RESUMEN
Popeye domain containing (POPDC) proteins are predominantly expressed in the heart and skeletal muscle, modulating the K2P potassium channel TREK-1 in a cAMP-dependent manner. POPDC1 and POPDC2 variants cause cardiac conduction disorders with or without muscular dystrophy. Searching for POPDC2-modulated ion channels using a functional co-expression screen in Xenopus oocytes, we found POPDC proteins to modulate the cardiac sodium channel Nav1.5. POPDC proteins downregulate Nav1.5 currents in a cAMP-dependent manner by reducing the surface expression of the channel. POPDC2 and Nav1.5 are both expressed in different regions of the murine heart and consistently POPDC2 co-immunoprecipitates with Nav1.5 from native cardiac tissue. Strikingly, the knock-down of popdc2 in embryonic zebrafish caused an increased upstroke velocity and overshoot of cardiac action potentials. The POPDC modulation of Nav1.5 provides a new mechanism to regulate cardiac sodium channel densities under sympathetic stimulation, which is likely to have a functional impact on cardiac physiology and inherited arrhythmias.
RESUMEN
The Popeye domain containing (POPDC) genes encode sarcolemma-localized cAMP effector proteins. Mutations in blood vessel epicardial substance (BVES) also known as POPDC1 and POPDC2 have been associated with limb-girdle muscular dystrophy and cardiac arrhythmia. Muscle biopsies of affected patients display impaired membrane trafficking of both POPDC isoforms. Biopsy material of patients carrying mutations in BVES were immunostained with POPDC antibodies. The interaction of POPDC proteins was investigated by co-precipitation, proximity ligation, bioluminescence resonance energy transfer and bimolecular fluorescence complementation. Site-directed mutagenesis was utilised to map the domains involved in protein-protein interaction. Patients carrying a novel homozygous variant, BVES (c.547G > T, p.V183F) displayed only a skeletal muscle pathology and a mild impairment of membrane trafficking of both POPDC isoforms. In contrast, variants such as BVES p.Q153X or POPDC2 p.W188X were associated with a greater impairment of membrane trafficking. Co-transfection analysis in HEK293 cells revealed that POPDC proteins interact with each other through a helix-helix interface located at the C-terminus of the Popeye domain. Site-directed mutagenesis of an array of ultra-conserved hydrophobic residues demonstrated that some of them are required for membrane trafficking of the POPDC1-POPDC2 complex. Mutations in POPDC proteins that cause an impairment in membrane localization affect POPDC complex formation while mutations which leave protein-protein interaction intact likely affect some other essential function of POPDC proteins.
Asunto(s)
Anticuerpos , Proteínas Musculares , Humanos , Células HEK293 , Mutación/genética , Biopsia , Homocigoto , Moléculas de Adhesión CelularRESUMEN
The establishment of macromolecular complexes by scaffolding proteins is key to the local production of cAMP by anchored adenylyl cyclase (AC) and the subsequent cAMP signaling necessary for cardiac functions. We identify a novel AC scaffold, the Popeye domain-containing (POPDC) protein. The POPDC family of proteins is important for cardiac pacemaking and conduction, due in part to their cAMP-dependent binding and regulation of TREK-1 potassium channels. We show that TREK-1 binds the AC9:POPDC1 complex and copurifies in a POPDC1-dependent manner with AC9 activity in heart. Although the AC9:POPDC1 interaction is cAMP-independent, TREK-1 association with AC9 and POPDC1 is reduced upon stimulation of the ß-adrenergic receptor (ßAR). AC9 activity is required for ßAR reduction of TREK-1 complex formation with AC9:POPDC1 and in reversing POPDC1 enhancement of TREK-1 currents. Finally, deletion of the gene-encoding AC9 (Adcy9) gives rise to bradycardia at rest and stress-induced heart rate variability, a milder phenotype than the loss of Popdc1 but similar to the loss of Kcnk2 (TREK-1). Thus, POPDC1 represents a novel adaptor for AC9 interactions with TREK-1 to regulate heart rate control.
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Adenilil Ciclasas , Canales de Potasio , Adenilil Ciclasas/genéticaRESUMEN
Extensive research has uncovered diverse forms of synaptic plasticity and an array of molecular signaling mechanisms that act as positive or negative regulators. Specifically, cyclic 3',5'-cyclic adenosine monophosphate (cAMP)-dependent signaling pathways are crucially implicated in long-lasting synaptic plasticity. In this study, we examine the role of Popeye domain-containing protein 1 (POPDC1) (or blood vessel epicardial substance (BVES)), a cAMP effector protein, in modulating hippocampal synaptic plasticity. Unlike other cAMP effectors, such as protein kinase A (PKA) and exchange factor directly activated by cAMP, POPDC1 is membrane-bound and the sequence of the cAMP-binding cassette differs from canonical cAMP-binding domains, suggesting that POPDC1 may have an unique role in cAMP-mediated signaling. Our results show that Popdc1 is widely expressed in various brain regions including the hippocampus. Acute hippocampal slices from Popdc1 knockout (KO) mice exhibit PKA-dependent enhancement in CA1 long-term potentiation (LTP) in response to weaker stimulation paradigms, which in slices from wild-type mice induce only transient LTP. Loss of POPDC1, while not affecting basal transmission or input-specificity of LTP, results in altered response during high-frequency stimulation. Popdc1 KO mice also show enhanced forskolin-induced potentiation. Overall, these findings reveal POPDC1 as a novel negative regulator of hippocampal synaptic plasticity and, together with recent evidence for its interaction with phosphodiesterases (PDEs), suggest that POPDC1 is involved in modulating activity-dependent local cAMP-PKA-PDE signaling.
Asunto(s)
Moléculas de Adhesión Celular , Hipocampo , Potenciación a Largo Plazo , Proteínas Musculares , Plasticidad Neuronal , Animales , Moléculas de Adhesión Celular/genética , AMP Cíclico/metabolismo , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Hipocampo/fisiología , Ratones , Ratones Endogámicos C57BL , Proteínas Musculares/genética , Transmisión SinápticaRESUMEN
BACKGROUND: Popeye domain-containing proteins 1 and 2 (POPDC1 and POPDC2) are transmembrane proteins involved in cyclic AMP-mediated signalling processes and are required for normal cardiac pacemaking and conduction. In order to identify novel protein interaction partners, POPDC1 and 2 proteins were attached to beads and compared by proteomic analysis with control beads in the pull-down of proteins from cultured human skeletal myotubes. RESULTS: There were highly-significant interactions of both POPDC1 and POPDC2 with XIRP1 (Xin actin binding repeat-containing protein 1), actin and, to a lesser degree, annexin A5. In adult human skeletal muscle, both XIRP1 and POPDC1/2 were present at the sarcolemma and in T-tubules. The interaction of POPDC1 with XIRP1 was confirmed in adult rat heart extracts. Using new monoclonal antibodies specific for POPDC1 and POPDC2, both proteins, together with XIRP1, were found mainly at intercalated discs but also at T-tubules in adult rat and human heart. CONCLUSIONS: Mutations in human POPDC1, POPDC2 and in human XIRP1, all cause pathological cardiac arrhythmias, suggesting a possible role for POPDC1/2 and XIRP1 interaction in normal cardiac conduction.
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Moléculas de Adhesión Celular/metabolismo , Proteínas de Unión al ADN/metabolismo , Cardiopatías/metabolismo , Proteínas de la Membrana/metabolismo , Proteínas Musculares/metabolismo , Proteínas Nucleares/metabolismo , Sarcolema/metabolismo , Actinas/metabolismo , Adulto , Animales , Anexina A5/metabolismo , Anticuerpos Monoclonales/metabolismo , Células COS , Chlorocebus aethiops , Humanos , Fibras Musculares Esqueléticas/metabolismo , Músculo Esquelético/metabolismo , Unión Proteica , Ratas Sprague-DawleyRESUMEN
Despite recent progress in the understanding of cardiac ion channel function and its role in inherited forms of ventricular arrhythmias, the molecular basis of cardiac conduction disorders often remains unresolved. We aimed to elucidate the genetic background of familial atrioventricular block (AVB) using a whole exome sequencing (WES) approach. In monozygotic twins with a third-degree AVB and in another, unrelated family with first-degree AVB, we identified a heterozygous nonsense mutation in the POPDC2 gene causing a premature stop at position 188 (POPDC2W188â), deleting parts of its cAMP binding-domain. Popeye-domain containing (POPDC) proteins are predominantly expressed in the skeletal muscle and the heart, with particularly high expression of POPDC2 in the sinoatrial node of the mouse. We now show by quantitative PCR experiments that in the human heart the POPDC-modulated two-pore domain potassium (K2P) channel TREK-1 is preferentially expressed in the atrioventricular node. Co-expression studies in Xenopus oocytes revealed that POPDC2W188â causes a loss-of-function with impaired TREK-1 modulation. Consistent with the high expression level of POPDC2 in the murine sinoatrial node, POPDC2W188â knock-in mice displayed stress-induced sinus bradycardia and pauses, a phenotype that was previously also reported for POPDC2 and TREK-1 knock-out mice. We propose that the POPDC2W188â loss-of-function mutation contributes to AVB pathogenesis by an aberrant modulation of TREK-1, highlighting that POPDC2 represents a novel arrhythmia gene for cardiac conduction disorders.
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Trastorno del Sistema de Conducción Cardíaco/genética , Moléculas de Adhesión Celular/genética , Predisposición Genética a la Enfermedad , Proteínas Musculares/genética , Potenciales de Acción , Animales , Bloqueo Atrioventricular/genética , Bradicardia/complicaciones , Moléculas de Adhesión Celular/metabolismo , Línea Celular , Estudios de Asociación Genética , Sistema de Conducción Cardíaco/metabolismo , Sistema de Conducción Cardíaco/patología , Heterocigoto , Homocigoto , Humanos , Leucocitos/metabolismo , Ratones Transgénicos , Proteínas Musculares/metabolismo , Mutación/genética , Canales de Potasio de Dominio Poro en Tándem/metabolismo , ARN/metabolismo , Nodo Sinoatrial/metabolismo , Estrés Fisiológico , Secuenciación del Exoma , Xenopus laevisRESUMEN
OBJECTIVE: The Popeye domain containing 3 (POPDC3) gene encodes a membrane protein involved in cyclic adenosine monophosphate (cAMP) signaling. Besides gastric cancer, no disease association has been described. We describe a new muscular dystrophy associated with this gene. METHODS: We screened 1,500 patients with unclassified limb girdle weakness or hyperCKemia for pathogenic POPDC3 variants. Five patients carrying POPDC3 variants were examined by muscle magnetic resonance imaging (MRI), muscle biopsy, and cardiac examination. We performed functional analyses in a zebrafish popdc3 knockdown model and heterologous expression of the mutant proteins in Xenopus laevis oocytes to measure TREK-1 current. RESULTS: We identified homozygous POPDC3 missense variants (p.Leu155His, p.Leu217Phe, and p.Arg261Gln) in 5 patients from 3 ethnically distinct families. Variants affected highly conserved residues in the Popeye (p.Leu155 and p.Leu217) and carboxy-terminal (p.Arg261) domains. The variants were almost absent from control populations. Probands' muscle biopsies were dystrophic, and serum creatine kinase levels were 1,050 to 9,200U/l. Muscle weakness was proximal with adulthood onset in most patients and affected lower earlier than upper limbs. Muscle MRI revealed fat replacement of paraspinal and proximal leg muscles; cardiac investigations were unremarkable. Knockdown of popdc3 in zebrafish, using 2 different splice-site blocking morpholinos, resulted in larvae with tail curling and dystrophic muscle features. All 3 mutants cloned in Xenopus oocytes caused an aberrant modulation of the mechano-gated potassium channel, TREK-1. INTERPRETATION: Our findings point to an important role of POPDC3 for skeletal muscle function and suggest that pathogenic variants in POPDC3 are responsible for a novel type of autosomal recessive limb girdle muscular dystrophy. ANN NEUROL 2019;86:832-843.
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Moléculas de Adhesión Celular/genética , Variación Genética/genética , Proteínas Musculares/genética , Músculo Esquelético/diagnóstico por imagen , Músculo Esquelético/fisiología , Distrofia Muscular de Cinturas/diagnóstico por imagen , Distrofia Muscular de Cinturas/genética , Adulto , Animales , Moléculas de Adhesión Celular/química , Estudios de Cohortes , Femenino , Técnicas de Silenciamiento del Gen/métodos , Humanos , Masculino , Persona de Mediana Edad , Proteínas Musculares/química , Linaje , Estructura Secundaria de Proteína , Xenopus laevis , Pez CebraRESUMEN
Popeye domain containing (Popdc) proteins are a unique family, which combine several different properties and functions in a surprisingly complex fashion. They are expressed in multiple tissues and cell types, present in several subcellular compartments, interact with different classes of proteins, and are associated with a variety of physiological and pathophysiological processes. Moreover, Popdc proteins bind the second messenger cAMP with high affinity and it is thought that they act as a novel class of cAMP effector proteins. Here, we will review the most important findings about the Popdc family, which accumulated since its discovery about 15 years ago. We will be focussing on Popdc protein interaction and function in striated muscle tissue. However, as a full picture only emerges if all aspects are taken into account, we will also describe what is currently known about the role of Popdc proteins in epithelial cells and in various types of cancer, and discuss these findings with regard to their relevance for cardiac and skeletal muscle.
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AMP Cíclico/metabolismo , Proteínas Musculares , Animales , Regulación de la Expresión Génica , Humanos , Proteínas Musculares/química , Proteínas Musculares/genética , Proteínas Musculares/metabolismoRESUMEN
The Popeye domain-containing 1 (POPDC1) gene encodes a plasma membrane-localized cAMP-binding protein that is abundantly expressed in striated muscle. In animal models, POPDC1 is an essential regulator of structure and function of cardiac and skeletal muscle; however, POPDC1 mutations have not been associated with human cardiac and muscular diseases. Here, we have described a homozygous missense variant (c.602C>T, p.S201F) in POPDC1, identified by whole-exome sequencing, in a family of 4 with cardiac arrhythmia and limb-girdle muscular dystrophy (LGMD). This allele was absent in known databases and segregated with the pathological phenotype in this family. We did not find the allele in a further screen of 104 patients with a similar phenotype, suggesting this mutation to be family specific. Compared with WT protein, POPDC1(S201F) displayed a 50% reduction in cAMP affinity, and in skeletal muscle from patients, both POPDC1(S201F) and WT POPDC2 displayed impaired membrane trafficking. Forced expression of POPDC1(S201F) in a murine cardiac muscle cell line (HL-1) increased hyperpolarization and upstroke velocity of the action potential. In zebrafish, expression of the homologous mutation (popdc1(S191F)) caused heart and skeletal muscle phenotypes that resembled those observed in patients. Our study therefore identifies POPDC1 as a disease gene causing a very rare autosomal recessive cardiac arrhythmia and LGMD, expanding the genetic causes of this heterogeneous group of inherited rare diseases.
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Arritmias Cardíacas/etiología , Proteínas de la Membrana/genética , Distrofia Muscular de Cinturas/etiología , Anciano , Anciano de 80 o más Años , Animales , Moléculas de Adhesión Celular , Niño , AMP Cíclico/metabolismo , Humanos , Masculino , Potenciales de la Membrana , Proteínas de la Membrana/fisiología , Persona de Mediana Edad , Proteínas Musculares , Mutación , Canales de Potasio de Dominio Poro en Tándem/análisis , Transporte de Proteínas , Pez CebraRESUMEN
Popdc (Popeye-domain-containing) genes encode membrane-bound proteins and are abundantly present in cardiac myocytes and in skeletal muscle fibres. Functional analysis of Popdc1 (Bves) and Popdc2 in mice and of popdc2 in zebrafish revealed an overlapping role for proper electrical conduction in the heart and maintaining structural integrity of skeletal muscle. Popdc proteins mediate cAMP signalling and modulate the biological activity of interacting proteins. The two-pore channel TREK-1 interacts with all three Popdc proteins. In Xenopus oocytes, the presence of Popdc proteins causes an enhanced membrane transport leading to an increase in TREK-1 current, which is blocked when cAMP levels are increased. Another important Popdc-interacting protein is caveolin 3, and the loss of Popdc1 affects caveolar size. Thus a family of membrane-bound cAMP-binding proteins has been identified, which modulate the subcellular localization of effector proteins involved in organizing signalling complexes and assuring proper membrane physiology of cardiac myocytes.
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Proteínas Musculares/metabolismo , Miocardio/metabolismo , Animales , Arritmias Cardíacas/metabolismo , Caveolina 3/metabolismo , Humanos , Canales de Potasio de Dominio Poro en Tándem/metabolismoRESUMEN
3'-5'-cyclic adenosine monophosphate (cAMP) is a second messenger, which plays an important role in the heart. It is generated in response to activation of G-protein-coupled receptors (GPCRs). Initially, it was thought that protein kinase A (PKA) exclusively mediates cAMP-induced cellular responses such as an increase in cardiac contractility, relaxation, and heart rate. With the identification of the exchange factor directly activated by cAMP (EPAC) and hyperpolarizing cyclic nucleotide-gated (HCN) channels as cAMP effector proteins it became clear that a protein network is involved in cAMP signaling. The Popeye domain containing (Popdc) genes encode yet another family of cAMP-binding proteins, which are prominently expressed in the heart. Loss-of-function mutations in mice are associated with cardiac arrhythmia and impaired skeletal muscle regeneration. Interestingly, the cardiac phenotype, which is present in both, Popdc1 and Popdc2 null mutants, is characterized by a stress-induced sinus bradycardia, suggesting that Popdc proteins participate in cAMP signaling in the sinuatrial node. The identification of the two-pore channel TREK-1 and Caveolin 3 as Popdc-interacting proteins represents a first step into understanding the mechanisms of heart rate modulation triggered by Popdc proteins.
RESUMEN
Popeye domain containing1 (Popdc1), also named Bves, is an evolutionary conserved membrane protein. Despite its high expression level in the heart little is known about its membrane localization and cardiac functions. The study examined the hypothesis that Popdc1 might be associated with the caveolae and play a role in myocardial ischemia tolerance. To address these issues, we analyzed hearts and cardiomyocytes of wild type and Popdc1-null mice. Immunoconfocal microscopy revealed co-localization of Popdc1 with caveolin3 in the sarcolemma, intercalated discs and T-tubules and with costameric vinculin. Popdc1 was co-immunoprecipitated with caveolin3 from cardiomyocytes and from transfected COS7 cells and was co-sedimented with caveolin3 in equilibrium density gradients. Caveolae disruption by methyl-ß-cyclodextrin or by ischemia/reperfusion (I/R) abolished the cellular co-localization of Popdc1 with caveolin3 and modified their density co-sedimentation. The caveolin3-rich fractions of Popdc1-null hearts redistributed to fractions of lower buoyant density. Electron microscopy showed a statistically significant 70% reduction in caveolae number and a 12% increase in the average diameter of the remaining caveolae in the mutant hearts. In accordance with these changes, Popdc1-null cardiomyocytes displayed impaired [Ca(+2)]i transients, increased vulnerability to oxidative stress and no pharmacologic preconditioning. In addition, induction of I/R injury to Langendorff-perfused hearts indicated a significantly lower functional recovery in the mutant compared with wild type hearts while their infarct size was larger. No improvement in functional recovery was observed in Popdc1-null hearts following ischemic preconditioning. The results indicate that Popdc1 is a caveolae-associated protein important for the preservation of caveolae structural and functional integrity and for heart protection.
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Caveolas/metabolismo , Proteínas de la Membrana/metabolismo , Isquemia Miocárdica/metabolismo , Animales , Western Blotting , Células COS , Calcio/metabolismo , Caveolina 3/metabolismo , Células Cultivadas , Chlorocebus aethiops , Inmunoprecipitación , Técnicas In Vitro , Masculino , Proteínas de la Membrana/genética , Ratones , Ratones Endogámicos C57BL , Microscopía Confocal , Isquemia Miocárdica/genética , Miocitos Cardíacos/citología , Miocitos Cardíacos/metabolismo , Reacción en Cadena de la Polimerasa , Unión Proteica , RatasRESUMEN
An intricate network of ion channels and pumps are involved in generating a diastolic pacemaker potential, which is transmitted to the working myocardium with the help of the cardiac conduction system. The principles of cardiac pacemaking are reasonably well understood, however, the mechanism by which the heart increases its beating frequency in response to adrenergic stimulation has not been fully worked out. The Popeye domain-containing (Popdc) genes encode plasma membrane-localized proteins that are able to bind cAMP with high affinity; mice with null mutations in Popdc1 or 2 have a stress-induced pacemaker dysfunction. The phenotype in both mutants develops in an age-dependent manner and thus may model pacemaker dysfunction in man, as well as provide novel mechanistic insights into the process of pacemaker adaptation to stress.
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Moléculas de Adhesión Celular , Sistema de Conducción Cardíaco/metabolismo , Proteínas Musculares , Miocardio/metabolismo , Adaptación Fisiológica , Animales , Moléculas de Adhesión Celular/genética , Moléculas de Adhesión Celular/metabolismo , Humanos , Canales Iónicos/metabolismo , Ratones , Modelos Biológicos , Proteínas Musculares/genética , Proteínas Musculares/metabolismo , Mutación , Estrés Fisiológico/fisiologíaRESUMEN
Cardiac pacemaker cells create rhythmic pulses that control heart rate; pacemaker dysfunction is a prevalent disorder in the elderly, but little is known about the underlying molecular causes. Popeye domain containing (Popdc) genes encode membrane proteins with high expression levels in cardiac myocytes and specifically in the cardiac pacemaking and conduction system. Here, we report the phenotypic analysis of mice deficient in Popdc1 or Popdc2. ECG analysis revealed severe sinus node dysfunction when freely roaming mutant animals were subjected to physical or mental stress. In both mutants, bradyarrhythmia developed in an age-dependent manner. Furthermore, we found that the conserved Popeye domain functioned as a high-affinity cAMP-binding site. Popdc proteins interacted with the potassium channel TREK-1, which led to increased cell surface expression and enhanced current density, both of which were negatively modulated by cAMP. These data indicate that Popdc proteins have an important regulatory function in heart rate dynamics that is mediated, at least in part, through cAMP binding. Mice with mutant Popdc1 and Popdc2 alleles are therefore useful models for the dissection of the mechanisms causing pacemaker dysfunction and could aid in the development of strategies for therapeutic intervention.
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Moléculas de Adhesión Celular/metabolismo , Proteínas Musculares/metabolismo , Canales de Potasio de Dominio Poro en Tándem/metabolismo , Animales , Relojes Biológicos , Bradicardia/genética , Electrocardiografía/métodos , Electrofisiología/métodos , Frecuencia Cardíaca , Proteínas de la Membrana/metabolismo , Ratones , Ratones Transgénicos , Fenotipo , Estructura Terciaria de Proteína , Telemetría/métodos , Factores de TiempoRESUMEN
The Popeye domain containing (Popdc) gene family displays preferential expression in skeletal muscle and heart. Only recently a significant gain in the understanding of the function of Popdc genes in the heart has been obtained. The Popdc genes encode membrane proteins harboring an evolutionary conserved Popeye domain, which functions as a binding domain for cyclic adenosine monophosphate (cAMP). Popdc proteins interact with the two-pore channel TREK-1 and enhance its current. This protein interaction is modulated by cAMP. Null mutations of members of the Popdc gene family in zebrafish and mouse are associated with severe cardiac arrhythmia phenotypes. While in zebrafish an atrioventricular block was prevalent, in mouse a stress-induced sinus bradycardia was observed, which was due to the presence of sinus pauses. Moreover, the phenotype develops in an age-dependent manner, being absent in the young animal and becoming increasingly severe, as the animals grow older. This phenotype is reminiscent of the sick sinus syndrome (SSS), which affects mostly the elderly and is characterized by the poor ability of the cardiac pacemaker to adapt the heart rate to the physiological demand. While being a prevalent disease, which is responsible for a large fraction of pacemaker implantations in Western countries, SSS is poorly understood at the molecular level. It is therefore expected that the study of the molecular basis of the stress-induced bradycardia in Popdc mice will shed new light on the etiology of pacemaker disease.