RESUMO
Loss or dysregulation of the normally precise control of heart rate via the autonomic nervous system plays a critical role during the development and progression of cardiovascular disease-including ischemic heart disease, heart failure, and arrhythmias. While the clinical significance of regulating changes in heart rate, known as the chronotropic effect, is undeniable, the mechanisms controlling these changes remain not fully understood. Heart rate acceleration and deceleration are mediated by increasing or decreasing the spontaneous firing rate of pacemaker cells in the sinoatrial node. During the transition from rest to activity, sympathetic neurons stimulate these cells by activating ß-adrenergic receptors and increasing intracellular cyclic adenosine monophosphate. The same signal transduction pathway is targeted by positive chronotropic drugs such as norepinephrine and dobutamine, which are used in the treatment of cardiogenic shock and severe heart failure. The cyclic adenosine monophosphate-sensitive hyperpolarization-activated current (If) in pacemaker cells is passed by hyperpolarization-activated cyclic nucleotide-gated cation channels and is critical for generating the autonomous heartbeat. In addition, this current has been suggested to play a central role in the chronotropic effect. Recent studies demonstrate that cyclic adenosine monophosphate-dependent regulation of HCN4 (hyperpolarization-activated cyclic nucleotide-gated cation channel isoform 4) acts to stabilize the heart rate, particularly during rapid rate transitions induced by the autonomic nervous system. The mechanism is based on creating a balance between firing and recently discovered nonfiring pacemaker cells in the sinoatrial node. In this way, hyperpolarization-activated cyclic nucleotide-gated cation channels may protect the heart from sinoatrial node dysfunction, secondary arrhythmia of the atria, and potentially fatal tachyarrhythmia of the ventricles. Here, we review the latest findings on sinoatrial node automaticity and discuss the physiological and pathophysiological role of HCN pacemaker channels in the chronotropic response and beyond.
Assuntos
Frequência Cardíaca , Canais Disparados por Nucleotídeos Cíclicos Ativados por Hiperpolarização , Nó Sinoatrial , Humanos , Animais , Nó Sinoatrial/metabolismo , Nó Sinoatrial/fisiopatologia , Nó Sinoatrial/fisiologia , Canais Disparados por Nucleotídeos Cíclicos Ativados por Hiperpolarização/metabolismo , Relógios BiológicosRESUMO
Cyclic adenosine monophosphate (cAMP)-protein kinase A (PKA) signaling controls sinoatrial node cell (SANC) function by affecting the degree of coupling between Ca2+ and membrane clocks. PKA is known to phosphorylate ionic channels, Ca2+ pump and release from the sarcoplasmic reticulum, and enzymes controlling ATP production in the mitochondria. While the PKA cytosolic targets in SANC have been extensively explored, its mitochondrial targets and its ability to maintain SANC energetic balance remain to be elucidated. To investigate the role of PKA in SANC energetics, we tested three hypotheses: (i) PKA is an important regulator of the ATP supply-to-demand balance, (ii) Ca2+ regulation of energetics is important for maintenance of NADH level and (iii) abrupt reduction in ATP demand first reduces the AP firing rate and, after dropping below a certain threshold, leads to a reduction in ATP. To gain mechanistic insights into the ATP supply-to-demand matching regulators, a modified model of mitochondrial energy metabolism was integrated into our coupled-clock model that describes ATP demand. Experimentally, increased ATP demand was accompanied by maintained ATP and NADH levels. Ca2+ regulation of energetics was found by the model to be important in the maintenance of NADH and PKA regulation was found to be important in the maintenance of intracellular ATP and the increase in oxygen consumption. PKA inhibition led to a biphasic reduction in AP firing rate, with the first phase being rapid and ATP-independent, while the second phase was slow and ATP-dependent. Thus, SANC energy balance is maintained by both Ca2+ and PKA signaling.
Assuntos
Sinalização do Cálcio , NAD , NAD/metabolismo , Sinalização do Cálcio/fisiologia , Miócitos Cardíacos/metabolismo , AMP Cíclico/metabolismo , Metabolismo Energético , Nó Sinoatrial/metabolismo , Trifosfato de Adenosina/metabolismo , Cálcio/metabolismoRESUMO
Abstract Background Dexmedetomidine (Dex) is widely used, and its most common side effect is bradycardia. The complete mechanism through which Dex induces bradycardia has not been elucidated. This research investigates the expression of gap junction proteins Connexin30.2 (Cx30.2) and Connexin40 (Cx40) within the sinoatrial node of rats with Dex-induced sinus bradycardia. Methods Eighty rats were randomly assigned to five groups. Saline was administered to rats in Group C. In the other four groups, the rats were administered Dex to induce bradycardia. In groups D1and D2, the rats were administered Dex at a loading dose of 30 μg.kg−1 and 100 μg.kg−1 for 10 min, then at 15 μg.kg−1.h−1 and 50 μg.kg−1.h−1 for 120 min separately. The rats in group D1A and D2A were administered Dex in the same way as in group D1and D2; however, immediately after the administration of the loading dose, 0.5 mg atropine was administered intravenously, and then at 0.5 mg.kg−1.h−1 for 120 min. The sinoatrial node was acquired after intravenous infusion was completed. Quantitative real-time polymerase chain reaction and western blot analyses were performed to measure mRNA and protein expression of Cx30.2 and Cx40, respectively. Results The expression of Cx30.2 increased, whereas the expression of Cx40 decreased within the sinoatrial node of rats with Dex-induced sinus bradycardia. Atropine reversed the effects of Dex on the expression of gap junction proteins. Conclusion Dex possibly altered the expression of gap junction proteins to slow down cardiac conduction velocity in the sinoatrial node.
Assuntos
Animais , Ratos , Nó Sinoatrial/metabolismo , Dexmedetomidina , Arritmias Cardíacas , Derivados da Atropina/metabolismo , Bradicardia/induzido quimicamente , Conexinas/genética , Conexinas/metabolismoRESUMO
BACKGROUND: The sinoatrial node (SAN) functions as the pacemaker of the heart, initiating rhythmic heartbeats. Despite its importance, the SAN is one of the most poorly understood cardiac entities because of its small size and complex composition and function. The Hippo signaling pathway is a molecular signaling pathway fundamental to heart development and regeneration. Although abnormalities of the Hippo pathway are associated with cardiac arrhythmias in human patients, the role of this pathway in the SAN is unknown. METHODS: We investigated key regulators of the Hippo pathway in SAN pacemaker cells by conditionally inactivating the Hippo signaling kinases Lats1 and Lats2 using the tamoxifen-inducible, cardiac conduction system-specific Cre driver Hcn4CreERT2 with Lats1 and Lats2 conditional knockout alleles. In addition, the Hippo-signaling effectors Yap and Taz were conditionally inactivated in the SAN. To determine the function of Hippo signaling in the SAN and other cardiac conduction system components, we conducted a series of physiological and molecular experiments, including telemetry ECG recording, echocardiography, Masson Trichrome staining, calcium imaging, immunostaining, RNAscope, cleavage under targets and tagmentation sequencing using antibodies against Yap1 or H3K4me3, quantitative real-time polymerase chain reaction, and Western blotting. We also performed comprehensive bioinformatics analyses of various datasets. RESULTS: We found that Lats1/2 inactivation caused severe sinus node dysfunction. Compared with the controls, Lats1/2 conditional knockout mutants exhibited dysregulated calcium handling and increased fibrosis in the SAN, indicating that Lats1/2 function through both cell-autonomous and non-cell-autonomous mechanisms. It is notable that the Lats1/2 conditional knockout phenotype was rescued by genetic deletion of Yap and Taz in the cardiac conduction system. These rescued mice had normal sinus rhythm and reduced fibrosis of the SAN, indicating that Lats1/2 function through Yap and Taz. Cleavage Under Targets and Tagmentation sequencing data showed that Yap potentially regulates genes critical for calcium homeostasis such as Ryr2 and genes encoding paracrine factors important in intercellular communication and fibrosis induction such as Tgfb1 and Tgfb3. Consistent with this, Lats1/2 conditional knockout mutants had decreased Ryr2 expression and increased Tgfb1 and Tgfb3 expression compared with control mice. CONCLUSIONS: We reveal, for the first time to our knowledge, that the canonical Hippo-Yap pathway plays a pivotal role in maintaining SAN homeostasis.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal , Proteínas de Ciclo Celular , Humanos , Camundongos , Animais , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas de Ciclo Celular/metabolismo , Fator de Crescimento Transformador beta3/metabolismo , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Nó Sinoatrial/metabolismo , Cálcio/metabolismo , Canal de Liberação de Cálcio do Receptor de Rianodina , Proteínas Serina-Treonina Quinases/genética , Homeostase , Fibrose , Proliferação de Células , Proteínas Supressoras de TumorRESUMO
Sinoatrial node (SAN) pacemaker cells originate from T-box transcription factor 18 (Tbx18)-expressing progenitor cells. The present study aimed to investigate whether overexpression of human transcription factor Tbx18 could reprogram human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) into SAN-like pacemaker cells (SANLPCs) in vitro. In the study, hiPSCs were first differentiated into hiPSC-CMs through regulating the Wnt/ß-catenin pathway, then purified hiPSC-CMs were transfected by Tbx18 adenovirus (Tbx18-CMs group) or green fluorescent protein (GFP) adenovirus (GFP-CMs group). The beating frequency of the Tbx18-CMs group was significantly higher than that of the hiPSC-CMs group and GFP-CMs group. Compared with the other two groups, the expression levels of hyperpolarization-activated cyclic nucleotide-gated potassium channel isoform 4, connexin-45 in the Tbx18-CMs group were markedly upregulated, while the expressions of transcription factor NKX2.5, CX43 were significantly downregulated. Whole-cell patch-clamp results illustrated that action potential and "funny" current (If ) similar to SAN pacemaker cells could be recorded in the Tbx18-CMs group. In conclusion, this present study demonstrated that overexpression of Tbx18 promoted the conversion of hiPSC-CMs into SANLPCs.
Assuntos
Células-Tronco Pluripotentes Induzidas , Diferenciação Celular , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Miócitos Cardíacos/metabolismo , Nó Sinoatrial/metabolismo , Proteínas com Domínio T/genética , Proteínas com Domínio T/metabolismoRESUMO
The sinoatrial (SA) node is the physiological pacemaker of the heart, and resting heart rate in humans is a well-known risk factor for cardiovascular disease and mortality. Consequently, the mechanisms of initiating and regulating the normal spontaneous SA node beating rate are of vital importance. Spontaneous firing of the SA node is generated within sinoatrial nodal cells (SANC), which is regulated by the coupled-clock pacemaker system. Normal spontaneous beating of SANC is driven by a high level of cAMP-mediated PKA-dependent protein phosphorylation, which rely on the balance between high basal cAMP production by adenylyl cyclases and high basal cAMP degradation by cyclic nucleotide phosphodiesterases (PDEs). This diverse class of enzymes includes 11 families and PDE3 and PDE4 families dominate in both the SA node and cardiac myocardium, degrading cAMP and, consequently, regulating basal cardiac pacemaker function and excitation-contraction coupling. In this review, we will demonstrate similarities between expression, distribution, and colocalization of various PDE subtypes in SANC and cardiac myocytes of different species, including humans, focusing on PDE3 and PDE4. Here, we will describe specific targets of the coupled-clock pacemaker system modulated by dual PDE3 + PDE4 activation and provide evidence that concurrent activation of PDE3 + PDE4, operating in a synergistic manner, regulates the basal cardiac pacemaker function and provides control over normal spontaneous beating of SANCs through (PDE3 + PDE4)-dependent modulation of local subsarcolemmal Ca2+ releases (LCRs).
Assuntos
Relógios Biológicos/fisiologia , Miócitos Cardíacos/metabolismo , Diester Fosfórico Hidrolases/metabolismo , Nó Sinoatrial/metabolismo , Animais , Cálcio/metabolismo , Sinalização do Cálcio/fisiologia , AMP Cíclico/metabolismo , HumanosRESUMO
Long non-coding RNA (lncRNA) is receiving increasing attention in embryonic stem cells (ESCs) research. However, the roles of lncRNA in the differentiation of ESCs into pacemaker-like cells are still unclear. Therefore, the present study aims to explore the roles and mechanisms of lncRNA in the differentiation of ESCs into pacemaker-like cells. ESCs were cultured and induced differentiation to pacemaker-like cells. RNA sequencing was used to identify the differential expression lncRNAs during the differentiation of ESCs into pacemaker-like cells. Cell morphology observation, flow cytometry, quantitative real-time polymerase chain reaction, western blot, and immunofluorescence were used to detect the differentiation of ESCs into pacemaker-like cells. LncRNA and genes overexpression or knockdown through transfected adenovirus in the differentiation process. The fluorescence in situ hybridization (FISH) detected the lncRNA location in the differentiated ESCs. Luciferase reporter gene assay, methylation-specific PCR, chromatin immunoprecipitation assay, and RNA immunoprecipitation assay were performed to reveal the mechanism of lncRNA-regulating HCN4 expression. Rescue experiments were used to confirm that lncRNA regulates the differentiation of ESCs into pacemaker-like cells through HCN4. We cultured the ESCs and induced the differentiation of ESCs into pacemaker-like cells successfully. The expression of lncRNA RCPCD was significantly decreased in the differentiation of ESCs into pacemaker-like cells. Overexpression of RCPCD inhibited the differentiation of ESCs into pacemaker-like cells. RCPCD inhibited the expression of HCN4 by increasing HCN4 methylation at the promoter region through DNMT1, DNMT2, and DNMT3. RCPCD inhibited the differentiation of ESCs into pacemaker-like cells by inhibiting the expression of HCN4. Our results confirm the roles and mechanism of lncRNA RCPCD in the differentiation of ESCs into pacemaker-like cells, which could pave the path for the development of a cell-based biological pacemaker.
Assuntos
Relógios Biológicos , Diferenciação Celular , Metilação de DNA , Canais Disparados por Nucleotídeos Cíclicos Ativados por Hiperpolarização/genética , Células-Tronco Embrionárias Murinas/metabolismo , Miócitos Cardíacos/metabolismo , Regiões Promotoras Genéticas , RNA Longo não Codificante/genética , Nó Sinoatrial/metabolismo , Animais , Células Cultivadas , DNA (Citosina-5-)-Metiltransferases/metabolismo , Regulação para Baixo , Regulação da Expressão Gênica no Desenvolvimento , Canais Disparados por Nucleotídeos Cíclicos Ativados por Hiperpolarização/metabolismo , Camundongos , RNA Longo não Codificante/metabolismo , Nó Sinoatrial/citologiaRESUMO
Background: The mechanisms underlying dysfunction in the sinoatrial node (SAN), the heart's primary pacemaker, are incompletely understood. Electrical and Ca2+-handling remodeling have been implicated in SAN dysfunction associated with heart failure, aging, and diabetes. Cardiomyocyte [Na+]i is also elevated in these diseases, where it contributes to arrhythmogenesis. Here, we sought to investigate the largely unexplored role of Na+ homeostasis in SAN pacemaking and test whether [Na+]i dysregulation may contribute to SAN dysfunction. Methods: We developed a dataset-specific computational model of the murine SAN myocyte and simulated alterations in the major processes of Na+ entry (Na+/Ca2+ exchanger, NCX) and removal (Na+/K+ ATPase, NKA). Results: We found that changes in intracellular Na+ homeostatic processes dynamically regulate SAN electrophysiology. Mild reductions in NKA and NCX function increase myocyte firing rate, whereas a stronger reduction causes bursting activity and loss of automaticity. These pathologic phenotypes mimic those observed experimentally in NCX- and ankyrin-B-deficient mice due to altered feedback between the Ca2+ and membrane potential clocks underlying SAN firing. Conclusions: Our study generates new testable predictions and insight linking Na+ homeostasis to Ca2+ handling and membrane potential dynamics in SAN myocytes that may advance our understanding of SAN (dys)function.
Assuntos
Potenciais de Ação , Simulação por Computador , Modelos Cardiovasculares , Miócitos Cardíacos/metabolismo , Nó Sinoatrial/metabolismo , Sódio/metabolismo , Animais , Camundongos , Trocador de Sódio e Cálcio/metabolismo , ATPase Trocadora de Sódio-Potássio/metabolismoRESUMO
BACKGROUND: The heart rate progressively increases throughout pregnancy, reaching a maximum in the third trimester. This elevated heart rate is also present in pregnant mice and is associated with accelerated automaticity, higher density of the pacemaker current If and changes in Ca2+ homeostasis in sinoatrial node (SAN) cells. Strong evidence has also been provided showing that 17ß-estradiol (E2) and estrogen receptor α (ERα) regulate heart rate. Accordingly, we sought to determine whether E2 levels found in late pregnancy cause the increased cardiac automaticity associated with pregnancy. METHODS AND RESULTS: Voltage- and current-clamp experiments were carried out on SAN cells isolated from female mice lacking estrogen receptor alpha (ERKOα) or beta (ERKOß) receiving chronic E2 treatment mimicking late pregnancy concentrations. E2 treatment significantly increased the action potential rate (284 ± 24 bpm, +E2 354 ± 23 bpm, p = 0.040) and the density of If (+52%) in SAN cells from ERKOß mice. However, If density remains unchanged in SAN cells from E2-treated ERKOα mice. Additionally, E2 also increased If density (+67%) in nodal-like human-induced pluripotent stem cell-derived cardiomyocytes (N-hiPSC-CM), recapitulating in a human SAN cell model the effect produced in mice. However, the L-type calcium current (ICaL) and Ca2+ transients, examined using N-hiPSC-CM and SAN cells respectively, were not affected by E2, indicating that other mechanisms contribute to changes observed in these parameters during pregnancy. CONCLUSION: The accelerated SAN automaticity observed in E2-treated ERKOß mice is explained by an increased If density mediated by ERα, demonstrating that E2 plays a major role in regulating SAN function during pregnancy.
Assuntos
Estrogênios/farmacologia , Coração/fisiologia , Potenciais de Ação/efeitos dos fármacos , Animais , Cafeína/farmacologia , Canais de Cálcio Tipo L/metabolismo , Estradiol/farmacologia , Receptor alfa de Estrogênio/metabolismo , Receptor beta de Estrogênio/metabolismo , Feminino , Coração/efeitos dos fármacos , Células-Tronco Pluripotentes Induzidas/efeitos dos fármacos , Células-Tronco Pluripotentes Induzidas/metabolismo , Camundongos Endogâmicos C57BL , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/metabolismo , Gravidez , Nó Sinoatrial/efeitos dos fármacos , Nó Sinoatrial/metabolismoRESUMO
How G protein-coupled receptors (GPCRs) evoke specific biological outcomes while utilizing a limited array of G proteins and effectors is poorly understood, particularly in native cell systems. Here, we examined signaling evoked by muscarinic (M2R) and adenosine (A1R) receptor activation in the mouse sinoatrial node (SAN), the cardiac pacemaker. M2R and A1R activate a shared pool of cardiac G protein-gated inwardly rectifying K+ (GIRK) channels in SAN cells from adult mice, but A1R-GIRK responses are smaller and slower than M2R-GIRK responses. Recordings from mice lacking Regulator of G protein Signaling 6 (RGS6) revealed that RGS6 exerts a GPCR-dependent influence on GIRK-dependent signaling in SAN cells, suppressing M2R-GIRK coupling efficiency and kinetics and A1R-GIRK signaling amplitude. Fast kinetic bioluminescence resonance energy transfer assays in transfected HEK cells showed that RGS6 prefers Gαo over Gαi as a substrate for its catalytic activity and that M2R signals preferentially via Gαo, while A1R does not discriminate between inhibitory G protein isoforms. The impact of atrial/SAN-selective ablation of Gαo or Gαi2 was consistent with these findings. Gαi2 ablation had minimal impact on M2R-GIRK and A1R-GIRK signaling in SAN cells. In contrast, Gαo ablation decreased the amplitude and slowed the kinetics of M2R-GIRK responses, while enhancing the sensitivity and prolonging the deactivation rate of A1R-GIRK signaling. Collectively, our data show that differences in GPCR-G protein coupling preferences, and the Gαo substrate preference of RGS6, shape A1R- and M2R-GIRK signaling dynamics in mouse SAN cells.
Assuntos
Canais de Potássio Corretores do Fluxo de Internalização Acoplados a Proteínas G/metabolismo , Proteínas RGS/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Nó Sinoatrial/metabolismo , Potenciais de Ação/fisiologia , Animais , Canais de Potássio Corretores do Fluxo de Internalização Acoplados a Proteínas G/genética , Células HEK293 , Frequência Cardíaca/fisiologia , Humanos , Preparação de Coração Isolado , Camundongos , Camundongos Knockout , Cultura Primária de Células , Proteínas RGS/genética , Receptor A1 de Adenosina/metabolismo , Receptor Muscarínico M2/metabolismo , Transdução de Sinais/fisiologia , Nó Sinoatrial/citologiaRESUMO
RATIONALE: The stress response of heart rate, which is determined by the plasticity of the sinoatrial node (SAN), is essential for cardiac function and survival in mammals. As an RNA-binding protein, CIRP (cold-inducible RNA-binding protein) can act as a stress regulator. Previously, we have documented that CIRP regulates cardiac electrophysiology at posttranscriptional level, suggesting its role in SAN plasticity, especially upon stress conditions. OBJECTIVE: Our aim was to clarify the role of CIRP in SAN plasticity and heart rate regulation under stress conditions. METHODS AND RESULTS: Telemetric ECG monitoring demonstrated an excessive acceleration of heart rate under isoprenaline stimulation in conscious CIRP-KO (knockout) rats. Patch-clamp analysis and confocal microscopic Ca2+ imaging of isolated SAN cells demonstrated that isoprenaline stimulation induced a faster spontaneous firing rate in CIRP-KO SAN cells than that in WT (wild type) SAN cells. A higher concentration of cAMP-the key mediator of pacemaker activity-was detected in CIRP-KO SAN tissues than in WT SAN tissues. RNA sequencing and quantitative real-time polymerase chain reaction analyses of single cells revealed that the 4B and 4D subtypes of PDE (phosphodiesterase), which controls cAMP degradation, were significantly decreased in CIRP-KO SAN cells. A PDE4 inhibitor (rolipram) abolished the difference in beating rate resulting from CIRP deficiency. The mechanistic study showed that CIRP stabilized the mRNA of Pde4b and Pde4d by direct mRNA binding, thereby regulating the protein expression of PDE4B and PDE4D at posttranscriptional level. CONCLUSIONS: CIRP acts as an mRNA stabilizer of specific PDEs to control the cAMP concentration in SAN, maintaining the appropriate heart rate stress response.
Assuntos
Proteínas e Peptídeos de Choque Frio/metabolismo , Nucleotídeo Cíclico Fosfodiesterase do Tipo 4/metabolismo , Frequência Cardíaca , Miócitos Cardíacos/metabolismo , Proteínas de Ligação a RNA/metabolismo , Agonistas Adrenérgicos beta/farmacologia , Animais , Células Cultivadas , Proteínas e Peptídeos de Choque Frio/genética , AMP Cíclico/metabolismo , Nucleotídeo Cíclico Fosfodiesterase do Tipo 4/genética , Isoproterenol/farmacologia , Masculino , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/fisiologia , Inibidores de Fosfodiesterase/farmacologia , Estabilidade de RNA , Proteínas de Ligação a RNA/genética , Ratos , Ratos Sprague-Dawley , Rolipram/farmacologia , Nó Sinoatrial/citologia , Nó Sinoatrial/metabolismo , Nó Sinoatrial/fisiologia , Estresse FisiológicoRESUMO
AIMS: Sinus venous valve (SVV) and sinoatrial node (SAN) develop together at the sinoatrial junction during embryogenesis. SVV ensures unidirectional cardiac input and SAN generates sinus rhythmic contraction, respectively; both functions are essential for embryonic survival. We aim to reveal the potential role of endocardial NOTCH signalling in SVV and SAN formation. METHODS AND RESULTS: We specifically deleted Notch1 in the endocardium using an Nfatc1Cre line. This deletion resulted in underdeveloped SVV and SAN, associated with reduced expression of T-box transcription factors, Tbx5 andTbx18, which are essential for the formation of SVV and SAN. The deletion also led to decreased expression of Wnt2 in myocardium of SVV and SAN. WNT2 treatment was able to rescue the growth defect of SVV and SAN resulted from the Notch1 deletion in whole embryo cultures. Furthermore, the Notch1 deletion reduced the expression of Nrg1 in the SVV myocardium and supplement of NRG1 restored the growth of SVV in cultured Notch1 knockout embryos. CONCLUSION: Our findings support that endocardial NOTCH1 controls the development of SVV and SAN by coordinating myocardial WNT and NRG1 signalling functions.
Assuntos
Seio Coronário/metabolismo , Miocárdio/metabolismo , Receptor Notch1/metabolismo , Nó Sinoatrial/metabolismo , Válvulas Venosas/metabolismo , Animais , Seio Coronário/embriologia , Regulação da Expressão Gênica no Desenvolvimento , Camundongos Knockout , Morfogênese , Neuregulina-1/genética , Neuregulina-1/metabolismo , Receptor Notch1/deficiência , Receptor Notch1/genética , Nó Sinoatrial/embriologia , Proteínas com Domínio T/genética , Proteínas com Domínio T/metabolismo , Válvulas Venosas/embriologia , Via de Sinalização Wnt , Proteína Wnt2/genética , Proteína Wnt2/metabolismoRESUMO
Heart rate in physiological conditions is set by the sinoatrial node (SN), the primary cardiac pacing tissue. Phosphoinositide 3-kinase (PI3K) signaling is a major regulatory pathway in all normal cells, and its dysregulation is prominent in diabetes, cancer, and heart failure. Here, we show that inhibition of PI3K slows the pacing rate of the SN in situ and in vitro and reduces the early slope of diastolic depolarization. Furthermore, inhibition of PI3K causes a negative shift in the voltage dependence of activation of the pacemaker current, I F, while addition of its second messenger, phosphatidylinositol 3,4,5-trisphosphate, induces a positive shift. These shifts in the activation of I F are independent of, and larger than, those induced by the autonomic nervous system. These results suggest that PI3K is an important regulator of heart rate, and perturbations in this signaling pathway may contribute to the development of arrhythmias.
Assuntos
Frequência Cardíaca , Fosfatidilinositol 3-Quinases/metabolismo , Sistemas do Segundo Mensageiro , Nó Sinoatrial/fisiologia , Potenciais de Ação , Animais , Relógios Biológicos , Células Cultivadas , Cães , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Fosfatos de Fosfatidilinositol/metabolismo , Coelhos , Nó Sinoatrial/metabolismoRESUMO
The sinus node is a collection of highly specialised cells constituting the heart's pacemaker. The molecular underpinnings of its pacemaking abilities are debated. Using high-resolution mass spectrometry, we here quantify >7,000 proteins from sinus node and neighbouring atrial muscle. Abundances of 575 proteins differ between the two tissues. By performing single-nucleus RNA sequencing of sinus node biopsies, we attribute measured protein abundances to specific cell types. The data reveal significant differences in ion channels responsible for the membrane clock, but not in Ca2+ clock proteins, suggesting that the membrane clock underpins pacemaking. Consistently, incorporation of ion channel expression differences into a biophysically-detailed atrial action potential model result in pacemaking and a sinus node-like action potential. Combining our quantitative proteomics data with computational modeling, we estimate ion channel copy numbers for sinus node myocytes. Our findings provide detailed insights into the unique molecular make-up of the cardiac pacemaker.
Assuntos
Relógios Biológicos/fisiologia , Peptídeos/química , Peptídeos/metabolismo , Proteômica , Nó Sinoatrial/metabolismo , Transcriptoma , Potenciais de Ação , Animais , Cromatografia Líquida , Regulação da Expressão Gênica/fisiologia , Concentração de Íons de Hidrogênio , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Peptídeos/genética , Espectrometria de Massas em TandemRESUMO
Conversion of intracellular Ca2+ signals to electrical activity results in multiple and differing physiological impacts depending on cell types. In some organs such as gastrointestinal and urinary systems, spontaneous Ca2+ oscillation in pacermaker cells can function essentially as a Ca2+ clock mechanism, which has been originally found in pacemaking in sinoatrial node cell of the heart. The conversion of discrete Ca2+ clock events to spontaneous electrical activity is an essential step for the initiation and propagation of pacemaker activity through the multicellular organs resulting in synchronized physiological functions. Here, a model of intracellular signal transduction from a Ca2+ oscillation to initiation of electrical slow waves and their propagation were reconstituted in HEK293â¯cells. This was accomplished based on ryanodine receptor (RyR) type 3, Ca2+-activated ion channels, i.e. small conductance Ca2+-activated K+ channel (SK2) or Ca2+-activated Cl- channel (TMEM16A), and connexin43 being heterologously co-expressed. The propagation of electrical waves was abolished or substantially reduced by treatment with selective blockers of the expressed channels and 18ß-glycyrrhetinic acid, a gap junction inhibitor, respectively. Thus, we demonstrated that the conversion of Ca2+ oscillation to electrical signals with cell to cell propagation can be reconstituted as a model of Ca2+ clock pacemaker activity by combinational expression of critical elements in heterologous expression system.
Assuntos
Sinalização do Cálcio , Cálcio/metabolismo , Conexina 43/metabolismo , Células Intersticiais de Cajal/metabolismo , Trocador de Sódio e Cálcio/metabolismo , Potenciais de Ação , Animais , Anoctamina-1/metabolismo , Relógios Biológicos , Células HEK293 , Humanos , Íons/metabolismo , Masculino , Potenciais da Membrana , Camundongos , Camundongos Endogâmicos BALB C , Proteínas de Neoplasias/metabolismo , Oscilometria , Canal de Liberação de Cálcio do Receptor de Rianodina/metabolismo , Nó Sinoatrial/metabolismoRESUMO
Store operated Ca2+ entry (SOCE) is an ancient and ubiquitous Ca2+ signaling pathway discovered decades ago, but the function of SOCE in human physiology is only now being revealed. The relevance of this pathway to striated muscle was solidified with the description of skeletal myopathies that result from mutations in STIM1 and Orai1, the two SOCE components. Here, we consider the evidence for STIM1 and SOCE in cardiac muscle and the sinoatrial node. We highlight recent studies revealing a role for STIM1 in cardiac growth in response to developmental and pathologic cues. We also review the role of STIM1 in the regulation of SOCE and Ca2+ store refilling in a non-Orai dependent manner. Finally, we discuss the importance of this pathway in ventricular cardiomyocytes where SOCE contribute to developmental growth and in pacemaker cells where SOCE likely has a fundamental to generating the cardiac rhythm.
Assuntos
Sinalização do Cálcio/fisiologia , Cálcio/metabolismo , Miocárdio/metabolismo , Proteínas de Neoplasias/metabolismo , Nó Sinoatrial/metabolismo , Molécula 1 de Interação Estromal/metabolismo , Animais , Humanos , Miócitos Cardíacos/metabolismo , Proteína ORAI1/metabolismoRESUMO
Aims: Diabetes, characterized by hyperglycaemia, causes sinus node dysfunction (SND) in several rodent models. Interleukin (IL)-10, which is a potent anti-inflammatory cytokine, has been reported to decrease in obese and diabetic patients. We tested the hypothesis that administration of IL-10 inhibits the development of SND caused by hyperglycaemia in streptozotocin (STZ)-induced diabetic mice. Methods and results: Six-week old CL57/B6 (WT) mice were divided into the following groups: control, STZ injection, and STZ injection with systemic administration of IL-10. IL-10 knockout mice were similarly treated. STZ-induced hyperglycaemia for 8 weeks significantly depressed serum levels of IL-10, but increased several proinflammatory cytokines in WT mice. STZ-induced hyperglycaemia-reduced resting heart rate (HR), and attenuated HR response to isoproterenol in WT mice. In isolated perfused heart experiments, corrected-sinus node recovery time was prolonged in WT mice with STZ injection. Sinus node tissue isolated from the WT-STZ group showed fibrosis, abundant infiltration of macrophages, increased production of reactive oxygen species (ROS), and depressed hyperpolarization activated cyclic nucleotide-gated potassium channel 4 (HCN4). However, the changes observed in the WT-STZ group were significantly attenuated by IL-10 administration and were further exaggerated in IL-10 knockout mice. In cultured cells, preincubation of IL-10 suppressed hyperglycaemia-induced apoptotic and profibrotic signals, and overproduction of ROS. IL-10 markedly inhibited the high glucose-induced p38 activation, and activated signal transducer and activator of transcription (STAT) 3 phosphorylation. Conclusions: Our results suggest that IL-10 attenuates ROS production, inflammation and fibrosis, and plays an important role in the inhibition of hyperglycaemia-induced SND by suppression of HCN4 downregulation. In addition, IL-10-mediated inhibition of p38 is dependent on STAT3 phosphorylation.
Assuntos
Antiarrítmicos/farmacologia , Glicemia/metabolismo , Diabetes Mellitus Experimental/tratamento farmacológico , Frequência Cardíaca/efeitos dos fármacos , Interleucina-10/farmacologia , Síndrome do Nó Sinusal/prevenção & controle , Nó Sinoatrial/efeitos dos fármacos , Animais , Apoptose/efeitos dos fármacos , Biomarcadores/sangue , Células Cultivadas , Diabetes Mellitus Experimental/sangue , Diabetes Mellitus Experimental/induzido quimicamente , Diabetes Mellitus Experimental/fisiopatologia , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Fibroblastos/patologia , Fibrose , Canais Disparados por Nucleotídeos Cíclicos Ativados por Hiperpolarização/metabolismo , Interleucina-10/sangue , Interleucina-10/genética , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Macrófagos/patologia , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/metabolismo , Miócitos Cardíacos/patologia , Fosforilação , Ratos Sprague-Dawley , Espécies Reativas de Oxigênio/metabolismo , Fator de Transcrição STAT3/metabolismo , Síndrome do Nó Sinusal/sangue , Síndrome do Nó Sinusal/induzido quimicamente , Síndrome do Nó Sinusal/fisiopatologia , Nó Sinoatrial/metabolismo , Nó Sinoatrial/patologia , Nó Sinoatrial/fisiopatologia , Estreptozocina , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
OBJECTIVE: Sick sinus syndrome (SSS) is one of the most common causes of cardiac impairment necessitating pacemaker implantation. However, studies of SSS pathogenesis are neither comprehensive nor conclusive due to limited success in achieving a stable rat SSS model. Here, we modified pinpoint press permeation to establish a stable rat SSS model. METHODS: We randomly assigned 138 male Sprague-Dawley rats into three groups: normal control (n = 8), sham (n = 10), and SSS (n = 120). Postoperatively, the SSS group was further divided into SSSA (n = 40), SSSB (n = 40), and SSSC (n = 40), based on reduction in heart rates by 20-30%, 31-40%, and 41-50%, respectively. We also assessed histomorphological characteristics and hyperpolarization-activated cyclic nucleotide-gated cation channel 4 (HCN4) expression in the sinoatrial node (SAN) at 1, 2, 3, and 4 weeks after surgery. RESULTS: Mortality was statistically higher in SSSC compared to SSSA and SSSB (7.5% versus 90.0% and 87.5%; P < 0.05). Heart rate in SSSA was gradually restored to preoperative levels by week 4 after surgery. In contrast, heart rate in SSSB was stable at 2-3 weeks after surgery. However, we observed that the tissues and cells in SAN were severely injured and also found a time-dependent increase in collagen content and atrium myocardium in SSSB. HCN4 expression was significantly reduced at all 4 time points in SSSB, with statistically significant differences among the groups (P < 0.01). CONCLUSION: We successfully developed a rat SSS model that was sustainable for up to 4 weeks.
Assuntos
Síndrome do Nó Sinusal/fisiopatologia , Nó Sinoatrial/fisiopatologia , Animais , Modelos Animais de Doenças , Átrios do Coração/metabolismo , Átrios do Coração/fisiopatologia , Frequência Cardíaca/fisiologia , Canais Disparados por Nucleotídeos Cíclicos Ativados por Hiperpolarização/metabolismo , Masculino , Ratos , Ratos Sprague-Dawley , Síndrome do Nó Sinusal/metabolismo , Nó Sinoatrial/metabolismoRESUMO
Binding of TRIP8b to the cyclic nucleotide binding domain (CNBD) of mammalian hyperpolarization-activated cyclic nucleotide-gated (HCN) channels prevents their regulation by cAMP. Since TRIP8b is expressed exclusively in the brain, we envisage that it can be used for orthogonal control of HCN channels beyond the central nervous system. To this end, we have identified by rational design a 40-aa long peptide (TRIP8bnano) that recapitulates affinity and gating effects of TRIP8b in HCN isoforms (hHCN1, mHCN2, rbHCN4) and in the cardiac current If in rabbit and mouse sinoatrial node cardiomyocytes. Guided by an NMR-derived structural model that identifies the key molecular interactions between TRIP8bnano and the HCN CNBD, we further designed a cell-penetrating peptide (TAT-TRIP8bnano) which successfully prevented ß-adrenergic activation of mouse If leaving the stimulation of the L-type calcium current (ICaL) unaffected. TRIP8bnano represents a novel approach to selectively control HCN activation, which yields the promise of a more targeted pharmacology compared to pore blockers.
Assuntos
AMP Cíclico/química , Canais Disparados por Nucleotídeos Cíclicos Ativados por Hiperpolarização/química , Miócitos Cardíacos/efeitos dos fármacos , Peptídeos/farmacologia , Canais de Potássio/química , Animais , Sítios de Ligação , Canais de Cálcio Tipo L/química , Canais de Cálcio Tipo L/genética , Canais de Cálcio Tipo L/metabolismo , Peptídeos Penetradores de Células/química , Peptídeos Penetradores de Células/genética , Peptídeos Penetradores de Células/metabolismo , AMP Cíclico/metabolismo , Expressão Gênica , Células HEK293 , Humanos , Canais Disparados por Nucleotídeos Cíclicos Ativados por Hiperpolarização/genética , Canais Disparados por Nucleotídeos Cíclicos Ativados por Hiperpolarização/metabolismo , Proteínas de Membrana/química , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Simulação de Acoplamento Molecular , Miócitos Cardíacos/citologia , Miócitos Cardíacos/metabolismo , Técnicas de Patch-Clamp , Peptídeos/síntese química , Peroxinas/química , Peroxinas/genética , Peroxinas/metabolismo , Canais de Potássio/genética , Canais de Potássio/metabolismo , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Coelhos , Nó Sinoatrial/citologia , Nó Sinoatrial/efeitos dos fármacos , Nó Sinoatrial/metabolismo , Produtos do Gene tat do Vírus da Imunodeficiência HumanaRESUMO
Aims: During pregnancy, there is a significant increase in heart rate (HR) potentially associated with an increased risk of arrhythmias or exacerbation of pre-existing cardiac conditions endangering both mother and foetus. Calcium homeostasis plays an important role in regulating automaticity of the sinoatrial node (SAN); however, its contribution to the accelerated HR during pregnancy remains unknown. Methods and results: Using murine SAN cells, we showed that pregnancy increased L-type Ca2+ current (ICaL) and CaV1.3 mRNA expression, whereas T-type Ca2+ current (ICaT) and its underlying channel were unchanged. Analysis of SAN intra-cellular Ca2+ oscillations showed that the rate of spontaneous Ca2+ transients was significantly higher in pregnant mice along with a higher mRNA expression of ryanodine receptor. Assessment of supra-ventricular arrhythmias using programmed electrical stimulation protocols on anaesthetized mice revealed higher susceptibility in pregnancy. Of note, the modifications associated with pregnancy were reversible following delivery. Furthermore, chronic administration of 17ß-estradiol (E2) to nodal-like human-induced pluripotent stem cell-derived cardiomyocytes (N-hiPSC-CM), control mice, oestrogen-receptor-ß knockout (ERKOß) but not ERKOα mice, accelerated cardiac automaticity, recapitulating the pregnancy phenotype in both mouse and human SAN cell models. Conclusion: Together, these results indicate that pregnancy considerably alters intra-cellular Ca2+ homeostasis sustaining faster HR during pregnancy. Importantly, these changes were dependent on an oestrogen receptor α (ERα) mechanism that resulted in increased ICaL and spontaneous Ca2+ release from the sarcoplasmic reticulum, highlighting a novel role for oestrogen in regulating HR.