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(Circulation Research. 2006;98:1422.)
© 2006 American Heart Association, Inc.

Integrative Physiology

Bradycardia and Slowing of the Atrioventricular Conduction in Mice Lacking Ca_V3.1/_1G T-Type Calcium Channels

Matteo E. Mangoni^*, Achraf Traboulsie^*, Anne-Laure Leoni, Brigitte Couette, Laurine Marger, Khai Le Quang, Elodie Kupfer, Anne Cohen-Solal, José Vilar, Hee-Sup Shin, Denis Escande, Flavien Charpentier, Joël Nargeot, Philippe Lory

From 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; and Center for Calcium and Learning (H.-S.S.), Korea Institute of Science and Technology, Cheongryang, Seoul, Republic of Korea.

Correspondence to Dr Matteo Mangoni, Institut de Génomique Fonctionnelle, CNRS UMR5203-INSERM U661, Université de Montpellier I–Université de Montpellier II, Département de Physiologie, 141, rue de la Cardonille, Montpellier cedex 05, F-34094 France. E-mail matteo.mangoni{at}igf.cnrs.fr

	Abstract

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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 Ca_v3.1/_1G T-type calcium channels (cacna1g) abolishes T-type calcium current (I_Ca,T) in isolated cells from the SAN and the atrioventricular node without affecting the L-type Ca²⁺ current (I_Ca,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 I_Ca,T was detected in right atrium myocytes in both wild-type and Ca_v3.1^–/– 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 Ca_v3.1/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

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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 I_f^6,7 and I_Ca,L^8–10 play major roles in controlling the diastolic depolarization after decaying of the fast component of delayed-rectifier K⁺ currents (I_Kr). Diastolic release of Ca²⁺ mediated by ryanodine receptors (RyRs) has been indicated as an important mechanism for controlling SAN pacemaking under activation of the ß-adrenergic receptor.¹¹ The SAN also expresses neuronal tetrodotoxin (TTX)-sensitive and cardiac TTX-resistant voltage-dependent Na⁺ currents (I_Na).¹² TTX-sensitive I_Na is involved in SAN pacemaking in the newborn rabbit¹³ and in the adult mouse.^12,14 The cardiac TTX-resistant SCN5A-mediated I_Na is important for conduction from the SAN to the atrium and in intranodal conduction.¹⁵ I_Na 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

I_Ca,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 I_Ca,T is expressed in pacemaker cells of mammals, including the rabbit²⁰ and the mouse.²² I_Ca,T is also expressed in the cardiac conduction system, the rabbit AVN,¹⁸ and in canine Purkinje cells.^23,24 The functional role of I_Ca,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 Ca_V3.1/_1G, Ca_V3.2/_1H, and Ca_V3.3/_1I subunits, has fostered investigation of the role of native I_Ca,T.²⁵ Here, we describe that Ca_V3.1 knockout (Ca_V3.1^–/–) mice lack I_Ca,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 Ca_v3.1 channels in pacemaking and cardiac conduction.

	Materials and Methods

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The generation of the Ca_V3.1^–/– 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

Top Abstract Introduction Materials and Methods Results Discussion References

 
Ca_V3.1^–/– SAN Pacemaker Cells Lack I_Ca,T
Electrophysiological recordings obtained on SAN cells of wild-type (WT) and Ca_V3.1^–/– animals revealed that I_Ca,T related to Ca_V3.1 is a major component of the total Ca²⁺ current (I_Ca) in SAN cells (Figure 1). Figure 1A shows representative I_Ca traces recorded from a holding potential (HP) of –90 mV in WT (Figure 1Aa) and Ca_V3.1^–/– (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). I_Ca,L was measured by applying depolarizing steps from a HP of –55 mV (Figure 1B, open symbols), revealing I_Ca,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 I_Ca voltage for half activation (V_0.5act) 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 V_0.5act of –47±1 mV for the net I_Ca,T (Figure 1Ba, dotted line). In contrast to WT SAN cells, none of the tested Ca_v3.1^–/– cells displayed detectable I_Ca,T (Figure 1Bb). Indeed, in Ca_V3.1^–/– SAN cells, switching to a HP of –55 mV did not alter significantly voltage dependence of I_Ca activation (HP, –90 mV: V_0.5act=–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 I_Ca,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 Ca_V3.1^–/– SAN cells at HP –90 mV is shown in Figure 1C (black and white bars, respectively) to estimate the I_Ca,T component related to Ca_v3.1. At –40 mV, I_Ca,T density was 6.8±1.6 pA/pF (+/+, N=9, n=18) and I_Ca,L density was 2.6±0.7 pA/pF, (–/–, N=10, n=14). I_Ca,L density was not significantly affected by inactivation of Ca_v3.1 channels (Figure 1D). Indeed, I_Ca,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 Ca_V3.1^–/– and WT SAN cells, respectively. Taken together, these data showed that inactivation of Ca_v3.1 channels abolished I_Ca,T and had no significant effect on I_Ca,L in SAN cells.

View larger version (34K): [in this window] [in a new window]   Figure 1. ICa,T in SAN cells from WT and CaV3.1–/– mice. A, Typical sample ICa traces obtained using a HP at –90 mV in a SAN cell from WT (a) and Cav3.1–/– (b) mice. B, Average I-V curves of the Ca2+ current obtained on WT (a) and Cav3.1–/– (b) SAN cells using a HP at –90 mV (filled symbols) and –55 mV (open symbols). Traces obtained at a test potential of –35 mV, from a HP of –90 and –55 mV, are shown in each inset in a and b, as indicated. The net ICa,T I-V curve recorded in WT SAN cells is indicated with a dotted line in a and b. C, Histogram showing the relative density of ICa,T and ICa,L at different test potentials as indicated. ICa,T was measured in SAN cells from WT (filled bar), and ICa,L was measured in SAN cells from CaV3.1–/– mice (open bar). HP was –90 mV for ICa,T and ICa,L. D, Density of ICa,L in SAN cells from WT (open bar) and Cav3.1–/– (filled bar) mice at different test potentials. ns indicates not significant.

To determine whether the pharmacological sensitivity of the SAN I_Ca,T is consistent with the expression of the Ca_v3.1 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 I_Ca,T, respectively. Because the IC₅₀ values for Ni²⁺ on recombinant Ca_V3.1 and Ca_V3.2 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 I_Ca,T in mouse adult SAN cells is generated by the Ca_V3.1 subunit. Consistent with these findings, real-time RT-PCR experiments showed that the Ca_v3.1 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 I_Ca,L blocker isradipine on Ca_v3.1^–/– SAN cells blocked I_Ca 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 I_Ca in SAN cells from Ca_V3.1^–/– mice was I_Ca,L.

View larger version (30K): [in this window] [in a new window]   Figure 2. A and B, Sample traces showing partial block of ICa,T by 200 µmol/L Ni2+ in WT SAN cells (A) and histogram of the percentage of inhibition at 50 and 200 µmol/L Ni2+ (B). C and D, Isradipine (Isr) block of ICa in Cav3.1–/– SAN cells measured from a HP of –90 mV (C) and –55 mV (D) as indicated. Note that no residual dihydropyridine-resistant ICa,T is recorded in cells lacking Cav3.1 channels at the corresponding peak of ICa,T recorded in WT cells (–35 mV). E and F, Corresponding averaged I-V curves in control conditions (filled boxes) and 2 µmol/L isradipine (open boxes).

Sedated Ca_V3.1^–/– Mice Have Intrinsic Slower Heart Rate and Prolonged AV Conduction
We next performed surface ECG recordings on sedated WT and Ca_V3.1^–/– 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 Ca_V3.1^–/– (–/–) mice before and after the ANS block are presented in Figure 3A. The loss of the Ca_V3.1 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 Ca_V3.1^–/– 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 Ca_V3.1^–/– 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 Ca_V3.1^–/– 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 Ca_V3.1^–/– animals, respectively; P<0.001; Figure 4B). The other ECG parameters were not significantly modified.

View larger version (23K): [in this window] [in a new window]   Figure 3. Surface ECGs obtained on sedated WT (+/+) and CaV3.1–/– (–/–) mice. A, Representative lead I ECG traces obtained from WT and CaV3.1–/– under baseline conditions (ANS+) (left panels) and after injection of atropine and propranolol to block the ANS (ANS–) (right panels). B, Histograms of the average RR interval, P wave duration, PQ interval, and QRS interval, respectively, obtained from WT (open bars) and CaV3.1–/– (filled bars) mice, before (ANS+) and after (ANS–) blockade of the ANS.

View larger version (25K): [in this window] [in a new window]   Figure 4. Pacemaker activity of isolated WT (+/+) and Cav3.1–/– (–/–) SAN cells. A, Representative sweeps of spontaneous action potentials obtained from SAN cells from WT (upper left trace) and CaV3.1–/– mice (lower left trace). Superimposition of typical action potentials from a WT (+/+) and from CaV3.1–/– SAN cell (–/–) is shown in phase in the right panel. B and C, Histograms of the average bpm value (B) and the slope of the diastolic depolarization (SDD) (C) SAN cells. D, Box histograms showing the average MDP and the Eth in SAN cells from WT (+/+) (open circles) and CaV3.1–/– (–/–) (filled circles). Corresponding 25th and 75th percentile define the box. E, Action potential duration (APD) measured in WT and CaV3.1–/– SAN cells.

SAN Cells From Ca_V3.1^–/– 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 Ca_V3.1^–/– mice was associated with dysfunction of SAN cell automaticity. Representative recordings obtained on WT and Ca_V3.1^–/– 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 Ca_V3.1^–/–, 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 (E_th) were observed (MDP: –56.9±1.3 mV in WT, n=15, and –58.2±1.4 mV for Ca_V3.1^–/– SAN cells, n=14; E_th: –45±1.3 mV in WT and –43±1.4 mV in Ca_V3.1^–/– SAN cells; Figure 4D). Also, the action potential duration (147±15 and 141±10 ms for Ca_V3.1^–/– and WT SAN cells, respectively; Figure 4E) and the action potential amplitude (APA) (92±8 and 90±8 mV for Ca_V3.1^–/– and WT SAN cells, respectively; not shown) were comparable in SAN cells from WT and Ca_v3.1^–/– mice.

Intracardiac Electrophysiology Study of Ca_V3.1^–/– Mice
The AV conduction delay in mice lacking Ca_v3.1 channels was measured under baseline intracardiac recording conditions. Eight of 10 WT and 7 of 9 Ca_V3.1^–/– mice had a distinctly visible His-bundle electrogram (Figure 5A). In this group of animals, atrial-His (AH) conduction times were significantly longer in Ca_V3.1^–/– 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 Ca_V3.1^–/– 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 Ca_v3.1^–/– mice. In contrast, the AVERP was significantly longer in Ca_V3.1^–/– 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 Ca_V3.1^–/– 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 Ca_V3.1^–/– mice than in WT mice (31±5 in Ca_v3.1^–/– and 17±2 ms in WT mice P<0.05; n=7 and 9, respectively).

View larger version (19K): [in this window] [in a new window]   Figure 5. Cardiac electrophysiology studies in anesthetized WT (+/+) and CaV3.1–/– (–/–) mice. A, Representative surface lead I ECG and intracardiac (intra.) recordings under baseline conditions. B, Histograms of the average AH and HV intervals measured in baseline conditions in WT (open bars; N=8) and CaV3.1–/– (filled bars; N=7) mice. RR intervals were 111±3 and 122±4 ms for WT and CaV3.1–/– mice, respectively (P<0.05). C, Histograms of the average atrial effective refractory period (AERP), atrioventricular ERP (AVERP), ventricular ERP (VERP), and AV Wenckebach cycle length (Wenck.) in WT (open bars; N=9 to 10) and CaV3.1–/– (filled bars; N=6 to 8) mice.

AVN Cells From Ca_V3.1^–/– Mice Lack I_Ca,T
We then tested whether dysfunction of AV conduction observed on inactivation of Ca_v3.1 channels was associated with downregulation of I_Ca,T in the AVN. I_Ca,T and I_Ca,L were recorded in isolated AVN cells using the same recording protocols as for SAN cells (Figure 6). I_Ca,T was found in all the WT AVN cells investigated (Figure 6A). Indeed, switching the HP from –90 to –60 mV significantly shifted the V_0.5act of the total I_Ca from –34±0.7 to –23±0.8 mV (n=7, P<0.05). In contrast, no I_Ca,T could be recorded in Ca_v3.1^–/– AVN cells (Figure 6Ab), and switching the HP from –90 to –60 mV did not shift the V_0.5act value (–25±1.3 mV to –27±0.8 mV, respectively; P=NS; Figure 6Bb). The peak density of the residual I_Ca measured from a HP of –60 mV in AVN cells was not altered by inactivation of Ca_v3.1 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 Ca_v3.1^–/–; P=NS). In addition, no I_Ca,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 V_0.5act (–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).

View larger version (27K): [in this window] [in a new window]   Figure 6. Recordings of ICa in isolated AVN and RA cells from WT and Cav3.1–/– mice. A, Sample ICa traces obtained using a HP at –90 mV in a AVN cell from WT (a) and Cav3.1–/– (b) mice. B, I-V curves of ICa obtained on WT (a) and Cav3.1–/– (b) SAN cells using a HP at –90 mV (filled symbols) and –60 mV (open symbols). The I-V curve of ICa,T calculated by subtracting records at HP of –60 mV from that at HP of –90 mV are shown as a dotted line. C and D, Lack of ICa,T in RA cells from WT mice. Sample traces recorded from a HP of –100 mV at test potential (TP) values of –30 and 0 mV are shown in C; Averaged I-V curves marked as in (B) are shown in D.

Bradycardia in Conscious Unrestrained Ca_V3.1^–/– Mice
Both the heart rate and the AV conduction were altered in freely moving Ca_V3.1^–/– mice (Figure 7A and 7B and the Table). The mean heart rate was significantly slowed in Ca_V3.1^–/– 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 I_Ca,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 Ca_V3.1^–/– mice (Figure 7C, data not shown for ULF and VLF), indicating that the degree of autonomic regulation of heart rate was not altered in Ca_V3.1^–/– mice.

View larger version (31K): [in this window] [in a new window]   Figure 7. Twenty-four-hour telemetric ECG recordings in conscious unrestrained WT (+/+) and CaV3.1–/– (–/–) mice. A, Representative ECG recordings obtained on WT and CaV3.1–/– animals during daytime. B, Examples of circadian variation of heart rate (in bpm) in WT and CaV3.1–/– mice over a 24-hour period. Dashed lines indicate mean day and night heart rates. C, Spectral analysis of heart rate variability. Results are illustrated for total power spectra (0 to 3.2 Hz) and specific frequency bands, ie, low frequency (LF) (0.32 to 1.2 Hz) and high frequency (HF) (1.2 to 3.2 Hz). Note that during the study period, heart rate was significantly slowed in CaV3.1–/– mice (455±13 bpm and 515±10 bpm for CaV3.1–/– and WT mice, respectively; P<0.005). TP indicates, test potential. D, Measurements of the systolic and diastolic blood pressure in WT (open bars; n=8) and CaV3.1–/– (filled bars; n=8) mice.

View this table: [in this window] [in a new window]   Table 1. Telemetric ECG Parameters in Freely Moving WT and 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 Ca_V3.1^–/– 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 Ca_V3.1^–/– 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 Ca_v3.1^–/– mice; P=NS) was observed between the 2 mouse strains (Figure 7D).

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
The major finding of this study is that genetic inactivation of the Ca_V3.1/_1G T-type Ca²⁺ channels in mice results in a significant slowing of the heart rate and AV conduction. Downregulation of I_Ca,T in both SAN and AVN cells of Ca_V3.1^–/– animals is documented, accounting for this in vivo phenotype. The lack of Ca_v3.1 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 Ca_v3.1 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.

I_Ca,T in SAN and AVN Cells Following Inactivation of Ca_v3.1 Channels
A striking observation of this study is that I_Ca,T in the mouse SAN is predominantly generated by the Ca_V3.1 subunit because we found no evidence for a residual I_Ca,T in Ca_v3.1^–/– mice in both SAN (Figure 1) and AVN cells (Figure 6). The low sensitivity of SAN I_Ca,T to Ni²⁺ is consistent with that observed on recombinant Ca_v3.1-mediated I_Ca,T.²⁷ Also consistent with our general findings is the recent observation that Ca_V3.2^–/– mice showed no ECG alterations.²⁹ Inactivation of Ca_v3.1 channels did not significantly modify the expression of I_Ca,L in Ca_v3.1^–/– in both SAN and AVN cells. Our data show, therefore, that expression of Ca_V3.2, Ca_v1.3, and Ca_v1.2 subunits do not compensate for the lack of Ca_v3.1 channels in Ca_v3.1^–/– mice.

Bradycardia and Atrioventricular Dysfunction in Ca_v3.1^–/– Mice
ECGs and intracardiac recordings document both bradycardia and slowing of the atrioventricular conduction in Ca_V3.1^–/– 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 Ca_v3.1^–/– mice is consistent with bradycardia being attributable to the lack of I_Ca,T in the SAN. The reduction of the mean heart rate measured on freely moving Ca_v3.1^–/– 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 Ca_v1.3-mediated I_Ca,L^9,10 and I_f⁶ and RyR-dependent diastolic release of Ca²⁺.¹¹ The observation that the maximal heart rate is comparable in WT and Ca_v3.1^–/– mice is consistent with this hypothesis. Comparison of mice lacking Ca_v1.3⁹ and Ca_v3.1 channels (this study) indicate that I_Ca,L and I_Ca,T play distinct roles in pacemaker activity in vivo. Indeed, knockout of Ca_v1.3 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 Ca_v3.1 channels in both freely moving and block of ANS conditions. These observations suggest that Ca_v3.1 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 Ca_v3.1 mice is consistent with this hypothesis.

We report that the slowing of the AV conduction in Ca_v3.1^–/– 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 Ca_v3.1^–/– mice. Consistently with ECGs and intracardiac recordings, we found no I_Ca,T in atrial myocytes from WT mice. Also, AVN cells display I_Ca,T related to Ca_V3.1 channels because it was no longer detectable in Ca_V3.1^–/– 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 Ca_v3.1 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 I_Na in intraatrial and His–Purkinje conduction. Also in contrast with SCN5A^+/–,¹⁹ Ca_v3.1^–/– mice do not present ventricular tachiarrhythmias. This SCN5A-mediated I_Na can possibly compensate for the lack of I_Ca,T in the His–Purkinje system.

Pacemaking in Ca_v3.1^–/– Mice
We describe that inactivation of Ca_V3.1 channels significantly prolongs the pacemaker cycle length in isolated SAN cells by reducing the slope of the diastolic depolarization, demonstrating the involvement of Ca_v3.1-mediated I_Ca,T in the setting of the diastolic depolarization in mouse SAN cells. In an attempt to compare the physiological roles of Ca_v3.1-mediated I_Ca,T with that of Ca_v1.3-mediated I_Ca,L and I_Na, 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 Ca_v3.1 channels contribute to pacemaking by activating during the diastolic depolarization for more negative voltages than TTX-sensitive I_Na and Ca_v1.3-mediated I_Ca,L (see supplemental Figure VB and VE). As a consequence, Ca_v3.1 channels can accelerate the diastolic depolarization rate in a voltage range in which I_Na is still not activated. This property can explain, at least in part, how Ca_v3.1 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 Ca_v3.1-mediated I_Ca,T compared with that of I_Ca,L and I_Na is available in the online data supplement.

Conclusion
In conclusion, our study demonstrates that Ca_V3.1 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 Ca_V3.1 and Ca_V3.2.^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 Ca_V3.1 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 Française 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.

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