Tiewei Lv

Inhibition of p300-HAT results in a reduced histone acetylation and down-regulation of gene expression in cardiac myocytes.

AIMS Histone acetylation plays an important role in cardiogenesis, but the underlying mechanism is unclear. In this study, we investigated the relationship between histone hypo-acetylation and the expression of cardiac-specific genes to explore the underlying mechanisms. MAIN METHODS Cardiac-specific genes that physically interacted with p300 protein in mouse hearts were analyzed using chromatin immunoprecipitation (ChIP) assays. The cultured mouse neonatal cardiac myocytes were treated with curcumin with different concentrations and durations. The changes of histone acetyltransferase (HAT) activities, histone acetylation, cardiac-specific genes expression, and structure of chromatin were assessed by ELISA, Western blotting, quantitative RT-PCR, and ChIP assays, respectively. KEY FINDINGS Results from the ChIP assay showed that GATA4, Nkx2.5, and Mef2c physically interacted with p300 protein. After treatment with 30 μM curcumin for 24h, the HAT activities of cardiac myocytes were inhibited significantly. And the acetylation of whole histone H3 was reduced by 0.3983-fold compared to control groups (P<0.05). Accordingly, the expression of cardiac-specific genes, GATA4, Nkx2.5, and Mef2c, were significantly down-regulated. Acetylation of histone H3 bound with promoter regions of these genes was significantly reduced. SIGNIFICANCE p300 interacts with cardiac-specific genes, GATA4, Nkx2.5 and Mef2c, and inhibition of p300-HAT by curcumin down-regulates their expression through the inhibition of histone H3 acetylation in the promoter regions. This finding indicates that p300-HAT mediated histone H3 acetylation plays an important role in the regulation of cardiac gene expression, which is a novel mechanism of epigenetic regulation in the heart during the development and in case of some congenital heart diseases.

Spatiotemporal expression of histone acetyltransferases, p300 and CBP, in developing embryonic hearts

Histone acetyltransferases (HATs), p300 and cAMP response element binding protein (CREB)-binding protein (CBP) are two structurally related transcriptional co-activators that activate expression of many eukaryotic genes involved in cellular growth and signaling, muscle differentiation and embryogenesis. However, whether these proteins play important and different roles in mouse cardiogenesis is not clear. Here, we investigate the protein distributions and mRNA expression of the two HATs in embryonic and adult mouse heart during normal heart development by using immunohistochemical and RT-PCR techniques. The data from immunohistochemical experiments revealed that p300 was extensively present in nearly every region of the hearts from embryonic stages to the adulthood. However, no CBP expression was detected in embryonic hearts at day E7.5. CBP expression appeared at the later stages, and the distribution of CBP was less than that of p300. In the developmental hearts after E10.5, both for p300 and CBP, the mRNA expression levels reached a peak on day E10.5, and then were gradually decreased afterwards. These results reveal that both p300 and CBP are related to embryonic heart development. The dynamic expression patterns of these two enzymes during mouse heart development indicate that they may play an important role on heart development. However, there is a difference in spatiotemporal expression patterns between these two enzymes during heart development. The expression of p300 is earlier and more predominate, suggesting that p300 may play a more important role in embryonic heart development especially during cardiac precursor cell induction and interventricular septum formation.

Alcohol Consumption During Gestation Causes Histone3 Lysine9 Hyperacetylation and an Alternation of Expression of Heart Development-Related Genes in Mice

BACKGROUND Alcohol abuse during gestation may cause congenital heart diseases (CHDs). The underlying mechanisms of alcohol-induced cardiac deformities are still not clear. Recent studies suggest that histone modification may play a crucial role in this pathological process. Moreover, our previous studies reported that ethanol could induce histone3 lysine9 (H3K9) hyperacetylation and overexpression of heart development-related genes in vitro. The aim of this study was to investigate the effect of alcohol consumption during gestation on the imbalance of H3K9 acetylation and the alternation of the expression of heart development-related genes during cardiogenesis. METHODS Pregnant mice were exposed to a single dose of alcohol (10 μl/g/d, 56% alcohol) by gavage every day in the morning from embryo day 7.5 (E7.5) to E15.5. Hematoxylin and eosin (H&E) staining was applied for observing the structure of the embryonic hearts. Western blotting and quantitative real-time polymerase chain reaction were used for detecting the level of H3K9 acetylation and gene expression. Histone acetyltransferase (HAT) and histone deacetylase (HDAC) activities were detected by colorimetric assay and fluorometric assay. RESULTS H&E staining of cardiac tissue showed abnormalities of embryonic hearts at E17.5. The level of H3K9 acetylation reached peak at E17.5 and decreased sharply to a low level at birth and maintained at low level afterward. Alcohol exposure increased H3K9 acetylation at E11.5, E14.5, E17.5, and E18.5, respectively (p < 0.05), and enhanced the expression of Gata4 in the embryonic hearts at E14.5 and E17.5, Mef2c at E14.5, and Nkx2.5 at E14.5 and E17.5, (p < 0.05) but not for Tbx5 (p > 0.05). On embryonic day 17.5, HAT activities of embryonic hearts increased significantly, however alcohol exposure did not alter HDAC activities. CONCLUSIONS These data indicate a time course of H3K9 acetylation change during heart development and demonstrate that alcohol exposure in utero may induce an increase of HAT activities, which results in H3K9 hyperacetylation and an increase of the expression of heart development-related genes. These findings reveal a novel epigenetic mechanism that connects the alcohol consumption during the pregnancy and the development of CHD in the fetus.

Inhibition of histone acetylation by curcumin reduces alcohol-induced fetal cardiac apoptosis.

BackgroundPrenatal alcohol exposure may cause cardiac development defects, however, the underlying mechanisms are not yet clear. In the present study we have investigated the roles of histone modification by curcumin on alcohol induced fetal cardiac abnormalities during the development.Methods and resultsQ-PCR and Western blot results showed that alcohol exposure increased gene and active forms of caspase-3 and caspase-8, while decreased gene and protein of bcl-2. ChIP assay results showed that, alcohol exposure increased the acetylation of histone H3K9 near the promoter region of caspase-3 and caspase-8, and decreased the acetylation of histone H3K9 near the promoter region of bcl-2. TUNEL assay data revealed that alcohol exposure increased the apoptosis levels in the embryonic hearts. In vitro experiments demonstrated that curcumin treatment could reverse the up-regulation of active forms of caspase-3 and caspase-8, and down-regulation of bcl-2 induced by alcohol treatment. In addition, curcumin also corrected the high level of histone H3K9 acetylation induced by alcohol. Moreover, the high apoptosis level induced by alcohol was reversed after curcumin treatment in cardiac cells.ConclusionsThese findings indicate that histone modification may play an important role in mediating alcohol induced fetal cardiac apoptosis, possibly through the up-regulation of H3K9 acetylation near the promoter regions of apoptotic genes. Curcumin treatment may correct alcohol-mediated fetal cardiac apoptosis, suggesting that curcumin may play a protective role against alcohol abuse caused cardiac damage during pregnancy.

Differentiation of mesenchymal stem cells into cardiomyocytes is regulated by miRNA-1-2 via WNT signaling pathway

BackgroundBone marrow derived stem cells (BMSCs) have the potential to differentiate into cardiomyocytes, but the rate of differentiation is low and the mechanism of differentiation is unclear completely. Here, we aimed to investigate the role of miR1-2 in differentiation of mouse BMSCs into cardiomyocyte-like cells and reveal the involved signaling pathways in the procedure.MethodsMouse BMSCs were treated with miR1-2 and 5-azacytine (5-aza). The expression of cardiac cell markers: NKx2.5, cTnI and GATA4 in BMSCs were examined by qPCR. The apoptosis rate was detected by flow cytometry and the activity of the Wnt/β-catenin signaling pathway was evaluated by measuring the upstream protein of this signaling pathway.ResultsAfter over-expression of miR1-2 in mouse BMSCs, the apoptosis rate was significantly lower than the 5-aza group, while the expressions of cardiac-specific genes: such as Nkx2.5, cTnI and GATA4 were significantly increased compared to the control group and the 5-aza group. Meanwhile, over-expression of miR1-2 in mouse BMSCs enhanced the expression of wnt11, JNK, β-catenin and TCF in the Wnt/β-catenin signaling pathway. Use of LGK-974, an inhibitor of Wnt/β-catenin signaling pathway, significantly reduced the expression of cardiac-specific genes and partially blocked the role of the miR1-2.ConclusionOver-expression of miR1-2 in mouse BMSCs can induce them toward promoted cardiomyocyte differentiation via the activation of the Wnt/β-catenin signaling pathway. Compared to 5-aza, miR1-2 can induce differentiation of BMSCs into cardiomyocytes more effectively with a less cytotoxicity.

Prenatal alcohol exposure causes the over-expression of DHAND and EHAND by increasing histone H3K14 acetylation in C57 BL/6 mice.

Prenatal alcohol exposure leads to congenital heart abnormal development, its mechanisms are still unknown. Recent reports have associated alcohol exposure with histone H3 acetylation. In the present study, we have performed the experiments to test the hypothesis that histone H3K14 acetylation is the key role in the fetal heart leads to over-expression of cardiac specific genes DHAND and EHAND caused by prenatal alcohol exposure. Seventy pregnant C57BL/6 mice were divided randomly into seven groups (n=10). They were the untreated group, dimethyl sulfoxide group, alcohol exposure group, curcumin treatment group, both alcohol and curcumin treatment group, SAHA treatment group, both alcohol and SAHA treatment group. Fetal mouse hearts were collected on embryonic day 14.5. The changes of HATs activities, the acetylation levels of histone H3K14 (H3K14ac), the expression levels of cardiac specific genes DHAND and EHAND, and structure of chromatin were determined. Our data indicates that curcumin and SAHA significantly reduces and increases the activities of HATs and the levels of histone H3K14ac in fetal hearts, respectively. The expression of DHAND and EHAND is significantly down-regulated and up-regulated in the groups treated with curcumin and SAHA. Furthermore, our results from ChIP assays have shown that the histone H3K14ac connects with the DHAND and EHAND genes are significantly inhibited by curcumin and simulated by SAHA. Our study suggests that prenatal alcohol exposure causes the over-expression of DHAND and EHAND by increasing H3K14ac in mice.

Measurements in Pediatric Patients with Cardiomyopathies: Comparison of Cardiac Magnetic Resonance Imaging and Echocardiography.

Aims: Cardiomyopathies are common cardiovascular diseases in children. Cardiac magnetic resonance imaging (cMRI) and echocardiography (Echo) are routinely applied in the detection and diagnosis of pediatric cardiomyopathies. In this study, we compared and explored the correlation between these two measurements in pediatric patients with various cardiomyopathies. Methods and Results: A total of 53 pediatric patients with cardiomyopathy hospitalized during the recent 3 years in our hospital were analyzed. All of them and 22 normal controls were assessed by both cMRI and Echo. Cardiac function of the patients was graded according to the New York Heart Association functional classification. The cardiac function indexes measured with both cMRI and Echo included left-ventricular (LV) end-diastolic volume (EDV), end-systolic volume, ejection fraction and fractional shortening. These parameters were somehow lower in cMRI measurements than in Echo measurements. The index of diastolic function, such as peak filling rate (PFR) measured with cMRI, had a good correlation with the clinical cardiac functional score, while the index of the diastolic function (early/atrial filling ratio and isovolumic relaxation time) measured with Echo was not well correlated with the clinical cardiac function score. Significant systolic dysfunction was detected by cMRI in 34 patients with dilated cardiomyopathy, LV noncompaction or endocardial fibroelastosis. Significant diastolic dysfunction was detected by cMRI in 19 patients with hypertrophic cardiomyopathy or restrictive cardiomyopathy showing an alteration in PFR and EDV. Conclusion: Both cMRI and Echo are of great value in the diagnosis and assessment of cardiac function in pediatric patients with cardiomyopathy. cMRI could accurately display the characteristic morphological changes in the hearts affected with cardiomyopathies, and late gadolinium enhancement on cMRI may reveal myocardial fibrosis, which has obvious advantages over Echo measurements in diagnosis. Furthermore, cMRI can quantitatively determine ventricular function because it does not make invalid geometrical assumptions.

Retrospective Studies on Pediatric Infective Endocarditis over 40 Years in a Mid-West Area of China

Objective: We have evaluated 106 pediatric cases of infective endocarditis (IE) to elucidate clinical manifestations and pathogenic microorganism profiling of IE in China. Methods: Clinical features, complications, pathogenic microorganisms, diagnosis and treatment of pediatric IE were reviewed in two groups of patients with IE from the different periods of time (group A, 34 cases obtained in the period from 2000 to 2011 and group B, 72 cases obtained in the period from 1964 to 1999). Results: A total of 106 pediatric patients with a definite diagnosis of IE based on the modified Duke criteria were enrolled and evaluated in this study. By comparing two groups of patients from different time periods, we found that the incidence of rheumatic heart disease was significantly reduced (from 19.4 to 5.9%), whereas congenital heart disease-associated IE had a tendency to increase (from 55.6 to 79.4%). Staphylococcus aureus was detected as the most common pathogenic microorganism, and its involvement tended to increase (from 32.0 to 58.5%), whereas the percentage of infections caused by Streptococcus viridans (8.0%) had not changed. It was interesting to note that the rate of vegetations detected was increased from 50.0 to 67.6% and the incidence of right-sided IE was also increased (from 35.0 to 60.9%). The most common valves involved in recent cases were tricuspid valves (increase from 30.0 to 47.8%), while mitral valve infection was reduced (from 60.0 to 39.1%). Penicillin was still the most commonly used antibiotic for the treatment of IE; the combination of penicillin plus cephalosporin has been recommended more and more recently. Conclusions: Comparing pediatric IE patients during the past 40 years, we found that the role of rheumatic heart disease as a predisposing factor is diminishing. Pediatric IE is still predominantly caused by staphylococci. The most commonly involved valves are tricuspid valves instead of mitral valves. Gram-positive bacteria showed an increased resistance to penicillin when used alone, and the use of combination treatment with antibiotics is increasing in the area.

Islet-1 may function as an assistant factor for histone acetylation and regulation of cardiac development-related transcription factor Mef2c expression.

Objective Islet-1 is an important transcription factor for cardiac development through mediating extensive interactions between DNA and proteins. The present study was to investigate the role of Islet-1 in regulating the expression of cardiac development-related transcription factors and mechanism. Methods and Results The expression of Islet-1 and histone acetylases (HATs) subtype p300 was determined in newborn mouse hearts and mouse embryonic hearts at different development stages using Western blot. The expression of Islet-1 and cardiac development-related transcription factors Mef2c, GATA4 and Tbx5 as well as histone H3 acetylation level were determined in cardiac progenitor cells with and without transfection of Islet-1 interference RNA (RNAi) in lentivirus using PCR and Western blot. Islet-1 peak expression occurred on day E14.5 in mouse embryonic heart, and was present in the promoter regions of Mef2c, GATA4 and Tbx5 that were precipitated with p300 antibody. When Islet-1 was inhibited with specific RNAi in cardiac progenitor cells, the expression of Mef2c and Tbx5, but not GATA4, was significantly suppressed along with selective reduction in histone H3 acetylation in the promoter region of Mef2c, but not GATA4 and Tbx5. The level of Mef2c DNA, not GATA4 and Tbx5, in the complex associated with p300 was significantly decreased in the cells with Islet-1 knockdown. Conclusions These data suggested that Islet-1 might function as an assistant factor that was involved in the regulation of histone acetylation and Mef2c expression via assisting p300 on specifically targeting the promoter of Mef2c.

Identification of target genes in cardiomyopathy with fibrosis and cardiac remodeling

BackgroundIdentify genes probably associated with chronic heart failure and predict potential target genes for dilated cardiomyopathy using bioinformatics analyses.MethodsGene expression profiles (series number GSE3585 and GSE42955) of cardiomyopathy patients and healthy controls were downloaded from the Expression Omnibus Gene (GEO) database. Differential expression of genes (DEGS) between the two groups of total 14 cardiomyopathy patients and 10 healthy controls were subsequently identified by limma package of R. Database for Annotation, Visualization, and Integrated Discovery (DAVID Tool), which is an analysis of enriched biological processes. Search Tool for the Retrieval Interacting Genes (STRING) was used as well for the analysis of protein-protein interaction network (PPI). Prediction of the potential drugs was suggested based on the preliminarily identified genes using Connectivity Map (CMap).ResultsEighty-nine DEGs were identified (57 up-regulated and 32 down-regulated). The most enrichment Gene Ontology (GO) terms (P < 0.05) contain genes involved in extracellular matrix (ECM) and biological adhesion signal pathways (P < 0.05, ES > 1.5) such as ECM-receptors, focal adhesion and transforming growth factor beta (TGF-β), etc. Fifty-one differentially expressed genes were found to encode interacting proteins. Eleven key genes along with related transcription factors were identified including CTGF, POSTN, CORIN, FIGF, etc.ConclusionBioinformatics-based analyses reveal the targeted genes probably associated with cardiomyopathy, which provide clues for pharmacological therapies aiming at the targets.