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1.
Aust N Z J Obstet Gynaecol ; 62(4): 500-505, 2022 08.
Artigo em Inglês | MEDLINE | ID: mdl-35220589

RESUMO

BACKGROUND: Women with a prior pregnancy at term are generally considered to be at reduced risk for subsequent spontaneous preterm birth (sPTB), whereas a previous sPTB is a major predictor for a future sPTB. AIMS: The objective of this study was to investigate the risk of recurrent sPTB in women with a prior term birth and a subsequent sPTB. MATERIALS AND METHODS: This is a retrospective cohort study conducted at St Thomas' Hospital in London, UK. There were 430 women included: 230 with a term birth (caesarean section or vaginal delivery) preceding a sPTB (term + sPTB group) and 200 with a prior sPTB only (sPTB only group). The primary outcome was sPTB, <37 weeks gestation. RESULTS: Of the term + sPTB group, 38.7% (89/230) had a recurrent sPTB compared to 20% (40/200) in the sPTB only group (P < 0.0001), with a relative risk (RR) of 1.9. Of women who had a term caesarean section and a subsequent PTB, 50% (30/60) had a further sPTB (RR 2.5 compared to the sPTB only group), while 34.7% (59/170) of women who had a term vaginal birth and subsequent sPTB, had a further sPTB (RR 1.7 compared to the sPTB only group). CONCLUSION: In women who have had a previous sPTB, the risk of a recurrence is much higher than in women with a prior term birth. The aetiology of PTB may be different in this subgroup of women and needs to be further elucidated to determine how best to identify and treat them.


Assuntos
Nascimento Prematuro , Cesárea/efeitos adversos , Feminino , Idade Gestacional , Humanos , Recém-Nascido , Gravidez , Nascimento Prematuro/epidemiologia , Nascimento Prematuro/etiologia , Estudos Retrospectivos
2.
J Muscle Res Cell Motil ; 41(2-3): 251-258, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32902830

RESUMO

Homozygous calsequestrin 2 (CASQ2) point mutations leads to catecholaminergic polymorphic ventricular tachycardia: a common pathogenetic feature appears to be the drastic reduction of mutant CASQ2 in spite of normal transcription. Comparative biochemical analysis of R33Q and D307H knock in mutant mice identifies different pathogenetic mechanisms for CASQ2 degradation and different molecular adaptive mechanisms. In particular, each CASQ2 point mutation evokes specific adaptive cellular and molecular processes in each of the four adaptive pathways investigated. Thus, similar clinical phenotypes and identical cellular mechanism for cardiac arrhythmia might imply different molecular adaptive mechanisms.


Assuntos
Calsequestrina/genética , Mutação Puntual , Taquicardia Ventricular/genética , Animais , Humanos , Masculino , Camundongos
3.
J Muscle Res Cell Motil ; 37(6): 225-233, 2016 12.
Artigo em Inglês | MEDLINE | ID: mdl-28130614

RESUMO

This study investigates the functional role of calsequestrin 2 (CASQ2) in both fast-twitch and slow-twitch skeletal muscles by using CASQ2-/- mice; CASQ2 is expressed throughout life in slow-twitch muscles, but only in the developmental and neonatal stages in fast-twitch muscles. CASQ2-/- causes increase in calsequestrin 1 (CASQ1) expression, but without functional changes in both muscle types. CASQ2-/- mice have ultrastructural changes in fast-twitch muscles only, i.e., formation of pentads and stacks in the sarcoplasmic reticulum.


Assuntos
Calsequestrina/metabolismo , Proteínas Musculares/metabolismo , Músculo Esquelético/metabolismo , Animais , Proteínas de Ligação ao Cálcio/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Músculo Esquelético/fisiologia , Retículo Sarcoplasmático/metabolismo
4.
Exp Cell Res ; 321(2): 178-89, 2014 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-24370574

RESUMO

Cardiac calsequestrin (CASQ2) contributes to intracellular Ca(2+) homeostasis by virtue of its low-affinity/high-capacity Ca(2+) binding properties, maintains sarcoplasmic reticulum (SR) architecture and regulates excitation-contraction coupling, especially or exclusively upon ß-adrenergic stimulation. Catecholaminergic polymorphic ventricular tachycardia (CPVT) is an inherited arrhythmogenic disease associated with cardiac arrest in children or young adults. Recessive CPVT variants are due to mutations in the CASQ2 gene. Molecular and ultra-structural properties were studied in hearts of CASQ2(R33Q/R33Q) and of CASQ2(-/-) mice from post-natal day 2 to week 8. The drastic reduction of CASQ2-R33Q is an early developmental event and is accompanied by down-regulation of triadin and junctin, and morphological changes of jSR and of SR-transverse-tubule junctions. Although endoplasmic reticulum stress is activated, no signs of either apoptosis or autophagy are detected. The other model of recessive CPVT, the CASQ2(-/-) mouse, does not display the same adaptive pattern. Expression of CASQ2-R33Q influences molecular and ultra-structural heart development; post-natal, adaptive changes appear capable of ensuring until adulthood a new pathophysiological equilibrium.


Assuntos
Adaptação Fisiológica/genética , Calsequestrina/genética , Coração/fisiologia , Taquicardia Ventricular/genética , Taquicardia Ventricular/fisiopatologia , Animais , Animais Recém-Nascidos , Modelos Animais de Doenças , Técnicas de Introdução de Genes , Genes Recessivos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Mutação de Sentido Incorreto/fisiologia
5.
Front Microbiol ; 13: 904451, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35774454

RESUMO

The cervicovaginal environment in pregnancy is proposed to influence risk of spontaneous preterm birth. The environment is shaped both by the resident microbiota and local inflammation driven by the host response (epithelia, immune cells and mucous). The contributions of the microbiota, metabolome and host defence peptides have been investigated, but less is known about the immune cell populations and how they may respond to the vaginal environment. Here we investigated the maternal immune cell populations at the cervicovaginal interface in early to mid-pregnancy (10-24 weeks of gestation, samples from N = 46 women), we confirmed neutrophils as the predominant cell type and characterised associations between the cervical neutrophil transcriptome and the cervicovaginal metagenome (N = 9 women). In this exploratory study, the neutrophil cell proportion was affected by gestation at sampling but not by birth outcome or ethnicity. Following RNA sequencing (RNA-seq) of a subset of neutrophil enriched cells, principal component analysis of the transcriptome profiles indicated that cells from seven women clustered closely together these women had a less diverse cervicovaginal microbiota than the remaining three women. Expression of genes involved in neutrophil mediated immunity, activation, degranulation, and other immune functions correlated negatively with Gardnerella vaginalis abundance and positively with Lactobacillus iners abundance; microbes previously associated with birth outcome. The finding that neutrophils are the dominant immune cell type in the cervix during pregnancy and that the cervical neutrophil transcriptome of pregnant women may be modified in response to the microbial cervicovaginal environment, or vice versa, establishes the rationale for investigating associations between the innate immune response, cervical shortening and spontaneous preterm birth and the underlying mechanisms.

6.
Biophys J ; 97(7): 1961-70, 2009 Oct 07.
Artigo em Inglês | MEDLINE | ID: mdl-19804727

RESUMO

Sarcoplasmic reticulum (SR) Ca(2+) release in striated muscle is mediated by a multiprotein complex that includes the ryanodine receptor (RyR) Ca(2+) channel and the intra-SR Ca(2+) buffering protein calsequestrin (CSQ). Besides its buffering role, CSQ is thought to regulate RyR channel function. Here, CSQ-dependent luminal Ca(2+) regulation of skeletal (RyR1) and cardiac (RyR2) channels is explored. Skeletal (CSQ1) or cardiac (CSQ2) calsequestrin were systematically added to the luminal side of single RyR1 or RyR2 channels. The luminal Ca(2+) dependence of open probability (Po) over the physiologically relevant range (0.05-1 mM Ca(2+)) was defined for each of the four RyR/CSQ isoform pairings. We found that the luminal Ca(2+) sensitivity of single RyR2 channels was substantial when either CSQ isoform was present. In contrast, no significant luminal Ca(2+) sensitivity of single RyR1 channels was detected in the presence of either CSQ isoform. We conclude that CSQ-dependent luminal Ca(2+) regulation of single RyR2 channels lacks CSQ isoform specificity, and that CSQ-dependent luminal Ca(2+) regulation in skeletal muscle likely plays a relatively minor (if any) role in regulating the RyR1 channel activity, indicating that the chief role of CSQ1 in this tissue is as an intra-SR Ca(2+) buffer.


Assuntos
Cálcio/metabolismo , Calsequestrina/metabolismo , Canal de Liberação de Cálcio do Receptor de Rianodina/metabolismo , Animais , Citosol/metabolismo , Músculo Esquelético/citologia , Miocárdio/citologia , Isoformas de Proteínas/metabolismo , Coelhos
7.
Biochem J ; 413(2): 291-303, 2008 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-18399795

RESUMO

Two missense mutations, R33Q and L167H, of hCASQ2 (human cardiac calsequestrin), a protein segregated to the lumen of the sarcoplasmic reticulum, are linked to the autosomal recessive form of CPVT (catecholaminergic polymorphic ventricular tachycardia). The effects of these mutations on the conformational, stability and Ca(2+) sensitivity properties of hCASQ2, were investigated. Recombinant WT (wild-type) and mutant CASQ2s were purified to homogeneity and characterized by spectroscopic (CD and fluorescence) and biochemical (size-exclusion chromatography and limited proteolysis) methods at 500 and 100 mM KCl, with or without Ca(2+) at a physiological intraluminal concentration of 1 mM; Ca(2+)-induced polymerization properties were studied by turbidimetry. In the absence of Ca(2+), mutations did not alter the conformation of monomeric CASQ2. For L167H only, at 100 mM KCl, emission fluorescence changes suggested tertiary structure alterations. Limited proteolysis showed that amino acid substitutions enhanced the conformational flexibility of CASQ2 mutants, which became more susceptible to tryptic cleavage, in the order L167H>R33Q>WT. Ca(2+) at a concentration of 1 mM amplified such differences: Ca(2+) stabilized WT CASQ2 against urea denaturation and tryptic cleavage, whereas this effect was reduced in R33Q and absent in L167H. Increasing [Ca(2+)] induced polymerization and precipitation of R33Q, but not that of L167H, which was insensitive to Ca(2+). Based on CASQ2 models, we propose that the Arg(33)-->Gln exchange made the Ca(2+)-dependent formation of front-to-front dimers more difficult, whereas the Leu(167)-->His replacement almost completely inhibited back-to-back dimer interactions. Initial molecular events of CPVT pathogenesis begin to unveil and appear to be different depending upon the specific CASQ2 mutation.


Assuntos
Cálcio/metabolismo , Calsequestrina/genética , Catecolaminas/metabolismo , Mutação , Miocárdio/metabolismo , Polimorfismo Genético , Taquicardia Ventricular/genética , Animais , Cálcio/química , Proteínas de Ligação ao Cálcio/química , Humanos , Conformação Proteica , Ratos , Proteínas Recombinantes/química , Espectrometria de Fluorescência/métodos , Espectrofotometria Ultravioleta/métodos
8.
Biomed Res Int ; 2019: 8748253, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31080832

RESUMO

Aging is related to a number of functional and morphological changes leading to progressive decline of the biological functions of an organism. Reactive Oxygen Species (ROS), released by several endogenous and exogenous processes, may cause important oxidative damage to DNA, proteins, and lipids, leading to important cellular dysfunctions. The imbalance between ROS production and antioxidant defenses brings to oxidative stress conditions and, related to accumulation of ROS, aging-associated diseases. The purpose of this review is to provide an overview of the most relevant data reported in literature on the natural compounds, mainly phytochemicals, with antioxidant activity and their potential protective effects on age-related diseases such as metabolic syndrome, diabetes, cardiovascular disease, cancer, neurodegenerative disease, and chronic inflammation, and possibly lower side effects, when compared to other drugs.


Assuntos
Envelhecimento/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , Compostos Fitoquímicos/farmacologia , Compostos Fitoquímicos/uso terapêutico , Envelhecimento/metabolismo , Animais , Antioxidantes/metabolismo , Antioxidantes/uso terapêutico , Humanos , Síndrome Metabólica/tratamento farmacológico , Síndrome Metabólica/metabolismo , Doenças Neurodegenerativas/tratamento farmacológico , Doenças Neurodegenerativas/metabolismo , Espécies Reativas de Oxigênio/metabolismo
9.
Biophys J ; 95(4): 2037-48, 2008 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-18469084

RESUMO

Cardiac calsequestrin (CASQ2) is an intrasarcoplasmic reticulum (SR) low-affinity Ca-binding protein, with mutations that are associated with catecholamine-induced polymorphic ventricular tachycardia (CPVT). To better understand how CASQ2 mutants cause CPVT, we expressed two CPVT-linked CASQ2 mutants, a truncated protein (at G112+5X, CASQ2(DEL)) or CASQ2 containing a point mutation (CASQ2(R33Q)), in canine ventricular myocytes and assessed their effects on Ca handling. We also measured CASQ2-CASQ2 variant interactions using fluorescence resonance transfer in a heterologous expression system, and evaluated CASQ2 interaction with triadin. We found that expression of CASQ2(DEL) or CASQ2(R33Q) altered myocyte Ca signaling through two different mechanisms. Overexpressing CASQ2(DEL) disrupted the CASQ2 polymerization required for high capacity Ca binding, whereas CASQ2(R33Q) compromised the ability of CASQ2 to control ryanodine receptor (RyR2) channel activity. Despite profound differences in SR Ca buffering strengths, local Ca release terminated at the same free luminal [Ca] in control cells, cells overexpressing wild-type CASQ2 and CASQ2(DEL)-expressing myocytes, suggesting that a decline in [Ca](SR) is a signal for RyR2 closure. Importantly, disrupting interactions between the RyR2 channel and CASQ2 by expressing CASQ2(R33Q) markedly lowered the [Ca](SR) threshold for Ca release termination. We conclude that CASQ2 in the SR determines the magnitude and duration of Ca release from each SR terminal by providing both a local source of releasable Ca and by effects on luminal Ca-dependent RyR2 gating. Furthermore, two CPVT-inducing CASQ2 mutations, which cause mechanistically different defects in CASQ2 and RyR2 function, lead to increased diastolic SR Ca release events and exhibit a similar CPVT disease phenotype.


Assuntos
Sinalização do Cálcio , Cálcio/metabolismo , Calsequestrina/metabolismo , Morte Súbita Cardíaca , Miócitos Cardíacos/metabolismo , Retículo Sarcoplasmático/metabolismo , Animais , Calsequestrina/genética , Células Cultivadas , Cães , Humanos
10.
Circ Res ; 98(9): 1151-8, 2006 May 12.
Artigo em Inglês | MEDLINE | ID: mdl-16601229

RESUMO

Catecholaminergic polymorphic ventricular tachycardia (CPVT) is a familial arrhythmogenic disorder associated with mutations in the cardiac ryanodine receptor (RyR2) and cardiac calsequestrin (CASQ2) genes. Previous in vitro studies suggested that RyR2 and CASQ2 interact as parts of a multimolecular Ca(2+)-signaling complex; however, direct evidence for such interactions and their potential significance to myocardial function remain to be determined. We identified a novel CASQ2 mutation in a young female with a structurally normal heart and unexplained syncopal episodes. This mutation results in the nonconservative substitution of glutamine for arginine at amino acid 33 of CASQ2 (R33Q). Adenoviral-mediated expression of CASQ2(R33Q) in adult rat myocytes led to an increase in excitation-contraction coupling gain and to more frequent occurrences of spontaneous propagating (Ca2+ waves) and local Ca2+ signals (sparks) with respect to control cells expressing wild-type CASQ2 (CASQ2WT). As revealed by a Ca2+ indicator entrapped inside the sarcoplasmic reticulum (SR) of permeabilized myocytes, the increased occurrence of spontaneous Ca2+ sparks and waves was associated with a dramatic decrease in intra-SR [Ca2+]. Recombinant CASQ2WT and CASQ2R33Q exhibited similar Ca(2+)-binding capacities in vitro; however, the mutant protein lacked the ability of its WT counterpart to inhibit RyR2 activity at low luminal [Ca2+] in planar lipid bilayers. We conclude that the R33Q mutation disrupts interactions of CASQ2 with the RyR2 channel complex and impairs regulation of RyR2 by luminal Ca2+. These results show that intracellular Ca2+ cycling in normal heart relies on an intricate interplay of CASQ2 with the proteins of the RyR2 channel complex and that disruption of these interactions can lead to cardiac arrhythmia.


Assuntos
Calsequestrina/metabolismo , Morte Súbita Cardíaca/etiologia , Exercício Físico , Canal de Liberação de Cálcio do Receptor de Rianodina/metabolismo , Taquicardia Ventricular/genética , Substituição de Aminoácidos , Animais , Arginina , Ligação Competitiva , Cálcio/metabolismo , Calsequestrina/genética , Estimulação Cardíaca Artificial/métodos , Catecolaminas/metabolismo , Feminino , Glutamina , Humanos , Membranas Intracelulares/metabolismo , Mutação , Miócitos Cardíacos/metabolismo , Técnicas de Patch-Clamp , Ratos , Proteínas Recombinantes/metabolismo , Retículo Sarcoplasmático/metabolismo , Síncope/genética , Taquicardia Ventricular/metabolismo , Taquicardia Ventricular/fisiopatologia
11.
Circulation ; 114(10): 1012-9, 2006 Sep 05.
Artigo em Inglês | MEDLINE | ID: mdl-16908766

RESUMO

BACKGROUND: Four distinct mutations in the human cardiac calsequestrin gene (CASQ2) have been linked to catecholaminergic polymorphic ventricular tachycardia (CPVT). The mechanisms leading to the clinical phenotype are still poorly understood because only 1 CASQ2 mutation has been characterized in vitro. METHODS AND RESULTS: We identified a homozygous 16-bp deletion at position 339 to 354 leading to a frame shift and a stop codon after 5aa (CASQ2(G112+5X)) in a child with stress-induced ventricular tachycardia and cardiac arrest. The same deletion was also identified in association with a novel point mutation (CASQ2(L167H)) in a highly symptomatic CPVT child who is the first CPVT patient carrier of compound heterozygous CASQ2 mutations. We characterized in vitro the properties of CASQ2 mutants: CASQ2(G112+5X) did not bind Ca2+, whereas CASQ2(L167H) had normal calcium-binding properties. When expressed in rat myocytes, both mutants decreased the sarcoplasmic reticulum Ca2+-storing capacity and reduced the amplitude of I(Ca)-induced Ca2+ transients and of spontaneous Ca2+ sparks in permeabilized myocytes. Exposure of myocytes to isoproterenol caused the development of delayed afterdepolarizations in CASQ2(G112+5X). CONCLUSIONS: CASQ2(L167H) and CASQ2(G112+5X) alter CASQ2 function in cardiac myocytes, which leads to reduction of active sarcoplasmic reticulum Ca2+ release and calcium content. In addition, CASQ2(G112+5X) displays altered calcium-binding properties and leads to delayed afterdepolarizations. We conclude that the 2 CASQ2 mutations identified in CPVT create distinct abnormalities that lead to abnormal intracellular calcium regulation, thus facilitating the development of tachyarrhythmias.


Assuntos
Calsequestrina/genética , Síncope/genética , Taquicardia Ventricular/genética , Substituição de Aminoácidos , Animais , Criança , Feminino , Técnicas de Transferência de Genes , Triagem de Portadores Genéticos , Humanos , Masculino , Células Musculares/fisiologia , Mutagênese Sítio-Dirigida , Mutação , Linhagem , Mutação Puntual , Ratos , Taquicardia Ventricular/fisiopatologia , Transfecção
12.
Biochem J ; 379(Pt 2): 505-12, 2004 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-14728599

RESUMO

CS (calsequestrin) is an acidic glycoprotein of the SR (sarcoplasmic reticulum) lumen and plays a crucial role in the storage of Ca2+ and in excitation-contraction coupling of skeletal muscles. CS is synthesized in the ER (endoplasmic reticulum) and is targeted to the TC (terminal cisternae) of SR via mechanisms still largely unknown, but probably involving vesicle transport through the Golgi complex. In the present study, two mutant forms of Sar1 and ARF1 (ADP-ribosylation factor 1) were used to disrupt cargo exit from ER-exit sites and intra-Golgi trafficking in skeletal-muscle fibres respectively. Co-expression of Sar1-H79G (His79-->Gly) and recombinant, epitope-tagged CS, CSHA1 (where HA1 stands for nine-amino-acid epitope of the viral haemagglutinin 1), barred segregation of CSHA1 to TC. On the other hand, expression of ARF1-N126I altered the subcellular localization of GM130, a cis -medial Golgi protein in skeletal-muscle fibres and myotubes, without interfering with CSHA1 targeting to either TC or developing SR. Thus active budding from ER-exit sites appears to be involved in CS targeting and routing, but these processes are insensitive to modification of intracellular vesicle trafficking and Golgi complex disruption caused by the mutant ARF1-N126I. It also appears that CS routing from ER to SR does not involve classical secretory pathways through ER-Golgi intermediate compartments, cis -medial Golgi and trans -Golgi network.


Assuntos
Calsequestrina/metabolismo , Retículo Endoplasmático/metabolismo , Músculo Esquelético/metabolismo , Retículo Sarcoplasmático/metabolismo , Vesículas Transportadoras/fisiologia , Fator 1 de Ribosilação do ADP/análise , Fator 1 de Ribosilação do ADP/genética , Animais , Animais Geneticamente Modificados , Autoantígenos , Calsequestrina/genética , Linhagem Celular , Hemaglutininas Virais/genética , Masculino , Proteínas de Membrana/análise , Camundongos , Microscopia de Fluorescência , Proteínas Monoméricas de Ligação ao GTP/análise , Proteínas Monoméricas de Ligação ao GTP/genética , Fibras Musculares Esqueléticas/metabolismo , Músculo Esquelético/química , Músculo Esquelético/ultraestrutura , Transporte Proteico , Ratos , Ratos Wistar , Proteínas Recombinantes de Fusão/análise , Proteínas Recombinantes de Fusão/metabolismo
13.
J Gen Physiol ; 142(2): 127-36, 2013 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-23858002

RESUMO

Release of Ca(2+) from the sarcoplasmic reticulum (SR) drives contractile function of cardiac myocytes. Luminal Ca(2+) regulation of SR Ca(2+) release is fundamental not only in physiology but also in physiopathology because abnormal luminal Ca(2+) regulation is known to lead to arrhythmias, catecholaminergic polymorphic ventricular tachycardia (CPVT), and/or sudden cardiac arrest, as inferred from animal model studies. Luminal Ca(2+) regulates ryanodine receptor (RyR)2-mediated SR Ca(2+) release through mechanisms localized inside the SR; one of these involves luminal Ca(2+) interacting with calsequestrin (CASQ), triadin, and/or junctin to regulate RyR2 function. CASQ2-RyR2 regulation was examined at the single RyR2 channel level. Single RyR2s were incorporated into planar lipid bilayers by the fusion of native SR vesicles isolated from either wild-type (WT), CASQ2 knockout (KO), or R33Q-CASQ2 knock-in (KI) mice. KO and KI mice have CPVT-like phenotypes. We show that CASQ2(WT) action on RyR2 function (either activation or inhibition) was strongly influenced by the presence of cytosolic MgATP. Function of the reconstituted CASQ2(WT)-RyR2 complex was unaffected by changes in luminal free [Ca(2+)] (from 0.1 to 1 mM). The inhibition exerted by CASQ2(WT) association with the RyR2 determined a reduction in cytosolic Ca(2+) activation sensitivity. RyR2s from KO mice were significantly more sensitive to cytosolic Ca(2+) activation and had significantly longer mean open times than RyR2s from WT mice. Sensitivity of RyR2s from KI mice was in between that of RyR2 channels from KO and WT mice. Enhanced cytosolic RyR2 Ca(2+) sensitivity and longer RyR2 open times likely explain the CPVT-like phenotype of both KO and KI mice.


Assuntos
Sinalização do Cálcio , Proteínas de Ligação ao Cálcio/metabolismo , Calsequestrina/metabolismo , Ventrículos do Coração/metabolismo , Canal de Liberação de Cálcio do Receptor de Rianodina/metabolismo , Trifosfato de Adenosina/metabolismo , Animais , Cálcio/metabolismo , Calsequestrina/genética , Ventrículos do Coração/patologia , Membranas Intracelulares/metabolismo , Ativação do Canal Iônico , Bicamadas Lipídicas/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Mutação , Ratos
14.
J Gen Physiol ; 131(4): 325-34, 2008 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-18347081

RESUMO

The luminal Ca2+ regulation of cardiac ryanodine receptor (RyR2) was explored at the single channel level. The luminal Ca2+ and Mg2+ sensitivity of single CSQ2-stripped and CSQ2-associated RyR2 channels was defined. Action of wild-type CSQ2 and of two mutant CSQ2s (R33Q and L167H) was also compared. Two luminal Ca2+ regulatory mechanism(s) were identified. One is a RyR2-resident mechanism that is CSQ2 independent and does not distinguish between luminal Ca2+ and Mg2+. This mechanism modulates the maximal efficacy of cytosolic Ca2+ activation. The second luminal Ca2+ regulatory mechanism is CSQ2 dependent and distinguishes between luminal Ca2+ and Mg2+. It does not depend on CSQ2 oligomerization or CSQ2 monomer Ca2+ binding affinity. The key Ca2+-sensitive step in this mechanism may be the Ca2+-dependent CSQ2 interaction with triadin. The CSQ2-dependent mechanism alters the cytosolic Ca2+ sensitivity of the channel. The R33Q CSQ2 mutant can participate in luminal RyR2 Ca2+ regulation but less effectively than wild-type (WT) CSQ2. CSQ2-L167H does not participate in luminal RyR2 Ca2+ regulation. The disparate actions of these two catecholaminergic polymorphic ventricular tachycardia (CPVT)-linked mutants implies that either alteration or elimination of CSQ2-dependent luminal RyR2 regulation can generate the CPVT phenotype. We propose that the RyR2-resident, CSQ2-independent luminal Ca2+ mechanism may assure that all channels respond robustly to large (>5 muM) local cytosolic Ca2+ stimuli, whereas the CSQ2-dependent mechanism may help close RyR2 channels after luminal Ca2+ falls below approximately 0.5 mM.


Assuntos
Cálcio/química , Calsequestrina/metabolismo , Proteínas Mutantes/metabolismo , Miócitos Cardíacos/metabolismo , Canal de Liberação de Cálcio do Receptor de Rianodina/química , Substituição de Aminoácidos , Animais , Cálcio/metabolismo , Sinalização do Cálcio , Calsequestrina/química , Citosol/química , Citosol/metabolismo , Dimerização , Condutividade Elétrica , Ativação do Canal Iônico/fisiologia , Bicamadas Lipídicas , Magnésio/química , Magnésio/metabolismo , Microssomos , Proteínas Mutantes/química , Técnicas de Patch-Clamp , Ratos , Canal de Liberação de Cálcio do Receptor de Rianodina/metabolismo , Retículo Sarcoplasmático/metabolismo , Relação Estrutura-Atividade , Taquicardia Ventricular/etiologia , Taquicardia Ventricular/fisiopatologia
15.
Am J Physiol Cell Physiol ; 291(2): C245-53, 2006 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-16571864

RESUMO

Calsequestrin (CS) is the low-affinity, high-capacity calcium binding protein segregated to the lumen of terminal cisternae (TC) of the sarcoplasmic reticulum (SR). The physiological role of CS in controlling calcium release from the SR depends on both its intrinsic properties and its localization. The mechanisms of CS targeting were investigated in skeletal muscle fibers and C2C12 myotubes, a model of SR differentiation, with four deletion mutants of epitope (hemagglutinin, HA)-tagged CS: CS-HA24NH2, CS-HA2D, CS-HA3D, and CS-HAHT, a double mutant of the NH2 terminus and domain III. As judged by immunofluorescence of transfected skeletal muscle fibers, only the double CS-HA mutant showed a homogeneous distribution at the sarcomeric I band, i.e., it did not segregate to TC. As shown by subfractionation of microsomes derived from transfected skeletal muscles, CS-HAHT was largely associated to longitudinal SR whereas CS-HA was concentrated in TC. In C2C12 myotubes, as judged by immunofluorescence, not only CS-HAHT but also CS-HA3D and CS-HA2D were not sorted to developing SR. Condensation competence, a property referable to CS oligomerization, was monitored for the several CS-HA mutants in C2C12 myoblasts, and only CS-HA3D was found able to condense. Together, the results indicate that 1) there are at least two targeting sequences at the NH2 terminus and domain III of CS, 2) SR-specific target and structural information is contained in these sequences, 3) heterologous interactions with junctional SR proteins are relevant for segregation, 4) homologous CS-CS interactions are involved in the overall targeting process, and 5) different targeting mechanisms prevail depending on the stage of SR differentiation.


Assuntos
Calsequestrina/metabolismo , Fibras Musculares Esqueléticas/metabolismo , Músculo Esquelético/metabolismo , Retículo Sarcoplasmático/metabolismo , Animais , Sítios de Ligação , Linhagem Celular , Camundongos , Ligação Proteica
16.
Biochem Biophys Res Commun ; 316(3): 884-92, 2004 Apr 09.
Artigo em Inglês | MEDLINE | ID: mdl-15033484

RESUMO

mRNA transcripts for Homer 1a and Homer 1c have been detected in skeletal muscle [Biochem. Biophys. Res. Commun. 279 (2000) 348]. Here, the subcellular distribution of recombinant HA1-tagged Homer 1c and HA1-tagged Homer 1a was investigated in C(2)C(12) myotubes and in transgenic skeletal muscle fibers of the adult rat by epifluorescent and confocal microscopy. In C(2)C(12) myotubes, Homer 1a was homogeneously localized in the cytosol and also labeled some nuclei whereas Homer 1c displayed a diffuse reticular/punctuate pattern in the cytosol with scattered punctuate labeling around nuclei; no co-localization was observed with the ryanodine receptor/Ca(2+) release channel (RYR1). The subcellular localization of the Homer 1 isoforms was markedly different in transgenic muscle fibers: Homer 1c was diffusely distributed at the I band and enlightened the Z line, whereas Homer 1a labeled both the I band and the A band with distinct reinforcement of the H line; neither Homer 1c nor Homer 1a co-localized with either calsequestrin or RYR1, two sarcoplasmic reticulum markers. Our findings are discussed in relation to reported effects of Homer 1 isoforms on RYR1 function.


Assuntos
Proteínas de Transporte/química , Fibras Musculares Esqueléticas/metabolismo , Músculo Esquelético/metabolismo , Neuropeptídeos/química , Animais , Animais Geneticamente Modificados , Western Blotting , Calsequestrina/metabolismo , Proteínas de Transporte/metabolismo , Linhagem Celular , Núcleo Celular/metabolismo , Clonagem Molecular , Citosol/metabolismo , DNA Complementar/metabolismo , Eletroforese em Gel de Poliacrilamida , Proteínas de Arcabouço Homer , Microscopia Confocal , Microscopia de Fluorescência , Músculo Esquelético/citologia , Neuropeptídeos/metabolismo , Ligação Proteica , Isoformas de Proteínas , Ratos , Ratos Wistar , Canal de Liberação de Cálcio do Receptor de Rianodina/metabolismo , Retículo Sarcoplasmático/metabolismo , Transfecção
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