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1.
Adv Exp Med Biol ; 1131: 131-161, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31646509

RESUMO

Calcium (Ca2+) is a fundamental regulator of cell fate and intracellular Ca2+ homeostasis is crucial for proper function of the nerve cells. Given the complexity of neurons, a constellation of mechanisms finely tunes the intracellular Ca2+ signaling. We are focusing on the sarco/endoplasmic reticulum (SR/ER) calcium (Ca2+)-ATPase (SERCA) pump, an integral ER protein. SERCA's well established role is to preserve low cytosolic Ca2+ levels ([Ca2+]cyt), by pumping free Ca2+ ions into the ER lumen, utilizing ATP hydrolysis. The SERCA pumps are encoded by three distinct genes, SERCA1-3, resulting in 12 known protein isoforms, with tissue-dependent expression patterns. Despite the well-established structure and function of the SERCA pumps, their role in the central nervous system is not clear yet. Interestingly, SERCA-mediated Ca2+ dyshomeostasis has been associated with neuropathological conditions, such as bipolar disorder, schizophrenia, Parkinson's disease and Alzheimer's disease. We summarize here current evidence suggesting a role for SERCA in the neurobiology of neuropsychiatric and neurodegenerative disorders, thus highlighting the importance of this pump in brain physiology and pathophysiology.


Assuntos
Encéfalo , Retículo Endoplasmático , Doenças do Sistema Nervoso , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático , Encéfalo/enzimologia , Encéfalo/patologia , Retículo Endoplasmático/enzimologia , Regulação Enzimológica da Expressão Gênica , Homeostase , Humanos , Doenças do Sistema Nervoso/enzimologia , Doenças do Sistema Nervoso/fisiopatologia , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/genética , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/metabolismo
2.
Zhonghua Yi Xue Yi Chuan Xue Za Zhi ; 36(8): 794-797, 2019 Aug 10.
Artigo em Chinês | MEDLINE | ID: mdl-31400130

RESUMO

OBJECTIVE: To explore the molecular basis for a pedigree affected with Darier-White disease. METHODS: Genomic DNA was isolated from 3 patients and 1 unaffected member from the pedigree, as well as 80 healthy controls. Targeted sequence capture and next-generation sequencing were used to screen mutations of skin disease-related genes. Candidate mutations were verified by Sanger sequencing, and co-segregation analysis was carried out to confirm the pathogenicity of mutation. Conservation analysis and protein structure and function were also predicted with Bioinformatic tools. RESULTS: A heterozygous mutation c.2246G>T (p.G749V) was identified in exon 15 of ATP2A2 gene in all 3 patients from the pedigree, but not in the unaffected member or 80 healthy controls. The corresponding amino acid was highly conserved, and mutation of which can lead to structural and functional changes of the protein. CONCLUSION: The c.2246G>T missense mutation of the ATP2A2 gene probably underlies the Darier-White disease in this pedigree by causing damages to the structure and function of sarcoplasmic/endoplasmic reticulum calcium ATPase 2 (SERCA2).


Assuntos
Doença de Darier/genética , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/genética , Heterozigoto , Humanos , Mutação de Sentido Incorreto , Linhagem
3.
BMC Cancer ; 19(1): 381, 2019 Apr 25.
Artigo em Inglês | MEDLINE | ID: mdl-31023247

RESUMO

BACKGROUND: Salinomycin is a monocarboxylic polyether antibiotic and is a potential chemotherapy drug. Our previous studies showed that salinomycin inhibited cell growth and targeted CSCs in prostate cancer. However, the precise target of salinomycin action is unclear. METHODS: In this work, we analyzed and identified differentially expressed genes (DEGs) after treatment with or without salinomycin using a gene expression microarray in vitro (PC-3 cells) and in vivo (NOD/SCID mice xenograft model generated from implanted PC-3 cells). Western blotting and immunohistochemical staining were used to analyze the expression of ATP2A3 and endoplasmic reticulum (ER) stress biomarkers. Flow cytometry was used to analyze the cell cycle, apoptosis and intracellular Ca2+ concentration. RESULTS: A significantly upregulated gene, ATPase sarcoplasmatic/endoplasmatic reticulum Ca2+ transporting 3 (ATP2A3), was successfully identified. In subsequent studies, we found that ATP2A3 overexpression could trigger ER stress and exert anti-cancer effects in PC-3 and DU145 cells. ATP2A3 was slightly expressed, but the ER stress biomarkers showed strong staining in prostate cancer tissues. We also found that salinomycin could trigger ER stress, which might be related to ATP2A3-mediated Ca2+ release in PC-3 cells. Furthermore, we found that salinomycin-triggered ER stress could promote apoptosis and thus exert anti-cancer effects in prostate cancer cells. CONCLUSION: This study demonstrates that ATP2A3 might be one of the potential targets for salinomycin, which can inhibit Ca2+ release and trigger ER stress to exert anti-cancer effects.


Assuntos
Estresse do Retículo Endoplasmático/efeitos dos fármacos , Neoplasias da Próstata/tratamento farmacológico , Piranos/administração & dosagem , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/genética , Animais , Apoptose/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Masculino , Camundongos , Células PC-3 , Neoplasias da Próstata/genética , Neoplasias da Próstata/patologia , Transdução de Sinais/efeitos dos fármacos , Ativação Transcricional/genética , Ensaios Antitumorais Modelo de Xenoenxerto
4.
Molecules ; 24(3)2019 Feb 02.
Artigo em Inglês | MEDLINE | ID: mdl-30717322

RESUMO

Arsenic trioxide (ATO) has been verified as a breakthrough with respect to the management of acute promyelocytic leukemia (APL) in recent decades but associated with some serious adverse phenomena, particularly cardiac functional abnormalities. Salvianolic acid A (Sal A) is a major effective component in treating ATO-induced cardiotoxicity. Therefore, the objective of our study was to assess whether Sal A had protective effects by the regulation of calcium homeostasis and endoplasmic reticulum (ER) stress. For the in vivo study, BALB/c mice were treated with ATO and/or Sal A via daily tail vein injections for two weeks. For the in vitro study, we detected the effects of ATO and/or Sal A in real time using adult rat ventricular myocytes (ARVMs) and an IonOptix MyoCam system. Our results showed that Sal A pretreatment alleviated cardiac dysfunction and Ca2+ overload induced by ATO in vivo and vitro. Moreover, Sal A increased sarcoplasmic reticulum (SR) Ca2+-ATPase (SERCA) activity and expression, alleviated [Ca2+]ER depletion, and decreased ER stress-related protein expression. Sal A protects the heart from ATO-induced injury and its administration correlates with the modulation of SERCA, the recovery of Ca2+ homeostasis, and the down-regulation of ER stress-mediated apoptosis.


Assuntos
Trióxido de Arsênio/efeitos adversos , Ácidos Cafeicos/administração & dosagem , Cardiotoxicidade/tratamento farmacológico , Lactatos/administração & dosagem , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/genética , Animais , Apoptose/efeitos dos fármacos , Trióxido de Arsênio/administração & dosagem , Cálcio/metabolismo , Cardiotoxicidade/etiologia , Modelos Animais de Doenças , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos dos fármacos , Homeostase/efeitos dos fármacos , Humanos , Leucemia Promielocítica Aguda/complicações , Leucemia Promielocítica Aguda/tratamento farmacológico , Camundongos , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/enzimologia , Ratos , Retículo Sarcoplasmático/efeitos dos fármacos
5.
J Agric Food Chem ; 67(11): 3242-3248, 2019 Mar 20.
Artigo em Inglês | MEDLINE | ID: mdl-30807139

RESUMO

The research was performed to investigate the difference of activity, expression, and S-nitrosylation of calcium transfer proteins between pale, soft, and exudative (PSE) and red, firm, and non-exudative (RFN) pork. Seven PSE and seven RFN pork longissimus thoracis (LT) muscles were chosen according to pH and L* at 1 h post-mortem and identified by drip loss at 24 h. The nitric oxide synthase (NOS) activity and neuronal nitric oxide synthase (nNOS) expression showed a significant difference between two groups ( p < 0.05). PSE meat had a considerably higher sarcoplasmic calcium concentration compared to RFN meat at 1 h post-mortem aging ( p < 0.05). In PSE meat, the expression of ryanodine receptor 1 (RyR1) and sarcoplasmic reticulum calcium ATPase 1 (SERCA1) was lower than that in RFN meat, while the relative S-nitrosylation level of RyR1 and SERCA1 was higher ( p < 0.05). In addition, a lower activity of SERCA was detected in PSE meat compared to RFN meat ( p < 0.05). Those results indicate that S-nitrosylation of RyR1 and SERCA1 can putatively play a crucial part in regulating calcium homeostasis. A high level of RyR1 and SERCA1 S-nitrosylation can induce the imbalance of calcium in cytoplasm, leading to accelerated pH decline and the development of PSE meat.


Assuntos
Carne/análise , Canal de Liberação de Cálcio do Receptor de Rianodina/genética , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/genética , Suínos/genética , Animais , Cálcio/metabolismo , Cor , Manipulação de Alimentos , Músculo Esquelético/química , Músculo Esquelético/metabolismo , Canal de Liberação de Cálcio do Receptor de Rianodina/metabolismo , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/metabolismo , Suínos/metabolismo
6.
J Vet Intern Med ; 33(2): 933-941, 2019 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-30720217

RESUMO

BACKGROUND: Sarcolipin (SLN), myoregulin (MRLN), and dwarf open reading frame (DWORF) are transmembrane regulators of the sarcoplasmic reticulum calcium transporting ATPase (SERCA) that we hypothesized played a role in recurrent exertional rhabdomyolysis (RER). OBJECTIVES: Compare coding sequences of SLN, MRLN, DWORF across species and between RER and control horses. Compare expression of muscle Ca2+ regulatory genes between RER and control horses. ANIMALS: Twenty Thoroughbreds (TB), 5 Standardbreds (STD), 6 Quarter Horses (QH) with RER and 39 breed-matched controls. METHODS: Sanger sequencing of SERCA regulatory genes with comparison of amino acid (AA) sequences among control, RER horses, human, mouse, and rabbit reference genomes. In RER and control gluteal muscle, quantitative real-time polymerase chain reaction of SERCA regulatory peptides, the calcium release channel (RYR1), and its accessory proteins calsequestrin (CASQ1), and calstabin (FKBP1A). RESULTS: The SLN gene was the highest expressed horse SERCA regulatory gene with a uniquely truncated AA sequence (29 versus 31) versus other species. Coding sequences of SLN, MRLN, and DWORF were identical in RER and control horses. A sex-by-phenotype effect occurred with lower CASQ1 expression in RER males versus control males (P < .001) and RER females (P = .05) and higher FKBP1A (P = .01) expression in RER males versus control males. CONCLUSIONS AND CLINICAL IMPORTANCE: The SLN gene encodes a uniquely truncated peptide in the horse versus other species. Variants in the coding sequence of SLN, MLRN, or DWORF were not associated with RER. Males with RER have differential gene expression that could reflect adaptations to stabilize RYR1.


Assuntos
Doenças dos Cavalos/genética , Rabdomiólise/veterinária , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/genética , Sequência de Aminoácidos , Animais , Estudos de Casos e Controles , Feminino , Expressão Gênica , Cavalos , Humanos , Masculino , Camundongos , Proteínas Musculares/genética , Proteínas Musculares/metabolismo , Músculo Esquelético , Coelhos , Rabdomiólise/genética
7.
Mol Genet Genomic Med ; 7(3): e552, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30688039

RESUMO

BACKGROUND: Pathogenic mutations causing aberrant splicing are often difficult to detect. Standard variant analysis of next-generation sequence (NGS) data focuses on canonical splice sites. Noncanonical splice sites are more difficult to ascertain. METHODS: We developed a bioinformatics pipeline that screens existing NGS data for potentially aberrant novel essential splice sites (PANESS) and performed a pilot study on a family with a myotonic disorder. Further analyses were performed via qRT-PCR, immunoblotting, and immunohistochemistry. RNAi knockdown studies were performed in Drosophila to model the gene deficiency. RESULTS: The PANESS pipeline identified a homozygous ATP2A1 variant (NC_000016.9:g.28905928G>A; NM_004320.4:c.1287G>A:p.(Glu429=)) that was predicted to cause the omission of exon 11. Aberrant splicing of ATP2A1 was confirmed via qRT-PCR, and abnormal expression of the protein product sarcoplasmic/endoplasmic reticulum Ca++ ATPase 1 (SERCA1) was demonstrated in quadriceps femoris tissue from the proband. Ubiquitous knockdown of SERCA led to lethality in Drosophila, as did knockdown targeting differentiating or fusing myoblasts. CONCLUSIONS: This study confirms the potential of novel in silico algorithms to detect cryptic mutations in existing NGS data; expands the phenotypic spectrum of ATP2A1 mutations beyond classic Brody myopathy; and suggests that genetic testing of ATP2A1 should be considered in patients with clinical myotonia.


Assuntos
Biologia Computacional/métodos , Testes Genéticos/métodos , Miotonia Congênita/genética , Sítios de Splice de RNA/genética , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/genética , Sequenciamento Completo do Exoma/métodos , Algoritmos , Animais , Células Cultivadas , Drosophila melanogaster , Humanos , Masculino , Músculo Esquelético/metabolismo , Mutação , Miotonia Congênita/patologia , Fenótipo , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/metabolismo , Adulto Jovem
8.
Nat Commun ; 9(1): 5227, 2018 12 07.
Artigo em Inglês | MEDLINE | ID: mdl-30531949

RESUMO

During drug development, tissue samples serve as indicators of disease activity and pharmacodynamic responses. Reliable non-invasive measures of drug target engagement will facilitate identification of promising new treatments. Here we develop and validate a novel bi-transgenic mouse model of myotonic dystrophy type 1 (DM1) in which expression of either DsRed or GFP is determined by alternative splicing of an upstream minigene that is mis-regulated in DM1. Using a novel in vivo fluorescence spectroscopy system, we show that quantitation of the DsRed/GFP ratio provides an accurate estimation of splicing outcomes in muscle tissue of live mice that nearly doubles throughput over conventional fluorescence imaging techniques. Serial in vivo spectroscopy measurements in mice treated with a C16 fatty acid ligand conjugated antisense (LICA) oligonucleotide reveal a dose-dependent therapeutic response within seven days, confirm a several-week duration of action, and demonstrate a two-fold greater target engagement as compared to the unconjugated parent oligonucleotide.


Assuntos
Processamento Alternativo , Músculos/efeitos dos fármacos , Distrofia Miotônica/tratamento farmacológico , Oligonucleotídeos Antissenso/farmacologia , Animais , Modelos Animais de Doenças , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Humanos , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Camundongos Transgênicos , Microscopia de Fluorescência , Músculos/metabolismo , Músculos/patologia , Distrofia Miotônica/genética , Distrofia Miotônica/metabolismo , Avaliação de Resultados (Cuidados de Saúde)/métodos , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/genética , Espectrometria de Fluorescência
9.
Life Sci ; 210: 29-39, 2018 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-30107170

RESUMO

AIM: Epidermal growth factor receptor (EGFR) related reactive oxygen species (ROS) generation results in myocardial damage. We aimed to investigate the role of gefitinib (EGFR-tyrosine kinase inhibitor) in diabetic cardiomyopathy (DbCM). METHOD: DbCM was induced by injecting streptozotocin (55 mg/kg for 5 consecutive days) to C57/BL6 mice intraperitoneally. Diabetic C57/BL6 mice (fasting blood glucose level ≥ 250 mg/dl) were allocated into four study groups and treated with two doses of gefitinib (30 mg/kg and 350 mg/kg per day) as well as ramipril (3 mg/kg/day) for four weeks. KEY FINDINGS: We observed a significant correlation between persistent hyperglycaemia with cardiac remodeling and alterations in myocardial architecture. Gefitinib significantly prevented lipid peroxidation (MDA), damage of antioxidant enzymes like superoxide dismutase (SOD), Catalase, glutathione (GSH) and thioredoxin reductase (TrxR). Gefitinib also prevented hypertrophy of myocardium evidenced by reduced heart weight to body weight ratio and TGF­ß related collagen deposition. Gefitinib maintained cardiac biomarkers like lactate dehydrogenase (LDH), Creatine Kinase­MB, brain natriuretic peptide (BNP) and cardiac Troponin­I (cTroponinI) indicating reduced myocardial damage. Decreased sarcoplasmic endoplasmic reticulum Ca2 + ATPase2a (SERCA2a) and sodium­calcium exchanger-1 (NCX1) protein depletion after gefitinib administration indicated improved Ca2+ homeostasis during myocardial contractility. Histopathology and transmission electron microscopy clearly showed almost normal myofibrils and mitochondrial arrangements in gefitinib treated mice. SIGNIFICANCE: Our findings suggest that gefitinib protects myocardial damage in DbCM via balancing oxidant-antioxidant system, decreased collagen deposition as well as improved CKMB, BNP, cTroponinI and SERCA2a/NCX-1. Thereby, it indicated that gefitinib may be a potential therapeutic drug for treating DbCM.


Assuntos
Diabetes Mellitus Experimental/complicações , Cardiomiopatias Diabéticas/tratamento farmacológico , Receptores ErbB/antagonistas & inibidores , Inibidores de Proteínas Quinases/farmacologia , Quinazolinas/farmacologia , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/metabolismo , Trocador de Sódio e Cálcio/metabolismo , Animais , Células Cultivadas , Cardiomiopatias Diabéticas/etiologia , Cardiomiopatias Diabéticas/patologia , Gefitinibe , Camundongos , Camundongos Endogâmicos C57BL , Miocárdio/metabolismo , Miocárdio/patologia , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/genética , Trocador de Sódio e Cálcio/genética
10.
Gene ; 673: 12-21, 2018 Oct 05.
Artigo em Inglês | MEDLINE | ID: mdl-29886036

RESUMO

Existing data indicate that a Ca2+ signal stimulates ecdysteroid hormone production by crustacean molting glands (Y-organs). Ca2+ signaling is dependent on a tightly regulated Ca2+ gradient, with intracellular free Ca2+ maintained at a low basal level (typically sub-micromolar). This is achieved through the action of proteins intrinsic to the plasma membrane and the membranes of organelles. One such protein, the sarco/endoplasmic reticulum Ca2+ ATPase (SERCA), pumps Ca2+ from cytosol to the lumen of the endoplasmic reticulum. As a step toward understanding Ca2+-mediated regulation of ecdysteroidogenesis, we have begun investigating Ca2+ transport proteins in Y-organs. In studies reported here, we used a PCR-based strategy to clone from Y-organs of the blue crab (Callinectes sapidus) a cDNA encoding a putative SERCA protein. The cloned Cas-SERCA cDNA (3806 bp) includes a 3057-bp open reading frame that encodes a 1019-residue protein (Cas-SERCA). The conceptually translated protein has a predicted molecular mass of 111.42 × 103 and contains all signature domains of an authentic SERCA, including ten transmembrane domains and a phosphorylation site at aspartate 351. A homology model of Cas-SERCA closely resembles models of related SERCA proteins. Phylogenetic analysis shows Cas-SERCA clusters with SERCA proteins from other arthropods. An assessment of tissue distribution indicates the Cas-SERCA transcript is widely distributed across tissues. Studies using quantitative PCR showed Cas-SERCA transcript abundance increased significantly in Y-organs activated by eyestalk ablation, a pattern consistent with the hypothesis that Cas-SERCA functions to maintain Ca2+ homeostasis in Y-organs.


Assuntos
Braquiúros/genética , Muda/genética , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/genética , Animais , Cálcio/metabolismo , Clonagem Molecular , Primers do DNA , Homeostase , Conformação Molecular , Filogenia , RNA/genética , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/metabolismo , Especificidade da Espécie , Distribuição Tecidual
11.
PLoS Genet ; 14(6): e1007433, 2018 06.
Artigo em Inglês | MEDLINE | ID: mdl-29879123

RESUMO

Circadian clocks impose daily periodicities to animal behavior and physiology. At their core, circadian rhythms are produced by intracellular transcriptional/translational feedback loops (TTFL). TTFLs may be altered by extracellular signals whose actions are mediated intracellularly by calcium and cAMP. In mammals these messengers act directly on TTFLs via the calcium/cAMP-dependent transcription factor, CREB. In the fruit fly, Drosophila melanogaster, calcium and cAMP also regulate the periodicity of circadian locomotor activity rhythmicity, but whether this is due to direct actions on the TTFLs themselves or are a consequence of changes induced to the complex interrelationship between different classes of central pacemaker neurons is unclear. Here we investigated this question focusing on the peripheral clock housed in the non-neuronal prothoracic gland (PG), which, together with the central pacemaker in the brain, controls the timing of adult emergence. We show that genetic manipulations that increased and decreased the levels of calcium and cAMP in the PG caused, respectively, a shortening and a lengthening of the periodicity of emergence. Importantly, knockdown of CREB in the PG caused an arrhythmic pattern of eclosion. Interestingly, the same manipulations directed at central pacemaker neurons caused arrhythmicity of eclosion and of adult locomotor activity, suggesting a common mechanism. Our results reveal that the calcium and cAMP pathways can alter the functioning of the clock itself. In the PG, these messengers, acting as outputs of the clock or as second messengers for stimuli external to the PG, could also contribute to the circadian gating of adult emergence.


Assuntos
Cálcio/fisiologia , Relógios Circadianos/fisiologia , AMP Cíclico/fisiologia , Drosophila melanogaster/fisiologia , Transdução de Sinais/genética , Animais , Encéfalo/fisiologia , Canais de Cálcio/genética , Ritmo Circadiano/fisiologia , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Glândulas Endócrinas/metabolismo , Feminino , Técnicas de Silenciamento de Genes , Locomoção/fisiologia , Masculino , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/genética , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/metabolismo
12.
Diab Vasc Dis Res ; 15(4): 322-335, 2018 07.
Artigo em Inglês | MEDLINE | ID: mdl-29762054

RESUMO

Sarco(endo)plasmic reticulum calcium adenosine triphosphatase is responsible for transporting cytosolic calcium into the sarcoplasmic reticulum and endoplasmic reticulum to maintain calcium homeostasis. Sarco(endo)plasmic reticulum calcium adenosine triphosphatase is the dominant isoform expressed in cardiac tissue, which is regulated by endogenous protein inhibitors, post-translational modifications, hormones as well as microRNAs. Dysfunction of sarco(endo)plasmic reticulum calcium adenosine triphosphatase is associated with heart failure, which makes sarco(endo)plasmic reticulum calcium adenosine triphosphatase a promising target for heart failure therapy. This review summarizes current approaches to ameliorate sarco(endo)plasmic reticulum calcium adenosine triphosphatase function and focuses on phospholamban, an endogenous inhibitor of sarco(endo)plasmic reticulum calcium adenosine triphosphatase, pharmacological tools and gene therapies.


Assuntos
Proteínas de Ligação ao Cálcio/metabolismo , Inibidores Enzimáticos/uso terapêutico , Terapia Genética/métodos , Insuficiência Cardíaca/terapia , Miócitos Cardíacos/efeitos dos fármacos , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/antagonistas & inibidores , Animais , Sinalização do Cálcio/efeitos dos fármacos , Proteínas de Ligação ao Cálcio/genética , Inibidores Enzimáticos/efeitos adversos , Regulação Neoplásica da Expressão Gênica , Insuficiência Cardíaca/enzimologia , Insuficiência Cardíaca/genética , Insuficiência Cardíaca/fisiopatologia , Humanos , Isoenzimas , Terapia de Alvo Molecular , Contração Miocárdica/efeitos dos fármacos , Miócitos Cardíacos/enzimologia , Miócitos Cardíacos/patologia , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/genética , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/metabolismo
13.
Subcell Biochem ; 87: 229-258, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29464562

RESUMO

The calcium pump (a.k.a. Ca2+-ATPase or SERCA) is a membrane transport protein ubiquitously found in the endoplasmic reticulum (ER) of all eukaryotic cells. As a calcium transporter, SERCA maintains the low cytosolic calcium level that enables a vast array of signaling pathways and physiological processes (e.g. synaptic transmission, muscle contraction, fertilization). In muscle cells, SERCA promotes relaxation by pumping calcium ions from the cytosol into the lumen of the sarcoplasmic reticulum (SR), the main storage compartment for intracellular calcium. X-ray crystallographic studies have provided an extensive understanding of the intermediate states that SERCA populates as it progresses through the calcium transport cycle. Historically, SERCA is also known to be regulated by small transmembrane peptides, phospholamban (PLN) and sarcolipin (SLN). PLN is expressed in cardiac muscle, whereas SLN predominates in skeletal and atrial muscle. These two regulatory subunits play critical roles in cardiac contractility. While our understanding of these regulatory mechanisms are still developing, SERCA and PLN are one of the best understood examples of peptide-transporter regulatory interactions. Nonetheless, SERCA appeared to have only two regulatory subunits, while the related sodium pump (a.k.a. Na+, K+-ATPase) has at least nine small transmembrane peptides that provide tissue specific regulation. The last few years have seen a renaissance in our understanding of SERCA regulatory subunits. First, structures of the SERCA-SLN and SERCA-PLN complexes revealed molecular details of their interactions. Second, an array of micropeptides concealed within long non-coding RNAs have been identified as new SERCA regulators. This chapter will describe our current understanding of SERCA structure, function, and regulation.


Assuntos
Sinalização do Cálcio/fisiologia , Cálcio , Retículo Endoplasmático , Células Musculares/enzimologia , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático , Animais , Cálcio/química , Cálcio/metabolismo , Proteínas de Ligação ao Cálcio/química , Proteínas de Ligação ao Cálcio/genética , Proteínas de Ligação ao Cálcio/metabolismo , Cristalografia por Raios X , Retículo Endoplasmático/enzimologia , Retículo Endoplasmático/genética , Humanos , Proteínas Musculares/química , Proteínas Musculares/genética , Proteínas Musculares/metabolismo , Proteolipídeos/química , Proteolipídeos/genética , Proteolipídeos/metabolismo , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/química , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/genética , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/metabolismo
14.
Sci Rep ; 8(1): 1158, 2018 01 18.
Artigo em Inglês | MEDLINE | ID: mdl-29348619

RESUMO

Cytokines present during low-grade inflammation contribute to ß-cell dysfunction and diabetes. Cytokine signaling disrupts ß-cell glucose-stimulated Ca2+ influx (GSCI) and endoplasmic reticulum (ER) Ca2+ ([Ca2+]ER) handling, leading to diminished glucose-stimulated insulin secretion (GSIS). However, cytokine-mediated changes in ion channel activity that alter ß-cell Ca2+ handling remain unknown. Here we investigated the role of K+ currents in cytokine-mediated ß-cell dysfunction. Kslow currents, which control the termination of intracellular Ca2+ ([Ca2+]i) oscillations, were reduced following cytokine exposure. As a consequence, [Ca2+]i and electrical oscillations were accelerated. Cytokine exposure also increased basal islet [Ca2+]i and decreased GSCI. The effect of cytokines on TALK-1 K+ currents were also examined as TALK-1 mediates Kslow by facilitating [Ca2+]ER release. Cytokine exposure decreased KCNK16 transcript abundance and associated TALK-1 protein expression, increasing [Ca2+]ER storage while maintaining 2nd phase GSCI and GSIS. This adaptive Ca2+ response was absent in TALK-1 KO islets, which exhibited decreased 2nd phase GSCI and diminished GSIS. These findings suggest that Kslow and TALK-1 currents play important roles in altered ß-cell Ca2+ handling and electrical activity during low-grade inflammation. These results also reveal that a cytokine-mediated reduction in TALK-1 serves an acute protective role in ß-cells by facilitating increased Ca2+ content to maintain GSIS.


Assuntos
Cálcio/metabolismo , Células Secretoras de Insulina/efeitos dos fármacos , Insulina/metabolismo , Interferon gama/farmacologia , Interleucina-1beta/farmacologia , Canais de Potássio de Domínios Poros em Tandem/genética , Fator de Necrose Tumoral alfa/farmacologia , Adulto , Animais , Feminino , Regulação da Expressão Gênica , Glucose/metabolismo , Humanos , Células Secretoras de Insulina/citologia , Células Secretoras de Insulina/metabolismo , Transporte de Íons , Ilhotas Pancreáticas/citologia , Ilhotas Pancreáticas/efeitos dos fármacos , Ilhotas Pancreáticas/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Potássio/metabolismo , Canais de Potássio de Domínios Poros em Tandem/metabolismo , Cultura Primária de Células , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/genética , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/metabolismo , Técnicas de Cultura de Tecidos
15.
J Mol Neurosci ; 64(1): 111-116, 2018 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-29214423

RESUMO

In this study, we evaluated the expression profile changes of genes that intervene in the calcium signaling pathway, in young and adult Wistar rats, using the animal model of neonatal lesion in ventral hippocampus (NLVH) (a recognized animal model for schizophrenia) and compared to the group of control animals (Sham). Through microarray technology, gene expression profiles were obtained from the three brain areas (nucleus accumbens, prefrontal cortex, and hippocampus) of young male Wistar rats (45 days) and adults (90 days) whether or not subjected to NLVH. The calcium signaling pathway reported a greater number of differentially expressed genes with z-score two values, > 2 (over-expression) and < - 2 (under-expression), in the three evaluated areas. The comparative analyses of this approach were performed in juvenile and adult rats with ventral hippocampal lesion in neonate rats (NLVH). NLVH influenced change expressions in various genes involved in Ca2+ homeostasis, including Cacna1d, Atp2a2, Adcy2, Ppp3cb, and Ptk2b. The expression of Adcy2, Ppp3cb, and Ptk2b genes changed in both age groups; therefore, the study of gene expression profiles between juvenile and adult rats may help to understand the molecular mechanisms of schizophrenia.


Assuntos
Sinalização do Cálcio/genética , Esquizofrenia/genética , Adenilil Ciclases/genética , Adenilil Ciclases/metabolismo , Animais , Calcineurina/genética , Calcineurina/metabolismo , Canais de Cálcio/genética , Canais de Cálcio/metabolismo , Quinase 2 de Adesão Focal/genética , Quinase 2 de Adesão Focal/metabolismo , Perfilação da Expressão Gênica , Hipocampo/metabolismo , Masculino , Ratos , Ratos Wistar , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/genética , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/metabolismo , Esquizofrenia/metabolismo
16.
J Immunol ; 200(2): 523-537, 2018 01 15.
Artigo em Inglês | MEDLINE | ID: mdl-29229678

RESUMO

Sarcoplasmic/endoplasmic reticulum Ca2+ adenosine triphosphatase (SERCA)2a, a critical regulator of calcium homeostasis, is known to be decreased in heart failure. Patients with myocarditis or dilated cardiomyopathy develop autoantibodies to SERCA2a suggesting that they may have pathogenetic significance. In this report, we describe epitope mapping analysis of SERCA2a in A/J mice that leads us to make five observations: 1) SERCA2a contains multiple T cell epitopes that induce varying degrees of myocarditis. One epitope, SERCA2a 971-990, induces widespread atrial inflammation without affecting noncardiac tissues; the cardiac abnormalities could be noninvasively captured by echocardiography, electrocardiography, and magnetic resonance microscopy imaging. 2) SERCA2a 971-990-induced disease was associated with the induction of CD4 T cell responses and the epitope preferentially binds MHC class II/IAk rather than IEk By creating IAk/and IEk/SERCA2a 971-990 dextramers, the T cell responses were determined by flow cytometry to be Ag specific. 3) SERCA2a 971-990-sensitized T cells produce both Th1 and Th17 cytokines. 4) Animals immunized with SERCA2a 971-990 showed Ag-specific Abs with enhanced production of IgG2a and IgG2b isotypes, suggesting that SERCA2a 971-990 can potentially act as a common epitope for both T cells and B cells. 5) Finally, SERCA2a 971-990-sensitized T cells were able to transfer disease to naive recipients. Together, these data indicate that SERCA2a is a critical autoantigen in the mediation of atrial inflammation in mice and that our model may be helpful to study the inflammatory events that underlie the development of conditions such as atrial fibrillation in humans.


Assuntos
Mapeamento de Epitopos , Epitopos/imunologia , Miocardite/imunologia , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/imunologia , Alelos , Animais , Proteínas de Bactérias , Citocinas/imunologia , Citocinas/metabolismo , Modelos Animais de Doenças , Mapeamento de Epitopos/métodos , Epitopos de Linfócito B/imunologia , Imunofluorescência , Expressão Gênica , Átrios do Coração/imunologia , Átrios do Coração/metabolismo , Átrios do Coração/patologia , Ventrículos do Coração/imunologia , Ventrículos do Coração/metabolismo , Ventrículos do Coração/patologia , Antígenos de Histocompatibilidade Classe II/genética , Antígenos de Histocompatibilidade Classe II/imunologia , Epitopos Imunodominantes/imunologia , Imuno-Histoquímica , Camundongos , Camundongos Endogâmicos , Miocardite/diagnóstico por imagem , Miocardite/patologia , Miócitos Cardíacos/imunologia , Miócitos Cardíacos/metabolismo , Miócitos Cardíacos/patologia , Peptídeos/imunologia , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/genética , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/metabolismo , Subpopulações de Linfócitos T/imunologia , Subpopulações de Linfócitos T/metabolismo
17.
Expert Opin Biol Ther ; 18(3): 237-249, 2018 03.
Artigo em Inglês | MEDLINE | ID: mdl-29202595

RESUMO

INTRODUCTION: Arrhythmias can cause symptoms ranging from simple dizziness to life-threatening circulatory collapse. Current management includes medical therapy and procedures such as catheter ablation or device implantation. However, these strategies still pose a risk of serious side effects, and some patients remain symptomatic. Advancement in our understanding of how arrhythmias develop on the cellular level has made more targeted approaches possible. In addition, contemporary studies have found that several genes are involved in the pathogenesis of arrhythmias. Areas covered: In the present review, the authors explore the cellular and genetic mechanisms leading to arrhythmias as well as the progress that has been made in using both gene and cell therapy to treat tachy- and bradyarrhythmias. They also consider why gene and cell therapy has resulted into a few clinical trials with promising results, however still not applicable in routine clinical practice. Expert opinion: The question currently is whether such biological therapies could replace current established approaches. The contemporary evidence suggests that despite recent advances in this field, it will need more work in experimental models before this is applied into clinical practice. Gene and cell studies targeting conduction and repolarization are promising, but still not ready for use in the clinical setting.


Assuntos
Arritmias Cardíacas/terapia , Animais , Arritmias Cardíacas/metabolismo , Arritmias Cardíacas/patologia , Bradicardia/metabolismo , Bradicardia/patologia , Bradicardia/terapia , Terapia Baseada em Transplante de Células e Tecidos , Terapia Genética , Humanos , Canais Disparados por Nucleotídeos Cíclicos Ativados por Hiperpolarização/genética , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/genética , Células-Tronco/citologia , Células-Tronco/metabolismo , Taquicardia/metabolismo , Taquicardia/patologia , Taquicardia/terapia
18.
Biochem Biophys Res Commun ; 500(1): 75-86, 2018 05 27.
Artigo em Inglês | MEDLINE | ID: mdl-28495532

RESUMO

Mitochondria are dynamic organelles involved in numerous physiological functions. Beyond their function in ATP production, mitochondria regulate cell death, reactive oxygen species (ROS) generation, immunity and metabolism. Mitochondria also play a key role in the buffering of cytosolic calcium, and calcium transported into the matrix regulates mitochondrial metabolism. Recently, the identification of the mitochondrial calcium uniporter (MCU) and associated regulators has allowed the characterization of new physiological roles for calcium in both mitochondrial and cellular homeostasis. Indeed, recent work has highlighted the importance of mitochondrial calcium homeostasis in regulating cell migration. Cell migration is a property common to all metazoans and is critical to embryogenesis, cancer progression, wound-healing and immune surveillance. Previous work has established that cytoplasmic calcium is a key regulator of cell migration, as oscillations in cytosolic calcium activate cytoskeletal remodelling, actin contraction and focal adhesion (FA) turnover necessary for cell movement. Recent work using animal models and in cellulo experiments to genetically modulate MCU and partners have shed new light on the role of mitochondrial calcium dynamics in cytoskeletal remodelling through the modulation of ATP and ROS production, as well as intracellular calcium signalling. This review focuses on MCU and its regulators in cell migration during physiological and pathophysiological processes including development and cancer. We also present hypotheses to explain the molecular mechanisms by which MCU may regulate mitochondrial dynamics and motility to drive cell migration.


Assuntos
Canais de Cálcio/metabolismo , Cálcio/metabolismo , Células Eucarióticas/metabolismo , Mitocôndrias/metabolismo , Neoplasias/metabolismo , Citoesqueleto de Actina , Animais , Canais de Cálcio/genética , Sinalização do Cálcio , Morte Celular , Movimento Celular , Proteínas do Citoesqueleto/genética , Proteínas do Citoesqueleto/metabolismo , Células Eucarióticas/citologia , Regulação da Expressão Gênica , Homeostase , Humanos , Neoplasias/genética , Neoplasias/patologia , Espécies Reativas de Oxigênio/metabolismo , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/genética , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/metabolismo
19.
Mol Cell Biochem ; 442(1-2): 19-28, 2018 May.
Artigo em Inglês | MEDLINE | ID: mdl-28884444

RESUMO

The cardiac sarco/endoplasmic reticulum Ca2+-ATPase-2a (SERCA2a) is vital for the correct handling of calcium concentration in cardiomyocytes. Recent studies showed that the induction of endoplasmic reticulum (ER) stress (ERS) with the SERCA2 inhibitor Thapsigargin (Tg) increases the mRNA and protein levels of SERCA2a. The SERCA2 gene promoter contains an ERS response element (ERSE) at position -78 bp that is conserved among species and might transcriptionally regulate SERCA2 gene expression. However, its involvement in SERCA2 basal and calcium-mediated transcriptional activation has not been elucidated. In this work, we show that in cellular cultures of neonatal rat ventricular myocytes, the treatment with Tg or the calcium ionophore A23187 increases the SERCA2a mRNA and protein abundance, as well as the transcriptional activity of two chimeric human SERCA2 gene constructs, containing -254 and -2579 bp of 5'-regulatory region cloned in the pGL3-basic vector and transiently transfected in cultured cardiomyocytes. We found that the ERSE present in the SERCA2 proximal promoter contains a CCAAT box that is involved in basal and ERS-mediated hSERCA2 transcriptional activation. The EMSA results showed that the CCAAT box present in the ERSE recruits the NF-Y transcription factor. Additionally, by ChIP assays, we confirmed in vivo binding of NF-Y and C/EBPß transcription factors to the SERCA2 gene proximal promoter.


Assuntos
Estresse do Retículo Endoplasmático/fisiologia , Regulação da Expressão Gênica/fisiologia , Miócitos Cardíacos/metabolismo , Elementos de Resposta , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/sangue , Transcrição Genética/fisiologia , Animais , Calcimicina/farmacologia , Ionóforos de Cálcio/farmacologia , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos dos fármacos , Ratos , Ratos Wistar , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/genética , Tapsigargina/farmacologia , Transcrição Genética/efeitos dos fármacos
20.
Biosci Rep ; 38(1)2018 02 28.
Artigo em Inglês | MEDLINE | ID: mdl-29208771

RESUMO

Iron-overload cardiomyopathy is prevalent on a worldwide basis and is a major comorbidity in patients with genetic hemochromatosis and secondary iron overload. Therapies are limited in part due to lack of a valid preclinical model, which recapitulates advanced iron-overload cardiomyopathy. Male hemojuvelin (HJV) knockout (HJVKO) mice, which lack HJV, a bone morphogenetic co-receptor protein required for hepcidin expression and systemic iron homeostasis, were fed a high-iron diet starting at 4 weeks of age for a duration of 1 year. Aged HJVKO mice in response to iron overload showed increased myocardial iron deposition and mortality coupled with oxidative stress and myocardial fibrosis culminating in advanced iron-overload cardiomyopathy. In a parallel group, iron-overloaded HJVKO mice received resveratrol (240 mg/day) at 9 months of age until 1 year of age. Echocardiography and invasive pressure-volume (PV) loop analyses revealed a complete normalization of iron-overload mediated diastolic and systolic dysfunction in response to resveratrol therapy. In addition, myocardial sarcoplasmic reticulum Ca2+ ATPase (SERCa2a) levels were reduced in iron-overloaded hearts and resveratrol therapy restored SERCa2a levels and suppressed up-regulation of the sodium-calcium exchanger (NCX1). Further, iron-mediated oxidative stress and myocardial fibrosis were suppressed by resveratrol treatment with concomitant activation of the p-Akt and p-AMP-activated protein kinase (AMPK) signaling pathways. A combination of ageing and high-iron diet in male HJVKO mice results in a valid preclinical model that recapitulates iron-overload cardiomyopathy in humans. Resveratrol therapy resulted in normalization of cardiac function demonstrating that resveratrol represents a feasible therapeutic intervention to reduce the burden of iron-overload cardiomyopathy.


Assuntos
Cardiomiopatias/tratamento farmacológico , Coração/efeitos dos fármacos , Sobrecarga de Ferro/tratamento farmacológico , Proteínas de Membrana/genética , Miocárdio/metabolismo , Animais , Cardiomiopatias/genética , Cardiomiopatias/metabolismo , Cardiomiopatias/patologia , Modelos Animais de Doenças , Coração/fisiopatologia , Hepcidinas/genética , Humanos , Ferro/metabolismo , Sobrecarga de Ferro/genética , Sobrecarga de Ferro/metabolismo , Sobrecarga de Ferro/patologia , Camundongos , Miocárdio/patologia , Estresse Oxidativo/efeitos dos fármacos , Proteínas Quinases/genética , Resveratrol , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/genética , Trocador de Sódio e Cálcio/genética , Estilbenos/administração & dosagem
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