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1.
Eur Rev Med Pharmacol Sci ; 25(7): 3136-3144, 2021 04.
Artigo em Inglês | MEDLINE | ID: mdl-33877683

RESUMO

OBJECTIVE: Disruption of intracellular Ca2+ homeostasis via excessive and pathological Ca2+ release from the endoplasmic reticulum (ER) and/or sarcoplasmic reticulum (SR) through ryanodine receptor (RyRs) Ca2+ channels play a critical role in the pathology of systemic inflammatory response syndrome (SIRS) and associated multiple organ dysfunction syndrome (MODS) in sepsis or septic shock. Dantrolene, a potent inhibitor of RyRs, is expected to ameliorate SIRS and MODS and decrease mortality in sepsis or septic shock patients. This review summarized the potential mechanisms of therapeutic effects of dantrolene in sepsis or septic shock at molecular, cell, and organ levels and provided suggestions and strategies for future clinical studies.


Assuntos
/tratamento farmacológico , Bloqueadores dos Canais de Cálcio/uso terapêutico , Dantroleno/uso terapêutico , Sepse/tratamento farmacológico , /metabolismo , Cálcio/metabolismo , Reposicionamento de Medicamentos , Retículo Endoplasmático/metabolismo , Humanos , Mortalidade , Insuficiência de Múltiplos Órgãos , Canal de Liberação de Cálcio do Receptor de Rianodina/metabolismo , Retículo Sarcoplasmático/metabolismo , Sepse/metabolismo , Choque Séptico/tratamento farmacológico , Choque Séptico/metabolismo
2.
Arch Biochem Biophys ; 701: 108817, 2021 04 15.
Artigo em Inglês | MEDLINE | ID: mdl-33626379

RESUMO

Protein kinase C is the superfamily of intracellular effector molecules which control crucial cellular functions. Here, we for the first time did the percentage estimation of all known PKC and PKC-related isozymes at the individual cadiomyocyte level. Broad spectrum of PKC transcripts is expressed in the left ventricular myocytes. In addition to the well-known 'heart-specific' PKCα, cardiomyocytes have the high expression levels of PKCN1, PKCδ, PKCD2, PKCε. In general, we detected all PKC isoforms excluding PKCη. In cardiomyocytes PKC activity tonically regulates voltage-gated Ca2+-currents, intracellular Ca2+ level and nitric oxide (NO) production. Imidazoline receptor of the first type (I1R)-mediated induction of the PKC activity positively modulates Ca2+ release through ryanodine receptor (RyR), increasing the Ca2+ leakage in the cytosol. In cardiomyocytes with the Ca2+-overloaded regions of > 9-10 µm size, the local PKC-induced Ca2+ signaling is transformed to global accompanied by spontaneous Ca2+ waves propagation across the entire cell perimeter. Such switching of Ca2+ signaling in cardiac cells can be important for the development of several cardiovascular pathologies and/or myocardial plasticity at the cardiomyocyte level.


Assuntos
Sinalização do Cálcio , Miócitos Cardíacos/enzimologia , Proteína Quinase C/metabolismo , Animais , Isoenzimas/metabolismo , Masculino , Ratos , Ratos Wistar , Canal de Liberação de Cálcio do Receptor de Rianodina/metabolismo
3.
Am J Physiol Cell Physiol ; 320(5): C806-C821, 2021 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-33596151

RESUMO

The purpose of this study was to investigate the mechanism underlying sarcoplasmic reticulum (SR) Ca2+ leakage after in vivo contractions. Rat gastrocnemius muscles were electrically stimulated in vivo, and then mechanically skinned fibers and SR microsomes were prepared from the muscles excised 30 min after repeated high-intensity contractions. The mechanically skinned fibers maintained the interaction between dihydropyridine receptors (DHPRs) and ryanodine receptors (RyRs), whereas the SR microsomes did not. Interestingly, skinned fibers from the stimulated muscles showed increased SR Ca2+ leakage, whereas Ca2+ leakage decreased in SR microsomes from the stimulated muscles. To enhance the orthograde signal of DHPRs, SR Ca2+ leakage in the skinned fiber was measured 1) under a continuously depolarized condition and 2) in the presence of nifedipine. As a result, in either of the two conditions, SR Ca2+ leakage in the rested fibers reached a level similar to that in the stimulated fibers. Furthermore, the increased SR Ca2+ leakage from the stimulated fibers was alleviated by treatment with 1 mM tetracaine (Tet) but not by treatment with 3 mM free Mg2+ (3 Mg). Tet exerted a greater inhibitory effect on the DHPR signal to RyR than 3 Mg, although their inhibitory effects on RyR were almost similar. These results suggest that the increased Ca2+ leakage after muscle contractions is mainly caused by the orthograde signal of DHPRs to RyRs.


Assuntos
Canais de Cálcio Tipo L/metabolismo , Sinalização do Cálcio , Cálcio/metabolismo , Contração Muscular , Fibras Musculares de Contração Rápida/metabolismo , Retículo Sarcoplasmático/metabolismo , Animais , Bloqueadores dos Canais de Cálcio/farmacologia , Canais de Cálcio Tipo L/efeitos dos fármacos , Sinalização do Cálcio/efeitos dos fármacos , Estimulação Elétrica , Masculino , Fibras Musculares de Contração Rápida/efeitos dos fármacos , Fosforilação , Ratos Wistar , Canal de Liberação de Cálcio do Receptor de Rianodina/metabolismo , Retículo Sarcoplasmático/efeitos dos fármacos , Fatores de Tempo
4.
Nucleic Acids Res ; 49(5): 2522-2536, 2021 03 18.
Artigo em Inglês | MEDLINE | ID: mdl-33561291

RESUMO

Simultaneous dysregulation of multiple microRNAs (miRs) affects various pathological pathways related to cardiac failure. In addition to being potential cardiac disease-specific markers, miR-23b/27b/24-1 were reported to be responsible for conferring cardiac pathophysiological processes. In this study, we identified a conserved guanine-rich RNA motif within the miR-23b/27b/24-1 cluster that can form an RNA G-quadruplex (rG4) in vitro and in cells. Disruption of this intragenic rG4 significantly increased the production of all three miRs. Conversely, a G4-binding ligand tetrandrine (TET) stabilized the rG4 and suppressed miRs production in human and rodent cardiomyocytes. Our further study showed that the rG4 prevented Drosha-DGCR8 binding and processing of the pri-miR, suppressing the biogenesis of all three miRs. Moreover, CRISPR/Cas9-mediated G4 deletion in the rat genome aberrantly elevated all three miRs in the heart in vivo, leading to cardiac contractile dysfunction. Importantly, loss of the G4 resulted in reduced targets for the aforementioned miRs critical for normal heart function and defects in the L-type Ca2+ channel-ryanodine receptor (LCC-RyR) coupling in cardiomyocytes. Our results reveal a novel mechanism for G4-dependent regulation of miR biogenesis, which is essential for maintaining normal heart function.


Assuntos
Quadruplex G , MicroRNAs/química , MicroRNAs/metabolismo , Contração Miocárdica/genética , Miócitos Cardíacos/metabolismo , Animais , Benzilisoquinolinas/farmacologia , Sistemas CRISPR-Cas , Células Cultivadas , Quadruplex G/efeitos dos fármacos , Regulação da Expressão Gênica , Miocárdio/metabolismo , Miócitos Cardíacos/fisiologia , Processamento Pós-Transcricional do RNA , Proteínas de Ligação a RNA/metabolismo , Ratos , Ratos Sprague-Dawley , Ribonuclease III/metabolismo , Canal de Liberação de Cálcio do Receptor de Rianodina/metabolismo
5.
Nat Commun ; 12(1): 807, 2021 02 05.
Artigo em Inglês | MEDLINE | ID: mdl-33547325

RESUMO

Ryanodine Receptors (RyRs) are massive channels that release Ca2+ from the endoplasmic and sarcoplasmic reticulum. Hundreds of mutations are linked to malignant hyperthermia (MH), myopathies, and arrhythmias. Here, we explore the first MH mutation identified in humans by providing cryo-EM snapshots of the pig homolog, R615C, showing that it affects an interface between three solenoid regions. We also show the impact of apo-calmodulin (apoCaM) and how it can induce opening by bending of the bridging solenoid, mediated by its N-terminal lobe. For R615C RyR1, apoCaM binding abolishes a pathological 'intermediate' conformation, distributing the population to a mixture of open and closed channels, both different from the structure without apoCaM. Comparisons show that the mutation primarily affects the closed state, inducing partial movements linked to channel activation. This shows that disease mutations can cause distinct pathological conformations of the RyR and facilitate channel opening by disrupting interactions between different solenoid regions.


Assuntos
Apoproteínas/química , Cálcio/química , Calmodulina/química , Hipertermia Maligna/metabolismo , Canal de Liberação de Cálcio do Receptor de Rianodina/química , Substituição de Aminoácidos , Animais , Apoproteínas/genética , Apoproteínas/metabolismo , Arginina/química , Arginina/metabolismo , Cálcio/metabolismo , Calmodulina/genética , Calmodulina/metabolismo , Microscopia Crioeletrônica , Cisteína/química , Cisteína/metabolismo , Expressão Gênica , Humanos , Transporte de Íons , Hipertermia Maligna/genética , Hipertermia Maligna/patologia , Modelos Moleculares , Músculo Esquelético/química , Músculo Esquelético/metabolismo , Mutação , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Canal de Liberação de Cálcio do Receptor de Rianodina/genética , Canal de Liberação de Cálcio do Receptor de Rianodina/metabolismo , Retículo Sarcoplasmático/química , Retículo Sarcoplasmático/metabolismo , Homologia de Sequência de Aminoácidos , Especificidade por Substrato , Suínos
6.
Ecotoxicol Environ Saf ; 213: 112025, 2021 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-33578098

RESUMO

The detailed molecular mechanism of wilforine, a novel botanical insecticidal component, remains unclear, except for the knowledge that it affects the calcium signaling pathway. The aim of the current study was to examine the underlying molecular mechanism of wilforine in Mythimna separata (Walker) by transcriptome and RNA interference (RNAi), with chlorantraniliprole as control. RNA sequencing showed that the relative expression of genes related to the calcium signaling pathway and muscle contraction in M. separata treated with wilforine significantly changed and was further validated by qRT-PCR. Interestingly, the expression level of the ryanodine receptor (MsRyR) gene was downregulated by wilforine at relatively high concentrations and long treatment time, contrary to that observed using chlorantraniliprole. Furthermore, a putative MsRyR was cloned using a 16,258-bp contiguous sequence containing a 308-bp 5'-untranslated region and 578-bp 3'-untranslated region by RT-PCR and RACE. The results of the RNAi experiment showed that injection of dsMsRyR significantly reduced MsRyR mRNA levels, and growth and development were inhibited. Importantly, silencing of the MsRyR gene resulted in decreased susceptibility to both wilforine and chlorantraniliprole. Together with the results of our previous studies on toxic symptoms and muscle tissue lesions between wilforine and chlorantraniliprole, we propose that RyR Ca2+ release channel dysfunction is closely related with significant lethal mechanisms of wilforine.


Assuntos
Inseticidas/toxicidade , Lactonas/toxicidade , Mariposas/fisiologia , Piridinas/toxicidade , Animais , Sinalização do Cálcio/efeitos dos fármacos , Larva/metabolismo , Mariposas/metabolismo , RNA Mensageiro/metabolismo , Rianodina , Canal de Liberação de Cálcio do Receptor de Rianodina/genética , Canal de Liberação de Cálcio do Receptor de Rianodina/metabolismo , Transcriptoma/efeitos dos fármacos , ortoaminobenzoatos
7.
Cell Calcium ; 94: 102342, 2021 03.
Artigo em Inglês | MEDLINE | ID: mdl-33444912

RESUMO

While various hypotheses surrounding the etiology of Alzheimer's disease (AD) have waxed and waned over the years, the calcium hypothesis of aging [1] has maintained its steady trajectory since the early 1990's, albeit often as the understudy. Here, Yao et al., [2] further implicate intracellular calcium dysregulation in AD pathogenesis, and focus the spotlight on the elusive ryanodine receptor-2 isoform.


Assuntos
Doença de Alzheimer , Canal de Liberação de Cálcio do Receptor de Rianodina , Peptídeos beta-Amiloides , Cálcio/metabolismo , Hipocampo/metabolismo , Humanos , Transtornos da Memória , Canal de Liberação de Cálcio do Receptor de Rianodina/metabolismo
8.
Life Sci ; 270: 119016, 2021 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-33515564

RESUMO

AIMS: Ryanodine receptor-1 (RyR1) is essential for skeletal muscle cell functions. However, its roles in vascular smooth muscle cells (SMCs) are well recognized. This study aims to determine the potential physiological importance and difference in systemic and pulmonary artery SMCs (SASMCs and PASMCs). METHODS: Local and global Ca2+ release were measured using a laser scanning confocal microscope and wide-field fluorescence microscope; membrane currents were recorded using a patch clamp recording; muscle contraction was determined using an organ bath system; RyR protein expression was assessed using immunofluorescence staining. Homozygous and heterozygous RyR1 gene knockout (RyR1-/- and RyR1+/-) mice were used to determine its specific functions. KEY FINDINGS: Ca2+ sparks were more prominently decreased in RyR1-/- ASMCs than in PASMCs. Caffeine induced a smaller increase in [Ca2+]i in both RyR1+/+ and RyR1-/- ASMCs than in PASMCs. High K+ produced a reduced [Ca2+]i increase in RyR1-/- PASMCs and ASMCs as well as a reduced contraction in RyR1+/- pulmonary artery and aortic tissues. ATP elicited a smaller increase in [Ca2+]i in RyR1-/- ASMCs and PASMCs with a greater inhibition in ASMCs. Norepinephrine-elicited muscle contraction was reduced in RyR1+/- aortic and pulmonary arteries. IP3 dialysis-induced Ca2+ release was much smaller in RyR1+/- ASMCs and PASMCs. Hypoxia-induced large Ca2+ and contractile responses were inhibited in RyR1+/- PASMCs. However, hypoxic exposure did not evoke a notable increase in [Ca2+]i in ASMCs. SIGNIFICANCE: Our findings for the first time provide clear genetic evidence for the functional importance and difference of RyR1 in systemic and pulmonary artery SMCs.


Assuntos
Sinalização do Cálcio/fisiologia , Miócitos de Músculo Liso/metabolismo , Canal de Liberação de Cálcio do Receptor de Rianodina/metabolismo , Animais , Cálcio/metabolismo , China , Masculino , Potenciais da Membrana/fisiologia , Contração Muscular/efeitos dos fármacos , Músculo Liso Vascular/metabolismo , Artéria Pulmonar/metabolismo , Ratos , Ratos Sprague-Dawley
9.
Eur J Med Chem ; 213: 113160, 2021 Mar 05.
Artigo em Inglês | MEDLINE | ID: mdl-33493827

RESUMO

The hypothesis of rescuing FKBP12/RyR1 interaction and intracellular calcium homeostasis through molecular "reshaping" of FKBP12 was investigated. To this end, novel 4-arylthioalkyl-1-carboxyalkyl-1,2,3-triazoles were designed and synthesized, and their efficacy was tested in human myotubes. A library of 17 compounds (10a-n) designed to dock the FKBP12/RyR1 hot-spot interface contact residues, was readily prepared from free α-amino acids and arylthioalkynes using CuAAC "click" protocols amenable to one-pot transformations in high overall yields and total configurational integrity. To model nitro-oxidative stress, human myotubes were treated with the peroxynitrite donor SIN1, and evidence was found that some triazoles 10 were able to normalize calcium levels, as well as FKBP12/RyR1 interaction. For example, compound 10 b at 150 nM rescued 46% of FKBP12/RyR1 interaction and up to 70% of resting cytosolic calcium levels in human myotubes under nitro-oxidative stress. All compounds 10 analyzed showed target engagement to FKBP12 and low levels of cytotoxicity in vitro. Compounds 10b, 10c, 10h, and 10iR were identified as potential therapeutic candidates to protect myotubes in muscle disorders with underlying nitro-oxidative stress, FKBP12/RyR1 dysfunction and calcium dysregulation.


Assuntos
Cálcio/metabolismo , Descoberta de Drogas , Músculo Esquelético/efeitos dos fármacos , Proteína 1A de Ligação a Tacrolimo/metabolismo , Triazóis/farmacologia , Relação Dose-Resposta a Droga , Células HEK293 , Humanos , Estrutura Molecular , Músculo Esquelético/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Canal de Liberação de Cálcio do Receptor de Rianodina/química , Canal de Liberação de Cálcio do Receptor de Rianodina/metabolismo , Relação Estrutura-Atividade , Proteína 1A de Ligação a Tacrolimo/química , Triazóis/síntese química , Triazóis/química
10.
Cells ; 9(12)2020 12 10.
Artigo em Inglês | MEDLINE | ID: mdl-33321866

RESUMO

Ca2+ homeostasis is essential for multiple neuronal functions and thus, Ca2+ dyshomeostasis can lead to widespread impairment of cellular and synaptic signaling, subsequently contributing to dementia and Alzheimer's disease (AD). While numerous studies implicate Ca2+ mishandling in AD, the cellular basis for loss of cognitive function remains under investigation. The process of synaptic degradation and degeneration in AD is slow, and constitutes a series of maladaptive processes each contributing to a further destabilization of the Ca2+ homeostatic machinery. Ca2+ homeostasis involves precise maintenance of cytosolic Ca2+ levels, despite extracellular influx via multiple synaptic Ca2+ channels, and intracellular release via organelles such as the endoplasmic reticulum (ER) via ryanodine receptor (RyRs) and IP3R, lysosomes via transient receptor potential mucolipin channel (TRPML) and two pore channel (TPC), and mitochondria via the permeability transition pore (PTP). Furthermore, functioning of these organelles relies upon regulated inter-organelle Ca2+ handling, with aberrant signaling resulting in synaptic dysfunction, protein mishandling, oxidative stress and defective bioenergetics, among other consequences consistent with AD. With few effective treatments currently available to mitigate AD, the past few years have seen a significant increase in the study of synaptic and cellular mechanisms as drivers of AD, including Ca2+ dyshomeostasis. Here, we detail some key findings and discuss implications for future AD treatments.


Assuntos
Doença de Alzheimer/metabolismo , Sinalização do Cálcio , Sinapses/fisiologia , Citosol/metabolismo , Retículo Endoplasmático/metabolismo , Humanos , Neurônios/metabolismo , Estresse Oxidativo , Canal de Liberação de Cálcio do Receptor de Rianodina/metabolismo
11.
Nat Commun ; 11(1): 5099, 2020 10 09.
Artigo em Inglês | MEDLINE | ID: mdl-33037202

RESUMO

Mutations in the skeletal muscle Ca2+ release channel, the type 1 ryanodine receptor (RYR1), cause malignant hyperthermia susceptibility (MHS) and a life-threatening sensitivity to heat, which is most severe in children. Mice with an MHS-associated mutation in Ryr1 (Y524S, YS) display lethal muscle contractures in response to heat. Here we show that the heat response in the YS mice is exacerbated by brown fat adaptive thermogenesis. In addition, the YS mice have more brown adipose tissue thermogenic capacity than their littermate controls. Blood lactate levels are elevated in both heat-sensitive MHS patients with RYR1 mutations and YS mice due to Ca2+ driven increases in muscle metabolism. Lactate increases brown adipogenesis in both mouse and human brown preadipocytes. This study suggests that simple lifestyle modifications such as avoiding extreme temperatures and maintaining thermoneutrality could decrease the risk of life-threatening responses to heat and exercise in individuals with RYR1 pathogenic variants.


Assuntos
Hipertermia Maligna/genética , Mutação , Canal de Liberação de Cálcio do Receptor de Rianodina/genética , Termogênese/fisiologia , Tecido Adiposo Marrom/metabolismo , Adolescente , Adulto , Animais , Criança , Pré-Escolar , Feminino , Resposta ao Choque Térmico/genética , Resposta ao Choque Térmico/fisiologia , Humanos , Lactente , Lactatos/sangue , Masculino , Hipertermia Maligna/etiologia , Hipertermia Maligna/mortalidade , Camundongos Endogâmicos C57BL , Camundongos Knockout , Pessoa de Meia-Idade , Estudos Retrospectivos , Canal de Liberação de Cálcio do Receptor de Rianodina/metabolismo , Termogênese/genética , Proteína Desacopladora 1/genética , Adulto Jovem
12.
PLoS Comput Biol ; 16(9): e1007728, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32970668

RESUMO

Calcium oscillations and waves induce depolarization in cardiac cells which are believed to cause life-threathening arrhythimas. In this work, we study the conditions for the appearance of calcium oscillations in both a detailed subcellular model of calcium dynamics and a minimal model that takes into account just the minimal ingredients of the calcium toolkit. To avoid the effects of homeostatic changes and the interaction with the action potential we consider the somewhat artificial condition of a cell without pacing and with no calcium exchange with the extracellular medium. Both the full subcellular model and the minimal model present the same scenarios depending on the calcium load: two stationary states, one with closed ryanodine receptors (RyR) and most calcium in the cell stored in the sarcoplasmic reticulum (SR), and another, with open RyRs and a depleted SR. In between, calcium oscillations may appear. The robustness of these oscillations is determined by the amount of calsequestrin (CSQ). The lack of this buffer in the SR enhances the appearance of oscillations. The minimal model allows us to relate the stability of the oscillating state to the nullcline structure of the system, and find that its range of existence is bounded by a homoclinic and a Hopf bifurcation, resulting in a sudden transition to the oscillatory regime as the cell calcium load is increased. Adding a small amount of noise to the RyR behavior increases the parameter region where oscillations appear and provides a gradual transition from the resting state to the oscillatory regime, as observed in the subcellular model and experimentally.


Assuntos
Cálcio/metabolismo , Miócitos Cardíacos/metabolismo , Animais , Calsequestrina/metabolismo , Modelos Biológicos , Canal de Liberação de Cálcio do Receptor de Rianodina/metabolismo , Retículo Sarcoplasmático/metabolismo , Processos Estocásticos , Frações Subcelulares/metabolismo
13.
Nat Commun ; 11(1): 4734, 2020 09 18.
Artigo em Inglês | MEDLINE | ID: mdl-32948759

RESUMO

A primary reason for the intense interest in structural biology is the fact that knowledge of structure can elucidate macromolecular functions in living organisms. Sustained effort has resulted in an impressive arsenal of tools for determining the static structures. But under physiological conditions, macromolecules undergo continuous conformational changes, a subset of which are functionally important. Techniques for capturing the continuous conformational changes underlying function are essential for further progress. Here, we present chemically-detailed conformational movies of biological function, extracted data-analytically from experimental single-particle cryo-electron microscopy (cryo-EM) snapshots of ryanodine receptor type 1 (RyR1), a calcium-activated calcium channel engaged in the binding of ligands. The functional motions differ substantially from those inferred from static structures in the nature of conformationally active structural domains, the sequence and extent of conformational motions, and the way allosteric signals are transduced within and between domains. Our approach highlights the importance of combining experiment, advanced data analysis, and molecular simulations.


Assuntos
Agonistas dos Canais de Cálcio/química , Substâncias Macromoleculares/química , Canal de Liberação de Cálcio do Receptor de Rianodina/química , Sítios de Ligação , Microscopia Crioeletrônica , Ligantes , Conformação Molecular , Simulação de Dinâmica Molecular , Canal de Liberação de Cálcio do Receptor de Rianodina/metabolismo
14.
Mol Pharmacol ; 98(4): 351-363, 2020 10.
Artigo em Inglês | MEDLINE | ID: mdl-32764093

RESUMO

Ryanodine receptor (RYR) mutations confer stress-triggered malignant hyperthermia (MH) susceptibility. Dietary caffeine (CAF) is the most commonly consumed psychoactive compound by humans. CAF-triggered Ca2+ release and its influences on skeletal muscle contractility are widely used as experimental tools to study RYR function/dysfunction and diagnose MH susceptibility. We hypothesize that dietary CAF achieving blood levels measured in human plasma exacerbates the penetrance of RYR1 MH susceptibility mutations triggered by gaseous anesthetic, affecting both central and peripheral adverse responses. Heterozygous R163C-RYR1 (HET) MH susceptible mice are used to investigate the influences of dietary CAF on both peripheral and central responses before and after induction of halothane (HAL) maintenance anesthesia under experimental conditions that maintain normal core body temperature. HET mice receiving CAF (plasma CAF 893 ng/ml) have significantly shorter times to respiratory arrest compared with wild type, without altering blood chemistry or displaying hyperthermia or muscle rigor. Intraperitoneal bolus dantrolene before HAL prolongs time to respiratory arrest. A pilot electrographic study using subcutaneous electrodes reveals that dietary CAF does not alter baseline electroencephalogram (EEG) total power, but significantly shortens delay to isoelectric EEG, which precedes respiratory and cardiac arrest. CAF ± HAL are studied on RYR1 single-channel currents and HET myotubes to define molecular mechanisms of gene-by-environment synergism. Strong pharmacological synergism between CAF and HAL is demonstrated in both single-channel and myotube preparations. Central and peripheral nervous systems mediate adverse responses to HAL in a HET model of MH susceptibility exposed to dietary CAF, a modifiable lifestyle factor that may mitigate risks of acute and chronic diseases associated with RYR1 mutations. SIGNIFICANCE STATEMENT: Dietary caffeine at a human-relevant dose synergizes adverse peripheral and central responses to anesthesia in malignant hyperthermia susceptible mice. Synergism of these drugs can be attributed to their actions at ryanodine receptors.


Assuntos
Cafeína/efeitos adversos , Dantroleno/efeitos adversos , Halotano/efeitos adversos , Hipertermia Maligna/fisiopatologia , Fibras Musculares Esqueléticas/fisiologia , Mutação , Canal de Liberação de Cálcio do Receptor de Rianodina/genética , Animais , Cafeína/farmacologia , Dantroleno/administração & dosagem , Modelos Animais de Doenças , Sinergismo Farmacológico , Eletroencefalografia/instrumentação , Feminino , Halotano/administração & dosagem , Heterozigoto , Humanos , Injeções Intraperitoneais , Masculino , Hipertermia Maligna/genética , Camundongos , Canal de Liberação de Cálcio do Receptor de Rianodina/metabolismo
15.
Nat Commun ; 11(1): 3527, 2020 07 15.
Artigo em Inglês | MEDLINE | ID: mdl-32669538

RESUMO

Ca2+ signaling in pulmonary arterial smooth muscle cells (PASMCs) plays an important role in pulmonary hypertension (PH). However, the underlying specific ion channel mechanisms remain largely unknown. Here, we report ryanodine receptor (RyR) channel activity and Ca2+ release both are increased, and association of RyR2 by FK506 binding protein 12.6 (FKBP12.6) is decreased in PASMCs from mice with chronic hypoxia (CH)-induced PH. Smooth muscle cell (SMC)-specific RyR2 knockout (KO) or Rieske iron-sulfur protein (RISP) knockdown inhibits the altered Ca2+ signaling, increased nuclear factor (NF)-κB/cyclin D1 activation and cell proliferation, and CH-induced PH in mice. FKBP12.6 KO or FK506 treatment enhances CH-induced PH, while S107 (a specific stabilizer of RyR2/FKBP12.6 complex) produces an opposite effect. In conclusion, CH causes RISP-dependent ROS generation and FKBP12.6/RyR2 dissociation, leading to PH. RISP inhibition, RyR2/FKBP12.6 complex stabilization and Ca2+ release blockade may be potentially beneficial for the treatment of PH.


Assuntos
Ciclina D1/metabolismo , Complexo III da Cadeia de Transporte de Elétrons/metabolismo , Hipertensão Pulmonar/metabolismo , NF-kappa B/metabolismo , Canal de Liberação de Cálcio do Receptor de Rianodina/metabolismo , Proteínas de Ligação a Tacrolimo/metabolismo , Animais , Sinalização do Cálcio , Proliferação de Células , Citosol/metabolismo , Humanos , Hipóxia/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mitocôndrias/metabolismo , Miócitos de Músculo Liso/metabolismo , Oxigênio/metabolismo , Artéria Pulmonar/patologia , Espécies Reativas de Oxigênio/metabolismo , Transtornos Respiratórios/metabolismo , Transdução de Sinais
16.
Nat Commun ; 11(1): 3624, 2020 07 17.
Artigo em Inglês | MEDLINE | ID: mdl-32681036

RESUMO

TRIM family proteins play integral roles in the innate immune response to virus infection. MG53 (TRIM72) is essential for cell membrane repair and is believed to be a muscle-specific TRIM protein. Here we show human macrophages express MG53, and MG53 protein expression is reduced following virus infection. Knockdown of MG53 in macrophages leads to increases in type I interferon (IFN) upon infection. MG53 knockout mice infected with influenza virus show comparable influenza virus titres to wild type mice, but display increased morbidity accompanied by more accumulation of CD45+ cells and elevation of IFNß in the lung. We find that MG53 knockdown results in activation of NFκB signalling, which is linked to an increase in intracellular calcium oscillation mediated by ryanodine receptor (RyR). MG53 inhibits IFNß induction in an RyR-dependent manner. This study establishes MG53 as a new target for control of virus-induced morbidity and tissue injury.


Assuntos
Influenza Humana/imunologia , Interferon beta/metabolismo , Proteínas de Membrana/metabolismo , Canal de Liberação de Cálcio do Receptor de Rianodina/metabolismo , Proteínas com Motivo Tripartido/metabolismo , Animais , Sinalização do Cálcio/imunologia , Linhagem Celular Tumoral , Modelos Animais de Doenças , Técnicas de Silenciamento de Genes , Técnicas de Inativação de Genes , Humanos , Imunidade Inata , Vírus da Influenza A Subtipo H1N1/imunologia , Influenza Humana/virologia , Interferon beta/imunologia , Macrófagos/imunologia , Macrófagos/metabolismo , Masculino , Proteínas de Membrana/genética , Camundongos , Camundongos Knockout , NF-kappa B/metabolismo , RNA Interferente Pequeno , Transdução de Sinais/imunologia , Proteínas com Motivo Tripartido/genética
17.
Proc Natl Acad Sci U S A ; 117(31): 18617-18626, 2020 08 04.
Artigo em Inglês | MEDLINE | ID: mdl-32675240

RESUMO

Genome-wide association studies have identified noncoding variants near TBX3 that are associated with PR interval and QRS duration, suggesting that subtle changes in TBX3 expression affect atrioventricular conduction system function. To explore whether and to what extent the atrioventricular conduction system is affected by Tbx3 dose reduction, we first characterized electrophysiological properties and morphology of heterozygous Tbx3 mutant (Tbx3 +/-) mouse hearts. We found PR interval shortening and prolonged QRS duration, as well as atrioventricular bundle hypoplasia after birth in heterozygous mice. The atrioventricular node size was unaffected. Transcriptomic analysis of atrioventricular nodes isolated by laser capture microdissection revealed hundreds of deregulated genes in Tbx3 +/- mutants. Notably, Tbx3 +/- atrioventricular nodes showed increased expression of working myocardial gene programs (mitochondrial and metabolic processes, muscle contractility) and reduced expression of pacemaker gene programs (neuronal, Wnt signaling, calcium/ion channel activity). By integrating chromatin accessibility profiles (ATAC sequencing) of atrioventricular tissue and other epigenetic data, we identified Tbx3-dependent atrioventricular regulatory DNA elements (REs) on a genome-wide scale. We used transgenic reporter assays to determine the functionality of candidate REs near Ryr2, an up-regulated chamber-enriched gene, and in Cacna1g, a down-regulated conduction system-specific gene. Using genome editing to delete candidate REs, we showed that a strong intronic bipartite RE selectively governs Cacna1g expression in the conduction system in vivo. Our data provide insights into the multifactorial Tbx3-dependent transcriptional network that regulates the structure and function of the cardiac conduction system, which may underlie the differences in PR duration and QRS interval between individuals carrying variants in the TBX3 locus.


Assuntos
Nó Atrioventricular , Proteínas com Domínio T , Transcriptoma/genética , Animais , Arritmias Cardíacas , Nó Atrioventricular/metabolismo , Nó Atrioventricular/fisiologia , Canais de Cálcio Tipo T/genética , Canais de Cálcio Tipo T/metabolismo , Camundongos , Camundongos Transgênicos , Mutação/genética , Canal de Liberação de Cálcio do Receptor de Rianodina/genética , Canal de Liberação de Cálcio do Receptor de Rianodina/metabolismo , Proteínas com Domínio T/genética , Proteínas com Domínio T/metabolismo
18.
Ann N Y Acad Sci ; 1475(1): 43-51, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32483859

RESUMO

There is a significant need to study the binding of active compounds to the specific sites on insect ryanodine receptors (RyRs) that are the targets of two novel classes of diamide insecticides to which insects are becoming increasingly resistant. Here, we describe a rapid assay to study the action of potential compounds on the flubendiamide (Flu) binding site of insect RyRs that uses a fluorescence polarization assay with the fluorescence probe Flu-R-L that we synthesized. The IC50 of Flu for inhibiting probe binding on insect RyR was 18.82 ng/mL. The binding of 86 novel phthalic diamide derivatives on insect RyRs was studied using this newly established assay, and the compounds that exhibited high-affinity binding in the assay also possessed in vivo insecticidal activity against Plutella xylostella. Thus, Flu-R-L is a highly selective and sensitive fluorescence probe for studying the binding affinity of novel compounds to the Flu binding site of insect RyRs. The assay based on Flu-R-L is a rapid, accurate, and sensitive method for the screening of potentially bioactive molecules that bind specifically to insect RyRs.


Assuntos
Corantes Fluorescentes/metabolismo , Mariposas/metabolismo , Canal de Liberação de Cálcio do Receptor de Rianodina/metabolismo , Animais , Benzamidas/química , Sítios de Ligação , Bioensaio , Corantes Fluorescentes/síntese química , Corantes Fluorescentes/química , Cinética , Pirazóis/química , Reprodutibilidade dos Testes , Sulfonas/química , ortoaminobenzoatos/química
19.
PLoS One ; 15(6): e0230465, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32559219

RESUMO

The slow afterhyperpolarising current, sIAHP, is a Ca2+-dependent current that plays an important role in the late phase of spike frequency adaptation. sIAHP is activated by voltage-gated Ca2+ channels, while the contribution of calcium from ryanodine-sensitive intracellular stores, released by calcium-induced calcium release (CICR), is controversial in hippocampal pyramidal neurons. Three types of ryanodine receptors (RyR1-3) are expressed in the hippocampus, with RyR3 showing a predominant expression in CA1 neurons. We investigated the specific role of CICR, and particularly of its RyR3-mediated component, in the regulation of the sIAHP amplitude and time course, and the activity-dependent potentiation of the sIAHP in rat and mouse CA1 pyramidal neurons. Here we report that enhancement of CICR by caffeine led to an increase in sIAHP amplitude, while inhibition of CICR by ryanodine caused a small, but significant reduction of sIAHP. Inhibition of ryanodine-sensitive Ca2+ stores by ryanodine or depletion by the SERCA pump inhibitor cyclopiazonic acid caused a substantial attenuation in the sIAHP activity-dependent potentiation in both rat and mouse CA1 pyramidal neurons. Neurons from mice lacking RyR3 receptors exhibited a sIAHP with features undistinguishable from wild-type neurons, which was similarly reduced by ryanodine. However, the lack of RyR3 receptors led to a faster and reduced activity-dependent potentiation of sIAHP. We conclude that ryanodine receptor-mediated CICR contributes both to the amplitude of the sIAHP at steady state and its activity-dependent potentiation in rat and mouse hippocampal pyramidal neurons. In particular, we show that RyR3 receptors play an essential and specific role in shaping the activity-dependent potentiation of the sIAHP. The modulation of activity-dependent potentiation of sIAHP by RyR3-mediated CICR contributes to plasticity of intrinsic neuronal excitability and is likely to play a critical role in higher cognitive functions, such as learning and memory.


Assuntos
Potenciais de Ação/efeitos dos fármacos , Cálcio/metabolismo , Cálcio/farmacologia , Hipocampo/citologia , Células Piramidais/citologia , Células Piramidais/efeitos dos fármacos , Canal de Liberação de Cálcio do Receptor de Rianodina/metabolismo , Animais , Hipocampo/efeitos dos fármacos , Cinética , Camundongos , Células Piramidais/metabolismo , Ratos
20.
Nat Rev Cardiol ; 17(11): 732-747, 2020 11.
Artigo em Inglês | MEDLINE | ID: mdl-32555383

RESUMO

Ca2+ is a fundamental second messenger in all cell types and is required for numerous essential cellular functions, including cardiac and skeletal muscle contraction. The intracellular concentration of free Ca2+ ([Ca2+]) is regulated primarily by ion channels, pumps (ATPases), exchangers and Ca2+-binding proteins. Defective regulation of [Ca2+] is found in a diverse spectrum of pathological states that affect all the major organs. In the heart, abnormalities in the regulation of cytosolic and mitochondrial [Ca2+] occur in heart failure (HF) and atrial fibrillation (AF), two common forms of heart disease and leading contributors to morbidity and mortality. In this Review, we focus on the mechanisms that regulate ryanodine receptor 2 (RYR2), the major sarcoplasmic reticulum (SR) Ca2+-release channel in the heart, how RYR2 becomes dysfunctional in HF and AF, and its potential as a therapeutic target. Inherited RYR2 mutations and/or stress-induced phosphorylation and oxidation of the protein destabilize the closed state of the channel, resulting in a pathological diastolic Ca2+ leak from the SR that both triggers arrhythmias and impairs contractility. On the basis of our increased understanding of SR Ca2+ leak as a shared Ca2+-dependent pathological mechanism in HF and AF, a new class of drugs developed in our laboratory, known as rycals, which stabilize RYR2 channels and prevent Ca2+ leak from the SR, are undergoing investigation in clinical trials.


Assuntos
Fibrilação Atrial/metabolismo , Cálcio/metabolismo , Insuficiência Cardíaca/metabolismo , Mitocôndrias Cardíacas/metabolismo , Miócitos Cardíacos/metabolismo , Canal de Liberação de Cálcio do Receptor de Rianodina/metabolismo , Proteínas de Ligação a Tacrolimo/metabolismo , Regulação Alostérica , Animais , Arritmias Cardíacas/metabolismo , Fibrilação Atrial/fisiopatologia , Sinalização do Cálcio , Diástole , Progressão da Doença , Acoplamento Excitação-Contração , Insuficiência Cardíaca/fisiopatologia , Humanos , Camundongos , Mutação , Oxirredução , Fosforilação , Estabilidade Proteica , Canal de Liberação de Cálcio do Receptor de Rianodina/genética , Retículo Sarcoplasmático/metabolismo
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