E. Nickel block of three cloned T-type calcium channels: low concentrations selectively block alpha1H. Biophys J. 1999; 77: 3034–3042.
Lande G, Demolombe S, Bammert A, Moorman A, Charpentier F, Escande D. Transgenic mice overexpressing human KvLQT1 dominant-negative isoform. Part II: pharmacological profile. Cardiovasc Res. 2001; 50: 328–334. [Order article via Infotrieve]
Chen CC, Lamping KG, Nuno DW, Barresi R, Prouty SJ, Lavoie JL, Cribbs LL, England SK, Sigmund CD, Weiss RM, Williamson RA, Hill JA, Campbell KP. Abnormal coronary function in mice deficient in 1H T-type Ca2+ channels. Science. 2003; 302: 1416–1418.
Royer A, van Veen TA, Le Bouter S, Marionneau C, Griol-Charhbili V, Leoni AL, Steenman M, van Rijen HV, Demolombe S, Goddard CA, Richer C, Escoubet B, Jarry-Guichard T, Colledge WH, Gros D, de Bakker JM, Grace AA, Escande D, Charpentier F. Mouse model of SCN5A-linked hereditary Lenegre’s disease: age-related conduction slowing and myocardial fibrosis. Circulation. 2005; 111: 1738–1746.
Cribbs LL, Lee JH, Yang J, Satin J, Zhang Y, Daud A, Barclay J, Williamson MP, Fox M, Rees M, Perez-Reyes E. Cloning and characterization of alpha1H from human heart, a member of the T-type Ca2+ channel gene family. Circ Res. 1998; 83: 103–109.
Monteil A, Chemin J, Bourinet E, Mennessier G, Lory P, Nargeot J. Molecular and functional properties of the human 1G subunit that forms T-type calcium channels. J Biol Chem. 2000; 275: 6090–6100.
Hu K, Qu Y, Yue Y, Boutjdir M. Functional basis of sinus bradycardia in congenital heart block. Circ Res. 2004; 94: e32–e38.
Birch PJ, Dekker LV, James IF, Southan A, Cronk D. Strategies to identify ion channel modulators: current and novel approaches to target neuropathic pain. Drug Discov Today. 2004; 9: 410–418. [Order article via Infotrieve]
您现在查看是摘要介绍页，详见ORG附件。