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1.
Circ Res ; 124(9): e63-e80, 2019 04 26.
Artigo em Inglês | MEDLINE | ID: mdl-30786847

RESUMO

RATIONALE: SERCA2a, sarco-endoplasmic reticulum Ca2+-ATPase, is a critical determinant of cardiac function. Reduced level and activity of SERCA2a are major features of heart failure. Accordingly, intensive efforts have been made to develop efficient modalities for SERCA2a activation. We showed that the activity of SERCA2a is enhanced by post-translational modification with SUMO1 (small ubiquitin-like modifier 1). However, the roles of other post-translational modifications on SERCA2a are still unknown. OBJECTIVE: In this study, we aim to assess the role of lysine acetylation on SERCA2a function and determine whether inhibition of lysine acetylation can improve cardiac function in the setting of heart failure. METHODS AND RESULTS: The acetylation of SERCA2a was significantly increased in failing hearts of humans, mice, and pigs, which is associated with the reduced level of SIRT1 (sirtuin 1), a class III histone deacetylase. Downregulation of SIRT1 increased the SERCA2a acetylation, which in turn led to SERCA2a dysfunction and cardiac defects at baseline. In contrast, pharmacological activation of SIRT1 reduced the SERCA2a acetylation, which was accompanied by recovery of SERCA2a function and cardiac defects in failing hearts. Lysine 492 (K492) was of critical importance for the regulation of SERCA2a activity via acetylation. Acetylation at K492 significantly reduced the SERCA2a activity, presumably through interfering with the binding of ATP to SERCA2a. In failing hearts, acetylation at K492 appeared to be mediated by p300 (histone acetyltransferase p300), a histone acetyltransferase. CONCLUSIONS: These results indicate that acetylation/deacetylation at K492, which is regulated by SIRT1 and p300, is critical for the regulation of SERCA2a activity in hearts. Pharmacological activation of SIRT1 can restore SERCA2a activity through deacetylation at K492. These findings might provide a novel strategy for the treatment of heart failure.


Assuntos
Insuficiência Cardíaca/metabolismo , Miócitos Cardíacos/metabolismo , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/metabolismo , Sirtuína 1/metabolismo , Acetilação , Trifosfato de Adenosina/metabolismo , Animais , Linhagem Celular , Células Cultivadas , Proteína p300 Associada a E1A/metabolismo , Insuficiência Cardíaca/enzimologia , Insuficiência Cardíaca/genética , Humanos , Lisina/genética , Lisina/metabolismo , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Miócitos Cardíacos/patologia , Processamento de Proteína Pós-Traducional , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/genética , Sirtuína 1/genética , Suínos
2.
Langmuir ; 34(15): 4494-4502, 2018 04 17.
Artigo em Inglês | MEDLINE | ID: mdl-29561155

RESUMO

Directed self-assembly (DSA) of block copolymer (BCP) thin films is a promising approach to enable next-generation patterning at increasingly smaller length scales. DSA utilizes interfacial wetting layers to force the BCP domains to self-assemble with the desired orientation with respect to the substrate. Here, we demonstrate that initiated chemical-vapor-deposited (iCVD) polydivinylbenzene (pDVB) ultrathin films can direct the self-assembly of poly(styrene- block-methylmethacrylate). We found that the methyl radicals formed at increased filament temperatures during the iCVD process result in the backbone methylation of pDVB. By tuning the degree of backbone methylation, we systematically changed the wetting properties of the iCVD pDVB from a slight poly(methylmethacrylate) preference to complete poly(styrene) preference. Additionally, we utilize the conformal nature of the iCVD to form a wetting layer over a topographical line and space pattern, which is subsequently used to produce self-assembled BCP films with both perpendicular orientation and long-range alignment.

3.
EMBO Rep ; 16(10): 1318-33, 2015 10.
Artigo em Inglês | MEDLINE | ID: mdl-26341627

RESUMO

The mitochondrial calcium uniporter (MCU) is responsible for mitochondrial calcium uptake and homeostasis. It is also a target for the regulation of cellular anti-/pro-apoptosis and necrosis by several oncogenes and tumour suppressors. Herein, we report the crystal structure of the MCU N-terminal domain (NTD) at a resolution of 1.50 Å in a novel fold and the S92A MCU mutant at 2.75 Å resolution; the residue S92 is a predicted CaMKII phosphorylation site. The assembly of the mitochondrial calcium uniporter complex (uniplex) and the interaction with the MCU regulators such as the mitochondrial calcium uptake-1 and mitochondrial calcium uptake-2 proteins (MICU1 and MICU2) are not affected by the deletion of MCU NTD. However, the expression of the S92A mutant or a NTD deletion mutant failed to restore mitochondrial Ca(2+) uptake in a stable MCU knockdown HeLa cell line and exerted dominant-negative effects in the wild-type MCU-expressing cell line. These results suggest that the NTD of MCU is essential for the modulation of MCU function, although it does not affect the uniplex formation.


Assuntos
Canais de Cálcio/química , Canais de Cálcio/metabolismo , Proteínas de Transporte da Membrana Mitocondrial/metabolismo , Cálcio/metabolismo , Canais de Cálcio/genética , Proteínas de Ligação ao Cálcio/metabolismo , Cristalografia por Raios X , Células HEK293 , Células HeLa , Humanos , Mitocôndrias/metabolismo , Proteínas de Transporte da Membrana Mitocondrial/genética , Modelos Moleculares , Mutação , Dobramento de Proteína , Domínios e Motivos de Interação entre Proteínas , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína
4.
Biochim Biophys Acta ; 1839(1): 25-32, 2014 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-24321384

RESUMO

Alternative splicing plays an important role in gene expression by producing different proteins from a gene. Caspase-2 pre-mRNA produces anti-apoptotic Casp-2S and pro-apoptotic Casp-2L proteins through exon 9 inclusion or skipping. However, the molecular mechanisms of exon 9 splicing are not well understood. Here we show that knockdown of SRSF3 (also known as SRp20) with siRNA induced significant increase of endogenous exon 9 inclusion. In addition, overexpression of SRSF3 promoted exon 9 skipping. Thus we conclude that SRSF3 promotes exon 9 skipping. In order to understand the functional target of SRSF3 on caspase-2 pre-mRNA, we performed substitution and deletion mutagenesis on the potential SRSF3 binding sites that were predicted from previous reports. We demonstrate that substitution mutagenesis of the potential SRSF3 binding site on exon 8 severely disrupted the effects of SRSF3 on exon 9 skipping. Furthermore, with the approach of RNA pulldown and immunoblotting analysis we show that SRSF3 interacts with the potential SRSF3 binding RNA sequence on exon 8 but not with the mutant RNA sequence. In addition, we show that a deletion of 26nt RNA from 5' end of exon 8, a 33nt RNA from 3' end of exon 10 and a 2225nt RNA from intron 9 did not compromise the function of SRSF3 on exon 9 splicing. Therefore we conclude that SRSF3 promotes exon 9 skipping of caspase-2 pre-mRNA by interacting with exon 8. Our results reveal a novel mechanism of caspase-2 pre-mRNA splicing.


Assuntos
Processamento Alternativo/genética , Apoptose/genética , Caspase 2/genética , Proteínas de Ligação a RNA/genética , Sítios de Ligação/genética , Caspase 2/biossíntese , Éxons/genética , Técnicas de Silenciamento de Genes , Células HEK293 , Humanos , Mutagênese , Precursores de RNA/genética , Proteínas de Ligação a RNA/metabolismo , Deleção de Sequência , Fatores de Processamento de Serina-Arginina
5.
Biochem Biophys Res Commun ; 463(4): 975-81, 2015 Aug 07.
Artigo em Inglês | MEDLINE | ID: mdl-26071359

RESUMO

The heart LIM protein (HLP) is a LIM-only protein family member that mediates protein-protein interactions. To date, no studies have yet been conducted regarding its function in the heart. In the present study, we have identified that HLP binds the cytosolic region of RyR2 in the heart using a bacterial two-hybrid system, LC-MS/MS, co-immunoprecipitation, and GST-pull down assays. Microscopy revealed that HLP forms a triple complex with RyR2 and caveolin-3. siRNA and adenovirus-mediated KD of HLP decreased the electrically evoked Ca(2+) release from the sarcoplasmic reticulum without directly affecting SERCA2 and RyR2 activities. Collectively, the HLP-RyR2 interaction in the cell surface caveolae region may be essential for efficient excitation-contraction coupling in the heart.


Assuntos
Cálcio/metabolismo , Caveolina 3/metabolismo , Proteínas com Domínio LIM/metabolismo , Miocárdio/metabolismo , Canal de Liberação de Cálcio do Receptor de Rianodina/metabolismo , Animais , Linhagem Celular , Masculino , Ligação Proteica , Ratos , Ratos Sprague-Dawley , Rianodina/metabolismo , Espectrometria de Massas em Tandem
6.
Biochem J ; 457(1): 151-62, 2014 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-24117217

RESUMO

Progressive cardiac hypertrophy owing to pathological stimuli, such as pressure overload, is frequently associated with the development of heart failure, a major cause of morbidity and mortality worldwide. Growing evidence has shown that miRNAs are extensively involved in the pathogenesis of cardiac hypertrophy. In the present study, we examined the hypothesis that the miR-19a/b family acts as a key regulator of cardiac hypertrophy and apoptosis. Forced overexpression of miR-19a/b was sufficient to induce hypertrophy in rat neonatal cardiomyocytes. Luciferase assays revealed that miR-19a/b directly target the anti-hypertrophic genes atrogin-1 and MuRF-1 (muscle RING-finger protein-1). The endogenous expressions of the target genes were down-regulated by miR-19a/b. Pro-hypertrophic calcineurin/NFAT (nuclear factor of activated T-cells) signalling was elevated markedly in the presence of miR-19b, and the calcineurin inhibitor CsA (cyclosporin A) and the PKC (protein kinase C) inhibitor GF10923X significantly attenuated the miR-19b-mediated increase in cell size and expression of hypertrophic markers. Furthermore, miR-19b led to increased cell survival through up-regulation of the NFAT target gene encoding α-crystallin-B and repression of the pro-apoptotic gene Bim (Bcl-2-interacting mediator of cell death) under ER (endoplasmic reticulum) stress conditions. Taken together, the results of the present study demonstrate that the miR-19a/b family regulates phenotypes of cardiomyocytes via suppression of multiple direct target genes.


Assuntos
Cardiomegalia/genética , MicroRNAs/fisiologia , Proteínas Musculares/genética , Miócitos Cardíacos/patologia , Proteínas Ligases SKP Culina F-Box/genética , Ubiquitina-Proteína Ligases/genética , Animais , Animais Recém-Nascidos , Células Cultivadas , Regulação da Expressão Gênica , Marcação de Genes , Células HEK293 , Humanos , Família Multigênica/fisiologia , Miócitos Cardíacos/metabolismo , Ratos , Ratos Sprague-Dawley , Proteínas com Motivo Tripartido
7.
BMC Genomics ; 15: 592, 2014 Jul 13.
Artigo em Inglês | MEDLINE | ID: mdl-25017872

RESUMO

BACKGROUND: RNA sequencing (RNA-seq) has revolutionized the detection of transcriptomic signatures due to its high-throughput sequencing ability. Therefore, genomic annotations on different animal species have been rapidly updated using information from tissue-enriched novel transcripts and novel exons. RESULTS: 34 putative novel transcripts and 236 putative tissue-enriched exons were identified using RNA-Seq datasets representing six tissues available in mouse databases. RT-PCR results indicated that expression of 21 and 2 novel transcripts were enriched in testes and liver, respectively, while 31 of the 39 selected novel exons were detected in the testes or heart. The novel isoforms containing the identified novel exons exhibited more dominant expression than the known isoforms in heart and testes. We also identified an example of pathology-associated exclusion of heart-enriched novel exons such as Sorbs1 and Cluh during pressure-overload cardiac hypertrophy. CONCLUSION: The present study depicted tissue-enriched novel transcripts, a tissue-specific isoform switch, and pathology-associated alternative splicing in a mouse model, suggesting tissue-specific genomic diversity and plasticity.


Assuntos
Fígado/metabolismo , Miocárdio/metabolismo , RNA/metabolismo , Testículo/metabolismo , Processamento Alternativo , Animais , Cardiomegalia/genética , Cardiomegalia/metabolismo , Cardiomegalia/patologia , Éxons , Sequenciamento de Nucleotídeos em Larga Escala , Masculino , Camundongos , Proteínas dos Microfilamentos/genética , Proteínas dos Microfilamentos/metabolismo , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , RNA/química , RNA/genética , Análise de Sequência de RNA , Transcriptoma
8.
Pflugers Arch ; 466(5): 987-1001, 2014 May.
Artigo em Inglês | MEDLINE | ID: mdl-24077737

RESUMO

Stromal interaction molecule 1 (STIM1) mediates Ca2+ movements from the extracellular space to the cytosol through a store-operated Ca2+ entry (SOCE) mechanism in various cells including skeletal muscle cells. In the present study, to reveal the unidentified functional role of the STIM1 C terminus from 449 to 671 amino acids in skeletal muscle, binding assays and quadrupole time-of-flight mass spectrometry were used to identify proteins binding in this region along with proteins that mediate skeletal muscle contraction and relaxation. STIM1 binds to sarcoplasmic/endoplasmic reticulum Ca2+-ATPase 1a (SERCA1a) via this region (called STIM1-SBR). The binding was confirmed in endogenous full-length STIM1 in rabbit skeletal muscle and mouse primary skeletal myotubes via co-immunoprecipitation assay and immunocytochemistry. STIM1 knockdown in mouse primary skeletal myotubes decreased Ca2+ uptake from the cytosol to the sarcoplasmic reticulum (SR) through SERCA1a only at micromolar cytosolic Ca2+ concentrations, suggesting that STIM1 could be required for the full activity of SERCA1a possibly during the relaxation of skeletal muscle. Various Ca2+ imaging experiments using myotubes expressing STIM1-SBR suggest that STIM1 is involved in intracellular Ca2+ distributions between the SR and the cytosol via regulating SERCA1a activity without affecting SOCE. Therefore, in skeletal muscle, STIM1 could play an important role in regulating Ca2+ movements between the SR and the cytosol.


Assuntos
Canais de Cálcio/metabolismo , Fibras Musculares Esqueléticas/metabolismo , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/metabolismo , Sequência de Aminoácidos , Animais , Sítios de Ligação , Cálcio/metabolismo , Canais de Cálcio/química , Canais de Cálcio/genética , Sinalização do Cálcio , Camundongos , Dados de Sequência Molecular , Ligação Proteica , Coelhos , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/genética , Molécula 1 de Interação Estromal
9.
Phys Chem Chem Phys ; 16(18): 8615-22, 2014 May 14.
Artigo em Inglês | MEDLINE | ID: mdl-24676056

RESUMO

Ambient humidity and high temperature are known to degrade dye-sensitized solar cells (DSSCs) via chromophore desorption. Recently, enhanced dye-attachment to TiO2 surfaces has been realized by coating molecularly functionalized surfaces with inorganic atomic layer deposition (ALD) coatings. Here, we apply this ALD approach to DSSCs and demonstrate that high energy conversion efficiencies can be maintained while significantly extending device lifetimes. While single component ALD layers show improved high-temperature stability, it significantly degraded up to 45% of initial DSSC performance right after ALD. We, however, find that mixed component ALD layers provide initial efficiencies within 90% of their untreated counterparts while still extending device lifetimes. Optimized ALD protection schemes maintain 80% of their initial efficiency after 500 h of thermal aging at 80 °C whereas efficiency of DSSCs with no ALD protection drop below 60% of their initial efficiencies. IR spectroscopy conducted in situ during ALD reveals that carboxylate linker groups transition from unbound or weakly-bound states, respectively, to more strongly bound bidentate structures. This strategy to improve dye-attachment by ALD while maintaining high performance is novel and promising for extending the functional lifetime for DSSCs and other related devices.

10.
Biochem J ; 453(2): 187-200, 2013 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-23668188

RESUMO

STIM1 (stromal interaction molecule 1) mediates SOCE (store-operated Ca²âº entry) in skeletal muscle. However, the direct role(s) of STIM1 in skeletal muscle, such as Ca²âº release from the SR (sarcoplasmic reticulum) for muscle contraction, have not been identified. The times required for the maximal expression of endogenous STIM1 or Orai1, or for the appearance of puncta during the differentiation of mouse primary skeletal myoblasts to myotubes, were all different, and the formation of puncta was detected with no stimulus during differentiation, suggesting that, in skeletal muscle, the formation of puncta is a part of the differentiation. Wild-type STIM1 and two STIM1 mutants (Triple mutant, missing Ca²âº-sensing residues but possessing the intact C-terminus; and E136X, missing the C-terminus) were overexpressed in the myotubes. The wild-type STIM1 increased SOCE, whereas neither mutant had an effect on SOCE. It was interesting that increases in the formation of puncta were observed in the Triple mutant as well as in wild-type STIM1, suggesting that SOCE-irrelevant puncta could exist in skeletal muscle. On the other hand, overexpression of wild-type or Triple mutant, but not E136X, attenuated Ca²âº releases from the SR in response to KCl [evoking ECC (excitation-contraction coupling) via activating DHPR (dihydropyridine receptor)] in a dominant-negative manner. The attenuation was removed by STIM1 knockdown, and STIM1 was co-immunoprecipitated with DHRP in a Ca²âº-independent manner. These results suggest that STIM1 negatively regulates Ca²âº release from the SR through the direct interaction of the STIM1 C-terminus with DHPR, and that STIM1 is involved in both ECC and SOCE in skeletal muscle.


Assuntos
Osso e Ossos/metabolismo , Cálcio/metabolismo , Proteínas de Membrana/fisiologia , Fibras Musculares Esqueléticas/metabolismo , Proteínas de Neoplasias/fisiologia , Retículo Sarcoplasmático/metabolismo , Animais , Sequência de Bases , Primers do DNA , DNA Complementar , Humanos , Camundongos , Reação em Cadeia da Polimerase em Tempo Real , Canal de Liberação de Cálcio do Receptor de Rianodina/metabolismo , Molécula 1 de Interação Estromal
11.
Nucleic Acids Res ; 40(Database issue): D803-8, 2012 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-22140115

RESUMO

Integrated Pathway Resources, Analysis and Visualization System (iPAVS) is an integrated biological pathway database designed to support pathway discovery in the fields of proteomics, transcriptomics, metabolomics and systems biology. The key goal of IPAVS is to provide biologists access to expert-curated pathways from experimental data belonging to specific biological contexts related to cell types, tissues, organs and diseases. IPAVS currently integrates over 500 human pathways (consisting of 24, 574 interactions) that include metabolic-, signaling- and disease-related pathways, drug-action pathways and several large process maps collated from other pathway resources. IPAVS web interface allows biologists to browse and search pathway resources and provides tools for data import, management, visualization and analysis to support the interpretation of biological data in light of cellular processes. Systems Biology Graphical Notations (SBGN) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway notations are used for the visual display of pathway information. The integrated datasets in IPAVS are made available in several standard data formats that can be downloaded. IPAVS is available at: http://ipavs.cidms.org.


Assuntos
Gráficos por Computador , Bases de Dados Factuais , Doença , Redes e Vias Metabólicas , Fenômenos Farmacológicos , Transdução de Sinais , Humanos , Internet , Biologia de Sistemas , Integração de Sistemas
12.
Nano Lett ; 13(4): 1481-8, 2013 Apr 10.
Artigo em Inglês | MEDLINE | ID: mdl-23537229

RESUMO

Photoelectrochemical (PEC) water splitting and solar fuels hold great promise for harvesting solar energy. TiO2-based photoelectrodes for water splitting have been intensively investigated since 1972. However, solar-to-fuel conversion efficiencies of TiO2 photoelectrodes are still far lower than theoretical values. This is partially due to the dilemma of a short minority carrier diffusion length, and long optical penetration depth, as well as inefficient electron collection. We report here the synthesis of TiO2 PEC electrodes by coating solution-processed antimony-doped tin oxide nanoparticle films (nanoATO) on FTO glass with TiO2 through atomic layer deposition. The conductive, porous nanoATO film-supported TiO2 electrodes, yielded a highest photocurrent density of 0.58 mA/cm(2) under AM 1.5G simulated sunlight of 100 mW/cm(2). This is approximately 3× the maximum photocurrent density of planar TiO2 PEC electrodes on FTO glass. The enhancement is ascribed to the conductive interconnected porous nanoATO film, which decouples the dimensions for light absorption and charge carrier diffusion while maintaining efficient electron collection. Transient photocurrent measurements showed that nanoATO films reduce charge recombination by accelerating transport of photoelectrons through the less defined conductive porous nanoATO network. Owing to the large band gap, scalable solution processed porous nanoATO films are promising as a framework to replace other conductive scaffolds for PEC electrodes.


Assuntos
Energia Solar , Compostos de Estanho/química , Titânio/química , Coloides/química , Eletroquímica/métodos , Eletrodos , Nanopartículas/química , Água/química
13.
J Biol Chem ; 287(18): 14336-48, 2012 Apr 27.
Artigo em Inglês | MEDLINE | ID: mdl-22389502

RESUMO

Junctophilins (JPs) play an important role in the formation of junctional membrane complexes (JMC) in striated muscle by physically linking the transverse-tubule and sarcoplasmic reticulum (SR) membranes. Researchers have found five JP2 mutants in humans with hypertrophic cardiomyopathy. Among these, Y141H and S165F are associated with severely altered Ca(2+) signaling in cardiomyocytes. We previously reported that S165F also induced both hypertrophy and altered intracellular Ca(2+) signaling in mouse skeletal myotubes. In the present study, we attempted to identify the dominant-negative role(s) of Y141H in primary mouse skeletal myotubes. Consistent with S165F, Y141H led to hypertrophy and altered Ca(2+) signaling (a decrease in the gain of excitation-contraction coupling and an increase in the resting level of myoplasmic Ca(2+)). However, unlike S165F, neither ryanodine receptor 1-mediated Ca(2+) release from the SR nor the phosphorylation of the mutated JP2 by protein kinase C was related to the altered Ca(2+) signaling by Y141H. Instead, abnormal JMC and increased SOCE via Orai1 were found, suggesting that the hypertrophy caused by Y141H progressed differently from S165F. Therefore JP2 can be linked to skeletal muscle hypertrophy via various Ca(2+) signaling pathways, and SOCE could be one of the causes of altered Ca(2+) signaling observed in muscle hypertrophy.


Assuntos
Canais de Cálcio/metabolismo , Sinalização do Cálcio , Cálcio/metabolismo , Proteínas de Membrana/metabolismo , Fibras Musculares Esqueléticas/metabolismo , Proteínas Musculares/metabolismo , Mutação de Sentido Incorreto , Substituição de Aminoácidos , Animais , Canais de Cálcio/genética , Cardiomiopatia Hipertrófica/genética , Cardiomiopatia Hipertrófica/metabolismo , Cardiomiopatia Hipertrófica/patologia , Humanos , Membranas Intracelulares/metabolismo , Membranas Intracelulares/patologia , Proteínas de Membrana/genética , Camundongos , Fibras Musculares Esqueléticas/patologia , Proteínas Musculares/genética , Miopatias Congênitas Estruturais/genética , Miopatias Congênitas Estruturais/metabolismo , Miopatias Congênitas Estruturais/patologia , Proteína ORAI1 , Canal de Liberação de Cálcio do Receptor de Rianodina/genética , Canal de Liberação de Cálcio do Receptor de Rianodina/metabolismo , Retículo Sarcoplasmático/genética , Retículo Sarcoplasmático/metabolismo , Retículo Sarcoplasmático/patologia
14.
J Biol Chem ; 287(3): 1679-87, 2012 Jan 13.
Artigo em Inglês | MEDLINE | ID: mdl-22123818

RESUMO

Calsequestrin (CSQ), the major intrasarcoplasmic reticulum calcium storage protein, undergoes dynamic polymerization and depolymerization in a Ca(2+)-dependent manner. However, no direct evidence of CSQ depolymerization in vivo with physiological relevance has been obtained. In the present study, live cell imaging analysis facilitated characterization of the in vivo dynamics of the macromolecular CSQ structure. CSQ2 appeared as speckles in the presence of normal sarcoplasmic reticulum (SR) Ca(2+) that were decondensed upon Ca(2+) depletion. Moreover, CSQ2 decondensation occurred only in the stoichiometric presence of junctin (JNT). When expressed alone, CSQ2 speckles remained unchanged, even after Ca(2+) depletion. FRET analysis revealed constant interactions between CSQ2 and JNT, regardless of the SR Ca(2+) concentration, implying that JNT is an essential component of the CSQ scaffold. In vitro solubility assay, electron microscopy, and atomic force microscopy studies using purified recombinant proteins confirmed Ca(2+) and JNT-dependent disassembly of the CSQ2 polymer. Accordingly, we conclude that reversible polymerization and depolymerization of CSQ are critical in SR Ca(2+) homeostasis.


Assuntos
Proteínas de Ligação ao Cálcio/metabolismo , Cálcio/metabolismo , Calsequestrina/metabolismo , Proteínas de Membrana/metabolismo , Oxigenases de Função Mista/metabolismo , Proteínas Musculares/metabolismo , Multimerização Proteica/fisiologia , Retículo Sarcoplasmático/metabolismo , Animais , Proteínas de Ligação ao Cálcio/genética , Calsequestrina/genética , Linhagem Celular , Homeostase/fisiologia , Humanos , Proteínas de Membrana/genética , Camundongos , Oxigenases de Função Mista/genética , Proteínas Musculares/genética , Retículo Sarcoplasmático/genética
15.
Biochem Biophys Res Commun ; 439(3): 327-32, 2013 Sep 27.
Artigo em Inglês | MEDLINE | ID: mdl-24012670

RESUMO

Disturbance of endoplasmic reticulum (ER) homeostasis causes ER stress (ERS), and triggers the unfolded protein response (UPR) that consequently reduces accumulation of unfolded proteins by increasing the quantity of ER chaperones. Calumenin, a Ca(2+)-binding protein with multiple EF hand motifs, which is located in the ER/SR, is highly expressed during the early developmental stage of the heart, similar to other ER-resident chaperones. The aim of this study was to investigate the functional role of calumenin during ERS in the heart. Like other chaperones (e.g., GRP94 and GRP78), calumenin expression was highly upregulated during ERS induced by 10 µg/ml tunicamycin, but attenuated in the presence of 500 µM PBA, the chemical chaperone in neonatal rat ventricular cardiomyocytes (NRVCs). Upon 7.5-fold overexpression of calumenin using a recombinant adenovirus system, the expression levels of ERS markers (GRP78, p-PERK, and p-elF2α) and ER-initiated apoptosis markers (CHOP and p-JNK) were reduced, whereas the survival protein BCL-2 was upregulated during ERS compared to the control. Evaluation of cell viability by TUNEL assay showed that apoptosis was also significantly reduced by calumenin overexpression in ERS-induced cells. Taken together, our results suggest that calumenin plays an essential role in the alleviation of ERS in neonatal rat cardiomyocytes.


Assuntos
Proteínas de Ligação ao Cálcio/metabolismo , Estresse do Retículo Endoplasmático , Miócitos Cardíacos/metabolismo , Animais , Apoptose , Proteínas de Ligação ao Cálcio/genética , Células Cultivadas , Retículo Endoplasmático/genética , Retículo Endoplasmático/metabolismo , Chaperona BiP do Retículo Endoplasmático , Células HEK293 , Humanos , Camundongos , Ratos , Ratos Sprague-Dawley , Regulação para Cima
16.
Basic Res Cardiol ; 108(3): 344, 2013 May.
Artigo em Inglês | MEDLINE | ID: mdl-23553082

RESUMO

The histidine-rich Ca(2+)-binding protein (HRC) is located in the lumen of the sarcoplasmic reticulum (SR) and exhibits high-capacity Ca(2+)-binding properties. Overexpression of HRC in the heart resulted in impaired SR Ca(2+) uptake and depressed relaxation through its interaction with SERCA2a. However, the functional significance of HRC in overall regulation of calcium cycling and contractility is not currently well defined. To further elucidate the role of HRC in vivo under physiological and pathophysiological conditions, we generated and characterized HRC-knockout (KO) mice. The KO mice were morphologically and histologically normal compared to wild-type (WT) mice. At the cellular level, ablation of HRC resulted in significantly enhanced contractility, Ca(2+) transients, and maximal SR Ca(2+) uptake rates in the heart. However, after-contractions were developed in 50 % of HRC-KO cardiomyocytes, compared to 11 % in WT mice under stress conditions of high-frequency stimulation (5 Hz) and isoproterenol application. A parallel examination of the electrical activity revealed significant increases in the occurrence of Ca(2+) spontaneous SR Ca(2+) release and delayed afterdepolarizations with ISO in HRC-KO, compared to WT cells. The frequency of Ca(2+) sparks was also significantly higher in HRC-KO cells with ISO, consistent with the elevated SR Ca(2+) load in the KO cells. Furthermore, HRC-KO cardiomyocytes showed significantly deteriorated cell contractility and Ca(2+)-cycling caused possibly by depressed SERCA2a expression after transverse-aortic constriction (TAC). Also HRC-null mice exhibited severe cardiac hypertrophy, fibrosis, pulmonary edema and decreased survival after TAC. Our results indicate that ablation of HRC is associated with poorly regulated SR Ca(2+)-cycling, and severe pathology under pressure-overload stress, suggesting an essential role of HRC in maintaining the integrity of cardiac function.


Assuntos
Sinalização do Cálcio , Proteínas de Ligação ao Cálcio/deficiência , Cardiomegalia/metabolismo , Hemodinâmica , Miócitos Cardíacos/metabolismo , Retículo Sarcoplasmático/metabolismo , Animais , Proteínas de Ligação ao Cálcio/genética , Estimulação Cardíaca Artificial , Cardiomegalia/etiologia , Cardiomegalia/genética , Cardiomegalia/patologia , Cardiomegalia/fisiopatologia , Modelos Animais de Doenças , Fibrose , Genótipo , Isoproterenol , Cinética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Contração Miocárdica , Miócitos Cardíacos/patologia , Fenótipo , Edema Pulmonar/etiologia , Edema Pulmonar/metabolismo , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/metabolismo , Índice de Gravidade de Doença
17.
Cerebrovasc Dis ; 36(1): 62-8, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23921172

RESUMO

BACKGROUND: Posterior circulation (PC) stroke, which was previously less well known than anterior circulation (AC) stroke, has become more identified due to the development of imaging equipment. Recently, the initial stroke severity assessed by the NIH Stroke Scale (NIHSS) was reported as a useful measure for predicting the outcome of PC as well as AC stroke. The aim of our study was to investigate the factors related to the stroke severity of PC ischemic stroke as assessed by the baseline NIHSS and the predictors of progressive neurological deficit and 3-month outcome. METHODS: All patients with first-time PC stroke (onset ≤ 7 days), admitted for a 5-year period and given a complete evaluation including brain MRI and angiographic studies, were enrolled. Patients were divided into two groups by the baseline NIHSS: moderate-to-severe stroke (MTSS, NIHSS > 5) and mild stroke (MS, NIHSS ≤ 5). Baseline characteristics, symptoms and progression, etiological subtypes, lesion characteristics from imaging, and patient 3-month outcome assessed by the modified Rankin Scale (mRS) were compared between the two groups. RESULTS: Among 604 enrolled patients with PC ischemic stroke, 143 belonged to the MTSS group and 461 to the MS group. In logistic regression analysis, MTSS was independently associated with white blood cell count (odds ratio, OR = 1.00, p = 0.001), high sensitivity C-reactive protein level (OR = 1.23, p = 0.004), dysarthria (OR = 2.59, p = 0.013), weakness (OR = 6.43, p < 0.001), dysphagia (OR = 5.77, p < 0.001) and decreased consciousness (OR = 10.54, p < 0.001). The independent predictors associated with progressive neurological deficit were MTSS (OR = 3.82, p = 0.001), the distal territory classified by lesion location (OR = 0.09, p = 0.004) and dysphagia (OR = 2.38, p = 0.010). The independent predictors associated with a 3-month mRS of 3-6 were MTSS (OR = 7.69, p < 0.001), diplopia (OR = 0.26, p = 0.023), visual field defect (OR = 4.87, p = 0.014), dysphagia (OR = 3.15, p < 0.001) and progressive neurological deficit (OR = 4.27, p < 0.001). CONCLUSIONS: The initial severity categorization of PC ischemic stroke by the NIHSS has provided several distinctions and could help with the prediction of neurological deficit progression and 3-month clinical outcome.


Assuntos
Circulação Cerebrovascular , Infarto da Artéria Cerebral Posterior/fisiopatologia , Índice de Gravidade de Doença , Idoso , Glicemia/análise , Sedimentação Sanguínea , Dano Encefálico Crônico/epidemiologia , Dano Encefálico Crônico/etiologia , Proteína C-Reativa/análise , Angiografia Cerebral/métodos , Comorbidade , Diabetes Mellitus/epidemiologia , Imagem de Difusão por Ressonância Magnética , Progressão da Doença , Feminino , Fibrinogênio/análise , Humanos , Hiperlipidemias/epidemiologia , Hipertensão/epidemiologia , Infarto da Artéria Cerebral Posterior/sangue , Infarto da Artéria Cerebral Posterior/classificação , Infarto da Artéria Cerebral Posterior/epidemiologia , Infarto da Artéria Cerebral Posterior/etiologia , Embolia Intracraniana/epidemiologia , Embolia Intracraniana/etiologia , Ataque Isquêmico Transitório/epidemiologia , Contagem de Leucócitos , Masculino , Pessoa de Meia-Idade , Sistema de Registros , República da Coreia/epidemiologia , Risco , Fatores de Risco , Fumar/efeitos adversos , Avaliação de Sintomas , Resultado do Tratamento
18.
Biochem J ; 447(3): 371-9, 2012 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-22867515

RESUMO

The structural proximity and functional coupling between the SR (sarcoplasmic reticulum) and mitochondria have been suggested to occur in the heart. However, the molecular architecture involved in the SR-mitochondrial coupling remains unclear. In the present study, we performed various genetic and Ca2+-probing studies to resolve the proteins involved in the coupling process. By using the bacterial 2-hybrid, glutathione transferase pull-down, co-immunoprecipitation and immunocytochemistry assays, we found that RyR2 (ryanodine receptor type 2), which is physically associated with VDAC2 (voltage-dependent anion channel 2), was co-localized in SR-mitochondrial junctions. Furthermore, a fractionation study revealed that VDAC2 was co-localized with RyR2 only in the subsarcolemmal region. VDAC2 knockdown by targeted short hairpin RNA led to an increased diastolic [Ca2+] (calcium concentration) and abolishment of mitochondrial Ca2+ uptake. Collectively, the present study suggests that the coupling of VDAC2 with RyR2 is essential for Ca2+ transfer from the SR to mitochondria in the heart.


Assuntos
Cálcio/metabolismo , Mitocôndrias Cardíacas/metabolismo , Canal de Liberação de Cálcio do Receptor de Rianodina/metabolismo , Retículo Sarcoplasmático/metabolismo , Canal de Ânion 2 Dependente de Voltagem/metabolismo , Animais , Linhagem Celular , Técnicas de Silenciamento de Genes , Humanos , Técnicas In Vitro , Transporte de Íons , Masculino , Camundongos , RNA Interferente Pequeno/genética , Ratos , Ratos Sprague-Dawley , Canal de Ânion 2 Dependente de Voltagem/genética
19.
Obes Surg ; 33(1): 94-104, 2023 01.
Artigo em Inglês | MEDLINE | ID: mdl-36319825

RESUMO

BACKGROUND: Bariatric surgery (BSx) is one of the most common surgical procedures performed in the USA. Nonetheless, data regarding 11-month period after BSx remain limited. METHODS: A retrospective cohort study using the 2016 National Readmission Database. Adult patients admitted for BSx in January were included. The follow-up period was 11 months (February-December). The primary outcome was all-cause 11-month readmission. Secondary outcomes were index admission (IA) and readmission in-hospital mortality rate and healthcare resource use associated with readmission. Multivariate regression was performed to identify independent risk factors for readmission. RESULTS: A total of 13,278 IA were included. The 11-month readmission rate was 11.1%. The mortality rate of readmission was 1.4% and 0.1% for IA (P < 0.01). The most common cause of readmission was hematemesis. Independent predictors were Charlson comorbidity index (CCI) score ≥ 3 (adjusted hazard ratio [aHR] 1.34; P = 0.05), increasing length of stay (aHR 1.01; P < 0.01), transfer to rehabilitation facilities (aHR 5.02; P < 0.01), undergoing laparoscopic Roux-en-Y gastric bypass (aHR 1.71; P = 0.02), adjustable gastric band (aHR 14.09; P < 0.01), alcohol use disorder (2.10; P = 0.01), and cannabis use disorder (aHR 3.37; P = 0.01). Private insurance as primary payer (aHR 0.65; P < 0.01) and BMI 45-49 kg/m2 (aHR 0.72; P < 0.01) were associated with less odds of readmission. The cumulative total hospitalization charges of readmission were $69.9 million. CONCLUSIONS: The 11-month readmission rate after BSx is 11.1%. Targeting modifiable predictors of readmission may help reduce the burden of readmissions on our healthcare system.


Assuntos
Cirurgia Bariátrica , Obesidade Mórbida , Adulto , Humanos , Readmissão do Paciente , Obesidade Mórbida/cirurgia , Estudos Retrospectivos , Complicações Pós-Operatórias/epidemiologia , Complicações Pós-Operatórias/etiologia , Incidência , Cirurgia Bariátrica/métodos , Fatores de Risco
20.
Adv Med Sci ; 68(2): 208-212, 2023 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-37329692

RESUMO

PURPOSE: The aim of this study was to build and validate modified score to be used in the healthcare cost and utilization project databases for further classification of acute pancreatitis (AP). MATERIALS AND METHODS: The National Inpatient Sample database for the years 2016-2019 was queried for all primary adult discharge diagnoses of AP. An mBISAP score system was created utilizing the ICD-10CM codes for pleural effusion, encephalopathy, acute kidney injury, systemic inflammatory response, and age >60. Each was assigned a 1-point score. A multivariable regression analysis was built to test for mortality. Sensitivity and specificity analyses were performed for mortality. RESULTS: A total of 1,160,869 primary discharges for AP were identified between 2016 and 2019. The pooled mortality rate was: 0.1%, 0.5%, 2.9%, 12.7%, 30.9% and 17.8% (P â€‹< â€‹0.01), respectively for scores 0 to 5. Multivariable regression analysis showed increasing odds of mortality with each one-point increment: mBISAP score of 1 (adjusted odds ratio [aOR] 6.67; 95% confidence interval [CI] 4.69-9.48), score of 2 (aOR 37.87; 95% CI 26.05- 55.03), score of 3 (aOR 189.38; 95% CI 127.47-281.38), score of 4 (aOR 535.38; 95% CI 331.74-864.02), score of 5 (aOR 184.38; 95% CI 53.91-630.60). Using a cut-off of ≥3, sensitivity and specificity analyses reported 27.0% and 97.7%, respectively, with an area under the curve (AUC) of 0.811. CONCLUSION: In this 4-year retrospective study of a US representative database, an mBISAP score was constructed showing increasing odds of mortality with each 1-point increase and a specificity of 97.7% for a cut-off of ≥3.


Assuntos
Pancreatite , Adulto , Humanos , Pancreatite/diagnóstico , Estudos Retrospectivos , Doença Aguda , Pacientes Internados , Índice de Gravidade de Doença , Prognóstico
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