RESUMO
OBJECTIVE: To determine the correlations of single nucleotide polymorphisms (SNPs) with atrial fibrillation (AF) in the Chinese Han population from the central plains.â© Methods: A total of 168 hospitalized patients, including 56 AF and 112 controls, were recruited in this case-control study. The clinical data were obtained from the medical records. All 5 SNPs, rs337711 in KCNN2, rs11264280 near KCNN3, rs17042171 near PITX2, rs6771157 and rs6795970 in SCN10A, were genotyped using amplification refractory mutation system-polymerase chain reaction or direct sequencing. The χ2 test was used to compare categorical variables and preliminarily examine correlations between the genotype frequencies and AF. Subsequently, a logistic regression model was constructed to determine the associations between the SNPs and AF based on the above screened results. Odds ratios (ORs) and 95% confidence interval (CI) were calculated to assess the strength of the correlations. Moreover, we downloaded the genotype data from the HapMap Project for linkage disequilibrium analysis of rs17042171.â© Results: AF patients were likely to be of older age and longer left atrial diameter and had more coronary artery disease and higher hypertension compared with the control group (P<0.05). Among the 5 SNPs, the frequency distribution of genotype AA for rs17042171 was significantly different between the AF and control groups (P<0.05). After adjusting for several covariates, there was still a high risk ratio in patients with the AA genotype compared with the AC+CC genotype (OR: 5.591, 95%CI 2.176 to 14.365, P-B<0.008). Similarly, stratification analysis on the AA genotype demonstrated significant differences between rs17042171 and persistent AF. However, there were not significant correlations between AF and the control groups for the other 4 SNPs (P<0.05).â© Conclusion: Rs17042171, near PITX2 on chromosome 4q25, is associated with AF susceptibility in the Chinese Han population from the central plains, suggesting that this SNP can provide a new strategy for clinical diagnosis in AF patients.
Assuntos
Fibrilação Atrial/genética , Polimorfismo de Nucleotídeo Único , Fatores Etários , Povo Asiático , Fibrilação Atrial/etnologia , Estudos de Casos e Controles , Distribuição de Qui-Quadrado , China/etnologia , Cromossomos Humanos 4-5 , Predisposição Genética para Doença , Genótipo , Geografia , Proteínas de Homeodomínio/genética , Humanos , Desequilíbrio de Ligação , Modelos Logísticos , Canal de Sódio Disparado por Voltagem NAV1.8/genética , Razão de Chances , Canais de Potássio Ativados por Cálcio de Condutância Baixa/genética , Fatores de Transcrição/genética , Proteína Homeobox PITX2RESUMO
OBJECTIVE: To explore the effects of membrane skeleton protein 4.1R on the efficiency of photodynamic therapy (PDT). METHODS: 4.1R gene knockout and wild-type mouse embryonic fibroblasts (MEFs) were incubated with various concentrations of 5-aminolevulinic acid (5-ALA) (0.25, 0.50, 1.00, 1.50 and 2.00 mmol/L), followed by exposure to 450 nm light at a dose of 72, 96, 120, 180, 240 mJ/cm(2). Cell counting kit 8 (CCK-8) assay was used to assess the survival rate after PDT treatment. Laser confocal microscopy was used to observe the location of photo-sensitizer protoporphyrin and fluorescence spectrophotometer for detecting the fluorescent intensity of intracellular protoporphyrin. The protein levels of rate-limiting enzyme of protoporphyrin synthesis, ferrochelatase (FECH) and hydroxymethylbilane synthase (HMBS) were determined by Western blot. RESULTS: Both cell lines were killed after 5-aminolevulinic acid (5-ALA)-PDT and its efficacy was dependent on 5-ALA concentration, incubation duration and light dose. The cell survival rates of 4.1R(-/-) MEF were significantly higher than those of 4.1R(+/+) MEF (46.9% ± 7.1% vs 12.5% ± 2.1%, P < 0.001) after PDT treatment with a light dose of 120 mJ/cm(2) mediated by 5-ALA 1.00 mmol/L. After incubation with 1.00 mmol/L 5-ALA, protoporphyrin was distributed throughout cytoplasm in both cell lines while the fluorescent intensity of 4.1R(+/+) MEF was higher than that of 4.1R(-/-) MEF (124.2 ± 3.5 vs 34.6 ± 3.8, P < 0.001). Western blot showed that no difference of FECH and HMBS protein level was found in two cell lines. CONCLUSIONS: A lack of protein 4.1R may attenuate the intracellular protoporphyrin level and the photo-cytotoxicity of PDT. No cellular change of ALA metabolic activity is found. Protein 4.1R may be involved in the ALA uptake in MEF cells so that the cellular level of protoporphyrin ultimately affects the PDT efficiency.
Assuntos
Fotoquimioterapia , Ácido Aminolevulínico , Animais , Linhagem Celular , Camundongos , Proteínas dos Microfilamentos , Microscopia Confocal , Fármacos Fotossensibilizantes , ProtoporfirinasRESUMO
The regulation of cardiac function by the nuclear transcription factor signal transducer and activator of transcription 4 (STAT4) has been recently recognized. Nevertheless, the role and mechanisms of action of STAT4 in myocardial ischemiareperfusion (I/R) injury remain unknown. Consequently, the present study constructed a rat model of I/R by ligation of the left anterior descending coronary artery. Following sacrifice, the rat hearts were excised and analyzed to investigated the effects of STAT4 on I/Rinduced myocardial injury. Western blotting demonstrated that expression of STAT4 decreased significantly in the rat model of cardiac I/R and in H9C2 cells that were subjected to hypoxia and reoxygenation (H/R). The overexpression of STAT4 in H9C2 cells reduced cell damage and apoptosis induced by H/R. Furthermore, both in vivo and in vitro, the level of PI3K decreased significantly. Although the AKT protein expression levels were not altered, the AKT phosphorylation levels decreased significantly. STAT4 overexpression enhanced the expression of PI3K and AKT in the H9C2 cells. On the whole, the present study demonstrated that STAT4 alleviated I/Rinduced myocardial injury through the PI3K/AKT signaling pathway.
Assuntos
Apoptose , Traumatismo por Reperfusão Miocárdica , Fosfatidilinositol 3-Quinases , Proteínas Proto-Oncogênicas c-akt , Fator de Transcrição STAT4 , Transdução de Sinais , Animais , Fator de Transcrição STAT4/metabolismo , Fator de Transcrição STAT4/genética , Traumatismo por Reperfusão Miocárdica/metabolismo , Traumatismo por Reperfusão Miocárdica/patologia , Ratos , Proteínas Proto-Oncogênicas c-akt/metabolismo , Masculino , Fosfatidilinositol 3-Quinases/metabolismo , Linhagem Celular , Modelos Animais de Doenças , Ratos Sprague-Dawley , Miócitos Cardíacos/metabolismo , Miócitos Cardíacos/patologia , FosforilaçãoRESUMO
AIMS: Diabetic cardiomyopathy (DCM) is one of the major cardiovascular complications of diabetes. However, the mechanism of DCM is not fully understood. Studies have confirmed that certain microRNAs (miRNAs/miRs) are key regulators of DCM. The aim of this study was to investigate the role and mechanism of microRNA (miR)-494 in cardiomyocyte apoptosis and autophagy induced by high glucose (HG). METHODS AND RESULTS: By establishing a rat DCM model and an HG-treated H9c2 cells injury model, cardiac function was detected by echocardiography, myocardial tissue was stained by immunohistochemistry, and Cell Counting Kit-8 assay and lactate dehydrogenase assay were used to detect the cardiomyocyte injury. Cell apoptosis was detected by terminal deoxynucleotidyl transferase dUTP nick end labelling staining, and western blotting was used to detect death and autophagy. The results showed that the expression level of miR-494 was higher in the myocardial tissue of DCM rats and the myocardial cells of H9c2 treated with HG. Compared with the corresponding negative control groups, miR-494 mimics enhanced HG-induced apoptosis and autophagy, whereas miR-494 inhibitors showed the opposite effect, corresponding PI3K, AKT, and mTOR phosphorylation level has changed. CONCLUSIONS: These findings identify that miR-494 could regulate cell apoptosis and autophagy through PI3K/AKT/mTOR signalling pathway, participating in the regulation of cardiomyocyte cell damage after HG. These findings provide new insights for the further study of the molecular mechanism and treatment of DCM.
Assuntos
MicroRNAs , Proteínas Proto-Oncogênicas c-akt , Animais , Ratos , Apoptose/genética , Autofagia/genética , Glucose/metabolismo , MicroRNAs/genética , MicroRNAs/metabolismo , Miócitos Cardíacos/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Fosfatidilinositol 3-Quinases/farmacologia , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteínas Proto-Oncogênicas c-akt/farmacologia , Serina-Treonina Quinases TOR/metabolismo , Serina-Treonina Quinases TOR/farmacologiaRESUMO
Heart failure (HF) is a major cause of death in cardiovascular diseases worldwide, and its molecular mechanisms and effective prevention strategies remain to be further studied. The myocardial cytoskeleton plays a pivotal role in many heart diseases. However, little is known about the function of the membrane cytoskeleton 4.1 protein family and related regulatory mechanisms in the pathogenesis of HF. In this study, we detected the localization and expression of the protein 4.1 family and ion channel proteins in a rat HF model induced by doxorubicin (DOX), and studied the interactions between them. Our results showed that compared with the control group, the HF group displayed an increased expression level of protein 4.1R and decreased levels of protein 4.1 G and 4.1 N. The Nav1.5 protein levels were significantly increased, while the SERCA2a and Cav1.2 protein levels were significantly decreased in the HF group. Furthermore, there is co-localization and interaction between protein 4.1R and Nav1.5, protein 4.1 G and SERCA2a, protein 4.1 N and Cav1.2, respectively. Taken together, the results indicated that the protein 4.1 family might be involved in the occurrence and development of HF through its interaction with ion channel proteins, suggesting that 4.1 proteins may serve as a novel therapeutic target for HF.
Assuntos
Canais de Cálcio Tipo L/metabolismo , Insuficiência Cardíaca/metabolismo , Proteínas dos Microfilamentos/metabolismo , Miocárdio/metabolismo , Canal de Sódio Disparado por Voltagem NAV1.5/metabolismo , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/metabolismo , Animais , Canais de Cálcio Tipo L/genética , Insuficiência Cardíaca/genética , Masculino , Proteínas dos Microfilamentos/genética , Canal de Sódio Disparado por Voltagem NAV1.5/genética , Ratos , Ratos Sprague-Dawley , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/genéticaRESUMO
Acute myocardial infarction can be caused by ischemia/reperfusion (I/R) injury; however, the mechanism underlying I/R is not completely understood. The present study investigated the functions and mechanisms underlying microRNA (miR)494 in I/Rinduced cardiomyocyte apoptosis and autophagy. Hypoxia/reoxygenation (H/R)treated H9c2 rat myocardial cells were used as an in vitro I/R injury model. Apoptosis and autophagy were analyzed by Cell Counting Kit8 assay, Lactic dehydrogenase and superoxide dismutase assay, flow cytometry, TUNEL staining and western blotting. Reverse transcriptionquantitative PCR demonstrated that, H9c2 cells treated with 12 h hypoxia and 3 h reoxygenation displayed significantly downregulated miR494 expression levels compared with control cells. Compared with the corresponding negative control (NC) groups, miR494 mimic reduced H/Rinduced cell apoptosis and autophagy, whereas miR494 inhibitor displayed the opposite effects. Silent information regulator 1 (SIRT1) was identified as a target gene of miR494. Furthermore, miR494 inhibitormediated effects on H/Rinduced cardiomyocyte apoptosis and autophagy were partially reversed by SIRT1 knockdown. Moreover, compared with siNC, SIRT1 knockdown significantly increased the phosphorylation levels of PI3K, AKT and mTOR in H/Rtreated and miR494 inhibitortransfected H9c2 cells. Collectively, the results indicated that miR494 served a protective role against H/Rinduced cardiomyocyte apoptosis and autophagy by directly targeting SIRT1, suggesting that miR494 may serve as a novel therapeutic target for myocardial I/R injury.
Assuntos
Hipóxia Celular/genética , MicroRNAs/genética , Miócitos Cardíacos/metabolismo , Animais , Apoptose/genética , Autofagia/genética , Hipóxia Celular/fisiologia , Linhagem Celular , Hipóxia/genética , Hipóxia/metabolismo , MicroRNAs/metabolismo , Infarto do Miocárdio/metabolismo , Traumatismo por Reperfusão Miocárdica/metabolismo , Miócitos Cardíacos/fisiologia , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Ratos , Transdução de Sinais/efeitos dos fármacos , Sirtuína 1/metabolismo , Serina-Treonina Quinases TOR/metabolismoRESUMO
5-aminolevulinic acid (5-ALA)-based photodynamic therapy (PDT) has been successfully used in the treatment of cancers. However, the mechanism of 5-ALA transportation into cancer cells is still not fully elucidated. Previous studies have confirmed that the efficiency of 5-ALA-PDT could be affected by the membrane skeleton protein 4.1R. In this study, we investigated the role of 4.1R in the transport of 5-ALA into cells. Wild-type (4.1R+/+ ) and 4.1R gene knockout (4.1R-/- ) mouse embryonic fibroblast (MEF) cells were incubated with 1 mm 5-ALA and different concentrations of specific inhibitors of GABA transporters GAT (1-3). Our results showed that the inhibition of GAT1 and GAT2 in particular markedly attenuated the intracellular PpIX production, reactive oxygen species (ROS) level and 5-ALA-induced photodamage. However, the inhibition of GAT3 did not show such effects. Further research showed that 4.1R-/- MEF cells had a lower expression of GAT1 and GAT2 than 4.1R+/+ MEF cells. Additionally, 4.1R directly bound to GAT1 and GAT2. Taken together, GAT1 and GAT2 transporters are involved in the uptake of 5-ALA in MEF cells. 4.1R plays an important role in transporting 5-ALA into cells via at least partly interaction with GAT1 and GAT2 transporters in 5-ALA-PDT.