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International audience; Background: Patients with metabolic syndrome (MetS) have an increased risk of atrial fibrillation (AF). Impaired Ca2+ homeostasis and mitochondrial dysfunction have emerged as an arrhythmogenic substrate in both patients and animal models of MetS. Whether impaired mitochondrial Ca2+ handling underlies AF associated with MetS remains poorly explored.Objectives: The aim of this study was to determine the initial mechanisms related to AF susceptibility and mitochondrial dysfunction encountered in metabolic cardiomyopathy.Methods: A total of 161 mice and 34 patients were studied. Mitochondrial Ca2+ and mitochondrial Ca2+ uniporter complex (MCUC) were investigated in right atrial tissue of patients with (n = 18) or without (n = 16) MetS and of C57Bl/6J mice fed with a high-fat sucrose diet (HFS) for 2 (n = 42) or 12 (n = 39) weeks. Susceptibility to AF was evaluated in isolated sinoatrial tissue and in vivo in mice.Results: Increased expression of the MICUs subunits of the MCUC (1.00 ± 0.33 AU vs 1.29 ± 0.23 AU; P = 0.034) was associated with impaired mitochondrial Ca2+ uptake in patients (168.7 ± 31.3 nmol/min/mg vs 127.3 ± 18.4 nmol/min/mg; P = 0.026) and HFS mice (0.10 ± 0.04 ΔF/F0 × ms-1 vs 0.06 ± 0.03 ΔF/F0 × ms-1; P = 0.0086, and 0.15 ± 0.07 ΔF/F0 × ms-1 vs 0.046 ± 0.03 ΔF/F0 × ms-1; P = 0.0076 in 2- and 12-week HFS mice, respectively). HFS mice elicited a 70% increased susceptibility to AF. The MCUC agonist kaempferol restored MCUC activity in vitro and abolished the occurrence of AF in HFS mice.Conclusions: Impaired MCUC activity and mitochondrial Ca2+ homeostasis from the early stage of metabolic cardiomyopathy in mice lead to AF. Given that similar defects in cardiac mitochondrial Ca2+ handling are present in MetS patients, the modulation of the MCUC activity represents an attractive antiarrhythmic strategy.

International audience; The heart automaticity is a fundamental physiological function in vertebrates. The cardiac impulse is generated in the sinus node by a specialized population of spontaneously active myocytes known as "pacemaker cells." Failure in generating or conducting spontaneous activity induces dysfunction in cardiac automaticity. Several families of ion channels are involved in the generation and regulation of the heart automaticity. Among those, voltage-gated L-type Cav1.3 (α1D) and T-type Cav3.1 (α1G) Ca2+ channels play important roles in the spontaneous activity of pacemaker cells. Ca2+ channel channelopathies specifically affecting cardiac automaticity are considered rare. Recent research on familial disease has identified mutations in the Cav1.3-encoding CACNA1D gene that underlie congenital sinus node dysfunction and deafness (OMIM # 614896). In addition, both Cav1.3 and Cav3.1 channels have been identified as pathophysiological targets of sinus node dysfunction and heart block, caused by congenital autoimmune disease of the cardiac conduction system. The discovery of channelopathies linked to Cav1.3 and Cav3.1 channels underscores the importance of Ca2+ channels in the generation and regulation of heart's automaticity.

BackgroundGray mouse lemurs (Microcebus murinus) are the smallest and most ancestral primates known. Their size falls in between that of mice and rats, and their genetic proximity to humans makes them an emerging model to study age-related neurodegeneration. Since mouse lemurs replicate similar senescence processes of humans, they constitute a useful model for studying cardiovascular dysfunctions. However, their cardiac physiology is unknown. Thus, we investigated the cardiac pacemaker activity generated by the sinoatrial node (SAN) of mouse lemurs, presenting the first characterization of heart automaticity in nonhuman primates.Methods and ResultsWe recorded cardiac automaticity in mouse lemurs and in their SAN tissues and pacemaker myocytes. Mouse lemurs have a heart rate (HR) in between those of mice and rats and a similar generation of the SAN electrical impulse. Their SAN myocytes express the main pacemaker currents at densities similar to mice: the hyperpolarization-activated current (If) and the L-type (Ica,L) and T-type (Ica,T) calcium currents. Conversely, their ventricular depolarization resembles that of large mammals and despite the small size of mouse lemurs, the total number of heartbeats in their life corresponds to what can be attained by humans.Using muscle-derived stem cells (MDSCs) from mouse lemurs, we also differentiated pacemaker-like (PML) cells showing spontaneous automaticity and expressing markers of native SAN myocytes (HCN4 and connexin-45).ConclusionsOur characterization of heart automaticity in Microcebus murinus provides new opportunities for comparative cardio-physiology studies in primates and with humans and for testing molecules that could modulate age-related dysfunctions of heart rate.

Funding Acknowledgements Type of funding sources: Foundation. Main funding source(s): ESC FRM Lefoulon Delalande Aims To investigate whether extracellular hypercalcemia alters the conduction through L-type Ca2+ channels (LTCCs), impairing the pacemaker activity of the heart. Introduction In the sino-atrial node (SAN), membrane currents and the dynamics of intracellular Ca2+ ([Ca2+]i) generate the pacemaker activity of the heart. SAN dysfunctions (SNDs) harm heart automaticity and have been associated with abnormal dynamics of [Ca2+]i. The LTCCs, Cav1.2 and Cav1.3 carry the main Ca2+ influx of SAN cells, which is necessary to sustain [Ca2+]i dynamics. Modified extracellular Ca2+ ([Ca2+]o) could alter Ca2+ influx through these channels. For example, cancer and hyperparathyroidism can raise [Ca2+]o, causing an extracellular hypercalcemia that could alter [Ca2+]i dynamics and impair SAN activity and heart automaticity. Methods and results To test this hypothesis, we measured contractions, [Ca2+]i release and L-type Ca2+ current (ICa,L) in spontaneous cells of the murine SAN. Then, we recorded rate and propagation of the spontaneous action potentials (APs) generated by the SAN tissue ex-vivo. In spontaneously beating SAN cells, we observed that the modification of [Ca2+]o affected [Ca2+]i and cell contractility through changes of ICa,L. In particular, the increase of [Ca2+]o dysregulated pacemaker activity, likely through excessive Ca2+ influx mediated by Cav1.2. [Ca2+]o increase to hypercalcemia induced arrhythmia also in the intact SAN tissues, activating ectopic leading regions of pacemaking and impairing conduction towards the atria. Conclusions Hypercalcemia causes excessive Cav1.2-mediated Ca2+ influx, which alters [Ca2+]I leading to pacemaker impairment. Modulation of LTCC may reduce pacemaker dysfunctions, preventing SND progression.

The spontaneous activity of the sinoatrial node initiates the heartbeat. Sino-atrial node dysfunction (SND) and sick sinoatrial (sick sinus) syndrome are caused by the heart's inability to generate a normal sinoatrial node action potential. In clinical practice, SND is generally considered an age-related pathology, secondary to degenerative fibrosis of the heart pacemaker tissue. However, other forms of SND exist, including idiopathic primary SND, which is genetic, and forms that are secondary to cardiovascular or systemic disease. The incidence of SND in the general population is expected to increase over the next half century, boosting the need to implant electronic pacemakers. During the last two decades, our knowledge of sino-atrial node physiology and of the pathophysiological mechanisms underlying SND has advanced considerably. This review summarizes the current knowledge about SND mechanisms and discusses the possibility of introducing new pharmacologic therapies for treating SND.

Cardiac automaticity is set by pacemaker activity of the sinus node (SAN). In addition to the ubiquitously expressed cardiac voltage-gated L-type Cav1.2 Ca2+ channel isoform, pacemaker cells within the SAN and the atrioventricular node co-express voltage-gated L-type Cav1.3 and T-type Cav3.1 Ca2+ channels (SAN-VGCCs). The role of SAN-VGCCs in automaticity is incompletely understood. We used knockout mice carrying individual genetic ablation of Cav1.3 (Cav1.3−/−) or Cav3.1 (Cav3.1−/−) channels and double mutant Cav1.3−/−/Cav3.1−/− mice expressing only Cav1.2 channels. We show that concomitant loss of SAN-VGCCs prevents physiological SAN automaticity, blocks impulse conduction and compromises ventricular rhythmicity. Coexpression of SAN-VGCCs is necessary for impulse formation in the central SAN. In mice lacking SAN-VGCCs, residual pacemaker activity is predominantly generated in peripheral nodal and extranodal sites by f-channels and TTX-sensitive Na+ channels. In beating SAN cells, ablation of SAN-VGCCs disrupted late diastolic local intracellular Ca2+ release, which demonstrates an important role for these channels in supporting the sarcoplasmic reticulum based “Ca2+clock” mechanism during normal pacemaking. These data implicate an underappreciated role for co-expression of SAN-VGCCs in heart automaticity and define an integral role for these channels in mechanisms that control the heartbeat.

The above article was published online with an error in Fig. 1b. There is a doubled action potential at the far right of the left panel of the figure.

International audience; Na/Ca exchange is the dominant calcium (Ca) efflux mechanism in cardiac myocytes. Although our knowledge of exchanger function (NCX1 in the heart) was originally established using biochemical and electrophysiological tools such as cardiac sarcolemmal vesicles and the giant patch technique [1-4], many advances in our understanding of the physiological/pathophysiological roles of NCX1 in the heart have been obtained using a suite of genetically modified mice. Early mouse studies focused on modification of expression levels of NCX1 in the ventricles, with transgenic overexpressors, global NCX1 knockout (KO) mice (which were embryonic lethal if homozygous), and finally ventricular-specific NCX1 KO [5-12]. We found, to our surprise, that ventricular cardiomyocytes lacking NCX1 can survive and function by engaging a clever set of adaptations to minimize Ca entry, while maintaining contractile function through an increase in excitation-contraction (EC) coupling gain [5,6,13]. Having studied ventricular NCX1 ablation in detail, we more recently focused on elucidating the role of NCX1 in the atria through altering NCX1 expression. Using a novel atrial-specific NCX1 KO mouse, we found unexpected changes in atrial cell morphology and calcium handling, together with dramatic alterations in the function of sinoatrial node (SAN) pacemaker activity. In this review, we will discuss these findings and their implications for cardiac disease.

Background: Endurance athletes are prone to bradyarrhythmias, which in the long-term may underscore the increased incidence of pacemaker implantation reported in this population. Our previous work in rodent models has shown training-induced sinus bradycardia to be due to microRNA (miR)-mediated transcriptional remodeling of the HCN4 channel, leading to a reduction of the “funny” (If) current in the sinoatrial node (SAN). Objective: To test if genetic ablation of G-protein-gated inwardly rectifying potassium channel, also known as IKACh channels prevents sinus bradycardia induced by intensive exercise training in mice. Methods: Control wild-type (WT) and mice lacking GIRK4 (Girk4–/–), an integral subunit of IKACh were assigned to trained or sedentary groups. Mice in the trained group underwent 1-h exercise swimming twice a day for 28 days, 7 days per week. We performed electrocardiogram recordings and echocardiography in both groups at baseline, during and after the training period. At training cessation, mice were euthanized and SAN tissues were isolated for patch clamp recordings in isolated SAN cells and molecular profiling by quantitative PCR (qPCR) and western blotting. Results: At swimming cessation trained WT mice presented with a significantly lower resting HR that was reversible by acute IKACh block whereas Girk4–/– mice failed to develop a training-induced sinus bradycardia. In line with HR reduction, action potential rate, density of If, as well as of T- and L-type Ca2+ currents (ICaT and ICaL) were significantly reduced only in SAN cells obtained from WT-trained mice. If reduction in WT mice was concomitant with downregulation of HCN4 transcript and protein, attributable to increased expression of corresponding repressor microRNAs (miRs) whereas reduced ICaL in WT mice was associated with reduced Cav1.3 protein levels. Strikingly, IKACh ablation suppressed all training-induced molecular remodeling observed in WT mice. Conclusion: Genetic ablation of cardiac IKACh in mice prevents exercise-induced sinus bradycardia by suppressing training induced remodeling of inward currents If, ICaT and ICaL due in part to the prevention of miR-mediated transcriptional remodeling of HCN4 and likely post transcriptional remodeling of Cav1.3. Strategies targeting cardiac IKACh may therefore represent an alternative to pacemaker implantation for bradyarrhythmias seen in some veteran athletes.

Summary Genetic deficiency of dystrophin leads to disability and premature death in Duchenne muscular dystrophy (DMD), affecting the heart as well as skeletal muscle. Here, we report that clinical-stage cardiac progenitor cells, known as cardiosphere-derived cells (CDCs), improve cardiac and skeletal myopathy in the mdx mouse model of DMD. Injection of CDCs into the hearts of mdx mice augments cardiac function, ambulatory capacity, and survival. Exosomes secreted by human CDCs reproduce the benefits of CDCs in mdx mice and in human induced pluripotent stem cell-derived Duchenne cardiomyocytes. Surprisingly, CDCs and their exosomes also transiently restored partial expression of full-length dystrophin in mdx mice. The findings further motivate the testing of CDCs in Duchenne patients, while identifying exosomes as next-generation therapeutic candidates., Graphical Abstract, Highlights • CDCs improve cardiac and skeletal myopathy in the mdx mouse model of DMD • CDC exosomes reproduce the benefits of CDCs • CDCs and their exosomes transiently restored partial expression of dystrophin, In this article, Marbán and colleagues show that exosomes mediate benefits of cell therapy in mouse and human models of Duchenne muscular dystrophy.

Key points Repolarizing currents through K+ channels are essential for proper sinoatrial node (SAN) pacemaking, but the influence of intracellular Ca2+ on repolarization in the SAN is uncertain. We identified all three isoforms of Ca2+-activated small conductance K+ (SK) channels in the murine SAN. SK channel blockade slows repolarization and subsequent depolarization of SAN cells. In the atrial-specific Na+/Ca2+ exchanger (NCX) knockout mouse, cellular Ca2+ accumulation during spontaneous SAN pacemaker activity produces intermittent hyperactivation of SK channels, leading to arrhythmic pauses alternating with bursts of pacing. These findings suggest that Ca2+-sensitive SK channels can translate changes in cellular Ca2+ into a repolarizing current capable of modulating pacemaking. SK channels are a potential pharmacological target for modulating SAN rate or treating SAN dysfunction, particularly under conditions characterized by abnormal increases in diastolic Ca2+. Abstract Small conductance K+ (SK) channels have been implicated as modulators of spontaneous depolarization and electrical conduction that may be involved in cardiac arrhythmia. However, neither their presence nor their contribution to sinoatrial node (SAN) pacemaker activity has been investigated. Using quantitative PCR (q-PCR), immunostaining and patch clamp recordings of membrane current and voltage, we identified all three SK isoforms (SK1, SK2 and SK3) in mouse SAN. Inhibition of SK channels with the specific blocker apamin prolonged action potentials (APs) in isolated SAN cells. Apamin also slowed diastolic depolarization and reduced pacemaker rate in isolated SAN cells and intact tissue. We investigated whether the Ca2+-sensitive nature of SK channels could explain arrhythmic SAN pacemaker activity in the atrial-specific Na+/Ca2+ exchange (NCX) knockout (KO) mouse, a model of cellular Ca2+ overload. SAN cells isolated from the NCX KO exhibited higher SK current than wildtype (WT) and apamin prolonged their APs. SK blockade partially suppressed the arrhythmic burst pacing pattern of intact NCX KO SAN tissue. We conclude that SK channels have demonstrable effects on SAN pacemaking in the mouse. Their Ca2+-dependent activation translates changes in cellular Ca2+ into a repolarizing current capable of modulating regular pacemaking. This Ca2+ dependence also promotes abnormal automaticity when these channels are hyperactivated by elevated Ca2+. We propose SK channels as a potential target for modulating SAN rate, and for treating patients affected by SAN dysfunction, particularly in the setting of Ca2+ overload.

Background The incidence of brady-arrhythmias is known to be higher in athletes. In rodent models of exercise training, bradycardia results from downregulation of f-(HCN4) channels leading to a reduction of the If current in the sino-atrial node (SAN). Objectives To test if genetic ablation of G-protein-gated inwardly rectifying potassium 4 (Girk4) channel prevents sinus bradycardia induced by intensive exercise training in mice. Methods Control (WT) and homozygous Girk4 knock-out (Girk4-/-) mice were assigned to trained or sedentary groups. Mice in the trained group underwent 1-hour exercise swimming twice a day for 28 days, 7 days per week whereas sedentary mice underwent 5-min swimming daily in the same period. We performed telemetric electrocardiogram recordings in both groups at baseline and during the training period. At training cessation, mice were euthanized and SAN tissues were isolated for patch clamp recordings in isolated SAN cells and transcriptional profiling by quantitative PCR (qPCR). Results At day 17 of the swimming regimen, heart rate (HR) reduction in trained WT mice was significantly different vs. WT sedentary mice, whereas Girk4-/- mice failed to develop sinus bradycardia. Action potential recordings in isolated SAN cells from trained WT mice showed lower rates of pacemaking in comparison to cells from mice of the other groups. In line with HR reduction, the density of If was significantly reduced only in SAN cells obtained from WT-trained mice. Correspondingly, qPCR analysis showed mRNA expression of HCN4 was significantly lower in WT-trained SAN relative to WT sedentary animals, but unchanged in Girk4-/- animals. This finding could be attributed to a significant increase in the expression of miR-423-5p (a transcriptional repressor of HCN4) observed in trained WT, but not trained Girk4-/- animals. Conclusion Genetic ablation of Girk4 channels prevents sinus bradycardia induced by down regulation of f-HCN4 channels in trained WT mice.

The above article was published online with an error in Fig. 1b. There is a doubled action potential at the far right of the left panel of the figure.

Cardiac differentiation of embryonic stem cells (ESCs) can give rise to de novo chamber cardiomyocytes and nodal pacemaker cells. Compared with our understanding of direct differentiation toward atrial and ventricular myocytes, the mechanisms for nodal pacemaker cell commitment are not well understood. Taking a cue from the prominence of canonical Wnt signaling during cardiac pacemaker tissue development in chick embryos, we asked if modulations of Wnt signaling influence cardiac progenitors to bifurcate to either chamber cardiomyocytes or pacemaker cells. Omitting an exogenous Wnt inhibitor, which is routinely added to maximize cardiac myocyte yield during differentiation of mouse and human ESCs, led to increased yield of spontaneously beating cardiomyocytes with action potential properties similar to those of native sinoatrial node pacemaker cells. The pacemaker phenotype was accompanied by enhanced expression of genes and gene products that mark nodal pacemaker cells such as Hcn4, Tbx18, Tbx3, and Shox2. Addition of exogenous Wnt3a ligand, which activates canonical Wnt/β-catenin signaling, increased the yield of pacemaker-like myocytes while reducing cTNT-positive pan-cardiac differentiation. Conversely, addition of inhibitors of Wnt/β-catenin signaling led to increased chamber myocyte lineage development at the expense of pacemaker cell specification. The positive impact of canonical Wnt signaling on nodal pacemaker cell differentiation was evidenced in direct differentiation of two human ESC lines and human induced pluripotent stem cells. Our data identify the Wnt/β-catenin pathway as a critical determinant of cardiac myocyte subtype commitment during ESC differentiation: endogenous Wnt signaling favors the pacemaker lineage, whereas its suppression promotes the chamber cardiomyocyte lineage.

Introduction We investigated whether abnormal activity of the L-type Ca2+ channels (LTCCs), Cav1.2 and Cav1.3, under extracellular hypercalcemia, impaired heart pacemaker activity. Membrane currents and intracellular Ca2+ ([Ca2 +]i) dynamics of the sino-atrial node (SAN) generate the heart pacemaker activity. Impairments of SAN activity, also called SAN dysfunction (SND), harms heart automaticity. Recent studies have associated abnormal [Ca2 +]i dynamics with SNDs. Cav1.2 and Cav1.3 carry the main Ca2+ influx of SAN cells to sustain their [Ca2 + ]i dynamics. Modified extracellular Ca2+ ([Ca2 +]o) could alter Ca2+ influx through these channels. For example, cancer and hyperparathyroidism can raise [Ca2 + ]o, causing an extracellular hypercalcemia that could alter [Ca2 + ]i dynamics and impair SAN activity and heart automaticity. Methods and results To test this hypothesis, we measured contractions, [Ca2 +]i release and L-type Ca2+ current (ICa,L) in spontaneous cells of the murine SAN. Then, we recorded rate and propagation of the spontaneous action potentials (APs) generated by the SAN tissue ex-vivo. In spontaneous SAN cells, we observed that the modification of [Ca2 +]o modulates [Ca2 +]i and cells contraction through changes of ICa,L. In particular, the increase of [Ca2 +]o dysregulated pacemaker activity, likely through excessive Ca2+ influx mediated by Cav1.2. [Ca2 +]o increase to hypercalcemic levels induced arrhythmia also in the intact SAN tissues, activating ectopic leading regions of pacemaking and impairing conduction towards the atria. We recovered these pacemaker dysfunctions by blocking Ca2 + -activated small-conductance K+ (SK) channels. Conclusions Hypercalcemia causes excessive Cav1.2-mediated Ca2+ influx, which alters [Ca2 +]i and could hyperactivate SK channels, leading to pacemaker impairment. Cav1.2 or SK modulation may reduce pacemaker dysfunctions, preventing SND progression.

Current treatments for heart automaticity disorders still lack a safe and efficient source of stem cells to restore normal biological pacemaking. Since adult Muscle-Derived Stem Cells (MDSC) show multi-lineage differentiation in vitro including into spontaneously beating cardiomyocytes, we questioned whether they could effectively differentiate into cardiac pacemakers, a specific population of cardiomyocytes producing electrical impulses in the sino-atrial node (SAN) of adult heart. We show here that beating cardiomyocytes, differentiated from MDSC in vitro, exhibit typical characteristics of cardiac pacemakers: expression of markers of the SAN lineage Hcn4, Tbx3 and Islet1, as well as spontaneous calcium transients and hyperpolarization-activated “funny” current and L-type Cav1.3 channels. Pacemaker-like myocytes differentiated in vitro from Cav1.3-deficient mouse stem cells produced slower rate of spontaneous Ca2+ transients, consistent with the reduced activity of native pacemakers in mutant mice. In vivo, undifferentiated wild type MDSC migrated and homed with increased engraftment to the SAN of bradycardic mutant Cav1.3-/- within 2-3 days after systemic I.P. injection. The increased homing of MDSCs corresponded to increased levels of the chemokine SDF1 and its receptor CXCR4 in mutant SAN tissue and was ensued by differentiation of MDSCs into Cav1.3-expressing pacemaker-like myocytes within 10 days and a significant improvement of the heart rate maintained for up to 40 days. Optical mapping and immunofluorescence analyses performed after 40 days on SAN tissue from transplanted wild type and mutant mice showed MDSCs integrated as pacemaking cells both electrically and functionally within recipient mouse SAN. These findings identify MDSCs as directly transplantable stem cells that efficiently home, differentiate and improve heart rhythm in mouse models of congenital bradycardia.

International audience; L'automatisme du coeur est un phénomène physiologique fascinant et constitue un défi remarquable pour les chercheurs. La rythmicité spontanée (automatisme) du coeur au stade adulte est générée dans le noeud sinusal (Fig. 1A) par une population spécialisée de myocytes connus sur le terme de « cellules pacemaker » (Fig. 1A). Les myocytes cardiaques automatiques se caractérisent par une phase de dépolarisation spontanée du potentiel de membrane connue sur le terme de « dépolarisation diastolique » car elle correspond à la phase diastolique du cycle cardiaque [1]. La dépolarisation diastolique (ou phase diastolique) est une dépolarisation lente, qui conduit le potentiel de membrane à la fin d'un potentiel d'action (PA) vers le seuil du potentiel d'action suivant (Fig. 1B). La dépolarisation diastolique nécessite à chaque instant un courant net entrant pour pouvoir maintenir la dépolarisation du potentiel de membrane. Ce courant entrant est généré par les canaux ioniques de la membrane plasmique [1]. Toutefois, dans l'embryon très précoce, avant la formation des chambres cardiaques, l'automatisme du « coeur » tubulaire n'implique pas de changement du potentiel de membrane. Les myocytes du coeur embryonnaire présentent des contractions spontanées rythmiques générées par une production constitutive d'inositol triphosphate (IP3). L'activation des récepteurs à l'IP3 (IP3R) induit la libération de Ca 2+ du réticulum endoplasmique, ce qui active des contractions périodiques du coeur tubulaire [2]. Lors de la formation des chambres cardiaques, l'expression des canaux ioniques de la membrane des cellules automatiques se met en place. À la fin du développement du coeur, l'automatisme est restreint au noeud sinusal et au myocytes du système de conduction atrio-ventriculaire. Suite à la maturation du système de conduction cardiaque et des branches sympathiques et parasympathi-ques du système nerveux autonome, les caté-cholamines et

Introduction Sinoatrial cells generate cardiac automaticity through functional interactions between membrane ionic currents and intracellular Ca2+ dynamics. Mitochondrial Ca2+ plays a role in ATP synthesis regulation and intracellular Ca2+ levels. Interestingly, both could modify AMPc levels and possibly regulate cardiac automatism. We then hypothesized that mitochondrial Ca2+ homeostasis interact with β-adrenergic pathways and regulates cardiac automatism. Objective To determine whether pharmacological inhibition of the mitochondrial Ca2+ uniporter (MCU), which is responsible for Ca2+ influx in mitochondria, affects the sinoatrial node function and β-adrenergic responses. Method Basal level and amplitude of Ca2+ transients were recorded using a ratiometric fluorescent probe (Fura-2/AM) in mice isolated sinoatrial cells. Optical voltage mapping was also used to study the spontaneous frequency of action potentials generated by intact sinoatrial tissue isolated from C57Bl6 mice. Results In basal conditions, inhibition of mitochondrial Ca2+ influx with Ru360 in isolated sinoatrial cells did not affect automaticity. On the other hand, Ru360 prevents intracellular Ca2+ increase after stimulation of L-type Ca2+ channels. Similarly, MCU inhibition applied to intact sinoatrial tissues did not show significant effects under basal conditions, but induced a significant reduction of the spontaneous frequency (PA/min) under β-adrenergic stimulation. Conclusion We here demonstrated that mitochondrial Ca2+ regulates intracellular Ca2+ dynamics, and that mitochondrial Ca2+ is needed to support cAMP production, by stimulating ATP synthesis during a β-adrenergic stimulation. We propose that this mechanism is required to maintain a high spontaneous frequency, especially under physiological stress conditions.

Genetic deficiency of dystrophin leads to disability and premature death in Duchenne muscular dystrophy, affecting the heart as well as skeletal muscle. Here we report that cardiosphere-derived cells (CDCs), which are being tested clinically for the treatment of Duchenne cardiomyopathy, improve cardiac and skeletal myopathy in the mdx mouse model of DMD and in human Duchenne cardiomyocytes. Injection of CDCs into the hearts of mdx mice augments cardiac function, ambulatory capacity and survival. Exosomes secreted by human CDCs reproduce the benefits of CDCs in mdx mice and in human Duchenne cardiomyocytes. The findings further motivate the testing of CDCs in Duchenne patients, while identifying exosomes as next-generation therapeutic candidates.

Repolarizing currents through K+ channels are essential for proper sinoatrial node (SAN) pacemaking, but the influence of intracellular Ca2+ on repolarization in the SAN is uncertain.We identified all three isoforms of Ca2+‐activated small conductance K+ (SK) channels in the murine SAN.SK channel blockade slows repolarization and subsequent depolarization of SAN cells.In the atrial‐specific Na+/Ca2+ exchanger (NCX) knockout mouse, cellular Ca2+ accumulation during spontaneous SAN pacemaker activity produces intermittent hyperactivation of SK channels, leading to arrhythmic pauses alternating with bursts of pacing.These findings suggest that Ca2+‐sensitive SK channels can translate changes in cellular Ca2+ into a repolarizing current capable of modulating pacemaking.SK channels are a potential pharmacological target for modulating SAN rate or treating SAN dysfunction, particularly under conditions characterized by abnormal increases in diastolic Ca2+.

Cardiac automaticity is a fundamental physiological function in vertebrates. Heart rate is under the control of several neurotransmitters and hormones and is permanently adjusted by the autonomic nervous system to match the physiological demand of the organism. Several classes of ion channels and proteins involved in intracellular Ca2+ handling contribute to pacemaker activity. Voltage-dependent T-type Ca2+ channels are an integral part of the complex mechanism underlying pacemaking. T-type channels also contribute to impulse conduction in mice and humans. Strikingly, T-type channel isoforms are co-expressed in the cardiac conduction system with other ion channels that play a major role in pacemaking such as f- (HCN4) and L-type Cav1.3 channels. Pharmacologic inhibition of T-type channels reduces the spontaneous activity of isolated pacemaker myocytes of the sino-atrial node, the dominant heart rhythmogenic centre. Target inactivation of T-type Cav3.1 channels abolishes I Ca,T in both sino-atrial and atrioventricular myocytes and reduces the daily heart rate of freely moving mice. Cav3.1 channels contribute also to automaticity of the atrioventricular node and to ventricular escape rhythms, thereby stressing the importance of these channels in automaticity of the whole cardiac conduction system. Accordingly, loss-of-function of Cav3.1 channels contributes to severe form of congenital bradycardia and atrioventricular block in paediatric patients.

Parasympathetic regulation of sinoatrial node (SAN) pacemaker activity modulates multiple ion channels to temper heart rate. The functional role of the G-protein–activated K+ current (IKACh) in the control of SAN pacemaking and heart rate is not completely understood. We have investigated the functional consequences of loss of IKACh in cholinergic regulation of pacemaker activity of SAN cells and in heart rate control under physiological situations mimicking the fight or flight response. We used knockout mice with loss of function of the Girk4 (Kir3.4) gene (Girk4−/− mice), which codes for an integral subunit of the cardiac IKACh channel. SAN pacemaker cells from Girk4−/− mice completely lacked IKACh. Loss of IKACh strongly reduced cholinergic regulation of pacemaker activity of SAN cells and isolated intact hearts. Telemetric recordings of electrocardiograms of freely moving mice showed that heart rate measured over a 24-h recording period was moderately increased (10%) in Girk4−/− animals. Although the relative extent of heart rate regulation of Girk4−/− mice was similar to that of wild-type animals, recovery of resting heart rate after stress, physical exercise, or pharmacological β-adrenergic stimulation of SAN pacemaking was significantly delayed in Girk4−/− animals. We conclude that IKACh plays a critical role in the kinetics of heart rate recovery to resting levels after sympathetic stimulation or after direct β-adrenergic stimulation of pacemaker activity. Our study thus uncovers a novel role for IKACh in SAN physiology and heart rate regulation.

International audience; Dysfunction of pacemaker activity in the sinoatrial node (SAN) underlies "sick sinus" syndrome (SSS), a common clinical condition characterized by abnormally low heart rate (bradycardia). If untreated, SSS carries potentially life-threatening symptoms, such as syncope and end-stage organ hypoperfusion. The only currently available therapy for SSS consists of electronic pacemaker implantation. Mice lacking L-type Cav1.3 Ca(2+) channels (Cav1.3(-/-)) recapitulate several symptoms of SSS in humans, including bradycardia and atrioventricular (AV) dysfunction (heart block). Here, we tested whether genetic ablation or pharmacological inhibition of the muscarinic-gated K(+) channel (IKACh) could rescue SSS and heart block in Cav1.3(-/-) mice. We found that genetic inactivation of IKACh abolished SSS symptoms in Cav1.3(-/-) mice without reducing the relative degree of heart rate regulation. Rescuing of SAN and AV dysfunction could be obtained also by pharmacological inhibition of IKACh either in Cav1.3(-/-) mice or following selective inhibition of Cav1.3-mediated L-type Ca(2+) (ICa,L) current in vivo. Ablation of IKACh prevented dysfunction of SAN pacemaker activity by allowing net inward current to flow during the diastolic depolarization phase under cholinergic activation. Our data suggest that patients affected by SSS and heart block may benefit from IKACh suppression achieved by gene therapy or selective pharmacological inhibition.

It is well established that Pacemaker activity of the sino-atrial node (SAN) initiates the heartbeat. However, the atrioventricular node (AVN) can generate viable pacemaker activity in case of SAN failure, but we have limited knowledge of the ionic bases of AVN automaticity. We characterized pacemaker activity and ionic currents in automatic myocytes of the mouse AVN. Pacemaking of AVN cells (AVNCs) was lower than that of SAN pacemaker cells (SANCs), both in control conditions and upon perfusion of isoproterenol (ISO). Block of I(Na) by tetrodotoxin (TTX) or of I(Ca,L) by isradipine abolished AVNCs pacemaker activity. TTX-resistant (I(Nar)) and TTX-sensitive (I(Nas)) Na(+) currents were recorded in mouse AVNCs, as well as T-(I(Ca,T)) and L-type (I(Ca,L)) Ca(2+) currents I(Ca,L) density was lower than in SANCs (51%). The density of the hyperpolarization-activated current, (I(f)) and that of the fast component of the delayed rectifier current (I(Kr)) were, respectively, lower (52%) and higher (53%) in AVNCs than in SANCs. Pharmacological inhibition of I(f) by 3 µM ZD-7228 reduced pacemaker activity by 16%, suggesting a relevant role for I(f) in AVNCs automaticity. Some AVNCs expressed also moderate densities of the transient outward K(+) current (I(to)). In contrast, no detectable slow component of the delayed rectifier current (I(Ks)) could be recorded in AVNCs. The lower densities of I(f) and I(Ca,L), as well as higher expression of I(Kr) in AVNCs than in SANCs may contribute to the intrinsically slower AVNCs pacemaking than that of SANCs.

Introduction Sino-atrial node (SAN) pacemaker activity is generated by ion channels of the plasma membrane, such as hyperpolarization-activated “funny” f-(HCN), Ca2+ channels and ryanodine receptor (RyR) – dependent Ca2+ release from the sarcoplasmic reticulum (SR). It is currently disputed whether Ca2+ release from RyRs could sustain viable pacemaker activity provided preserved SR Ca2+ content. While working myocytes express L-type Cav1.2 channels to maintain SR Ca2+ content, SAN cells express also L-type Cav1.3 and T-type Cav3.1 channels to generate pacemaking. Objectives We used mutant mice carrying concomitant ablation of Cav1.3 and Cav3.1 (Cav1.3−/−/Cav3.1−/−) to study the importance of these channels in automaticity. We also investigated the role of f-HCN channels and RyR-dependent Ca2+ release in residual pacemaker activity of mutant mice. Methods We employed in vivo telemetric recordings of heart rate (HR) in Cav1.3−/−, Cav3.1−/− and Cav1.3−/−/Cav3.1−/− mice. We studied the consequences of pharmacologic inhibition of f-HCN and TTX-sensitive Na+ channels in mutant mice using Langendorff perfused hearts or optical mapping (OM) of the pacemaker impulse in intact SAN preparations (SANs). Results Cav ablation reduced HR in mice: Cav3.1−/− (−7.6%, n = 11), Cav1.3−/− (−24.4%, n = 8), Cav1.3−/−/Cav3.1−/− (−35%, n = 11). In OM experiments on SANs, concomitant inhibition of f-HCN and Nav1.1 channels slowed pacemaking in wild-type (−48%, n = 7) and Cav3.1−/− (−37%, n = 7), while arresting automaticity in 4/6 of Cav1.3−/−, 3/6 of Cav1.3−/−/Cav3.1−/−. When present, residual pacemaking was reduced by 82%. Similar results were obtained using isolated Cav1.3−/−/Cav3.1−/− pacemaker cells were automaticity arrested in 5/9 cells tested, or was reduced by 80% in 4/9 cells. Conclusion Heart automaticity is primarily generated by Cav1.3 and f-HCN channels. RyR-dependent Ca2+ release cannot sustain automaticity following concomitant targeting of Cav1.3 and f-HCN channels.

Duchenne muscular dystrophy, a crippling genetic disease leading to premature death, affects the heart as well as skeletal muscle. Indeed, cardiomyopathy is the leading cause of death in Duchenne patients. There are no approved treatments for the cardiomyopathy, and novel Duchenne-specific experimental approaches such as exon skipping do not benefit the heart. Here we demonstrate that cardiosphere-derived cells (CDCs), which are in advanced clinical testing for therapeutic regeneration after myocardial infarction, reverse the key pathophysiological hallmarks of Duchenne cardiomyopathy (oxidative stress, inflammation, fibrosis and mitochondrial dysfunction) in mdx mice. Exosomes secreted by human CDCs reproduce the benefits of CDCs in mdx mice, and reverse mitochondrial dysfunction in human Duchenne cardiomyocytes. Both CDCs and their exosomes improve heart function in mdx mice (P

In sinoatrial node (SAN) cells, electrogenic sodium-calcium exchange (NCX) is the dominant calcium (Ca) efflux mechanism. However, the role of NCX in the generation of SAN automaticity is controversial. To investigate the contribution of NCX to pacemaking in the SAN, we performed optical voltage mapping and high-speed 2D laser scanning confocal microscopy (LSCM) of Ca dynamics in an ex vivo intact SAN/atrial tissue preparation from atrial-specific NCX knockout (KO) mice. These mice lack P waves on electrocardiograms, and isolated NCX KO SAN cells are quiescent. Voltage mapping revealed disorganized and arrhythmic depolarizations within the NCX KO SAN that failed to propagate into the atria. LSCM revealed intermittent bursts of Ca transients. Bursts were accompanied by rising diastolic Ca, culminating in long pauses dominated by Ca waves. The L-type Ca channel agonist BayK8644 reduced the rate of Ca transients and inhibited burst generation in the NCX KO SAN whereas the Ca buffer 1,2-Bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (acetoxymethyl ester) (BAPTA AM) did the opposite. These results suggest that cellular Ca accumulation hinders spontaneous depolarization in the NCX KO SAN, possibly by inhibiting L-type Ca currents. The funny current (If) blocker ivabradine also suppressed NCX KO SAN automaticity. We conclude that pacemaker activity is present in the NCX KO SAN, generated by a mechanism that depends upon If. However, the absence of NCX-mediated depolarization in combination with impaired Ca efflux results in intermittent bursts of pacemaker activity, reminiscent of human sinus node dysfunction and "tachy-brady" syndrome.

Sino-atrial node (SAN) automaticity is an essential mechanism of heart rate generation that is still not completely understood. Recent studies highlighted the importance of intracellular Ca(2+) ([Ca(2+)]i) dynamics during SAN pacemaker activity. Nevertheless, the functional role of voltage-dependent L-type Ca(2+) channels in controlling SAN [Ca(2+)]i release is largely unexplored. Since Cav1.3 is the predominant L-type Ca(2+) channel isoform in SAN cells, we studied [Ca(2+)]i dynamics in isolated cells and ex vivo SAN preparations explanted from wild-type (WT) and Cav1.3 knockout (KO) mice (Cav1.3(-/-)).We found that Cav1.3 deficiency strongly impaired [Ca(2+)]i dynamics, reducing the frequency of local [Ca(2+)]i release events and preventing their synchronization. This impairment inhibited the generation of Ca(2+) transients and delayed spontaneous activity. We also used action potentials recorded in WT SAN cells as voltage-clamp commands for Cav1.3(-/-) cells. Although these experiments showed abolished Ca(2+) entry through L-type Ca(2+) channels in the diastolic depolarization range of KO SAN cells, their sarcoplasmic reticulum Ca(2+) load remained normal. β-Adrenergic stimulation enhanced pacemaking of both genotypes, though, Cav1.3(-/-) SAN cells remained slower than WT. Conversely, we rescued pacemaker activity in Cav1.3(-/-) SAN cells and intact tissues through caffeine-mediated stimulation of Ca(2+)-induced Ca(2+) release.Cav1.3 channels play a critical role in the regulation of [Ca(2+)]i dynamics, providing an unanticipated mechanism for triggering local [Ca(2+)]i releases and thereby controlling pacemaker activity. Our study also provides an additional pathophysiological mechanism for congenital SAN dysfunction and heart block linked to Cav1.3 loss of function in humans.

The mechanisms underlying cardiac automaticity are still incompletely understood and controversial. Here we report the complete conditional and time-controlled silencing of the 'funny' current (If) by expression of a dominant-negative, non-conductive HCN4-channel subunit (hHCN4-AYA). Heart-specific If silencing caused altered [Ca(2+)]i release and Ca(2+) handling in the sinoatrial node, impaired pacemaker activity and symptoms reminiscent of severe human disease of pacemaking. The effects of If silencing critically depended on the activity of the autonomic nervous system. We were able to rescue the failure of impulse generation and conduction by additional genetic deletion of cardiac muscarinic G-protein-activated (GIRK4) channels in If-deficient mice without impairing heartbeat regulation. Our study establishes the role of f-channels in cardiac automaticity and indicates that arrhythmia related to HCN loss-of-function may be managed by pharmacological or genetic inhibition of GIRK4 channels, thus offering a new therapeutic strategy for the treatment of heart rhythm diseases.

Background— Catecholaminergic polymorphic ventricular tachycardia is characterized by stress-triggered syncope and sudden death. Patients with catecholaminergic polymorphic ventricular tachycardia manifest sinoatrial node (SAN) dysfunction, the mechanisms of which remain unexplored. Methods and Results— We investigated SAN [Ca 2+ ] i handling in mice carrying the catecholaminergic polymorphic ventricular tachycardia–linked mutation of ryanodine receptor (RyR2 R4496C ) and their wild-type (WT) littermates. In vivo telemetric recordings showed impaired SAN automaticity in RyR2 R4496C mice after isoproterenol injection, analogous to what was observed in catecholaminergic polymorphic ventricular tachycardia patients after exercise. Pacemaker activity was explored by measuring spontaneous [Ca 2+ ] i transients in SAN cells within the intact SAN by confocal microscopy. RyR2 R4496C SAN presented significantly slower pacemaker activity and impaired chronotropic response under β-adrenergic stimulation, accompanied by the appearance of pauses (in spontaneous [Ca 2+ ] i transients and action potentials) in 75% of the cases. Ca 2+ spark frequency was increased by 2-fold in RyR2 R4496C SAN. Whole-cell patch-clamp experiments performed on isolated RyR2 R4496C SAN cells showed that L-type Ca 2+ current ( I Ca,L ) density was reduced by ≈50%, an effect blunted by internal Ca 2+ buffering. Isoproterenol dramatically increased the frequency of Ca 2+ sparks and waves by ≈5 and ≈10-fold, respectively. Interestingly, the sarcoplasmic reticulum Ca 2+ content was significantly reduced in RyR2 R4496C SAN cells in the presence of isoproterenol, which may contribute to stopping the “Ca 2+ clock” rhythm generation, originating SAN pauses. Conclusion— The increased activity of RyR2 R4496C in SAN leads to an unanticipated decrease in SAN automaticity by a Ca 2+ -dependent decrease of I Ca,L and sarcoplasmic reticulum Ca 2+ depletion during diastole, identifying subcellular pathophysiological alterations contributing to the SAN dysfunction in catecholaminergic polymorphic ventricular tachycardia patients.

The atrioventricular node controls cardiac impulse conduction and generates pacemaker activity in case of failure of the sino-atrial node. Understanding the mechanisms of atrioventricular automaticity is important for managing human pathologies of heart rate and conduction. However, the physiology of atrioventricular automaticity is still poorly understood. We have investigated the role of three key ion channel-mediated pacemaker mechanisms namely, Ca(v)1.3, Ca(v)3.1 and HCN channels in automaticity of atrioventricular node cells (AVNCs). We studied atrioventricular conduction and pacemaking of AVNCs in wild-type mice and mice lacking Ca(v)3.1 (Ca(v)3.1(-/-)), Ca(v)1.3 (Ca(v)1.3(-/-)), channels or both (Ca(v)1.3(-/-)/Ca(v)3.1(-/-)). The role of HCN channels in the modulation of atrioventricular cells pacemaking was studied by conditional expression of dominant-negative HCN4 channels lacking cAMP sensitivity. Inactivation of Ca(v)3.1 channels impaired AVNCs pacemaker activity by favoring sporadic block of automaticity leading to cellular arrhythmia. Furthermore, Ca(v)3.1 channels were critical for AVNCs to reach high pacemaking rates under isoproterenol. Unexpectedly, Ca(v)1.3 channels were required for spontaneous automaticity, because Ca(v)1.3(-/-) and Ca(v)1.3(-/-)/Ca(v)3.1(-/-) AVNCs were completely silent under physiological conditions. Abolition of the cAMP sensitivity of HCN channels reduced automaticity under basal conditions, but maximal rates of AVNCs could be restored to that of control mice by isoproterenol. In conclusion, while Ca(v)1.3 channels are required for automaticity, Ca(v)3.1 channels are important for maximal pacing rates of mouse AVNCs. HCN channels are important for basal AVNCs automaticity but do not appear to be determinant for β-adrenergic regulation.

Catecholaminergic Polymorphic Ventricular Tachycardia (CPVT) is an arrhythmogenic disease characterized by stress-triggered syncope and sudden death. While ventricular tachycardia appears only during stress conditions, CPVT patients manifest basal sino-atrial node (SAN) dysfunction, but the underlying mechanism remains unknown. We investigated Ca2+ handling on SAN from transgenic mice expressing the CPVT ryanodine receptor R4496C mutation (RyR2R4496C) and on their wild type (WT) littermates. A 2-fold increase of Ca2+ spark frequency in RyRR4496C SAN cells was recorded in basal conditions during the diastolic periods. beta-adrenergic stimulation with 20 nM isoproterenol, multiplied by ∼10-fold the occurrence of Ca2+ sparks and Ca2+ waves. Spontaneous [Ca2+]i transients recorded by confocal microscope in isolated SAN from RyR2R4496C mice presented a slower beating rate than WT SAN and an impaired positive chronotropic response to beta-adrenergic stimulation. Moreover, isoproterenol induced the appearance of [Ca2+]i transients and action potentials pauses in 75% of RyR2R4496C SAN cells. Caffeine experiments showed that the sarcoplasmic reticulum (SR) Ca2+ load was significantly reduced in the RyR2R4496C SAN cells. In vivo telemetric recordings of electrocardiograms (ECG) identified atrial and junctional escape beats overcoming sinus pauses in RyR2R4496C mice following isoproterenol injection consistent with the pauses recorded in isolated SAN. Similar ECGs were observed in CPVT patients during exercise testing, validating the animal model. We conclude that the increased activity of the RyR2R4496C SAN cells during diastolic periods unloads the SR with Ca2+, participating in SAN dysfunction in CPVT.

Within the family of voltage-gated calcium channels (VGCCs), L-type channels (L-VGCCs) represent a well-established therapeutic target for calcium channel blockers, which are widely used to treat hypertension and myocardial ischemia. L-VGCCs outside the cardiovascular system also control key physiological processes such as neuronal plasticity, sensory cell function (e.g. in the inner ear and retina) and endocrine function (e.g. in pancreatic beta cells and adrenal chromaffin cells). Research into L-VGCCs was stimulated by the discovery that the known L-VGCC isoforms (Ca(V)1.1, Ca(V)1.2, Ca(V)1.3 and Ca(V)1.4) possess different biophysical properties. However, no L-VGCC-isoform-selective drugs have yet been identified. In this review, we examine Ca(V)1.2 and Ca(V)1.3 isoforms at the level of genetic structure, splice variants, post-translational modifications and functional protein coupling. We discuss candidate Ca(V)1.2- and Ca(V)1.3-specific characteristics as future therapeutic targets in individual organs.

Cardiac automaticity, in normal conditions, is generated by the Sinoatrial Node (SAN) tissue.In the last years, two pacemaker mechanisms are proposed: the “ion channels clock”, based on the If current and the intracellular SR-dependent “Ca2+ clock”, based on spontaneous diastolic Ca2+ release. In our opinion a relevant role is played also by the Cav1.3 (L-type Ca2+ current) and Cav3.1 (T-type Ca2+ current) channels. For this reason we studied the two mouse models Cav1.3 KO and Cav1.3/Cav3.1 double KO in vivo and in vitro. Electrocardiograms analysis showed a strong bradycardia (p

The spontaneous activity of pacemaker myocytes controls the heartbeat. Automaticity is due to the presence of the slow diastolic depolarization phase, which leads the membrane potential from the end of the repolarization phase to the threshold of the following action potential. f- (HCN) channels underlying the hyperpolarization-activated “funny” current (If) are thought to play a key role in the generation and autonomic regulation of the diastolic depolarization and heart rate, but their role is still subject of controversy. Here we show that conditional and time-controlled expression of a dominant-negative non-conductive human HCN4 channel subunit (hHCN4-AYA) in the mouse heart leads to virtually complete silencing of If current (>95%) in the diastolic depolarization range in the sino-atrial node and in the conduction system. Heart-specific If silencing induced sino-atrial bradycardia, sinus pauses, severe dysfunction of atrioventricular conduction and ventricular arrhythmias. In comparison to control myocytes, the basal automaticity of hHCN4-AYA SAN myocytes was reduced by 76% and by 67% in myocytes of the conduction system. However, the relative maximal positive chronotropic effect of badrenergic activation on in vivo heart rate, isolated atria or pacemaking of individual SAN and conduction myocytes was preserved showing that If does not play an exclusive role in heart rate regulation. Unexpectedly, crossing hHCN4-AYA mutant mice with mice lacking the cardiac muscarinic G-protein-activated channel Girk4 (Girk4-/-) eliminated atrioventricular blocks and ventricular arrhythmias without preventing the autonomic regulation of heart rate. Our study shows, for the first time, the functional consequences of If silencing on heart rate and rhythm and indicates the possibility of managing cardiac disease related to HCN loss-of-function in humans by pharmacologic or genetic inhibition Girk4 channels.

Objectives We aimed to test if genetic inactivation of the cardiac IKACh current could improve the phenotype of mice lacking L-type Cav1.3 Ca2+ channels (Cav1.3-/-), a mouse model recapitulating the sino-atrial node dysfunction and supraventricular arrhythmias typical of the “sick sinus” syndrome (SSS) in humans. Background SSS is an invalidating disease. The only currently available therapy consists in the implantation of an electronic pacemaker. Cav1.3-/- mice present several characteristics of the SSS, including bradycardia associated with atrial fibrillation or flutter, as well as type I and II atrioventricular conduction blocks. IKACh is involved in autonomic regulation of heart rate and in the triggering of atrial fibrillation by the parasympathetic nervous system, but its role in establishing the symptoms of SSS is unknown. Methods We crossed IKACh-deficient mice (Kir3.4-/-), with Cav1.3-/- mice to obtain double knockout (Cav1.3-/-/Kir3.4-/-) animals. Telemetric recordings of heart rate and ECG waveforms of wild-type, Cav1.3-/-, Kir3.4-/- and Cav1.3-/-/ Kir3.4-/- mice were performed. The frequency of occurrence of episodes of atrial arrhythmias was evaluated by intracardiac recordings. Results Genetic inactivation of IKACh in Cav1.3-/-/Kir3.4-/- mice significantly improved the mean heart rate of Cav1.3-/- mice and completely abolished atrioventricular conduction blocks. Atrial fibrillation and flutter that were typical of Cav1.3-/- mice could not be observed or triggered in Cav1.3-/-/Kir3.4-/- mice. Conclusion Inactivation of IKACh effectively improved SSS in Cav1.3-/- mice. Our study indicates that SSS patients can benefit of IKACh suppression by gene therapy or selective pharmacologic inhibitors.

AimTo investigate the effects of different agonist for the muscarinic receptor on cardiac automaticity in Cav1.3-/-, Kir 3.4-/- and Cav1.3-/-Kir3.4-/- mice.MethodIn vivo: mice received methoxamine or CCPA once intraperitoneally and telemetric recordings were run continuously over 8h. Quantitative ECG analyses were performed. In vitro: action potentials were recorded in isolated SAN cells before and after application of different doses of Ach, the variation in the spontaneous firing rate was evaluated.ResultIn vivo: in WT, in Cav1.3-/- and in Cav1.3-/-Kir3.4-/- mice, methoxamine (6mg/kg) reduces the HR (p0.05) at each of the three doses tested. Concerning Kir3.4-/- cells, we have seen a reduction (p

Pacemaker activity of the sino-atrial node (SAN) controls heart rate. However, the mechanism underling SAN pacemaker activity is not completely understood and in particular, the respective physiological importance of ion channels and ryanodine receptor (RyR)-dependent Ca2+ release in pacemaking is hotly debated. We have investigated Ca2+ handling in SAN pacemaker cells of wild-type (WT) and mice lacking L-type Cav1.3 (Cav1.3-/-) channels. In isolated Cav1.3-/- SAN cells the frequency of Ca2+ transients was reduced by 45% compared to WT pacemaker cells. Loss of Cav1.3 channels also blunted by about 47% the positive chronotropic effect induced by 0.01 μM isoproterenol (ISO). Furthermore, in Cav1.3-/- pacemaker cells, local Ca2+ release (LCR) occurring during the diastolic phase was reduced by 71%. In SAN cells from mice in which L-type Cav1.2 channels have been rendered insensitive to dihydropyridines (Cav1.2DHP-/-), application of 0.3 μM isradipine decreased diastolic LCR by 78 %, thus showing that Cav1.3 channels are major regulators of RyR-dependent LCR during the diastolic phase. In individual cells of isolated intact SAN, pacemaking of Cav1.3-/- cells was characterized by reduced (37%) frequency of Ca2+ transients and an increase in Ca2+ waves. Normal pacemaking in Cav1.3-/- isolated SAN cells and intact tissue could be observed only after direct activation of RyR-dependent Ca2+ release by low doses of caffeine (200 μM). Experiments with high doses of caffeine (10 mM) in Cav1.3-/- cells, showed that the reduction in diastolic LCR and in the frequency Ca2+ transients could not be ascribed to a decrease in sarcoplasmic reticulum (SR) Ca2+ content. Our results show that in SAN pacemaker cell, LCR Ca2+ release is tightly controlled by Cav1.3 channels and that such a control is critical for promoting the formation of whole-cell Ca2+ transients.Support: FWF (P20670, P22528), ANR-06-PHYSIO-004-01.

The atrioventricular node (AVN) can generate pacemaker activity in case of failure of the sino-atrial node (SAN). However, the mechanisms underlying pacemaking in AVN cells (AVNCs) are poorly understood. Voltage-dependent ion channels such as hyperpolarization-activated HCN channels, L-type Cav1.3 and T-type Cav3.1 channels are known to play a role in pacemaking of sino-atrial node cells (SANCs). Here, we investigate the role of these channels in AVNCs pacemaker activity using genetically modified mouse strains and show that they differentially impact pacemaking of AVNCs than of SANCs. Indeed, contrary to SANCs, Cav1.3 channels are necessary for pacemaking of AVNCs and accounted for the predominant fraction of ICa,L. Inactivation of Cav3.1 channels impaired automaticity in AVNCs by promoting sporadic block of automaticity and spontaneous cellular arrhythmia.Abolition of the cAMP sensitivity of HCN channels shifted the If activation to voltages negative to that spanning the diastolic depolarization and prevented AVNCs automaticity in basal conditions. However pacemaker activity could be restored to control levels by adrenergic receptor stimulation.Inactivation of both Cav1.3 and Cav3.1 results in abolishment of pacemaking. Inhibition of TTX-resistant (INar) Na+ current showed that this is a key contributor of the action potential (AP) threshold and upstroke velocity.Conclusion: 1. Spontaneous firing rate in AVNCs is strongly dependent from Cav1.3-mediated L type calcium current and from TTX resistant sodium current (INa,r). 2. In AVNCs the Cav1.3 isoform seems to be predominant compared to Cav1.2 isoform. 3. The fact that hyperpolarization of Cav1.3-/-AVNCs pacemaking can be observed suggests that the absence of pacemaker activity is not due to the impossibility to generate the upstroke phase of the AP but probably due to an imbalance between outward and inward currents during the diastolic depolarization.

The cardiac Sodium-Calcium exchanger (NCX1) is the dominant Ca efflux protein in SinoAtrial Node (SAN) cells. NCX1 has also been implicated in the generation of pacemaker activity, through the “Ca clock”. To study SAN pacing in the absence of NCX1, we created an atrial-specific NCX1 KO mouse using a Cre/loxP system under the control of the endogenous sarcolipin promoter. NCX1 KO mice have complete AV block and no P waves. The latter is caused by conduction interference between SAN and atria. To test the hypothesis that SAN pacemaking can be maintained by funny current (If) in the absence of NCX1, we recorded Ca transients (using Cal 520 and high speed 2D confocal imaging) in an ex vivo tissuepreparation that included the SAN, right atrium and left atrium. At ∼36°C, the SAN pacemaker rate was 5.3±0.3Hz in WT (n=14) but only 2±0.2Hz (n=17) in KO. The pattern of pacing in KO was characterized by frequent bursts of Ca transients alternating with long pauses. During bursts in KO, the rate was comparable to WT (4.1±0.3Hz). WT SANs (n=5) showed a 70.5±6.1% increase in rate after exposure to the β-adrenergic agonist ISO (10μM), while KO showed no increase in average rate. However, when considering only rate during burst activity in KO, 6 out of 10 KO SANs responded significantly to ISO 10μM (52±16% increase). The specific If inhibitor ivabradine (IVA, 9μM) reduced the spontaneous pacemaker rate in both WT (n=6) and KO (n=8) SANs (44±9% and 58.9±6.8% decrease, respectively), and even during the bursts in the KO (36±17.9% decrease). Thus, NCX1 KO SANs can generate bursts of pacemaker activity similar to WT. These bursts are responsive to both ISO and IVA, consistent with If-mediated pacemaker activity despite the absence of NCX.