RESUMO
Ca2+ release from the sarcoplasmic reticulum (SR) plays a central role in excitation-contraction coupling (ECC) in skeletal muscles. However, the mechanism by which activation of the voltage-sensors/dihydropyridine receptors (DHPRs) in the membrane of the transverse tubular system leads to activation of the Ca2+-release channels/ryanodine receptors (RyRs) in the SR is not fully understood. Recent observations showing that a very small Ca2+ leak through RyR1s in mammalian skeletal muscle can markedly raise the background [Ca2+] in the junctional space (JS) above the Ca2+ level in the bulk of the cytosol indicate that there is a diffusional barrier between the JS and the cytosol at large. Here, I use a mathematical model to explore the hypothesis that a sudden rise in Ca2+ leak through DHPR-coupled RyR1s, caused by reduced inhibition at the RyR1 Ca2+/Mg2+ inhibitory I1-sites when the associated DHPRs are activated, is sufficient to enable synchronized responses that trigger a regenerative rise of Ca2+ release that remains under voltage control. In this way, the characteristic response to Ca2+ of RyR channels is key not only for the Ca2+ release mechanism in cardiac muscle and other tissues, but also for the DHPR-dependent Ca2+ release in skeletal muscle.
Assuntos
Canais de Cálcio Tipo L , Cálcio , Músculo Esquelético , Canal de Liberação de Cálcio do Receptor de Rianodina , Canal de Liberação de Cálcio do Receptor de Rianodina/metabolismo , Músculo Esquelético/metabolismo , Animais , Cálcio/metabolismo , Canais de Cálcio Tipo L/metabolismo , Retículo Sarcoplasmático/metabolismo , Sinalização do Cálcio/fisiologia , Acoplamento Excitação-Contração , Modelos Biológicos , HumanosRESUMO
Micro and nanoscale patterning of surface features and biochemical cues have emerged as tools to precisely direct neurite growth into close proximity with next generation neural prosthesis electrodes. Biophysical cues can exert greater influence on neurite pathfinding compared to the more well studied biochemical cues; yet the signaling events underlying the ability of growth cones to respond to these microfeatures remain obscure. Intracellular Ca2+ signaling plays a critical role in how a growth cone senses and grows in response to various cues (biophysical features, repulsive peptides, chemo-attractive gradients). Here, we investigate the role of inositol triphosphate (IP3) and ryanodine-sensitive receptor (RyR) signaling as sensory neurons (spiral ganglion neurons, SGNs, and dorsal root ganglion neurons, DRGNs) pathfind in response to micropatterned substrates of varied geometries. We find that IP3 and RyR signaling act in the growth cone as they navigate biophysical cues and enable proper guidance to biophysical, chemo-permissive, and chemo-repulsive micropatterns. In response to complex micropatterned geometries, RyR signaling appears to halt growth in response to both topographical features and chemo-repulsive cues. IP3 signaling appears to play a more complex role, as growth cones appear to sense the microfeatures in the presence of xestospongin C but are unable to coordinate turning in response to them. Overall, key Ca2+ signaling elements, IP3 and RyR, are found to be essential for SGNs to pathfind in response to engineered biophysical and biochemical cues. These findings inform efforts to precisely guide neurite regeneration for improved neural prosthesis function, including cochlear implants.
Assuntos
Neuritos , Canal de Liberação de Cálcio do Receptor de Rianodina , Transdução de Sinais , Canal de Liberação de Cálcio do Receptor de Rianodina/metabolismo , Neuritos/metabolismo , Animais , Gânglios Espinais/metabolismo , Gânglios Espinais/citologia , Cones de Crescimento/metabolismo , Cones de Crescimento/efeitos dos fármacos , Sinalização do Cálcio , Ratos , Propriedades de Superfície , Células Cultivadas , Oxazóis , Compostos MacrocíclicosRESUMO
Atrial fibrillation (AF) and heart failure with preserved ejection fraction (HFpEF) often coexist; however, clinically available anti-AF drugs can exacerbate symptoms of HFpEF. M201-A suppressed ryanodine receptor-mediated diastolic Ca2+ leakage, possibly inhibiting common pathological processes toward AF and HFpEF. To bridge the basic information to clinical practice, we assessed its cardiohemodynamic, anti-AF and ventricular proarrhythmic profile using halothane-anesthetized dogs (n = 4). M201-A hydrochloride in doses of 0.03, 0.3 and 3 mg/kg/10 min was intravenously administered, providing peak plasma concentrations of 0.09, 0.81 and 5.70 µg/mL, respectively. The high dose of M201-A showed various cardiovascular actions. Namely, M201-A increased mean blood pressure and tended to enhance isovolumetric ventricular relaxation without suppressing ventricular contraction or decreasing cardiac output. M201-A enhanced atrioventricular conduction, but hardy affected intra-atrial/ventricular conduction. Importantly, M201-A prolonged effective refractory period more potently in the atrium than in the ventricle, indicating that it may become an atrial-selective antiarrhythmic drug. Meanwhile, M201-A prolonged QT interval/QTcV, and showed reverse frequency-dependent delay of ventricular repolarization. M201-A prolonged J-Tpeakc without prolonging Tpeak-Tend or terminal repolarization period, indicating the risk of causing torsade de pointes is negligible. Thus, M201-A is expected to become a hopeful therapeutic strategy for patients having pathology of both AF and HFpEF.
Assuntos
Antiarrítmicos , Fibrilação Atrial , Insuficiência Cardíaca , Canal de Liberação de Cálcio do Receptor de Rianodina , Volume Sistólico , Animais , Cães , Canal de Liberação de Cálcio do Receptor de Rianodina/metabolismo , Fibrilação Atrial/tratamento farmacológico , Fibrilação Atrial/fisiopatologia , Insuficiência Cardíaca/tratamento farmacológico , Insuficiência Cardíaca/fisiopatologia , Volume Sistólico/efeitos dos fármacos , Antiarrítmicos/farmacologia , Masculino , Átrios do Coração/efeitos dos fármacos , Átrios do Coração/fisiopatologia , Relação Dose-Resposta a Droga , Humanos , FemininoRESUMO
Upon stimulation of membrane receptors, nicotinic acid adenine dinucleotide phosphate (NAADP) is formed as second messenger within seconds and evokes Ca2+ signaling in many different cell types. Here, to directly stimulate NAADP signaling, MASTER-NAADP, a Membrane permeAble, STabilized, bio-rEversibly pRotected precursor of NAADP is synthesized and release of its active NAADP mimetic, benzoic acid C-nucleoside, 2'-phospho-3'F-adenosine-diphosphate, by esterase digestion is confirmed. In the presence of NAADP receptor HN1L/JPT2 (hematological and neurological expressed 1-like protein, HN1L, also known as Jupiter microtubule-associated homolog 2, JPT2), this active NAADP mimetic releases Ca2+ and increases the open probability of type 1 ryanodine receptor. When added to intact cells, MASTER-NAADP initially evokes single local Ca2+ signals of low amplitude. Subsequently, also global Ca2+ signaling is observed in T cells, natural killer cells, and Neuro2A cells. In contrast, control compound MASTER-NADP does not stimulate Ca2+ signaling. Likewise, in cells devoid of HN1L/JPT2, MASTER-NAADP does not affect Ca2+ signaling, confirming that the product released from MASTER-NAADP is a bona fide NAADP mimetic.
Assuntos
Sinalização do Cálcio , Cálcio , NADP , NADP/análogos & derivados , NADP/metabolismo , Animais , Humanos , Cálcio/metabolismo , Camundongos , Sistemas do Segundo Mensageiro , Permeabilidade da Membrana Celular , Canal de Liberação de Cálcio do Receptor de Rianodina/metabolismo , Células Matadoras Naturais/metabolismo , Linfócitos T/metabolismoRESUMO
The atrioventricular node (AVN) is a key component of the cardiac conduction system and takes over pacemaking of the ventricles if the sinoatrial node fails. IP3 (inositol 1,4,5 trisphosphate) can modulate excitability of myocytes from other regions of the heart, but it is not known whether IP3 receptor (IP3-R) activation modulates AVN cell pacemaking. Consequently, this study investigated effects of IP3 on spontaneous action potentials (APs) from AVN cells isolated from rabbit hearts. Immunohistochemistry and confocal imaging demonstrated the presence of IP3-R2 in isolated AVN cells, with partial overlap with RyR2 ryanodine receptors seen in co-labelling experiments. In whole-cell recordings at physiological temperature, application of 10 µM membrane-permeant Bt3-(1,4,5)IP3-AM accelerated spontaneous AP rate and increased diastolic depolarization rate, without direct effects on ICa,L, IKr, If or INCX. By contrast, application via the patch pipette of 5 µM of the IP3-R inhibitor xestospongin C led to a slowing in spontaneous AP rate and prevented 10 µM Bt3-(1,4,5)IP3-AM application from increasing the AP rate. UV excitation of AVN cells loaded with caged-IP3 led to an acceleration in AP rate, the magnitude of which increased with the extent of UV excitation. 2-APB slowed spontaneous AP rate, consistent with a role for constitutive IP3-R activity; however, it was also found to inhibit ICa,L and IKr, confounding its use for studying IP3-R. Under AP voltage clamp, UV excitation of AVN cells loaded with caged IP3 activated an inward current during diastolic depolarization. Collectively, these results demonstrate that IP3 can modulate AVN cell pacemaking rate.
Assuntos
Potenciais de Ação , Nó Atrioventricular , Receptores de Inositol 1,4,5-Trifosfato , Inositol 1,4,5-Trifosfato , Miócitos Cardíacos , Animais , Coelhos , Potenciais de Ação/efeitos dos fármacos , Nó Atrioventricular/efeitos dos fármacos , Nó Atrioventricular/metabolismo , Receptores de Inositol 1,4,5-Trifosfato/metabolismo , Inositol 1,4,5-Trifosfato/metabolismo , Miócitos Cardíacos/metabolismo , Miócitos Cardíacos/efeitos dos fármacos , Canal de Liberação de Cálcio do Receptor de Rianodina/metabolismo , Oxazóis/farmacologia , MasculinoRESUMO
S100A1, a calcium-binding protein, plays a crucial role in regulating Ca2+ signaling pathways in skeletal and cardiac myocytes via interactions with the ryanodine receptor (RyR) to affect Ca2+ release and contractile performance. Biophysical studies strongly suggest that S100A1 interacts with RyRs but have been inconclusive about both the nature of this interaction and its competition with another important calcium-binding protein, calmodulin (CaM). Thus, high-resolution cryo-EM studies of RyRs in the presence of S100A1, with or without additional CaM, were needed. The elegant work by Weninger et al. demonstrates the interaction between S100A1 and RyR1 through various experiments and confirms that S100A1 activates RyR1 at sub-micromolar Ca2+ concentrations, increasing the open probability of RyR1 channels.
Assuntos
Canal de Liberação de Cálcio do Receptor de Rianodina , Proteínas S100 , Canal de Liberação de Cálcio do Receptor de Rianodina/metabolismo , Humanos , Animais , Proteínas S100/metabolismo , Proteínas S100/química , Cálcio/metabolismo , Calmodulina/metabolismoRESUMO
Heart failure, the leading cause of mortality and morbidity in the developed world, is characterized by cardiac ryanodine receptor 2 channels that are hyperphosphorylated, oxidized, and depleted of the stabilizing subunit calstabin-2. This results in a diastolic sarcoplasmic reticulum Ca2+ leak that impairs cardiac contractility and triggers arrhythmias. Genetic mutations in ryanodine receptor 2 can also cause Ca2+ leak, leading to arrhythmias and sudden cardiac death. Here, we solved the cryogenic electron microscopy structures of ryanodine receptor 2 variants linked either to heart failure or inherited sudden cardiac death. All are in the primed state, part way between closed and open. Binding of Rycal drugs to ryanodine receptor 2 channels reverts the primed state back towards the closed state, decreasing Ca2+ leak, improving cardiac function, and preventing arrhythmias. We propose a structural-physiological mechanism whereby the ryanodine receptor 2 channel primed state underlies the arrhythmias in heart failure and arrhythmogenic disorders.
Assuntos
Arritmias Cardíacas , Cálcio , Microscopia Crioeletrônica , Insuficiência Cardíaca , Canal de Liberação de Cálcio do Receptor de Rianodina , Canal de Liberação de Cálcio do Receptor de Rianodina/metabolismo , Canal de Liberação de Cálcio do Receptor de Rianodina/genética , Canal de Liberação de Cálcio do Receptor de Rianodina/química , Insuficiência Cardíaca/metabolismo , Insuficiência Cardíaca/genética , Arritmias Cardíacas/metabolismo , Arritmias Cardíacas/genética , Humanos , Animais , Cálcio/metabolismo , Mutação , Retículo Sarcoplasmático/metabolismo , Morte Súbita Cardíaca/etiologiaRESUMO
The type 1 ryanodine receptor (RyR1) is a Ca2+ release channel in the sarcoplasmic reticulum that is essential for skeletal muscle contraction. RyR1 forms a channel with six transmembrane segments, in which S5 is the fifth segment and is thought to contribute to pore formation. However, its role in channel gating remains unclear. Here, we performed a functional analysis of several disease-associated mutations in S5 and interpreted the results with respect to the published RyR1 structures to identify potential interactions associated with the mutant phenotypes. We demonstrate that S5 plays a dual role in channel gating: the cytoplasmic side interacts with S6 to reduce the channel activity, whereas the luminal side forms a rigid structural base necessary for S6 displacement in channel opening. These results deepen our understanding of the molecular mechanisms of RyR1 channel gating and provide insight into the divergent disease phenotypes caused by mutations in S5.
Assuntos
Ativação do Canal Iônico , Mutação , Canal de Liberação de Cálcio do Receptor de Rianodina , Canal de Liberação de Cálcio do Receptor de Rianodina/metabolismo , Canal de Liberação de Cálcio do Receptor de Rianodina/genética , Humanos , Células HEK293 , Animais , Cálcio/metabolismoRESUMO
ATP and benzoylbenzoyl-ATP (BzATP) increase free cytosolic Ca2+ concentration ([Ca2+]i) in conjunctival goblet cells (CGCs) resulting in mucin secretion. The purpose of this study was to investigate the source of the Ca2+i mobilized by ATP and BzATP. First-passage cultured rat CGCs were incubated with Fura-2/AM, and [Ca2+]i was measured under several conditions with ATP and BzATP stimulation. The following conditions were used: 1) preincubation with the Ca2+ chelator EGTA, 2) preincubation with the SERCA inhibitor thapsigargin (10-6 M), which depletes ER Ca2+ stores, 3) preincubation with phospholipase C (PLC) or protein kinase A (PKA) inhibitor, or 4) preincubation with the voltage-gated calcium channel antagonist nifedipine (10-5 M) and the ryanodine receptor (RyR) antagonist dantrolene (10-5 M). Immunofluorescence microscopy (IF) and quantitative reverse transcription polymerase chain reaction (RT-qPCR) were used to investigate RyR presence in rat and human CGCs. ATP-stimulated peak [Ca2+]i was significantly lower after chelating Ca2+i with 2 mM EGTA in Ca2+-free buffer. The peak [Ca2+]i increase in CGCs preincubated with thapsigargin, the PKA inhibitor H89, nifedipine, and dantrolene, but not the PLC inhibitor, was reduced for ATP at 10-5 M and BzATP at 10-4 M. Incubating CGCs with dantrolene alone decreased [Ca2+]i and induced CGC cell death at a high concentration. RyR3 was detected in rat and human CGCs with IF and RT-qPCR. We conclude that ATP- and BzATP-induced Ca2+i increases originate from the ER and that RyR3 may be an essential regulator of CGC [Ca2+]i. This study contributes to the understanding of diseases arising from defective Ca2+ signaling in nonexcitable cells.NEW & NOTEWORTHY ATP and benzoylbenzoyl-ATP (BzATP) induce mucin secretion through an increase in free cytosolic calcium concentration ([Ca2+]i) in conjunctival goblet cells (CGCs). The mechanisms through which ATP and BzATP increase [Ca2+]i in CGCs are unclear. Ryanodine receptors (RyRs) are fundamental in [Ca2+]i regulation in excitable cells. Herein, we find that ATP and BzATP increase [Ca2+]i through the activation of protein kinase A, voltage-gated calcium channels, and RyRs, and that RyRs are crucial for nonexcitable CGCs' Ca2+i homeostasis.
Assuntos
Trifosfato de Adenosina , Cálcio , Células Caliciformes , Canal de Liberação de Cálcio do Receptor de Rianodina , Animais , Canal de Liberação de Cálcio do Receptor de Rianodina/metabolismo , Canal de Liberação de Cálcio do Receptor de Rianodina/genética , Cálcio/metabolismo , Trifosfato de Adenosina/metabolismo , Trifosfato de Adenosina/análogos & derivados , Células Caliciformes/efeitos dos fármacos , Células Caliciformes/metabolismo , Ratos , Células Cultivadas , Túnica Conjuntiva/metabolismo , Túnica Conjuntiva/efeitos dos fármacos , Agonistas Purinérgicos/farmacologia , Ratos Sprague-Dawley , Sinalização do Cálcio/efeitos dos fármacos , Humanos , Masculino , Fosfolipases Tipo C/metabolismoRESUMO
Mutations in the nuclear envelope (NE) protein lamin A/C (encoded by LMNA), cause a severe form of dilated cardiomyopathy (DCM) with early-onset life-threatening arrhythmias. However, molecular mechanisms underlying increased arrhythmogenesis in LMNA-related DCM (LMNA-DCM) remain largely unknown. Here we show that a frameshift mutation in LMNA causes abnormal Ca2+ handling, arrhythmias and disformed NE in LMNA-DCM patient-specific iPSC-derived cardiomyocytes (iPSC-CMs). Mechanistically, lamin A interacts with sirtuin 1 (SIRT1) where mutant lamin A/C accelerates degradation of SIRT1, leading to mitochondrial dysfunction and oxidative stress. Elevated reactive oxygen species (ROS) then activates the Ca2+/calmodulin-dependent protein kinase II (CaMKII)-ryanodine receptor 2 (RYR2) pathway and aggravates the accumulation of SUN1 in mutant iPSC-CMs, contributing to arrhythmias and NE deformation, respectively. Taken together, the lamin A/C deficiency-mediated ROS disorder is revealed as central to LMNA-DCM development. Manipulation of impaired SIRT1 activity and excessive oxidative stress is a potential future therapeutic strategy for LMNA-DCM.
Assuntos
Cardiomiopatia Dilatada , Células-Tronco Pluripotentes Induzidas , Lamina Tipo A , Miócitos Cardíacos , Estresse Oxidativo , Espécies Reativas de Oxigênio , Sirtuína 1 , Cardiomiopatia Dilatada/metabolismo , Cardiomiopatia Dilatada/genética , Cardiomiopatia Dilatada/patologia , Lamina Tipo A/metabolismo , Lamina Tipo A/genética , Células-Tronco Pluripotentes Induzidas/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Humanos , Sirtuína 1/metabolismo , Sirtuína 1/genética , Miócitos Cardíacos/metabolismo , Miócitos Cardíacos/patologia , Fenótipo , Arritmias Cardíacas/metabolismo , Arritmias Cardíacas/genética , Arritmias Cardíacas/patologia , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/metabolismo , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/genética , Mutação da Fase de Leitura , Cálcio/metabolismo , Canal de Liberação de Cálcio do Receptor de Rianodina/metabolismo , Canal de Liberação de Cálcio do Receptor de Rianodina/genética , Membrana Nuclear/metabolismo , Mitocôndrias/metabolismo , Masculino , Proteínas Associadas aos Microtúbulos/metabolismo , Proteínas Associadas aos Microtúbulos/genéticaRESUMO
BACKGROUND: Transverse (t)-tubules drive the rapid and synchronous Ca2+ rise in cardiac myocytes. The virtual complete atrial t-tubule loss in heart failure (HF) decreases Ca2+ release. It is unknown if or how atrial t-tubules can be restored and how this affects systolic Ca2+. METHODS: HF was induced in sheep by rapid ventricular pacing and recovered following termination of rapid pacing. Serial block-face scanning electron microscopy and confocal imaging were used to study t-tubule ultrastructure. Function was assessed using patch clamp, Ca2+, and confocal imaging. Candidate proteins involved in atrial t-tubule recovery were identified by western blot and expressed in rat neonatal ventricular myocytes to determine if they altered t-tubule structure. RESULTS: Atrial t-tubules were lost in HF but reappeared following recovery from HF. Recovered t-tubules were disordered, adopting distinct morphologies with increased t-tubule length and branching. T-tubule disorder was associated with mitochondrial disorder. Recovered t-tubules were functional, triggering Ca2+ release in the cell interior. Systolic Ca2+, ICa-L, sarcoplasmic reticulum Ca2+ content, and sarcoendoplasmic reticulum Ca2+ ATPase function were restored following recovery from HF. Confocal microscopy showed fragmentation of ryanodine receptor staining and movement away from the z-line in HF, which was reversed following recovery from HF. Acute detubulation, to remove recovered t-tubules, confirmed their key role in restoration of the systolic Ca2+ transient, the rate of Ca2+ removal, and the peak L-type Ca2+ current. The abundance of telethonin and myotubularin decreased during HF and increased during recovery. Transfection with these proteins altered the density and structure of tubules in neonatal myocytes. Myotubularin had a greater effect, increasing tubule length and branching, replicating that seen in the recovery atria. CONCLUSIONS: We show that recovery from HF restores atrial t-tubules, and this promotes recovery of ICa-L, sarcoplasmic reticulum Ca2+ content, and systolic Ca2+. We demonstrate an important role for myotubularin in t-tubule restoration. Our findings reveal a new and viable therapeutic strategy.
Assuntos
Átrios do Coração , Insuficiência Cardíaca , Miócitos Cardíacos , Animais , Insuficiência Cardíaca/metabolismo , Insuficiência Cardíaca/fisiopatologia , Insuficiência Cardíaca/patologia , Miócitos Cardíacos/metabolismo , Miócitos Cardíacos/patologia , Miócitos Cardíacos/ultraestrutura , Átrios do Coração/metabolismo , Átrios do Coração/patologia , Átrios do Coração/fisiopatologia , Ovinos , Cálcio/metabolismo , Sinalização do Cálcio , Ratos , Retículo Sarcoplasmático/metabolismo , Retículo Sarcoplasmático/ultraestrutura , Retículo Sarcoplasmático/patologia , Recuperação de Função Fisiológica , Mitocôndrias Cardíacas/metabolismo , Mitocôndrias Cardíacas/ultraestrutura , Mitocôndrias Cardíacas/patologia , Células Cultivadas , Sístole , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/metabolismo , Canal de Liberação de Cálcio do Receptor de Rianodina/metabolismo , Ratos Sprague-Dawley , FemininoRESUMO
The functioning of the cardiovascular system is critical for embryo survival. Cardiac contractions depend on the sequential activation of different classes of voltage-gated ion channels. Understanding the fundamental features of these interactions is important for identifying the mechanisms of pathologies development in the myocardium. However, at present there is no consensus on which ion channels are involved in the formation of automaticity in the early embryonic stages. The aim of this study was to elucidate the expression of genes encoding various types of ion channels that are involved in the generation of electrical activity chicken heart at different stages of ontogenesis. We analyzed the expression of 14 genes from different families of ion channels. It was revealed that the expression profiles of ion channel genes change depending on the stages of ontogenesis. The HCN4, CACNA1D, SCN1A, SCN5A, KCNA1 genes have maximum expression at the tubular heart stage. In adult, a switch occurs to the higher expression of CACNA1C, KCNH6, RYR and SLC8A1 genes. This data correlated with the results obtained by the microelectrode method. It can be assumed that the automaticity of the tubular heart is mainly due to the mechanism of the «membrane-clock¼ (hyperpolarization-activated current (If), Ca2+-current L-type (ICaL), Na+-current (INa) and the slow component of the delayed rectifier K+-current (IKs)). Whereas in adult birds, the mechanism for generating electrical impulses is determined by both « membrane- clock¼ and «Ca2+-clock¼.
Assuntos
Galinhas , Regulação da Expressão Gênica no Desenvolvimento , Coração , Miocárdio , Animais , Embrião de Galinha , Miocárdio/metabolismo , Galinhas/genética , Coração/embriologia , Canal de Liberação de Cálcio do Receptor de Rianodina/genética , Canal de Liberação de Cálcio do Receptor de Rianodina/metabolismo , Trocador de Sódio e Cálcio/genética , Trocador de Sódio e Cálcio/metabolismo , Canais Disparados por Nucleotídeos Cíclicos Ativados por Hiperpolarização/genética , Canais Disparados por Nucleotídeos Cíclicos Ativados por Hiperpolarização/metabolismo , Canais Iônicos/genética , Canais Iônicos/metabolismoRESUMO
Seizures are increasingly being recognized as the hallmark of Alzheimer's disease (AD). Neuronal hyperactivity can be a consequence of neuronal damage caused by abnormal amyloid ß (Aß) depositions. However, it can also be a cell-autonomous phenomenon causing AD by Aß-independent mechanisms. Various studies using animal models have shown that Ca2+ is released from the endoplasmic reticulum (ER) via type 1 inositol triphosphate receptors (InsP3R1s) and ryanodine receptors (RyRs). To investigate which is the main pathophysiological mechanism in human neurons, we measured Ca2+ signaling in neural cells derived from three early-onset AD patients harboring Presenilin-1 variants (PSEN1 p.A246E, p.L286V, and p.M146L). Of these, it has been reported that PSEN1 p.A246E and p.L286V did not produce a significant amount of abnormal Aß. We found all PSEN1-mutant neurons, but not wild-type, caused abnormal Ca2+-bursts in a manner dependent on the calcium channel, Ryanodine Receptor 2 (RyR2). Indeed, carvedilol, an RyR2 inhibitor, and VK-II-86, an analog of carvedilol without the ß-blocking effects, sufficiently eliminated the abnormal Ca2+ bursts. In contrast, Dantrolene, an inhibitor of RyR1 and RyR3, and Xestospongin c, an IP3R inhibitor, did not attenuate the Ca2+-bursts. The Western blotting showed that RyR2 expression was not affected by PSEN1 p.A246E, suggesting that the variant may activate the RyR2. The RNA-Seq data revealed that ER-stress responsive genes were increased, and mitochondrial Ca2+-transporter genes were decreased in PSEN1A246E cells compared to the WT neurons. Thus, we propose that aberrant Ca2+ signaling is a key link between human pathogenic PSEN1 variants and cell-intrinsic hyperactivity prior to deposition of abnormal Aß, offering prospects for the development of targeted prevention strategies for at-risk individuals.
Assuntos
Doença de Alzheimer , Sinalização do Cálcio , Cálcio , Carvedilol , Neurônios , Presenilina-1 , Canal de Liberação de Cálcio do Receptor de Rianodina , Feminino , Humanos , Masculino , Doença de Alzheimer/metabolismo , Doença de Alzheimer/genética , Doença de Alzheimer/patologia , Cálcio/metabolismo , Sinalização do Cálcio/efeitos dos fármacos , Retículo Endoplasmático/metabolismo , Retículo Endoplasmático/efeitos dos fármacos , Neurônios/metabolismo , Neurônios/efeitos dos fármacos , Presenilina-1/genética , Presenilina-1/metabolismo , Canal de Liberação de Cálcio do Receptor de Rianodina/metabolismo , Canal de Liberação de Cálcio do Receptor de Rianodina/genética , Carvedilol/farmacologiaRESUMO
The key mutations, such as the Gly-4891-Glu substitution and the Ile-4734 multiple substitutions within the ryanodine receptors (RyR), are linked to diamide resistance in fall armyworm (FAW), Spodoptera frugiperda. In this study, we found that FAW remained sensitive to cyantraniliprole and chlorantraniliprole, while its sensitivity to flubendiamide was reduced. Moreover, a low level of heterozygous mutation at I4743 was observed. To facilitate the detection procedure of these mutations, a simple and efficient loop-mediated isothermal amplification (LAMP) protocol was developed for operation. The reaction for detecting the G4891E and I4743 single or multiple mutations was carried out at 68 °C for 85 min and 68 °C for 85 min or 68 °C for 65 min, respectively. These LAMP reactions can be easily observed via visualization of the color change from pink to yellow. This assay provides a simple, convenient, and effective means of detecting mutations in the RyR of FAW for pest management purposes.
Assuntos
Proteínas de Insetos , Mutação , Técnicas de Amplificação de Ácido Nucleico , Canal de Liberação de Cálcio do Receptor de Rianodina , Spodoptera , Animais , Canal de Liberação de Cálcio do Receptor de Rianodina/genética , Canal de Liberação de Cálcio do Receptor de Rianodina/metabolismo , Canal de Liberação de Cálcio do Receptor de Rianodina/química , Spodoptera/genética , Spodoptera/efeitos dos fármacos , Técnicas de Amplificação de Ácido Nucleico/métodos , Proteínas de Insetos/genética , Proteínas de Insetos/metabolismo , Proteínas de Insetos/química , Inseticidas/farmacologia , ortoaminobenzoatos/farmacologia , Benzamidas/farmacologia , Sulfonas/farmacologia , Pirazóis/farmacologia , Resistência a Inseticidas/genética , Fluorocarbonos , Ftalimidas , Técnicas de Diagnóstico MolecularRESUMO
The expression excitation-contraction (EC) coupling in skeletal muscle was coined in 1952 (Sandow A. Yale J Biol Med 25: 176-201, 1952). The term evolved narrowly to include only the processes at the triad that intervene between depolarization of the transverse tubular (T-tubular) membrane and Ca2+ release from the sarcoplasmic reticulum (SR). From 1970 to 1988, the foundation of EC coupling was elucidated. The channel through which Ca2+ was released during activation was located in the SR by its specific binding to the plant insecticide ryanodine. This channel was called the ryanodine receptor (RyR). The RyR contained four subunits that together constituted the "SR foot" structure that traversed the gap between the SR and the T-tubular membrane. Ca2+ channels, also called dihydropyridine receptors (DHPRs), were located in the T-tubular membrane at the triadic junction and shown to be essential for EC coupling. There was a precise relationship between the two channels. Four DHPRs, organized as tetrads, were superimposed on alternate RyRs. This structure was consistent with the proposal that EC coupling was mediated via a movement of intramembrane charge in the T-tubular system. The speculation was that the DHPR acted as a voltage sensor transferring information to the RyRs of the SR by protein-protein interaction causing the release of Ca2+ from the SR. A great deal of progress was made by 1988 toward understanding EC coupling. However, the ultimate question of how voltage sensing is coupled to the opening of the SR Ca2+ release channel remains unresolved.NEW & NOTEWORTHY The least understood part of the series of events in excitation-contraction coupling in skeletal muscle was how information was transmitted from the transverse tubules to the sarcoplasmic (SR) and how Ca2+ was released from the SR. Through an explosion of technical approaches including physiological, biochemical, structural, pharmacological, and molecular genetics, much was discovered between 1970 and 1988. By the end of 1988, the foundation of EC coupling in skeletal muscle was established.
Assuntos
Acoplamento Excitação-Contração , Músculo Esquelético , Retículo Sarcoplasmático , Retículo Sarcoplasmático/metabolismo , Retículo Sarcoplasmático/fisiologia , Humanos , Músculo Esquelético/metabolismo , Músculo Esquelético/fisiologia , Acoplamento Excitação-Contração/fisiologia , Animais , Canal de Liberação de Cálcio do Receptor de Rianodina/metabolismo , Fisiologia , Cálcio/metabolismoRESUMO
Calmodulin (CaM) is a ubiquitous, small cytosolic calcium (Ca2+)-binding sensor that plays a vital role in many cellular processes by binding and regulating the activity of over 300 protein targets. In cardiac muscle, CaM modulates directly or indirectly the activity of several proteins that play a key role in excitation-contraction coupling (ECC), such as ryanodine receptor type 2 (RyR2), l-type Ca2+ (Cav1.2), sodium (NaV1.5) and potassium (KV7.1) channels. Many recent clinical and genetic studies have reported a series of CaM mutations in patients with life-threatening arrhythmogenic syndromes, such as long QT syndrome (LQTS) and catecholaminergic polymorphic ventricular tachycardia (CPVT). We recently showed that four arrhythmogenic CaM mutations (N98I, D132E, D134H, and Q136P) significantly reduce the binding of CaM to RyR2. Herein, we investigate in vivo functional effects of these CaM mutations on the normal zebrafish embryonic heart function by microinjecting complementary RNA corresponding to CaMN98I, CaMD132E, CaMD134H, and CaMQ136P mutants. Expression of CaMD132E and CaMD134H mutants results in significant reduction of the zebrafish heart rate, mimicking a severe form of human bradycardia, whereas expression of CaMQ136P results in an increased heart rate mimicking human ventricular tachycardia. Moreover, analysis of cardiac ventricular rhythm revealed that the CaMD132E and CaMN98I zebrafish groups display an irregular pattern of heart beating and increased amplitude in comparison to the control groups. Furthermore, circular dichroism spectroscopy experiments using recombinant CaM proteins reveals a decreased structural stability of the four mutants compared to the wild-type CaM protein in the presence of Ca2+. Finally, Ca2+-binding studies indicates that all CaM mutations display reduced CaM Ca2+-binding affinities, with CaMD132E exhibiting the most prominent change. Our data suggest that CaM mutations can trigger different arrhythmogenic phenotypes through multiple and complex molecular mechanisms.
Assuntos
Arritmias Cardíacas , Calmodulina , Peixe-Zebra , Animais , Calmodulina/metabolismo , Calmodulina/genética , Arritmias Cardíacas/genética , Arritmias Cardíacas/metabolismo , Mutação , Proteínas de Peixe-Zebra/genética , Proteínas de Peixe-Zebra/metabolismo , Humanos , Canal de Liberação de Cálcio do Receptor de Rianodina/genética , Canal de Liberação de Cálcio do Receptor de Rianodina/metabolismo , Mutação de Sentido Incorreto , Taquicardia Ventricular/genética , Taquicardia Ventricular/metabolismo , Cálcio/metabolismoRESUMO
Heart Failure with preserved ejection fraction (HFpEF) has a high rate of sudden cardiac death (SCD) and empirical treatment is ineffective. We developed a novel preclinical model of metabolic HFpEF that presents with stress-induced ventricular tachycardia (VT). Mechanistically, we discovered arrhythmogenic changes in intracellular Ca2+ handling distinct from the changes pathognomonic for heart failure with reduced ejection fraction. We further show that dantrolene, a stabilizer of the ryanodine receptor Ca2+ channel, attenuates HFpEF-associated arrhythmogenic Ca2+ handling in vitro and suppresses stress-induced VT in vivo. We propose ryanodine receptor stabilization as a mechanistic approach to mitigation of malignant VT in metabolic HFpEF.
Assuntos
Arritmias Cardíacas , Cálcio , Dantroleno , Modelos Animais de Doenças , Insuficiência Cardíaca , Canal de Liberação de Cálcio do Receptor de Rianodina , Canal de Liberação de Cálcio do Receptor de Rianodina/metabolismo , Animais , Cálcio/metabolismo , Insuficiência Cardíaca/metabolismo , Insuficiência Cardíaca/tratamento farmacológico , Insuficiência Cardíaca/fisiopatologia , Arritmias Cardíacas/metabolismo , Arritmias Cardíacas/tratamento farmacológico , Dantroleno/farmacologia , Volume Sistólico/efeitos dos fármacos , Miócitos Cardíacos/metabolismo , Miócitos Cardíacos/efeitos dos fármacos , Humanos , Taquicardia Ventricular/metabolismo , Taquicardia Ventricular/tratamento farmacológico , Camundongos , Masculino , Sinalização do Cálcio/efeitos dos fármacosRESUMO
Endocrine-disrupting chemicals (EDCs) are toxic pollutants generated by artificial activities. Moreover, their hormone-like structure induces disturbances, such as mimicking or blocking metabolic activity. Previous studies on EDCs have focused on the adverse effect of the endocrine system in vertebrates, with limited investigations conducted on ion channels in invertebrates. Thus, in this study, we investigated the potential adverse effects of exposure to bisphenol-A (BPA) and di-(2-ethylhexyl) phthalate (DEHP) at the molecular level on the ryanodine receptor (RyR), a calcium ion channel receptor in Macrophthalmus japonicus. In the phylogenetic analysis, the RyR amino acid sequences in M. japonicus clustered with those in the Crustacean and formed separated branches for RyR in insects and mammals. When exposed to 1 µg L-1 BPA, a significant increase in RyR mRNA expression was observed in the gills on day 1, although a similar level to the control group was observed from day 4 to day 7. However, the RyR expression due to DEHP exposure decreased on days 1 and 4, although it increased on day 7 following exposure to 10 µg L-1. The RyR expression pattern in the hepatopancreas increased for up to 4 days, depending on the BPA concentration. However, there was a tendency for the expression to decrease gradually after the statistical significance increased during the early stage of DEHP exposure (D1). Hence, the transcriptional alterations in the M. japonicus RyR gene observed in the study suggest that exposure toxicities to EDCs, such as BPA and DEHP, have the potential to disrupt calcium ion channel signaling in the gills and hepatopancreas of M. japonicus crabs.
Assuntos
Compostos Benzidrílicos , Braquiúros , Disruptores Endócrinos , Fenóis , Canal de Liberação de Cálcio do Receptor de Rianodina , Poluentes Químicos da Água , Animais , Disruptores Endócrinos/toxicidade , Canal de Liberação de Cálcio do Receptor de Rianodina/genética , Canal de Liberação de Cálcio do Receptor de Rianodina/metabolismo , Braquiúros/efeitos dos fármacos , Braquiúros/genética , Braquiúros/metabolismo , Compostos Benzidrílicos/toxicidade , Fenóis/toxicidade , Poluentes Químicos da Água/toxicidade , Filogenia , Dietilexilftalato/toxicidade , Brânquias/metabolismo , Brânquias/efeitos dos fármacos , Proteínas de Artrópodes/genética , Proteínas de Artrópodes/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Sequência de AminoácidosRESUMO
Cyclic oligomeric depsipeptides (COD) are a structural class within naturally occurring compounds with a wide range of biological activity. Verticilide is a COD (24-membered ring) that was identified by its inhibition of insect ryanodine receptor (RyR). We have since found that the enantiomer of verticilide (ent-verticilide, 1) is a potent inhibitor of mammalian RyR2, a cardiac calcium channel, and therefore a potential antiarrhythmic agent. Oddly, nat-verticilide does not inhibit RyR2. To further develop ent-verticilide as an antiarrhythmic, we explored potential SAR through systematic modification of the ester's functionality to both N-H and N-Me amides. The syntheses of these ent-verticilide-inspired analogs are detailed using a monomer-based platform enabled by enantioselective catalysis. Two analogs among 23 exhibited measurable reduction of calcium sparks in a functional assay of RyR2 activity. These findings illustrate the value of natural product-inspired therapeutic development, but the less-studied approach where the non-natural enantiomeric series harbors important SAR.
Assuntos
Antiarrítmicos , Produtos Biológicos , Depsipeptídeos , Canal de Liberação de Cálcio do Receptor de Rianodina , Depsipeptídeos/química , Depsipeptídeos/farmacologia , Depsipeptídeos/síntese química , Relação Estrutura-Atividade , Canal de Liberação de Cálcio do Receptor de Rianodina/metabolismo , Canal de Liberação de Cálcio do Receptor de Rianodina/química , Produtos Biológicos/química , Produtos Biológicos/farmacologia , Produtos Biológicos/síntese química , Humanos , Antiarrítmicos/farmacologia , Antiarrítmicos/química , Antiarrítmicos/síntese química , Animais , EstereoisomerismoRESUMO
BACKGROUND: Oxidative stress may contribute to cardiac ryanodine receptor (RyR2) dysfunction in diabetic cardiomyopathy. Ginsenoside Rb1 (Rb1) is a major pharmacologically active component of ginseng to treat cardiovascular diseases. Whether Rb1 treat diabetes injured heart remains unknown. This study was to investigate the effect of Rb1 on diabetes injured cardiac muscle tissue and to further investigate its possible molecular pharmacology mechanisms. METHODS: Male Sprague-Dawley rats were injected streptozotocin solution for 2 weeks, followed 6 weeks Rb1 or insulin treatment. The activity of SOD, CAT, Gpx, and the levels of MDA was measured; histological and ultrastructure analyses, RyR2 activity and phosphorylated RyR2(Ser2808) protein expression analyses; and Tunel assay were performed. RESULTS: There was decreased activity of SOD, CAT, Gpx and increased levels of MDA in the diabetic group from control. Rb1 treatment increased activity of SOD, CAT, Gpx and decreased the levels of MDA as compared with diabetic rats. Neutralizing the RyR2 activity significantly decreased in diabetes from control, and increased in Rb1 treatment group from diabetic group. The expression of phosphorylation of RyR2 Ser2808 was increased in diabetic rats from control, and were attenuated with insulin and Rb1 treatment. Diabetes increased the apoptosis rate, and Rb1 treatment decreased the apoptosis rate. Rb1 and insulin ameliorated myocardial injury in diabetic rats. CONCLUSIONS: These data indicate that Rb1 could be useful for mitigating oxidative damage, reduced phosphorylation of RyR2 Ser2808 and decreased the apoptosis rate of cardiomyocytes in diabetic cardiomyopathy.