Angelo G Torrente

Pietro Mesirca

Isabelle Bidaud

PhD Matteo E Mangoni
email: matteo.mangoni@igf.cnrs.fr

Channelopathies of voltage-gated L-type Cav1.3/α1D and T-type Cav3.1/α1G Ca 2+ channels in dysfunction of heart automaticity

Keywords: Sinus Node Dysfunction, sinus node, Ca 2+ channels, Cav1.3, Cav3.1

    Channelopathies of voltage-gated L-type Cav1.3/α1D and T-type Cav3.1/α1G Ca2+ channels in dysfunction of heart automaticity

Introduction

Heart automaticity is a highly complex function. It integrates the pacemaker activity of the sino-atrial node (SAN) with impulse conduction through the atrioventricular node (AVN) and the ventricular conduction system. The structural and ionic mechanisms underlying heart automaticity are intrinsically robust and redundant. However, SAN dysfunction (SND) and heart block constitute a relatively widespread clinical condition in cardiology, especially in the elder population [START_REF] Jensen | Incidence of and risk factors for sick sinus syndrome in the general population[END_REF]. Several forms of SND and atrioventricular conduction block (heart block) have been described. SND may manifest in the absence of other accompanying such as heart failure, drug intoxication or diabetes and showing genetic legacy. This condition is defined as idiopathic or primary SND. In contrast, acquired forms of SND are generally secondary to other clinical conditions or co-morbidities [START_REF] Kusumoto | ACC/AHA/HRS Guideline on the Evaluation and Management of Patients With Bradycardia and Cardiac Conduction Delay: Executive Summary: A Report of the American College of Cardiology/American Heart Association Task Force on Clinical Practice Guidelines, and the Heart Rhythm Society[END_REF]. Acquired SND forms account for the majority of patients. A substantial amount of research over the last 10 years has helped identify mechanisms of secondary SND (see Monfredi and Boyett for review [START_REF] Monfredi | Sick sinus syndrome and atrial fibrillation in older persons -A view from the sinoatrial nodal myocyte[END_REF]). Similarly, an increasing number of studies have described new mechanisms underlying primary forms of SND and, more specifically, inheritable forms of SND and heart block. Genetic investigation of SND patients has identified mutation in ion channels responsible for a wide array of familial idiopathic SND. These include mutation in Na + channels [START_REF] Butters | Mechanistic links between Na+ channel (SCN5A) mutations and impaired cardiac pacemaking in sick sinus syndrome[END_REF], hyperpolarization-activated channels [START_REF] Baruscotti | HCN-related channelopathies[END_REF], G-protein-gated K + channels [START_REF] Kuss | Familial Sinus Node Disease Caused by a Gain of GIRK (G-Protein Activated Inwardly Rectifying K(+) Channel) Channel Function[END_REF], as well as L-and T-type voltage-gated Ca 2+ channels (VGCCs) [START_REF] Hu | Functional basis of sinus bradycardia in congenital heart block[END_REF][START_REF] Baig | Loss of Ca(v)1.3 (CACNA1D) function in a human channelopathy with bradycardia and congenital deafness[END_REF].

Adult cardiac myocytes of the SAN and the AVN express three isoforms of VGCCs: Ttype Cav3.1, L-type Cav1.3 and Cav1.2 channels [START_REF] Mangoni | Voltagedependent calcium channels and cardiac pacemaker activity: From ionic currents to genes[END_REF]. Idiopathic forms of SND and heart block induced by pathogenic mutation of the Cav1.3-encoding gene CACNA1D are known [START_REF] Baig | Loss of Ca(v)1.3 (CACNA1D) function in a human channelopathy with bradycardia and congenital deafness[END_REF][START_REF] Liaqat | Identification of CACNA1D variants associated with sinoatrial node dysfunction and deafness in additional Pakistani families reveals a clinical significance[END_REF]. Furthermore, autoimmune idiopathic forms of SND due to disruption of Cav1.3 and Cav3.1 channel activity have been described [START_REF] Hu | Functional basis of sinus bradycardia in congenital heart block[END_REF] (see Qu et al. [START_REF] Qu | Autoimmune Calcium Channelopathies and Cardiac Electrical Abnormalities[END_REF] for review). In this manuscript, we will review the current knowledge about channelopathies of Cav3.1 and Cav1.3 channels leading to dysfunction of SAN pacemaker activity and atrioventricular conduction. In particular, we will first summarize the general mechanism of cardiac pacemaking, highlighting the functional role of VGCCs. Then, we will review the cardiac phenotype of individuals carrying channelopathies of VGCCs and discuss the mechanisms leading to SND and heart block. Finally, we will discuss the possibility to introduce new therapies against idiopathic SND mediated by loss-of-function of VGCCs.

Current models of SAN automaticity: the "coupled-clock" model of pacemaking

The SAN is a heterogeneous tissue [START_REF] Boyett | The sinoatrial node, a heterogeneous pacemaker structure[END_REF][START_REF] Bleeker | Functional and morphological organization of the rabbit sinus node[END_REF], composed by pacemaker cells, atrial myocytes [START_REF] Verheijck | Distribution of atrial and nodal cells within the rabbit sinoatrial node: models of sinoatrial transition[END_REF] and fibroblasts [START_REF] De Maziere | Spatial and functional relationship between myocytes and fibroblasts in the rabbit sinoatrial node[END_REF]. In addition, the SAN contains also adipocytes, epithelial cells, vascular endothelial cells, macrophages and neurons [START_REF] Linscheid | Quantitative proteomics and single-nucleus transcriptomics of the sinus node elucidates the foundation of cardiac pacemaking[END_REF]. Pacemaker cells inside the SAN region are sparse and their mutual electrical coupling is low [START_REF] Ten Velde | Spatial distribution of connexin43, the major cardiac gap junction protein, visualizes the cellular network for impulse propagation from sinoatrial node to atrium[END_REF]. This characteristic is due to high expression of low conductance (Cx) 45 and poor expression of intermediate-conductance of Cx43 channels [START_REF] Ten Velde | Spatial distribution of connexin43, the major cardiac gap junction protein, visualizes the cellular network for impulse propagation from sinoatrial node to atrium[END_REF][START_REF] Verheijck | Electrophysiological features of the mouse sinoatrial node in relation to connexin distribution[END_REF][START_REF] Boyett | Connexins in the sinoatrial and atrioventricular nodes[END_REF].

The automaticity of pacemaker cells is initiated by diastolic depolarization, a spontaneous depolarization phase of driving the membrane voltage from the end of the repolarization to the threshold of the following action potential upstroke (Figure 1). The diastolic depolarization is regulated in opposite ways by the two branches of the autonomic nervous system. The sympathetic branch increases the slope of the depolarization and the parasympathetic branch decreases it. Catecholamines positively regulate the slope of the diastolic depolarization via the activation of ß-adrenergic receptors (ßARs) and consequent stimulation of cAMP synthesis by adenylate cyclases. Elevation of cAMP concentration positively regulates the activity of several plasmalemmal ion channels and stimulates Ca 2+ release from ryanodine receptors (RyR2) of the sarcoplasmic reticulum (SR) [START_REF] Mangoni | Genesis and regulation of the heart automaticity[END_REF][START_REF] Lakatta | A coupled SYSTEM of intracellular Ca2+ clocks and surface membrane voltage clocks controls the timekeeping mechanism of the heart's pacemaker[END_REF] (Figure 1A, upper halve). In antagonism with adrenergic activation, muscarinic type 2 receptors (M2Rs) decrease pacemaking by promoting down-regulation of intracellular cAMP and inducing opening of G protein activated K + channels (GIRK1/GIRK4) underlying cardiac IKACh [START_REF] Wickman | Abnormal heart rate regulation in GIRK4 knockout mice[END_REF][START_REF] Mesirca | The G-protein-gated K+ channel, IKACh, is required for regulation of pacemaker activity and recovery of resting heart rate after sympathetic stimulation[END_REF].

Several aspects of pacemaker mechanism are not fully understood. However, a substantial part of the current knowledge on the generation of the diastolic depolarization has been included in the so called "coupled-clock" model of pacemaking [START_REF] Lakatta | A coupled SYSTEM of intracellular Ca2+ clocks and surface membrane voltage clocks controls the timekeeping mechanism of the heart's pacemaker[END_REF]. The coupled-clock model of pacemaking indicates that the generation of diastolic depolarization results from a functional association between the activation of the hyperpolarization-activated "funny" current (If) and a phenomenon of local diastolic RyR2-dependent Ca 2+ release, also know as the "Ca 2+ clock" [START_REF] Lakatta | A coupled SYSTEM of intracellular Ca2+ clocks and surface membrane voltage clocks controls the timekeeping mechanism of the heart's pacemaker[END_REF].

If is activated at the end of the repolarization phase of the action potential and supplies inward current throughout the range of the diastolic depolarization [START_REF] Difrancesco | The role of the funny current in pacemaker activity[END_REF] (Figure 1B). Adrenergic and cholinergic agonists exert opposite effects on If voltage-dependence. Norepinephrine shifts If activation curve to more positive voltages, while acetylcholine negatively shifts it. These opposing effects are explained by the direct sensitivity of f-channels to cAMP [START_REF] Difrancesco | Direct activation of cardiac pacemaker channels by intracellular cyclic AMP[END_REF], which increases the probability of channel opening at a given voltage [START_REF] Difrancesco | Modulation of single hyperpolarization-activated channels (i(f)) by cAMP in the rabbit sino-atrial node[END_REF]. F-channels are encoded by the hyperpolarization activated cyclic nucleotide gated channel (HCN) family, which comprises four distinct isoforms HCN1-HCN4. The predominant HCN isoform expressed in the SAN and AVN is HCN4, which accounts for 80% of the total HCN mRNAs [START_REF] Shi | Distribution and prevalence of hyperpolarization-activated cation channel (HCN) mRNA expression in cardiac tissues[END_REF].

The concept of Ca 2+ clock as a pacemaker mechanism proposes that SAN RyR2 have high spontaneous open probability. This spontaneous, voltage-independent activity generates pulsatile diastolic Ca 2+ release. These events of Ca 2+ release are coupled to diastolic depolarization via activation of the electrogenic Na + /Ca 2+ exchange mediated by the cardiac type 1 exchanger (NCX1; lower halve of Figure 1A) [START_REF] Lakatta | A coupled SYSTEM of intracellular Ca2+ clocks and surface membrane voltage clocks controls the timekeeping mechanism of the heart's pacemaker[END_REF][START_REF] Monfredi | Modern concepts concerning the origin of the heartbeat[END_REF]. In the Ca 2+ clock model of automaticity, intracellular cAMP increases the slope of the diastolic depolarization phase by facilitating and synchronizing spontaneous Ca 2+ release events to increase NCX1 activity. In the Ca 2+ clock model of pacemaking, L-type Ca 2+ channels provide Ca 2+ influx during the action potential upstroke phase to ensure SR Ca 2+ load. However, both the Ca 2+ clock or the coupled-clock model of automaticity cannot explain the phenotype of mouse models and of individuals with SND induced by loss-of-function of VGCCs [START_REF] Mangoni | Functional role of L-type Cav1.3 Ca2+ channels in cardiac pacemaker activity[END_REF][START_REF] Torrente | L-type Cav1.3 channels regulate ryanodine receptor-dependent Ca2+ release during sino-atrial node pacemaker activity[END_REF][START_REF] Baig | Loss of Ca(v)1.3 (CACNA1D) function in a human channelopathy with bradycardia and congenital deafness[END_REF].

Voltage-gated Ca 2+ channels in the generation of SAN automaticity

Several ion channels including VGCCs contribute to the generation of diastolic depolarization. SAN pacemaker cells express both T-and L-type Ca 2+ channels. The mRNAs of the T-type isoforms Cav3.1 and Cav3.2 are expressed in the SAN [START_REF] Mangoni | Bradycardia and slowing of the atrioventricular conduction in mice lacking CaV3.1/alpha1G T-type calcium channels[END_REF]. However, the predominant T-type isoform in the adult SAN is Cav3.1 (see ref. [START_REF] Mangoni | Bradycardia and slowing of the atrioventricular conduction in mice lacking CaV3.1/alpha1G T-type calcium channels[END_REF] for review). The threshold for activation of the Cav3.1 -mediated ICaT is -55 mV however, 10% of the total steady-state Cav3.1-mediated ICaT is available at the maximum diastolic potential [START_REF] Mangoni | Bradycardia and slowing of the atrioventricular conduction in mice lacking CaV3.1/alpha1G T-type calcium channels[END_REF] (Figure 1B). The relative position of the steadystate activation and inactivation curves for Cav3.1-mediated ICaT does not suggest significant window current component [START_REF] Mangoni | Voltagedependent calcium channels and cardiac pacemaker activity: From ionic currents to genes[END_REF]. However, further studies using selective blockade of ICaT will address this potentially important aspect of ICaT in SAN cells.

Despite the low steady-state availability of T-type channels at diastolic voltages, Cav3.1 knockout mice (Cav3.1 -/-) show moderate SAN rate reduction (-10%) and prolonged atrioventricular (PR) conduction interval [START_REF] Mangoni | Bradycardia and slowing of the atrioventricular conduction in mice lacking CaV3.1/alpha1G T-type calcium channels[END_REF]. In addition, recent data indicate that Cav3.1mediated ICaT is positively modulated by ßARs agonists in the SAN [START_REF] Li | Increasing T-type calcium channel activity by beta-adrenergic stimulation contributes to beta-adrenergic regulation of heart rates[END_REF]. In conclusion, consistent results indicate that T-type Cav3.1 channels contribute to set the SAN firing rate.

Adult ventricular myocytes express the L-type Cav1.2 isoform, which couples excitation to contraction in the working myocardium. In contrast, SAN pacemaker cells co-express two distinct L-type Ca 2+ channel isoforms, Cav1.3 and Cav1.2 [START_REF] Mangoni | Functional role of L-type Cav1.3 Ca2+ channels in cardiac pacemaker activity[END_REF][START_REF] Zhang | Functional Roles of Ca(v)1.3 (alpha(1D)) calcium channel in sinoatrial nodes: insight gained using gene-targeted null mutant mice[END_REF]. Cav1.3-mediated ICaL is characterized by a more negative threshold for activation than Cav1.2-mediated ICaL (-45 mV for Cav1.3 vs. -25 mV for Cav1.2-mediated ICaL, respectively; Figure 1B) [START_REF] Mangoni | Functional role of L-type Cav1.3 Ca2+ channels in cardiac pacemaker activity[END_REF]. Thanks to the lower threshold of activation, the L-type Cav1.3 channels play a major role in the generation of the diastolic depolarization [START_REF] Mangoni | Functional role of L-type Cav1.3 Ca2+ channels in cardiac pacemaker activity[END_REF]. In addition, recent work has showed that Cav1.3 is an obligatory molecular component of the sustained inward Na + current (Ist) [START_REF] Toyoda | CaV1.3 L-type Ca2+ channel contributes to the heartbeat by generating a dihydropyridinesensitive persistent Na+ current[END_REF]. Ist supplies inward current that sums to Cav1.3-mediated ICaL during the diastolic depolarization (Figure 1B). Moreover, Cav1.3 channels stimulate RyRdependent local Ca 2+ release, possibly enhancing NCX1 activity during the phase of diastolic depolarization. In summary, SAN cells express 3 distinct voltage-gated Ca 2+ channel isoforms, whose voltage dependencies of activation span the range of the diastolic depolarization (Cav3.1 and Cav1.3) and the upstroke phase of the action potential (Cav1.3, Cav1.2, see Figure 1B) (see [START_REF] Mangoni | Voltagedependent calcium channels and cardiac pacemaker activity: From ionic currents to genes[END_REF] and [START_REF] Mesirca | Functional role of voltage gated Ca(2+) channels in heart automaticity[END_REF], for review).

Voltage-gated Na + channels mediate the TTX-sensitive Na + current (INaTTX), which contribute to pacemaking and intra-nodal conduction in the mouse, rabbit and human SAN [START_REF] Maier | An unexpected requirement for brain-type sodium channels for control of heart rate in the mouse sinoatrial node[END_REF][START_REF] Baruscotti | A TTX-sensitive inward sodium current contributes to spontaneous activity in newborn rabbit sino-atrial node cells[END_REF][START_REF] Lei | Requirement of neuronal-and cardiac-type sodium channels for murine sinoatrial node pacemaking[END_REF][START_REF] Li | Impaired neuronal sodium channels cause intranodal conduction failure and reentrant arrhythmias in human sinoatrial node[END_REF] (Figure 1B). In the human SAN, the TTX-sensitive Nav1.6 isoform plays an important role in intra-nodal conduction and SND [START_REF] Li | Impaired neuronal sodium channels cause intranodal conduction failure and reentrant arrhythmias in human sinoatrial node[END_REF]. In the SAN, the "cardiac" Nav1.5 isoform underlies the TTX-resistant INa. However, the contribution of Nav1.5-mediated INa may be limited to intranodal or nodal-atrial impulse conduction. Consistently with this hypothesis, mice haplo-insufficient for Nav1.5 show atrio-ventricular and intra-ventricular conduction defects, rather than SND [START_REF] Lei | Sinus node dysfunction following targeted disruption of the murine cardiac sodium channel gene Scn5a[END_REF].

Ion channels of the transient receptor potential channels family (TRP) are expressed in the SAN and involved in pacemaker activity [START_REF] Demion | TRPM4, a Ca(2+)-activated nonselective cation channel in mouse sino-atrial node cells[END_REF][START_REF] Sah | Ion channel-kinase TRPM7 is required for maintaining cardiac automaticity[END_REF][START_REF] Ju | Store-operated Ca2+ influx and expression of TRPC genes in mouse sinoatrial node[END_REF]. TRPC channels mediate store-operated Ca 2+ entry in pacemaker cells [START_REF] Ju | Store-operated Ca2+ influx and expression of TRPC genes in mouse sinoatrial node[END_REF]. The channel-kinase TRPM7 isoform contributes to pacemaking by regulating HCN4 expression in the SAN and AVN. In addition, it contributes to Ca 2+ influx in SAN cells [START_REF] Sah | Timing of myocardial trpm7 deletion during cardiogenesis variably disrupts adult ventricular function, conduction, and repolarization[END_REF]. TRPM4 channels have mixed Na + /K + selectivity and contribute to the basal beating rate of SAN cells [START_REF] Demion | TRPM4, a Ca(2+)-activated nonselective cation channel in mouse sino-atrial node cells[END_REF]. Since these channels are sensitive to intracellular Ca 2+ , it is possible that RyR2 -dependent Ca 2+ release positively regulates TRPM4 in SAN cells.

The mouse and rabbit SAN express also Ca 2+ activated K + channels (KCa) [START_REF] Gueguinou | KCa and Ca(2+) channels: the complex thought[END_REF]. Three classes of KCa have been identified: Big [START_REF] Lai | BK channels regulate sinoatrial node firing rate and cardiac pacing in vivo[END_REF], Intermediate [START_REF] Haron-Khun | SK4 K(+) channels are therapeutic targets for the treatment of cardiac arrhythmias[END_REF] and Small [START_REF] Chen | Apamin modulates electrophysiological characteristics of the pulmonary vein and the Sinoatrial Node[END_REF][START_REF] Torrente | Contribution of small conductance K+ channels to sinoatrial node pacemaker activity: insights from atrial-specific Na+ /Ca2+ exchange knockout mice[END_REF] conductance K + channels. These channels show differential sensitivity to intracellular Ca 2+ . Ca 2+ positively regulates Big KCa (BK), even if these channels are mainly voltage-dependent [START_REF] Gueguinou | KCa and Ca(2+) channels: the complex thought[END_REF]. Intermediate and Small KCa (IK and SK, respectively) are voltage independent and highly sensitive to Ca 2+ [START_REF] Haron-Khun | SK4 K(+) channels are therapeutic targets for the treatment of cardiac arrhythmias[END_REF][START_REF] Torrente | Contribution of small conductance K(+) channels to sinoatrial node pacemaker activity: insights from atrial-specific Na(+) /Ca(2+) exchange knockout mice[END_REF]. In atrial cardiomyocytes such sensitivity to Ca 2+ is mediated by the association with calmodulin (CaM) [START_REF] Gueguinou | KCa and Ca(2+) channels: the complex thought[END_REF]. SK channels coupling with L-type Ca 2+ channels has been demonstrated also in atrial and atrioventricular myocytes [START_REF] Zhang | Functional roles of a Ca2+-activated K+ channel in atrioventricular nodes[END_REF][START_REF] Zhang | Coupling of SK channels, L-type Ca(2+) channels, and ryanodine receptors in cardiomyocytes[END_REF].

The Sinus Node Dysfunction and Deafness Syndrome: a Cav1.3 channelopathy

Transgenic mice carrying ablation of Cav1.3 have provided significant insights in the importance of these channels in pacemaker activity. To date only one pathogenic mutation of the CACNA1D gene underlying SND has been identified: the Sinus Node Dysfunction and Deafness (SANDD OMIM # 614896)) syndrome. Together with the Jervell-Lange-Nielsen syndrome caused by mutations in either KCNQ1 [START_REF] Neyroud | A novel mutation in the potassium channel gene KVLQT1 causes the Jervell and Lange-Nielsen cardioauditory syndrome[END_REF] (OMIM # 220400) or KCNE1 [START_REF] Drici | Involvement of IsK-associated K+ channel in heart rate control of repolarization in a murine engineered model of Jervell and Lange-Nielsen syndrome[END_REF] (OMIM # 612347) channels encoding the IKs current, SANDD constitutes a syndromic channelopathy, in which deafness is associated with heart arrhythmia. In addition, this channelopathy was the first to provide direct genetic evidence of the importance of Cav1.3 in SAN automaticity in humans [START_REF] Baig | Loss of Ca(v)1.3 (CACNA1D) function in a human channelopathy with bradycardia and congenital deafness[END_REF].

At present, SANDD has been identified in seven consanguineous Pakistani families, all from the Khyber Pakhtunkhwa province [START_REF] Baig | Loss of Ca(v)1.3 (CACNA1D) function in a human channelopathy with bradycardia and congenital deafness[END_REF][START_REF] Liaqat | Identification of CACNA1D variants associated with sinoatrial node dysfunction and deafness in additional Pakistani families reveals a clinical significance[END_REF]. The SANDD mutation of CACNA1D gene consists in a 3 base pair insertion adding a glycine residue at position 403-404 (p.403-404InsGly) in the alternatively spliced exon 8B. The related sequence in exon 8A does not contain the insertion. Expression of exons 8A and 8B is mutually exclusive. The 8B exon is preferentially expressed in inner hair cells and in the SAN. Addition of this glycine residue affects the folding of one of the four S6 helices probably forming the inner pore of the 8B isoform of Cav1.3. The glycine residue lies in the cytoplasmic side of the pore [START_REF] Baig | Loss of Ca(v)1.3 (CACNA1D) function in a human channelopathy with bradycardia and congenital deafness[END_REF] (Figure 2A). This region of S6 is highly conserved among VGCCs and is very sensitive functionally. Indeed, missense mutations in this region of the CACNA1F (Cav1.4) or CACNA1C (Cav1.2) genes cause stationary night blindness [START_REF] Hoda | Congenital stationary night blindness type 2 mutations S229P, G369D, L1068P, and W1440X alter channel gating or functional expression of Ca(v)1.4 L-type Ca2+ channels[END_REF] and Timothy syndrome [START_REF] Splawski | Ca(V)1.2 calcium channel dysfunction causes a multisystem disorder including arrhythmia and autism[END_REF], respectively. Nevertheless, these mutations induce channel gain-of-function by hyperpolarizing the channel voltage-dependency of activation and/or by slowing the channel inactivation kinetics [START_REF] Calorio | Impaired chromaffin cell excitability and exocytosis in autistic Timothy syndrome TS2neo mouse rescued by L-type calcium channel blockers[END_REF]. Instead, the p.403-404InsGly silences the conductance of mutant Cav1.3 (Cav1.3 403Gly ).

Non-conducting Cav1.3 403Gly channels are targeted to the plasma membrane, but fail to produce functional Ca 2+ current [START_REF] Baig | Loss of Ca(v)1.3 (CACNA1D) function in a human channelopathy with bradycardia and congenital deafness[END_REF] (Figure 2B). The exact mechanism leading to silencing of the channel's conductance is not fully understood. However, recordings of the voltage-dependence of gating currents of mutant Cav1.3 channels provided some insights into how the mutation affects the channel's gating. Indeed, ON-gating currents show faster time-to-peak in comparison to wildtype counterparts, suggesting that this mutation induces uncoupling between gating charge movement of the S4 segment and channel opening. This hypothesis is consistent with the observation that the time integral of the gating currents of the mutated channels is smaller than wild-type, while the ON-rise time of gating currents is faster. Accessorily, this observation shows that the glycine residue locates outside the S4 segment, although it can still influence its movement preventing the channel to enter in the open conductive state. Another non-exclusive hypothesis could be that the inserted glycine residue, which lays close to inner side of the channel pore could sterically interfere with the flow of Ca 2+ ions.

While homozygous 403-404InsGly individuals develop SANDD, heterozygous subjects belonging to the same family are normal. A complete clinical correlative rhythmologic follow-up according to current guidelines [START_REF] Kusumoto | ACC/AHA/HRS Guideline on the Evaluation and Management of Patients With Bradycardia and Cardiac Conduction Delay: Executive Summary: A Report of the American College of Cardiology/American Heart Association Task Force on Clinical Practice Guidelines, and the Heart Rhythm Society[END_REF] could not be performed in these patients. However, the ECG analysis and retrospective history of homozygous individuals carrying mutant Cav1.3 InsGly channels is consistent with symptomatic SND. Indeed, SND in homozygous SANDD individuals was manifest as sinus bradycardia at day time (heart rate between 38 and 52 bpm) and severe atrioventricular conduction dysfunction [START_REF] Baig | Loss of Ca(v)1.3 (CACNA1D) function in a human channelopathy with bradycardia and congenital deafness[END_REF]. Sinus bradycardia was particularly prominent at night-time with resting heart rates below 35 bpm (Figure 3A). Other ECG hallmarks of SND include sinus pauses and SAN exit block. Hallmarks of atrioventricular dysfunction in SANDD individuals range from Mobitz 1, 2 nd -degree block to episodes of complete heart block with junctional escape rhythms [START_REF] Baig | Loss of Ca(v)1.3 (CACNA1D) function in a human channelopathy with bradycardia and congenital deafness[END_REF]. Importantly, similar hallmarks of SND and atrioventricular block are present in homozygous Cav1.3 knockout (Cav1.3 -/-) mice (Figure 3B) [START_REF] Mesirca | G protein-gated IKACh channels as therapeutic targets for treatment of sick sinus syndrome and heart block[END_REF]. Three of the commonest SND symptoms, dizziness, fatigue and syncope during physical exercise have been also reported in young homozygous SANDD individuals [START_REF] Baig | Loss of Ca(v)1.3 (CACNA1D) function in a human channelopathy with bradycardia and congenital deafness[END_REF][START_REF] Liaqat | Identification of CACNA1D variants associated with sinoatrial node dysfunction and deafness in additional Pakistani families reveals a clinical significance[END_REF]. Instead, atrial fibrillation has not been directly reported in SANDD patients. Cav1.3 -/-mice show inducible atrial fibrillation, probably due to shorter duration of the atrial action potential and to alteration in intracellular Ca 2+ handling (Figure 3B) [START_REF] Mancarella | Impaired Ca2+ homeostasis is associated with atrial fibrillation in the alpha1D L-type Ca2+ channel KO mouse[END_REF]. The reasons for this difference between SANDD patients and Cav1.3 -/-mice are unclear. They may be explained by structural differences between the mouse and the human heart or to a differential functional impact between deletion of Cav1.3 -/-mice versus silencing of channels' conductance in SANDD patients. Another mutation in CACNA1D has been identified in a family carrying p.403-404InsGly and presenting SND (missense mutation p.(A376V)) [START_REF] Liaqat | Identification of CACNA1D variants associated with sinoatrial node dysfunction and deafness in additional Pakistani families reveals a clinical significance[END_REF]. This variant is also predicted to be pathogenic, but has not been characterized functionally [START_REF] Liaqat | Identification of CACNA1D variants associated with sinoatrial node dysfunction and deafness in additional Pakistani families reveals a clinical significance[END_REF].

The ANK2 syndrome: inheritable depletion of Cav1.3 contributes to SND

Besides SANDD, which is generated by specific mutation in the CACNA1D gene, another form of inherited SND has been linked to a change in the functional state of Cav1.3 channels. Ankyrin-B is a cytoskeletal adapter protein encoded by the ANK2 gene (OMIM # 600919). Ankyrin-B is important for proper membrane localization of multiple ion channels and transporters. Mutations in the ANK2 gene were initially linked to inheritable forms of ventricular tachycardia and sudden death [START_REF] Mohler | Ankyrin-B mutation causes type 4 long-QT cardiac arrhythmia and sudden cardiac death[END_REF]. Further work on ANK2 variants has identified French families showing that this gene is also important for the normal functioning of the SAN pacemaker mechanism [START_REF] Scouarnec | Dysfunction in ankyrin-B-dependent ion channel and transporter targeting causes human sinus node disease[END_REF][START_REF] Hund | Ankyrin-based targeting pathway regulates human sinoatrial node automaticity[END_REF]. Indeed, these variants in ANK2 are linked to arrhythmia syndrome and severe SND requiring pacemaker implantation [START_REF] Scouarnec | Dysfunction in ankyrin-B-dependent ion channel and transporter targeting causes human sinus node disease[END_REF]. Importantly, mice haplo-insufficient for Ankyrin-B (ANK2 +/-) show severe bradycardia and highly-variable heart rate compared to wild-type littermates [START_REF] Scouarnec | Dysfunction in ankyrin-B-dependent ion channel and transporter targeting causes human sinus node disease[END_REF]. This phenotype is explained by the capability of Ankyrin-B to control membrane localization of critical players of pacemaker activity. Indeed, Ankyrin-B haplo-insufficiency reduces the membrane localization of the NCX, the Na + /K + ATPase, IP3 receptors and of Cav1.3 channels [START_REF] Scouarnec | Dysfunction in ankyrin-B-dependent ion channel and transporter targeting causes human sinus node disease[END_REF]. Because the localization of both ion channels and transporters is affected by Ankyrin-B haploinsufficiency, it is difficult to quantitate the specific contribution of Cav1.3 to SND observed in this mouse model. However, inherited forms of SND linked to ANK2 loss-of-function constitute further evidence of the importance of Cav1.3 channels in the generation of SAN automaticity in humans.

Congenital heart block: an autoimmune Cav1.3 and Cav3.1 channelopathies

Congenital heart block (CHB, OMIM # 234700) is a autoimmune neonatal lupus disease of the cardiac conduction system affecting newborns and infants (see refs. [START_REF] Boutjdir | Molecular and ionic basis of congenital complete heart block[END_REF][START_REF] Karnabi | Role of calcium channels in congenital heart block[END_REF][START_REF] Qu | Autoimmune Calcium Channelopathies and Cardiac Electrical Abnormalities[END_REF] for review). It has been proposed that CHB belongs to a specific class of channelopathies linked to autoimmune disease [START_REF] Qu | Autoimmune Calcium Channelopathies and Cardiac Electrical Abnormalities[END_REF]. The general incidence of CHB is estimated to about 1 in 11,000 births [START_REF] Buyon | Autoimmune-associated congenital heart block: demographics, mortality, morbidity and recurrence rates obtained from a national neonatal lupus registry[END_REF]. CHB carries severe consequences. Indeed, mortality among siblings may score up to 30% during the first three months after birth and over 60% of surviving affected children require lifelong electronic pacemaker implantation [START_REF] Buyon | Autoimmune-associated congenital heart block: demographics, mortality, morbidity and recurrence rates obtained from a national neonatal lupus registry[END_REF][START_REF] Boutjdir | Molecular and ionic basis of congenital complete heart block[END_REF].

Familial recurrence of CHB is high and influences the decision making of having another child [START_REF] Boutjdir | Molecular and ionic basis of congenital complete heart block[END_REF]. Indeed, the probability of having the disease at the second birth is more than twofold that of the first birth. The disease originates from transplacental transport of maternal autoantibodies that severely affects the heart of the fetus before and after birth, while having milder or no effects on the mother's heart. Maternal autoantibodies target intracellular ribonucleoproteins, as well as membrane receptors and ion channels [START_REF] Boutjdir | Molecular and ionic basis of congenital complete heart block[END_REF]. The principal hallmark of CHB is atrioventricular block [START_REF] Crittenden | Familial Congenital Heart Block[END_REF], even if sinus bradycardia and SND often accompanies heart block [START_REF] Boutjdir | Molecular and ionic basis of congenital complete heart block[END_REF]. The severity of heart block is variable ranging from prolongation of the PR interval to 3 rd degree block with dissociated rhythm. While 1 st and 2 nd degree blocks may spontaneously reverse after birth, complete 3 rd degree block is irreversible and requires pacemaker implantation. In addition, some CHB children develop heart failure later in life despite the implanted pacemaker [START_REF] Boutjdir | Molecular and ionic basis of congenital complete heart block[END_REF].

The mechanisms underlying CHB are multiple and complex. However, a substantial amount of studies over the last two decades demonstrate a prominent involvement of both L-type and T-type VGCCs in the development of SND and heart block in CHB infants. Maternal autoantibodies were characterized as being directed against soluble ribonucleoproteins 48kD SSB/La, 52kD SSA/Ro, and 60kD SSA/Ro. Bioinformatics analysis conducted in early studies suggested that the 52 kD SSA/Ro shared significant homology with the α1-subunit of Cav1.2. In contrast, the homology between Cav1.2 and 60kD SSA/Ro or 48 kD SSB/La ribonucleoproteins are minor [START_REF] Boutjdir | Molecular and ionic basis of congenital complete heart block[END_REF].

Experimental work in vivo has shown that female mice injected with purified autoantibodies from mothers of children with CHB produced a highly-significant number of pups presenting with sinus bradycardia and prolongation of PR interval [START_REF] Brucato | Prevention of recurrences of corticosteroid-dependent idiopathic pericarditis by colchicine in an adolescent patient[END_REF][START_REF] Boutjdir | Molecular and ionic basis of congenital complete heart block[END_REF]. In addition, female mice immunized with 52kD SSA/Ro gave birth to pups showing 2 nd degree atrioventricular block and sinus bradycardia [START_REF] Boutjdir | Arrhythmogenicity of IgG and anti-52-kD SSA/Ro affinity-purified antibodies from mothers of children with congenital heart block[END_REF]. These same antibodies slowed the beating rate of Langendorff-perfused human fetal hearts and reduced ICaL in ventricular myocytes [START_REF] Boutjdir | Arrhythmogenicity of IgG and anti-52-kD SSA/Ro affinity-purified antibodies from mothers of children with congenital heart block[END_REF] and in myocytes from Purkinje fibres [START_REF] Qu | Autoantibodies from mothers of children with congenital heart block downregulate cardiac L-type Ca channels[END_REF]. More specific investigation of the supraventricular conduction system showed that maternal autoantibodies slowed pacemaker activity of intact rat AVN preparations [START_REF] Boutjdir | Molecular and ionic basis of congenital complete heart block[END_REF] and induced sinus bradycardia and atrioventricular block [START_REF] Restivo | Optical mapping of activation patterns in an animal model of congenital heart block[END_REF].

The effects of autoantibodies on ICaL were first explained by inhibition of Cav1.2 channels [START_REF] Qu | Autoantibodies from mothers of children with congenital heart block downregulate cardiac L-type Ca channels[END_REF]. Deeper analysis of the effects of maternal antibodies on SAN ionic currents showed significant inhibition of ICaL and ICaT and no significant effect on IKr and If [START_REF] Hu | Functional basis of sinus bradycardia in congenital heart block[END_REF]. Consistent with current inhibition, autoantibodies slowed the pacemaker activity of isolated SAN cells [START_REF] Hu | Functional basis of sinus bradycardia in congenital heart block[END_REF].

Given the major role of Cav1.3 in determining SAN ICaL, pacemaker activity and atrioventricular conduction, the effects of autoantibodies on Cav1.3-mediated ICaL has been tested in Xenopus oocytes [START_REF] Qu | Novel molecular mechanism involving alpha1D (Cav1.3) L-type calcium channel in autoimmune-associated sinus bradycardia[END_REF]. Interestingly, autoantibodies inhibit heterologously expressed Cav1.3mediated ICaL, while application of Bay K8644 rescues current density [START_REF] Qu | Novel molecular mechanism involving alpha1D (Cav1.3) L-type calcium channel in autoimmune-associated sinus bradycardia[END_REF]. Recently, the binding sites of autoantibodies on the α1-subunit of Cav1.3 [START_REF] Karnabi | Congenital heart block: identification of autoantibody binding site on the extracellular loop (domain I, S5-S6) of alpha(1D) L-type Ca channel[END_REF] and Cav3.1 [START_REF] Strandberg | Congenital heart block maternal sera autoantibodies target an extracellular epitope on the alpha1G T-type calcium channel in human fetal hearts[END_REF] channels have been identified. In both channels, the binding site lays in the extracellular loop linking the S5 and S6 domains of α1-subunit, in close proximity to the channel's pore. The positioning of the binding sites in Cav1.3 and Cav3.1 channels is consistent with the observation that acutely perfused autoantibodies quickly induce inhibition of Cav1.3-mediated ICaL and Cav3.1-mediated ICaT.

The mechanism leading to CHB in the fetal heart may involve a two-stages process. In the first stage, maternal autoantibodies bind to the α1 subunit inhibiting channel's permeability. In the second chronic and irreversible stage of the disease, VGCCs are internalized in the myocyte [START_REF] Karnabi | Role of calcium channels in congenital heart block[END_REF].

It has also been proposed that chronic exposure to maternal autoantibodies and channels internalization trigger myocyte death and consequent cell loss in the conduction system. This hypothesis is consistent with post-mortem observations in CHB hearts which reported fibrosis and calcification of the SAN and AVN [START_REF] Karnabi | Role of calcium channels in congenital heart block[END_REF]. In addition, heart failure could be a consequence of chronic block and internalization of Cav1.2 channels [START_REF] Karnabi | Role of calcium channels in congenital heart block[END_REF].

It has been shown that overexpression of Cav1.2 in the heart prevent the CHB phenotype of pups born to mice immunized with SSA/Ro or SSB/La antigens [START_REF] Karnabi | Rescue and worsening of congenital heart block-associated electrocardiographic abnormalities in two transgenic mice[END_REF]. It could thus be inferred that, given that adult cardiomyocytes express higher densities of ICaL, compared to fetal counterparts, they may be protected from cell death processes triggered by the loss of Ca 2+ currents. These data may also explain, in part, why mothers are protected from CHB despite being exposed to the same circulating autoantibodies as fetuses [START_REF] Karnabi | Role of calcium channels in congenital heart block[END_REF].

Two transgenic mouse models of CHB have been proposed. The first employs Cav1.3 -/- female mice immunized with SSA/Ro or SSB/La antigens. Pups born to these mice present with sinus bradycardia and high degree atrioventricular blocks, sometimes with several isolated P waves not followed by a QRS complex. In comparison, pups born to immunized wild-type mice, in which Cav1.3 is still expressed, present with less pronounced sinus bradycardia and lower severity atrioventricular dysfunction [START_REF] Karnabi | Rescue and worsening of congenital heart block-associated electrocardiographic abnormalities in two transgenic mice[END_REF]. The phenotype of pups born to immunized Cav1.3 -/-mothers is consistent with mice lacking both Cav1.3 and Cav3.1 channels (Cav1.3 -/-/Cav3.1 -/-). Indeed, these mice show high-degree atrioventricular block and sinus bradycardia [START_REF] Marger | Functional roles of Ca(v)1.3, Ca(v)3.1 and HCN channels in automaticity of mouse atrioventricular cells: insights into the atrioventricular pacemaker mechanism[END_REF]. These results underscore the importance of Cav1.3 and Cav3.1 channels inhibition and down-regulation in the establishment of atrioventricular lesion in CHB.

In conclusion, the physiopathological mechanism of CHB are consistent with the importance of Cav1.3 and Cav3.1 channels in the generation of SAN pacemaking and atrioventricular conduction in both model mammals and in humans.

Mechanistic considerations: how do channelopathies of Cav1.3 or Cav3.1 lead to SND?

In its current formulation, the coupled clock model of SAN pacemaking gives the primary roles in the generation of automaticity to activation of f-channels and to diastolic spontaneous RyR2-dependent Ca 2+ release. This is often in marked contrast with the observed functional impact of Cav1.3 loss-of-function in heart automaticity of mouse models and humans. Thus, questions arise as to the mechanisms linking Cav1.3 channels to the generation of the diastolic depolarization and about how Cav1.3 loss-of-function prevents normal pacemaking in human SANDD.

Our group has identified three Cav1.3-dependent pathways contributing to diastolic depolarization. First, Cav1.3 channels drive inward Ca 2+ current in the range of the diastolic depolarization (Figure 4). In this respect, under activation of ßARs the recorded threshold for activation of Cav1.3-mediated ICaL lays between -55 mV and -60 mV [START_REF] Mangoni | Functional role of L-type Cav1.3 Ca2+ channels in cardiac pacemaker activity[END_REF][START_REF] Torrente | L-type Cav1.3 channels regulate ryanodine receptor-dependent Ca2+ release during sino-atrial node pacemaker activity[END_REF]. Second, Cav1.3mediated ICaL contributes to pacemaking by stimulating and synchronizing diastolic RyR2dependent Ca 2+ release (Figure 5) [START_REF] Torrente | L-type Cav1.3 channels regulate ryanodine receptor-dependent Ca2+ release during sino-atrial node pacemaker activity[END_REF]. Third, Cav1.3 channels are a necessary molecular component of the sustained inward Na + current (Ist) [START_REF] Toyoda | CaV1.3 L-type Ca2+ channel contributes to the heartbeat by generating a dihydropyridinesensitive persistent Na+ current[END_REF]. Furthermore, selective pharmacologic inhibition of L-type Cav1.3 channels reduces heart rate in vivo in Cav1.2 DHP-/-mice [START_REF] Sinnegger-Brauns | Isoform-specific regulation of mood behavior and pancreatic beta cell and cardiovascular function by L-type Ca 2+ channels[END_REF][START_REF] Mesirca | G protein-gated IKACh channels as therapeutic targets for treatment of sick sinus syndrome and heart block[END_REF].

In conclusion, Cav1.3 channels contribute to the generation of the diastolic depolarization by driving inward current at diastolic voltages and by coordinating RyR2-dependent Ca 2+ release. Loss of these two downstream pathways may explain the phenotype of SANDD, ANK2 and CHB patients. Cav1.3 channels thus constitute an ideal functional bridge between changes in membrane voltages and intracellular Ca 2+ release during pacemaking [START_REF] Torrente | L-type Cav1.3 channels regulate ryanodine receptor-dependent Ca2+ release during sino-atrial node pacemaker activity[END_REF]. In comparison to Cav1.3, T-type Cav3.1 channel steady-state availability is reduced at diastolic voltages. However, work on genetically-modified mice have showed that these channels play a role in SAN activity, atrioventricular conduction and junctional automaticity [START_REF] Mangoni | Bradycardia and slowing of the atrioventricular conduction in mice lacking CaV3.1/alpha1G T-type calcium channels[END_REF][START_REF] Marger | Functional roles of Ca(v)1.3, Ca(v)3.1 and HCN channels in automaticity of mouse atrioventricular cells: insights into the atrioventricular pacemaker mechanism[END_REF][START_REF] Quang | T-type calcium current contributes to escape automaticity and governs the occurrence of lethal arrhythmias after atrioventricular block in mice[END_REF]. In perspective, recent work has described pathogenic variants of CACNA1D (OMIM, # 615474), CACNA1G (OMIM#618087) and CACNA1H (OMIM # 617027) genes inducing gain of function in Cav1.3 [START_REF] Pinggera | CACNA1D de novo mutations in autism spectrum disorders activate Cav1.3 L-type calcium channels[END_REF][START_REF] Pinggera | New gain-of-function mutation shows CACNA1D as recurrently mutated gene in autism spectrum disorders and epilepsy[END_REF][START_REF] Tan | Aldosterone-Producing Adenomas: Histopathology-Genotype Correlation and Identification of a Novel CACNA1D Mutation[END_REF], Cav3.1 [START_REF] Chemin | De novo mutation screening in childhood-onset cerebellar atrophy identifies gain-of-function mutations in the CACNA1G calcium channel gene[END_REF] and Cav3.2 [START_REF] Scholl | Recurrent gain of function mutation in calcium channel CACNA1H causes early-onset hypertension with primary aldosteronism[END_REF] channels. Gain-of-function is characterized by dramatic slowing of Cav1.3mediated ICaL or Cav3.1-mediated ICaT inactivation kinetics and negative shift of the current steadystate inactivation curve. These gain-of-function variants are linked to primary aldosteronism [START_REF] Tan | Aldosterone-Producing Adenomas: Histopathology-Genotype Correlation and Identification of a Novel CACNA1D Mutation[END_REF], early-onset neurodevelopmental defects [START_REF] Chemin | De novo mutation screening in childhood-onset cerebellar atrophy identifies gain-of-function mutations in the CACNA1G calcium channel gene[END_REF] and autism [START_REF] Pinggera | New gain-of-function mutation shows CACNA1D as recurrently mutated gene in autism spectrum disorders and epilepsy[END_REF]. It will be very important to pursue the screening for similar variants affecting the function of Cav1.3 or Cav3.1 channels in the cardiac pacemaker tissue, as they could explain primary forms of inappropriate sinus tachycardia.

Rescuing dysfunction of heart automaticity in Cav1.3 and Cav3.1 channelopathies

The pacemaker mechanism is a highly-integrated physiological function. To date, it has not been possible to arrest pacemaking by targeting an individual ion channel isoform. A similar observation stands also for adrenergic or cholinergic regulation of pacemaker activity. We may thus wonder if we could take advantage of the functional redundancy between SAN ion channels to rescue SND. In particular, we should consider that the diastolic depolarization is the result of an equilibrium between inward and outward currents that give rise to a net inward current, which in turn allow membrane depolarization. Cav1.3-mediated ICaL and Cav3.1-mediated ICaT generate inward currents thereby participating in the equilibrium as accelerator of the diastolic depolarization.

Since the heart rate is under the constant action of the autonomic nervous system, one may expect that the parasympathetic input could worsen SND induced by loss-of-function of SAN VGCCs channels. First recordings of heart rhythm in Cav1.3 -/-mice indicated that pharmacologic inhibition of the anticholinergic nervous system input by atropine negated the difference in heart rate between wild-type and knockout mice [START_REF] Platzer | Congenital deafness and sinoatrial node dysfunction in mice lacking class D L-type Ca2+ channels[END_REF]. This was the first evidence suggesting that sympatholytic drugs could be effective in rescuing SND following Cav1.3 loss-of-function. In following studies by our group, it was showed that the cardiac IKACh carried by GIRK1/GIRK4 channels acts as a tonic inhibitor of SAN activity [START_REF] Mesirca | The G-protein-gated K+ channel, IKACh, is required for regulation of pacemaker activity and recovery of resting heart rate after sympathetic stimulation[END_REF]. Indeed, genetic ablation of IKACh obtained by cross-breeding Girk4 -/-with Cav1.3 -/-mice restored the heart rate and rhythm of double mutant animals in comparison to Cav1.3 -/-counterparts (Figure 6) [START_REF] Mesirca | G protein-gated IKACh channels as therapeutic targets for treatment of sick sinus syndrome and heart block[END_REF]. Importantly, the effect of genetic ablation of IKACh could be mimicked by administration to Cav1.3 -/-mice of tertiapine-Q, a peptide inhibitor of IKACh. This rescuing effect is attributable to restore of the equilibrium between inward and outward currents in the SAN and AVN of Cav1.3 -/-mice.

Taken together, these results suggest that selective IKACh inhibitors could be effective in managing SND due to channelopathies of Cav1.3 and Cav3.1 [START_REF] Mesirca | G protein-gated IKACh channels as therapeutic targets for treatment of sick sinus syndrome and heart block[END_REF]. In addition, it has been proposed that bait peptides mimicking the binding region of maternal autoantibodies to the S5-S6 loop of VGCCs could be used to prevent channel inhibition and internalization in CHB patients [START_REF] Karnabi | Congenital heart block: identification of autoantibody binding site on the extracellular loop (domain I, S5-S6) of alpha(1D) L-type Ca channel[END_REF][START_REF] Strandberg | Congenital heart block maternal sera autoantibodies target an extracellular epitope on the alpha1G T-type calcium channel in human fetal hearts[END_REF]. This interesting approach could be combined with targeting of IKACh in CHB. Both these approaches could be complementary to pacemaker implantation in chronic forms of SND and CHB.

Conclusions and perspectives

L-type Cav1.3 and T-type Cav3.1 Ca 2+ channels play an important role in the generation of heart automaticity. Since the beginning of the new century [START_REF] Mangoni | Pacemaker currents in mouse pacemaker cells[END_REF][START_REF] Mangoni | Properties of the hyperpolarization-activated current (I(f)) in isolated mouse sino-atrial cells[END_REF], genetically-modified mice have helped identify the role of ion channels isoforms in pacemaker activity [START_REF] Mangoni | Voltagedependent calcium channels and cardiac pacemaker activity: From ionic currents to genes[END_REF]. Importantly a number of these murine lines faithfully recapitulated some forms of inherited SND due to mutation in Cav1.3 channels and autoimmune loss-of-function of Cav3.1 channels. This enabled in depth mechanistic analysis of VGCCs-mediated SND mechanisms and provided precious insights into potential therapeutic strategies.

In perspective, we expect that further research on how Cav1.3 and Cav3.1 channels promote pacemaking will further deepen our knowledge on heart automaticity as a whole. Future exploration of genetic registries of patients with history of SND and associated arrhythmias will help evaluate the real incidence of Cav1.3 and Cav3.1 channelopathies leading to dysfunction in heart automaticity in the general population.

Figure 1 .

 1 Figure 1. Summary representation of SAN pacemaker activity. (A) Schematic drawing of a mouse SAN pacemaker cell showing the principal membrane receptors and ion channels together with RyR2, the SR calcium pump SERCA and the Na + /Ca 2+ exchanger (NCX1). Two of the most important protein kinases involved in regulation of pacemaker activity by the autonomic nervous system, the PKA and CaMKII, are shown. (B) Simulated transmembrane voltage during a spontaneous action potential (AP) generated by a computational model of mouse SAN pacemaking [15]. The other panels show calculated ionic currents during the AP. The following ionic currents are shown: funny current (If), (mediated by HCN family members), Cav1.3-and Cav1.2-mediated ICa,L, sustained inward Na + current Ist, Cav3.1-mediated ICa,T, fast TTX-sensitive Na + current (INa) (mediated by Nav1.1), and delayed rectifier K + current (mediated by the cardiac ERG1 gene). The current scale of ICaL, Ist and ICaT have been expanded to show the predicted current flow in the diastolic depolarization (DD). Note activation of Cav1.3-mediated ICaL and Ist at diastolic voltages. The simulation shows the sum of Cav1.3-mediated ICaL and Ist because these two currents share Cav1.3 as a common molecular determinant [80].

Figure 2 .

 2 Figure 2. The SANDD p.403-404InsGly mutation silences heterologously expressed Cav1.3-mediated ICaL. (A) General transmembrane topology of the α1 subunit of VGCCs. The approximate position of the insertion mutation p.403-404InsGly is indicated by the red dot. (B) Current-to-voltage relationships of Ba 2+ currents for wild-type containing regular exon 8B (black) or mutant 8Bm variant (gray) channels, and mock-transfected controls (triangles) tsA cells. From ref. [1]: Baig et al. (2011) Loss of Cav1.3 (CACNA1D) function in a human channelopathy with bradycardia and congenital deafness. Nat Neurosci 14:77-84.

Figure 3 .

 3 Figure 3. SND and atrioventricular dysfunctions of SANDD patients are recapitulated in Cav1.3 -/-mice. (A). Sample ECG recordings from an unaffected family member (a) and three individuals who are homozygous for the CACNA1D mutation (b-d). In b and c, asterisks mark P waves that precede QRS complexes; arrows indicate waveforms that suggest P waves coinciding with T waves. Numbers indicate heart rate (bpm) calculated from the corresponding beat-to-beat RR interval. From ref. [1]: Baig et al. (2011) Loss of Cav1.3 (CACNA1D) function in a human channelopathy with bradycardia and congenital deafness. Nat Neurosci 14:77-84. (B). Original ECG recordings from a wild-type (a) and Cav1.3 -/-(b) mouse. Asterisks represent conducted P waves as in (A), while (#) symbols mark non conducted atrial depolarisations. Arrow indicate P waves falling in correspondence of T waves. These ECG hallmarks in mice are predominantly generated by IKACh activation (see Figure 6).

Figure 4 .

 4 Figure 4. Sample recordings of ICa aligned with line scan imaging of intracellular Ca 2+ ([Ca 2+ ]i) release in single SAN cells. (A) Sample of mouse SAN cell AP applied as voltage command. The right panels show close-up view of the diastolic AP phase. (B) Sample traces of the corresponding net ICa in wild-type (WT) and Cav1.3 -/-SAN cells before and after perfusion of Nifedipine (Nife, 3 µM). The right panels show the diastolic Ca 2+ influx in faster time scale. Note the strong decrease of ICa in recordings from Cav1.3 -/-cells, due to ablation of diastolic Cav1.3mediated ICaL. (C) Time-integral of [Ca 2+ ]i fluorescence corresponding to the line scans below. Note the presence of an ascending ramp phase of [Ca 2+ ]i release in the wild-type cell. The ramp is generated by local Ca 2+ release (LCR) preceding cell-wide [Ca 2+ ]i transients. (D) 2D line-scan of

Figure 5 .

 5 Figure 5. Cav1.3-mediated synchronization of LCRs under β-adrenergic stimulation of pacemaking. (A) and (B): Line-scans (upper panel) and corresponding time-integrals (bottom panels) of fluorescence before (Contr) and after perfusion of the β-adrenergic agonist isoproterenol (ISO 10 nM) in single SAN cells of wild-type and Cav1.3 -/-mice. Note the ramp slope before and after β-adrenergic stimulation of wild-type cells (short lines in red and gray indicated ramps of wildtype and Cav1.3 -/-mice. (C) and (D): Cav1.3-mediated synchronization of LCRs events measured as the correlation between ramp amplitude (defined as the ratio between the fluorescence at the end and at the beginning of the ramp) and ramp length (interval between the beginning and the end of the ramp) in wild-type and Cav1.3 -/-cells. The straight line show linear regression of data. Pearson's coefficient calculation indicates significant correlation between ramp amplitude and length following ISO in wild-type cells but not in Cav1.3 -/-cells. From: ref. [77]: Torrente et al. (2016) L-type Cav1.3 channels regulate ryanodine receptor-dependent Ca 2+ release during sino-atrial node pacemaker activity. Cardiovasc Res 109:451-461.

Figure 6 .

 6 Figure 6. Rescuing SND and atrioventricular dysfunction by IKACh targeting. (A). This schematic representation depicts the balance between inward and outward currents determining the SAN diastolic depolarization and heart rhythm. In each panel, a sample of SAN spontaneous action potentials (Top), ion channels contributing to the diastolic depolarization (Middle), and sample of ECG recordings (Bottom) are shown. Green channels represents IKACh, red channel represents Cav1.3-mediated ICaL (ICav1.3), and the light-blue circles shows the idealized sum of other inward currents involved in automaticity (e.g. NCX1, If, ICaT, TRPM4…). Adapted from reference (35), with permission by Whiley and Son. From: ref. [54] Mesirca et al. (2016) G protein-gated IKACh channels as therapeutic targets for treatment of sick sinus syndrome and heart block. Proc Natl Acad Sci U S A 113:E932-941.

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Acknowledgments

The group is a member of the Laboratory of Excellence "Ion Channel Science and Therapeutics" supported by a grant from ANR (ANR-11-LABX-0015). Research supported by the Fondation pour la Recherche Medicale "Physiopathologie Cardiovasculaire" (DPC20171138970, M.E.M.), by the Agence Nationale de la Recherche (ANR-15-CE14-0004-01, M.E.M.). We also thank the Fondation Leducq (TNE 19CVD03; to Matteo E. Mangoni and Peter J. Mohler) for supporting the "Fighting Against Sinus Node Dysfunction and Associated Arrhythmias" (FANTASY) network.