RESUMO
Atrial fibrillation (AF) is the most common sustained cardiac arrhythmia. Excessive stimulation of the IP3 signalling pathway has been linked to AF through abnormal calcium handling. However, little is known about the mechanisms involved in this process. We expressed the fluorescence resonance energy transfer (FRET) based cytosolic cAMP sensor EPAC-SH187 in neonatal rat atrial myocytes (NRAMs) and neonatal rat ventricular myocytes (NRVMs). In NRAMs, addition of the α-1 agonist phenylephrine (PE, 3 µM) resulted in a FRET change 21.20 ± 7.43 % and addition of membrane permeant IP3 derivative, 2,3,6-tri-O-Butyryl-myo-IP3(1,4,5)-hexakis(acetoxymethyl)ester (IP3-AM, 20 µM) resulted in a peak of 20.31 ± 6.74 %. These FRET changes imply an increase in cAMP. Prior application of IP3 receptor (IP3R) inhibitors 2-Aminoethyl diphenylborinate (2-APB, 2.5µM) or Xestospongin-C (0.3 µM) significantly inhibited the change in FRET in NRAMs in response to PE. Xestospongin-C (0.3 µM) significantly inhibited the change in FRET in NRAMs in response to IP3-AM. The FRET change in response to PE in NRVMs were not inhibited by 2-APB or Xestospongin-C. Finally, the localisation of cAMP signals was tested by expressing the FRET-based cAMP sensor, AKAP79-CUTie, which targets the intracellular surface of the plasmalemma. We found in NRAMs that PE led to FRET change corresponding to an increase in cAMP that was inhibited by 2-APB and Xestospongin C. These data support further investigation of the pro-arrhythmic nature and components of IP3 induced cAMP signalling to identify potential pharmacological targets.
RESUMO
Inositol trisphosphate (IP3) is a Ca2+-mobilizing second messenger shown to modulate atrial muscle contraction and is thought to contribute to atrial fibrillation. Cellular pathways underlying IP3 actions in cardiac tissue remain poorly understood, and the work presented here addresses the question whether IP3-mediated Ca2+ release from the sarcoplasmic reticulum is linked to adenylyl cyclase activity including Ca2+-stimulated adenylyl cyclases (AC1 and AC8) that are selectively expressed in atria and sinoatrial node (SAN). Immunocytochemistry in guinea pig atrial myocytes identified colocalization of type 2 IP3 receptors with AC8, while AC1 was located in close vicinity. Intracellular photorelease of IP3 by UV light significantly enhanced the amplitude of the Ca2+ transient (CaT) evoked by electrical stimulation of atrial myocytes (31 ± 6% increase 60 s after photorelease, n = 16). The increase in CaT amplitude was abolished by inhibitors of adenylyl cyclases (MDL-12,330) or protein kinase A (H89), showing that cAMP signaling is required for this effect of photoreleased IP3. In mouse, spontaneously beating right atrial preparations, phenylephrine, an α-adrenoceptor agonist with effects that depend on IP3-mediated Ca2+ release, increased the maximum beating rate by 14.7 ± 0.5%, n = 10. This effect was substantially reduced by 2.5 µmol/L 2-aminoethyl diphenylborinate and abolished by a low dose of MDL-12,330, observations which are again consistent with a functional interaction between IP3 and cAMP signaling involving Ca2+ stimulation of adenylyl cyclases in the SAN pacemaker. Understanding the interaction between IP3 receptor pathways and Ca2+-stimulated adenylyl cyclases provides important insights concerning acute mechanisms for initiation of atrial arrhythmias.NEW & NOTEWORTHY This study provides evidence supporting the proposal that IP3 signaling in cardiac atria and sinoatrial node involves stimulation of Ca2+-activated adenylyl cyclases (AC1 and AC8) by IP3-evoked Ca2+ release from junctional sarcoplasmic reticulum. AC8 and IP3 receptors are shown to be located close together, while AC1 is nearby. Greater understanding of these novel aspects of the IP3 signal transduction mechanism is important for future study in atrial physiology and pathophysiology, particularly atrial fibrillation.
Assuntos
Adenilil Ciclases/metabolismo , Relógios Biológicos , Sinalização do Cálcio , Átrios do Coração/enzimologia , Frequência Cardíaca , Receptores de Inositol 1,4,5-Trifosfato/metabolismo , Inositol 1,4,5-Trifosfato/metabolismo , Miócitos Cardíacos/enzimologia , Nó Sinoatrial/enzimologia , Potenciais de Ação , Animais , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Cobaias , Átrios do Coração/citologia , Isoenzimas , Masculino , Camundongos , Retículo Sarcoplasmático/enzimologia , Fatores de TempoRESUMO
The aim of this chapter is to discuss evidence concerning the many roles of calcium ions, Ca2+, in cell signaling pathways that control heart function. Before considering details of these signaling pathways, the control of contraction in ventricular muscle by Ca2+ transients accompanying cardiac action potentials is first summarized, together with a discussion of how myocytes from the atrial and pacemaker regions of the heart diverge from this basic scheme. Cell signaling pathways regulate the size and timing of the Ca2+ transients in the different heart regions to influence function. The simplest Ca2+ signaling elements involve enzymes that are regulated by cytosolic Ca2+. Particularly important examples to be discussed are those that are stimulated by Ca2+, including Ca2+-calmodulin-dependent kinase (CaMKII), Ca2+ stimulated adenylyl cyclases, Ca2+ stimulated phosphatase and NO synthases. Another major aspect of Ca2+ signaling in the heart concerns actions of the Ca2+ mobilizing agents, inositol trisphosphate (IP3), cADP-ribose (cADPR) and nicotinic acid adenine dinucleotide phosphate, (NAADP). Evidence concerning roles of these Ca2+ mobilizing agents in different regions of the heart is discussed in detail. The focus of the review will be on short term regulation of Ca2+ transients and contractile function, although it is recognized that Ca2+ regulation of gene expression has important long term functional consequences which will also be briefly discussed.
Assuntos
Sinalização do Cálcio , Coração , Animais , Cálcio/metabolismo , ADP-Ribose Cíclica , Coração/fisiologia , Humanos , Contração Muscular/fisiologia , NADPRESUMO
BACKGROUND: Among his major cardiac electrophysiological contributions, Miles Vaughan Williams (1918-2016) provided a classification of antiarrhythmic drugs that remains central to their clinical use. METHODS: We survey implications of subsequent discoveries concerning sarcolemmal, sarcoplasmic reticular, and cytosolic biomolecules, developing an expanded but pragmatic classification that encompasses approved and potential antiarrhythmic drugs on this centenary of his birth. RESULTS: We first consider the range of pharmacological targets, tracking these through to cellular electrophysiological effects. We retain the original Vaughan Williams Classes I through IV but subcategorize these divisions in light of more recent developments, including the existence of Na+ current components (for Class I), advances in autonomic (often G protein-mediated) signaling (for Class II), K+ channel subspecies (for Class III), and novel molecular targets related to Ca2+ homeostasis (for Class IV). We introduce new classes based on additional targets, including channels involved in automaticity, mechanically sensitive ion channels, connexins controlling electrotonic cell coupling, and molecules underlying longer-term signaling processes affecting structural remodeling. Inclusion of this widened range of targets and their physiological sequelae provides a framework for a modernized classification of established antiarrhythmic drugs based on their pharmacological targets. The revised classification allows for the existence of multiple drug targets/actions and for adverse, sometimes actually proarrhythmic, effects. The new scheme also aids classification of novel drugs under investigation. CONCLUSIONS: We emerge with a modernized classification preserving the simplicity of the original Vaughan Williams framework while aiding our understanding and clinical management of cardiac arrhythmic events and facilitating future developments in this area.
Assuntos
Antiarrítmicos/classificação , Antiarrítmicos/uso terapêutico , Arritmias Cardíacas/tratamento farmacológico , Sistema de Condução Cardíaco/efeitos dos fármacos , Frequência Cardíaca/efeitos dos fármacos , Moduladores de Transporte de Membrana/classificação , Moduladores de Transporte de Membrana/uso terapêutico , Terminologia como Assunto , Potenciais de Ação/efeitos dos fármacos , Animais , Antiarrítmicos/efeitos adversos , Arritmias Cardíacas/diagnóstico , Arritmias Cardíacas/metabolismo , Arritmias Cardíacas/fisiopatologia , Bloqueadores dos Canais de Cálcio/classificação , Bloqueadores dos Canais de Cálcio/uso terapêutico , Sistema de Condução Cardíaco/metabolismo , Sistema de Condução Cardíaco/fisiopatologia , Humanos , Canais Iônicos/efeitos dos fármacos , Canais Iônicos/metabolismo , Moduladores de Transporte de Membrana/efeitos adversos , Neurotransmissores/classificação , Neurotransmissores/uso terapêutico , Bloqueadores dos Canais de Potássio/classificação , Bloqueadores dos Canais de Potássio/uso terapêutico , Bloqueadores do Canal de Sódio Disparado por Voltagem/classificação , Bloqueadores do Canal de Sódio Disparado por Voltagem/uso terapêuticoRESUMO
KEY POINTS: A robust cardiac slicing approach was developed for optical mapping of transmural gradients in transmembrane potential (Vm ) and intracellular Ca2+ transient (CaT) of murine heart. Significant transmural gradients in Vm and CaT were observed in the left ventricle. Frequency-dependent action potentials and CaT alternans were observed in all ventricular regions with rapid pacing, with significantly greater incidence in the endocardium than epicardium. The observations demonstrate the feasibility of our new approach to cardiac slicing for systematic analysis of intrinsic transmural and regional gradients in Vm and CaT. ABSTRACT: Transmural and regional gradients in membrane potential and Ca2+ transient in the murine heart are largely unexplored. Here, we developed and validated a robust approach which combines transverse ultra-thin cardiac slices and high resolution optical mapping to enable systematic analysis of transmural and regional gradients in transmembrane potential (Vm ) and intracellular Ca2+ transient (CaT) across the entire murine ventricles. The voltage dye RH237 or Ca2+ dye Rhod-2 AM were loaded through the coronary circulation using a Langendorff perfusion system. Short-axis slices (300 µm thick) were prepared from the entire ventricles (from the apex to the base) by using a high-precision vibratome. Action potentials (APs) and CaTs were recorded with optical mapping during steady-state baseline and rapid pacing. Significant transmural gradients in Vm and CaT were observed in the left ventricle, with longer AP duration (APD50 and APD75 ) and CaT duration (CaTD50 and CaTD75 ) in the endocardium compared with that in the epicardium. No significant regional gradients were observed along the apico-basal axis of the left ventricle. Interventricular gradients were detected with significantly shorter APD50 , APD75 and CaTD50 in the right ventricle compared with left ventricle and ventricular septum. During rapid pacing, AP and CaT alternans were observed in most ventricular regions, with significantly greater incidence in the endocardium in comparison with epicardium. In conclusion, these observations demonstrate the feasibility of our new approach to cardiac slicing for systematic analysis of intrinsic transmural and regional gradients in Vm and CaT in murine ventricular tissue.
Assuntos
Sinalização do Cálcio , Endocárdio/metabolismo , Ventrículos do Coração/metabolismo , Coração/fisiologia , Potenciais da Membrana , Imagem Óptica/métodos , Pericárdio/metabolismo , Animais , Endocárdio/diagnóstico por imagem , Ventrículos do Coração/diagnóstico por imagem , Camundongos , Pericárdio/diagnóstico por imagemRESUMO
Nicotinic acid adenine dinucleotide phosphate (NAADP) and cyclic ADP-ribose (cADPR) are Ca2+-mobilizing messengers important for modulating cardiac excitation-contraction coupling and pathophysiology. CD38, which belongs to the ADP-ribosyl cyclase family, catalyzes synthesis of both NAADP and cADPR in vitro However, it remains unclear whether this is the main enzyme for their production under physiological conditions. Here we show that membrane fractions from WT but not CD38-/- mouse hearts supported NAADP and cADPR synthesis. Membrane permeabilization of cardiac myocytes with saponin and/or Triton X-100 increased NAADP synthesis, indicating that intracellular CD38 contributes to NAADP production. The permeabilization also permitted immunostaining of CD38, with a striated pattern in WT myocytes, whereas CD38-/- myocytes and nonpermeabilized WT myocytes showed little or no staining, without striation. A component of ß-adrenoreceptor signaling in the heart involves NAADP and lysosomes. Accordingly, in the presence of isoproterenol, Ca2+ transients and contraction amplitudes were smaller in CD38-/- myocytes than in the WT. In addition, suppressing lysosomal function with bafilomycin A1 reduced the isoproterenol-induced increase in Ca2+ transients in cardiac myocytes from WT but not CD38-/- mice. Whole hearts isolated from CD38-/- mice and exposed to isoproterenol showed reduced arrhythmias. SAN4825, an ADP-ribosyl cyclase inhibitor that reduces cADPR and NAADP synthesis in mouse membrane fractions, was shown to bind to CD38 in docking simulations and reduced the isoproterenol-induced arrhythmias in WT hearts. These observations support generation of NAADP and cADPR by intracellular CD38, which contributes to effects of ß-adrenoreceptor stimulation to increase both Ca2+ transients and the tendency to disturb heart rhythm.
Assuntos
ADP-Ribosil Ciclase 1/metabolismo , Sinalização do Cálcio , ADP-Ribose Cíclica/metabolismo , Glicoproteínas de Membrana/metabolismo , Miócitos Cardíacos/metabolismo , NADP/análogos & derivados , Retículo Sarcoplasmático/metabolismo , ADP-Ribosil Ciclase 1/antagonistas & inibidores , Agonistas Adrenérgicos beta/farmacologia , Animais , Antiarrítmicos/química , Antiarrítmicos/metabolismo , Antiarrítmicos/farmacologia , Sinalização do Cálcio/efeitos dos fármacos , Permeabilidade da Membrana Celular/efeitos dos fármacos , Células Cultivadas , Detergentes/farmacologia , Inibidores Enzimáticos/farmacologia , Coração/efeitos dos fármacos , Técnicas In Vitro , Masculino , Glicoproteínas de Membrana/antagonistas & inibidores , Camundongos Endogâmicos C57BL , Camundongos Knockout , Simulação de Acoplamento Molecular , Contração Miocárdica/efeitos dos fármacos , Miócitos Cardíacos/citologia , Miócitos Cardíacos/efeitos dos fármacos , NADP/metabolismo , Transporte Proteico/efeitos dos fármacos , Coelhos , Retículo Sarcoplasmático/efeitos dos fármacos , Retículo Sarcoplasmático/enzimologia , Análise de Célula ÚnicaRESUMO
Ca(2+)-permeable type 2 two-pore channels (TPC2) are lysosomal proteins required for nicotinic acid adenine dinucleotide phosphate (NAADP)-evoked Ca(2+) release in many diverse cell types. Here, we investigate the importance of TPC2 proteins for the physiology and pathophysiology of the heart. NAADP-AM failed to enhance Ca(2+) responses in cardiac myocytes from Tpcn2(-/-) mice, unlike myocytes from wild-type (WT) mice. Ca(2+)/calmodulin-dependent protein kinase II inhibitors suppressed actions of NAADP in myocytes. Ca(2+) transients and contractions accompanying action potentials were increased by isoproterenol in myocytes from WT mice, but these effects of ß-adrenoreceptor stimulation were reduced in myocytes from Tpcn2(-/-) mice. Increases in amplitude of L-type Ca(2+) currents evoked by isoproterenol remained unchanged in myocytes from Tpcn2(-/-) mice showing no loss of ß-adrenoceptors or coupling mechanisms. Whole hearts from Tpcn2(-/-) mice also showed reduced inotropic effects of isoproterenol and a reduced tendency for arrhythmias following acute ß-adrenoreceptor stimulation. Hearts from Tpcn2(-/-) mice chronically exposed to isoproterenol showed less cardiac hypertrophy and increased threshold for arrhythmogenesis compared with WT controls. Electron microscopy showed that lysosomes form close contacts with the sarcoplasmic reticulum (separation â¼ 25 nm). We propose that Ca(2+)-signaling nanodomains between lysosomes and sarcoplasmic reticulum dependent on NAADP and TPC2 comprise an important element in ß-adrenoreceptor signal transduction in cardiac myocytes. In summary, our observations define a role for NAADP and TPC2 at lysosomal/sarcoplasmic reticulum junctions as unexpected but major contributors in the acute actions of ß-adrenergic signaling in the heart and also in stress pathways linking chronic stimulation of ß-adrenoceptors to hypertrophy and associated arrhythmias.
Assuntos
Canais de Cálcio/fisiologia , Lisossomos/metabolismo , Miocárdio/metabolismo , NADP/análogos & derivados , Receptores Adrenérgicos beta/metabolismo , Retículo Sarcoplasmático/metabolismo , Transdução de Sinais , Animais , Canais de Cálcio/genética , Cobaias , Masculino , Camundongos , Camundongos Knockout , NADP/fisiologiaRESUMO
Rhythms of electrical activity in all regions of the heart can be influenced by a variety of intracellular membrane bound organelles. This is true both for normal pacemaker activity and for abnormal rhythms including those caused by early and delayed afterdepolarizations under pathological conditions. The influence of the sarcoplasmic reticulum (SR) on cardiac electrical activity is widely recognized, but other intracellular organelles including lysosomes and mitochondria also contribute. Intracellular organelles can provide a timing mechanism (such as an SR clock driven by cyclic uptake and release of Ca2+, with an important influence of intraluminal Ca2+), and/or can act as a Ca2+ store involved in signalling mechanisms. Ca2+ plays many diverse roles including carrying electric current, driving electrogenic sodium-calcium exchange (NCX) particularly when Ca2+ is extruded across the surface membrane causing depolarization, and activation of enzymes which target organelles and surface membrane proteins. Heart function is also influenced by Ca2+ mobilizing agents (cADP-ribose, nicotinic acid adenine dinucleotide phosphate and inositol trisphosphate) acting on intracellular organelles. Lysosomal Ca2+ release exerts its effects via calcium/calmodulin-dependent protein kinase II to promote SR Ca2+ uptake, and contributes to arrhythmias resulting from excessive beta-adrenoceptor stimulation. A separate arrhythmogenic mechanism involves lysosomes, mitochondria and SR. Interacting intracellular organelles, therefore, have profound effects on heart rhythms and NCX plays a central role. This article is part of the theme issue 'The heartbeat: its molecular basis and physiological mechanisms'.
Assuntos
Arritmias Cardíacas , Cálcio , Humanos , Cálcio/metabolismo , Arritmias Cardíacas/metabolismo , Coração , Transdução de Sinais , Organelas/metabolismo , Sinalização do CálcioRESUMO
Atrial fibrillation (AF) is the most common chronic arrhythmia presenting a heavy disease burden. We report a new approach for generating cardiomyocytes (CMs) resembling atrial cells from human-induced pluripotent stem cells (hiPSCs) using a combination of Gremlin 2 and retinoic acid treatment. More than 40% of myocytes showed rod-shaped morphology, expression of CM proteins (including ryanodine receptor 2, α-actinin-2 and F-actin) and striated appearance, all of which were broadly similar to the characteristics of adult atrial myocytes (AMs). Isolated myocytes were electrically quiescent until stimulated to fire action potentials with an AM profile and an amplitude of approximately 100 mV, arising from a resting potential of approximately -70 mV. Single-cell RNA sequence analysis showed a high level of expression of several atrial-specific transcripts including NPPA, MYL7, HOXA3, SLN, KCNJ4, KCNJ5 and KCNA5. Amplitudes of calcium transients recorded from spontaneously beating cultures were increased by the stimulation of α-adrenoceptors (activated by phenylephrine and blocked by prazosin) or ß-adrenoceptors (activated by isoproterenol and blocked by CGP20712A). Our new approach provides human AMs with mature characteristics from hiPSCs which will facilitate drug discovery by enabling the study of human atrial cell signalling pathways and AF. This article is part of the theme issue 'The heartbeat: its molecular basis and physiological mechanisms'.
Assuntos
Fibrilação Atrial , Células-Tronco Pluripotentes Induzidas , Adulto , Humanos , Miócitos Cardíacos/metabolismo , Diferenciação Celular/fisiologia , Fibrilação Atrial/metabolismo , Receptores Adrenérgicos/metabolismo , Canais de Potássio Corretores do Fluxo de Internalização Acoplados a Proteínas G/metabolismoRESUMO
Ca(2+)-stimulated adenylyl cyclases (ACs) have recently been shown to play important roles in pacemaking in the sino-atrial node. Here we present evidence that Ca(2+)-stimulated ACs are functionally active in guinea-pig atrial myocytes. Basal activity of an AC in isolated atrial myocytes was demonstrated by the observations that MDL 12,330A (10 µm), an AC inhibitor, reduced L-type Ca(2+) current (I(CaL)) amplitude, while inhibition of phosphodiesterases with IBMX (100 µm) increased I(CaL) amplitude. Buffering of cytosolic Ca(2+) by exposure of myocytes to BAPTA-AM (5 µm) reduced I(CaL) amplitude, as did inhibition of Ca(2+) release from the sarcoplasmic reticulum with ryanodine (2 µm) and thapsigargin (1 µm). [Ca(2+)]i-activated calmodulin kinase II (CaMKII) inhibition with KN-93 (1 µm) reduced I(CaL), but subsequent application of BAPTA-AM further reduced I(CaL). This effect of BAPTA-AM, in the presence of CaMKII inhibition, demonstrates that there is an additional Ca(2+)-modulated pathway (not dependent on CaMKII) that regulates I(CaL) in atrial myocytes. The effects of BAPTA could be reversed by forskolin (10 µm), a direct stimulator of all AC isoforms, which would restore cAMP levels. In the presence of BAPTA-AM, the actions of IBMX were reduced. In addition, inclusion of cAMP in the patch electrode in the whole-cell configuration prevented the effects of BAPTA. These effects are all consistent with a role for Ca(2+)-stimulated AC in the regulation of atrial myocyte I(CaL).
Assuntos
Adenilil Ciclases/metabolismo , Canais de Cálcio Tipo L/metabolismo , Cálcio/metabolismo , Miócitos Cardíacos/metabolismo , Animais , Artérias/citologia , Artérias/metabolismo , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/antagonistas & inibidores , AMP Cíclico/metabolismo , Citosol/metabolismo , Cobaias , Átrios do Coração/citologia , Átrios do Coração/metabolismo , Masculino , Diester Fosfórico Hidrolases/metabolismo , Canal de Liberação de Cálcio do Receptor de Rianodina/metabolismo , Retículo Sarcoplasmático/metabolismoRESUMO
Multi-parametric electrophysiological measurements using optical methods have become a highly valued standard in cardiac research. Most published optical mapping systems are expensive and complex. Although some applications demand high-cost components and complex designs, many can be tackled with simpler solutions. Here, we describe (1) a camera-based voltage and calcium imaging system using a single 'economy' electron-multiplying charge-coupled device camera and demonstrate the possibility of using a consumer camera for imaging calcium transients of the heart, and (2) a photodiode-based voltage and calcium high temporal resolution measurement system using single-element photodiodes and an optical fibre. High-throughput drug testing represents an application where system scalability is particularly attractive. Therefore, we tested our systems on tissue exposed to a well-characterized and clinically relevant calcium channel blocker, nifedipine, which has been used to treat angina and hypertension. As experimental models, we used the Langendorff-perfused whole-heart and thin ventricular tissue slices, a preparation gaining renewed interest by the cardiac research community. Using our simplified systems, we were able to monitor simultaneously the marked changes in the voltage and calcium transients that are responsible for the negative inotropic effect of the compound.
Assuntos
Eletrofisiologia Cardíaca/métodos , Avaliação Pré-Clínica de Medicamentos/métodos , Coração/efeitos dos fármacos , Coração/fisiologia , Miocárdio/metabolismo , Imagens com Corantes Sensíveis à Voltagem/métodos , Animais , Cálcio/metabolismo , Bloqueadores dos Canais de Cálcio/farmacologia , Cobaias , Nifedipino/farmacologiaRESUMO
Evidence suggests that ß-Adrenergic receptor signaling increases heart rate and force through not just cyclic AMP but also the Ca(2+)-releasing second messengers NAADP (nicotinic acid adenine dinucleotide phosphate) and cADPR (cyclic ADP-ribose). Nevertheless, proof of the physiological relevance of these messengers requires direct measurements of their levels in response to receptor stimulation. Here we report that in intact Langendorff-perfused hearts ß-adrenergic stimulation increased both messengers, with NAADP being transient and cADPR being sustained. Both NAADP and cADPR have physiological and therefore pathological relevance by providing alternative drug targets in the ß-adrenergic receptor signaling pathway.
Assuntos
ADP-Ribose Cíclica/metabolismo , Miocárdio/metabolismo , NADP/análogos & derivados , Receptores Adrenérgicos beta/metabolismo , Agonistas Adrenérgicos beta/farmacologia , Animais , Cobaias , Coração/efeitos dos fármacos , Técnicas In Vitro , NADP/metabolismo , Transdução de SinaisRESUMO
Calcium ions play a central role in determining the timing and magnitude of the pumping action of heart muscle in a process which couples electrical activity of action potentials to muscle contraction. Regulation of this excitation-contraction coupling is achieved by Ca2+ signalling mechanisms that include activation of Ca2+ mobilising agents which influence the movement of Ca2+ between intracellular membrane-bound compartments. Research discussed here concerns endolysosomes, which play diverse signalling roles throughout the body. In the heart, a population of endolysosomes is strategically placed close to two other important membrane bound organelles, sarcoplasmic reticulum (SR) and mitochondria. In each case this proximity provides a structural basis for highly localised Ca2+ signalling in nanodomains between endolysosomes and the organelle. Ca2+ is released from endolysosomes via at least two varieties of two-pore domain channels (TPCs) in mammalian cardiac cells, TPC1 determining the interaction with mitochondria, while TPC2 controls the influence on SR. Ca2+ release via both TPC1 and TPC2 is enhanced by the Ca2+ mobilising agent, nicotinic acid adenine dinucleotide phosphate (NAADP) which is synthesised in the heart primarily by CD38. In normal physiology, NAADP plays an important regulatory role in which Ca2+ is released from endolysosomes via TPC2 channels into a nanodomain next to SR, and an amplification mechanism resulting from Ca2+ activation of CaMKII enhances SR Ca2+ uptake by the enzyme SERCA to increase the amplitude of the Ca2+ transient accompanying action potentials. A separate mechanism underlies pathology associated with reperfusion after ischaemia, when NAADP-mediated endolysosomal calcium release via TPC1 acts on nearby mitochondria resulting in abnormal SR Ca2+ release and extreme disruption to the normal excitation-contraction coupling process, causing muscle damage. There are different roles for PKA in the two pathways dependant on TPC1 or TPC2. Oxidising conditions during reperfusion following ischaemia promote disulphide bond formation in PKAIalpha causing accumulation of PKAI holoenzyme in endolysosomes and cardioprotective inhibition of TPC1 channels. In the case of TPC2, PKAII actions are thought to enhance NAADP synthesis by CD38 therefore promoting the endolysosomal influence on SR Ca2+. Excessive activation of this pathway leads to cardiac arrhythmias and hypertrophy.
Assuntos
Canais de Cálcio , Sinalização do Cálcio , Cálcio , Endossomos , Coração , Animais , Cálcio/metabolismo , Canais de Cálcio/metabolismo , Endossomos/metabolismo , Coração/fisiologia , Humanos , Lisossomos/metabolismo , NADP/farmacologia , Retículo Sarcoplasmático/metabolismoRESUMO
Atrial arrhythmias, such as atrial fibrillation (AF), are a major mortality risk and a leading cause of stroke. The IP3 signalling pathway has been proposed as an atrial-specific target for AF therapy, and atrial IP3 signalling has been linked to the activation of calcium sensitive adenylyl cyclases AC1 and AC8. We investigated the involvement of AC1 in the response of intact mouse atrial tissue and isolated guinea pig atrial and sino-atrial node (SAN) cells to the α-adrenoceptor agonist phenylephrine (PE) using the selective AC1 inhibitor ST034307. The maximum rate change of spontaneously beating mouse right atrial tissue exposed to PE was reduced from 14.5% to 8.2% (p = 0.005) in the presence of 1 µM ST034307, whereas the increase in tension generated in paced left atrial tissue in the presence of PE was not inhibited by ST034307 (Control = 14.2%, ST034307 = 16.3%; p > 0.05). Experiments were performed using isolated guinea pig atrial and SAN cells loaded with Fluo-5F-AM to record changes in calcium transients (CaT) generated by 10 µM PE in the presence and absence of 1 µM ST034307. ST034307 significantly reduced the beating rate of SAN cells (0.34-fold decrease; p = 0.003) but did not inhibit changes in CaT amplitude in response to PE in atrial cells. The results presented here demonstrate pharmacologically the involvement of AC1 in the downstream response of atrial pacemaker activity to α-adrenoreceptor stimulation and IP3R calcium release.
RESUMO
Calcium handling is vital to normal physiological function in the heart. Human atrial arrhythmias, eg. atrial fibrillation, are a major morbidity and mortality burden, yet major gaps remain in our understanding of how calcium signaling pathways function and interact. Inositol trisphosphate (IP3) is a calcium-mobilizing second messenger and its agonist-induced effects have been observed in many tissue types. In the atria IP3 receptors (IR3Rs) residing on junctional sarcoplasmic reticulum augment cellular calcium transients and, when over-stimulated, lead to arrhythmogenesis. Recent studies have demonstrated that the predominant pathway for IP3 actions in atrial myocytes depends on stimulation of calcium-dependent forms of adenylyl cyclase (AC8 and AC1) by IP3-evoked calcium release from the sarcoplasmic reticulum. AC8 shows co-localisation with IP3Rs and AC1 appears to be nearby. These observations support crosstalk between calcium and cAMP pathways in nanodomains in atria. Similar mechanisms also appear to operate in the pacemaker region of the sinoatrial node. Here we discuss these significant advances in our understanding of atrial physiology and pathology, together with implications for the identification of potential novel targets and modulators for the treatment of atrial arrhythmias.
RESUMO
Phosphorylation of ion channels plays an important role in the regulation of cardiac function, but signaling mechanisms controlling dephosphorylation are not well understood. We have tested the hypothesis that p(21)-activated kinase-1 (Pak1), a serine-threonine protein kinase regulated by Ras-related small G proteins, regulates sinoatrial node (SAN) ion channel activity through a mechanism involving protein phosphatase 2A. We report a novel role of Pak1-mediated signaling in attenuating isoproterenol-induced enhancement of L-type Ca(2+) current (I(CaL)) and delayed rectifier potassium current (I(K)) in guinea pig SAN pacemaker cells. We demonstrate that in guinea pig SAN: (1) there is abundant expression of endogenous Pak1 in pacemaker cells; (2) expression of constitutively active Pak1 depresses isoproterenol-induced upregulation of I(CaL) and I(K); (3) inhibition of protein phosphatase 2A increases the enhancement of I(K) and I(CaL) by isoproterenol in Ad-Pak1-infected cells; (4) protein phosphatase 2A coimmunoprecipitates with endogenous Pak1 in SAN tissue; and (5) expression of constitutively active Pak1 suppresses the chronotropic action of isoproterenol on pacemaker activity of intact SAN preparations. In conclusion, our data demonstrate that a Pak1 signaling pathway exists in cardiac pacemaker cells and that this novel pathway plays a role in the regulation of ion channel activity.
Assuntos
Canais de Cálcio Tipo L/fisiologia , Canais de Potássio de Retificação Tardia/fisiologia , Proteínas Serina-Treonina Quinases/fisiologia , Nó Sinoatrial/metabolismo , Animais , Feminino , Cobaias , Frequência Cardíaca/efeitos dos fármacos , Isoproterenol/farmacologia , Fosfoproteínas Fosfatases/fisiologia , Proteína Fosfatase 2 , Transfecção , Quinases Ativadas por p21RESUMO
In this mini-review, we provide an overview of phenylethanolamine-N-methyl transferase (Pnmt)-derived cardiomyocytes (PdCMs), a recently discovered cardiomyocyte subpopulation. We discuss their anatomical localization, physiological characteristics, possible function, and future perspectives. Their unique distribution in the heart, electrical activity, Ca2+ transient properties, and potential role in localized adrenergic signaling are discussed.
RESUMO
The actions of cyclic ADP-ribose (cADPR), a regulator of Ca2+-induced Ca2+ release (CICR), were investigated on Ca2+ release and sarcoplasmic reticulum (SR) Ca2+ loading in cardiac myocytes at physiological temperature. In guinea-pig ventricular cells, cADPR, applied via patch pipette or from photorelease of its caged derivative, increased contraction amplitude and whole-cell Ca2+ transients, without affecting SR Ca2+ load (measured in response to rapid caffeine application). Under voltage-clamp conditions, photorelease of caged cADPR enhanced Ca2+ transient magnitude without affecting the peak amplitude of L-type Ca2+ current or its rate of decay, indicative of an increase in CICR gain. In rat permeabilised ventricular myocytes, rapid application of cADPR increased Ca2+ spark frequency within 30 s, and this effect was maintained over a 10 min exposure. Enhancement of spark frequency was not associated with changes in SR Ca2+ load at 30 s and 3 min of exposure to cADPR; however, prolonged exposure (10 min) was associated with an increased SR Ca2+ load (32+/-7%). The observations are consistent with dual actions of cADPR: a rapid effect on CICR that does not depend on an increased SR Ca2+ load, and an additional slower effect that is associated with enhanced SR Ca2+ levels.