Yanhui Sheng

Nicotine Promotes Cardiomyocyte Apoptosis via Oxidative Stress and Altered Apoptosis-Related Gene Expression

Objective: To investigate the effect of nicotine on cardiomyocyte apoptosis in vitro and explore the potential mechanisms involved. Methods: The MTT assay was used to detect the viability of cultured cardiomyocytes exposed to different concentrations of nicotine (0.1–100 µM). Laser confocal microscopy, TUNEL assay and flow cytometry were utilized to detect cardiomyocyte apoptosis. Oxidative stress was evaluated by the levels of lactic dehydrogenase, malondialdehyde and superoxide dismutase in the supernatant of culture media. Real-time PCR was conducted to identify mRNA expression changes in apoptosis-related genes between the nicotine and the control group. Results: Nicotine was found to inhibit cardiomyocyte viability in a concentration-dependent manner. Our results demonstrated that nicotine can promote cardiomyocyte apoptosis and the antioxidant glutathione can protect cardiomyocytes from apoptosis via inhibition of nicotine-induced oxidative stress. Real-time PCR indicated that the expression of Bcl-2, Pax3, Bmp4 and Slug was down-regulated in the nicotine group, while the expression of P53, Bax and Msx1 was up-regulated. Conclusion: Nicotine promotes cardiomyocyte apoptosis by inducing oxidative stress and disrupting apoptosis-related gene expression.

Transcatheter closure of perimembranous ventricular septal defect in children: safety and efficiency with symmetric and asymmetric occluders.

Objectives: This study was designed to determine the safety and efficiency of asymmetric and symmetric ventricular septal occluders (AVSDOs and SVSDOs, respectively) for closure perimembranous ventricular septal defect (PMVSD) in children. Methods: Between January 2003 and December 2007, 142 children with PMVSD were treated with occluders (64 with AVSDOs and 78 with SVSDOs). Results: The defect diameter was 5.3 ± 1.1 mm in the AVSDO group and 5.4 ± 1.3 mm in the SVSDO group (P > 0.05). The success rates were similar between two groups [93.8% (AVSDO) vs. 94.9% (SVSDO), P > 0.05]. Two patients in the AVSDO group were switched to the SVSDO group due to residual shunts, and one patient in the SVSDO group was switched due to aortic regurgitation after deployment of the occluder. After procedure, 17 patients [seven with AVSDOs and nine with SVSDOs (P > 0.05)] developed various types of heart block (HB). Among them, 13 patients converted to the normal sinus rhythm. The remaining four cases had not recovered at the end of the study. Conclusions: Transcatheter closure of PMVSD using both AVSDO and SVSDO was safe and effective. Development of HB was the main complication for both devices.

Risk Factors and Outcomes of Post-Procedure Heart Blocks After Transcatheter Device Closure of Perimembranous Ventricular Septal Defect

OBJECTIVES The aim of this study was to analyze the risk factors and mid-term outcomes associated with post-procedure heart blocks (PPHBs) after transcatheter closure of perimembranous ventricular septal defect (pmVSD). BACKGROUND The development of heart blocks remains a major challenge for transcatheter closure of pmVSD. METHODS Transcatheter closure of pmVSD was carried out in 228 patients. Electrocardiography and 24-h Holter monitoring were performed before the procedure, within 1 week after the procedure, then 1, 3, 6, and 12 months, and every year thereafter. RESULTS Thirty-three patients (14.5%) who received transcatheter closure of pmVSD developed PPHBs. PPHBs included right bundle branch block (57.6%), left bundle branch block (24.2%), and atrioventricular block (18.2%). High-degree atrioventricular blocks occurred in 4 patients and recovered to normal conduction after intravenous administration of hydrocortisone. PPHBs recovered to normal conduction in 21 patients by the time of hospital discharge. Compared with the patients without PPHBs, the patients suffering PPHBs were characterized by a significantly longer distance between the aortic valve and the defect (DAVD), a shorter distance from the lower rim of the defect to the septal leaflet of the tricuspid valve (DLRD-SLTV), and a larger diameter difference between the occluder and ventricular septal defect (DDOV). The earlier the PPHBs developed after the procedure, the more difficult the recovery to normal conduction. CONCLUSIONS The outcome of PPHBs after transcatheter closure of pmVSD was satisfactory, as most patients recovered to normal conduction. Measurements of DLRD-SLTV, DAVD, and DDOV may be useful in predicting the incidence of PPHBs.

Inhibition of proliferation and differentiation and promotion of apoptosis by cyclin L2 in mouse embryonic carcinoma P19 cells

Cyclin L2 (CCNL2) is a novel member of the cyclin gene family. In a previous study, we demonstrated that CCNL2 expression was upregulated in ventricular septum tissues from patients with ventricular septal defect compared to healthy controls. In the present study, we established a stable CCNL2-overexpressing P19 cell line that can differentiate to myocardial cells when treated with 1% dimethyl sulfoxide (DMSO). Our data showed that stable CCNL2-overexpressing P19 cells were less differentiated after treatment with 1% DMSO and that expression of myocardial cell differentiation-related genes (such as cardiac actin, GATA4, Mef2C, Nkx2.5, and BNP) were reduced compared to vector-only transfected P19. Moreover, P19 cells overexpressing the CCNL2 gene had a reduced growth rate and a remarkably decreased S phase. We also found that these cells underwent apoptosis, as detected by two different apoptosis assays. The anti-apoptotic Bcl-2 protein was also downregulated in these cells. In addition, real-time PCR analysis revealed that expression of Wnt and beta-catenin was suppressed and GSK3beta was induced in the CCNL2-overexpressing P19 cells. These data suggest that overexpression of CCNL2 inhibited proliferation and differentiation of mouse embryonic carcinoma P19 cells and induced them to undergo apoptosis, possibly through the Wnt signal transduction pathway.

Silencing of FABP3 promotes apoptosis and induces mitochondrion impairment in embryonic carcinoma cells

Fatty acid binding protein 3 (FABP3) (also known as H-FABP) is a member of the intracellular lipid-binding protein family, and is mainly expressed in cardiac muscle tissue. The in vivo function of FABP3 is proposed to be in fatty acid metabolism, trafficking, and cell signaling. Our previous study found that FABP3 is highly regulated in patients with ventricular septal defect (VSD), and may play a significant role in the development of human VSD. In the present study, we aimed to investigate the impact of FABP3 knockdown by RNA interference (RNAi) on apoptosis and mitochondrial function of embryonic carcinoma (P19) cells. The results revealed that downregulated FABP3 expression promoted apoptosis, and resulted in mitochondrial deformation, increased mitochondrial membrane potential (MMP), and decreased intracellular ATP synthesis. In addition, the knockdown of FABP3 also led to excess intracellular ROS production. However, there was no obvious influence on the amount of mitochondrial DNA. Collectively, our results indicated that FABP3 knockdown promoted apoptosis and caused mitochondrial dysfunction in P19 cells, which might be responsible for the development of human VSD.

Relationship between brachial-ankle pulse wave velocity and metabolic syndrome components in a Chinese population.

Abstract The purpose of this study was to assess the relationship between arterial stiffness, as measured by brachial-ankle pulse wave velocity (baPWV), and the presence of the metabolic syndrome (MS) in a Chinese population. A total of 4,445 subjects were enrolled. The prevalence of MS in our study population was 21.7%, 17.2% and 25.6% for the general population, males and females, respectively. With adjustments for age, gender, cigarette smoking, heart rate, total cholesterol, low-density lipoprotein (LDL) cholesterol, and the use of anti-hypertensive drug, the stepwise regression analysis showed that baPWV had a significant relationship with components of MS, including systolic blood pressure (P < 0.001), diastolic blood pressure (P < 0.001), glucose (P < 0.001), high-density lipoprotein (HDL) cholesterol (P  =  0.04), and triglycerides (P < 0.001), but no relationship with waist circumference (P  =  0.25). With an increase in the number of the MS components, baPWV increased significantly both in women and men. This study indicated that the MS is indeed a risk factor for arterial stiffness. Monitoring of baPWV in patients with MS may help in identifying persons at high risk for cardiovascular disease.

Silencing of FABP3 Inhibits Proliferation and Promotes Apoptosis in Embryonic Carcinoma Cells

Fatty acid–binding protein 3 (FABP3) facilitates the movement of fatty acids in cardiac muscle. Previously, we reported that FABP3 is highly upregulated in the myocardium of ventricular septal defect patients and overexpression of FABP3 inhibited proliferation and promoted apoptosis in embryonic carcinoma cells (P19 cells). In this study, we aimed to investigate the effect of FABP3 gene silencing on P19 cell differentiation, proliferation and apoptosis. We used RNA interference and a lentiviral-based vector system to create a stable FABP3-silenced P19 cell line; knockdown of FABP3 was confirmed by quantitative real-time PCR. Expression analysis of specific differentiation marker genes using quantitative real-time PCR and observation of morphological changes using an inverted microscope revealed that knockdown of FABP3 did not significantly affect the differentiation of P19 cells into cardiomyocytes. CCK-8 proliferation assays and cell cycle analysis demonstrated that FABP3 gene silencing significantly inhibited P19 cell proliferation. Furthermore, Annexin V-FITC/propidium iodide staining and the caspase-3 activity assay revealed that FABP3 gene silencing significantly promoted serum starvation–induced apoptosis in P19 cells. In agreement with our previous research, these results demonstrate that FABP3 may play an important role during embryonic heart development, and that either overexpression or silencing of FABP3 will lead to an imbalance between proliferation and apoptosis, which may result in embryonic cardiac malformations.

Dynamic mitochondrial changes during differentiation of P19 embryonic carcinoma cells into cardiomyocytes

Murine P19 embryonal carcinoma cells are multipotent cells that can differentiate into cardiomyocytes when treated with dimethyl sulfoxide. This experimental model provides an invaluable tool to study different aspects of cardiac differentiation, such as the function of cardiac‑specific transcription factors and signaling pathways, and the regulation of contractile protein expression. The role of mitochondria during cardiac differentiation is unclear. In this context, we have examined the mitochondrial-related changes in undifferentiated and differentiated P19 cells. We observed that mitochondrial DNA content sharply decreased in P19 cell aggregates compared to undifferentiated cells, accompanied by decreased levels of adenosine triphosphate (ATP) and reactive oxygen species (ROS). Following the aggregation stage, the mitochondrial DNA content reached its highest level on day 7 of the differentiation process, with the intracellular ROS level showing a trend to increase, similar to cellular ATP production. In conclusion, our study on differentiating P19 embryonal carcinoma cells provides new insights into the role of mitochondria in the differentiation of P19 stem cells into beating cardiomyocytes.

Differential expression of microRNAs in cardiac myocytes compared to undifferentiated P19 cells.

microRNA (miRNA) expression is tightly controlled in a tissue-specific and developmental stage-specific manner; some are highly and specifically expressed in cardiovascular tissues. miRNA expression profiling, using miRNA microarrays facilitates studying the biological function of miRNAs. We investigated changes in miRNA expression profiles during differentiation of P19 cells into cardiac myocytes in order to elucidate the mechanisms of heart development. The morphology of P19 cells during differentiation was observed using an inverted microscope. Western blot analysis was performed to detect cardiac troponin I (cTnI) expression. Total RNA was extracted from P19 cells for microarray and real-time quantitative reverse transcription-polymerase chain reaction (real-time qRT-PCR) analyses to determine the miRNA expression profile. The miRNA microarray revealed differential expression of 49 miRNAs, of which 26 were down-regulated and 23 were up-regulated in differentiated cardiac myocytes, compared to normal P19 cells. This was confirmed by real-time qRT-PCR. We also utilized target prediction analysis to identify gene targets. Some miRNAs may have important roles in cardiac development and congenital heart defects (CHDs). Further analysis of miRNA function to confirm their target genes during cardiac development will determine the potential for novel miRNA-based therapeutic strategies.

Quality control of the blood pressure phenotype in the Gaoyou population study.

Abstract The Korotkoff approach is the only blood pressure (BP) measurement technique that allows contemporary data to be compared with decades of research. We randomly recruited 4483 people (53.3% women; mean age 52.1 years) from Gaoyou County, Jiangsu Province, China. Nine observers recorded the participants™ BP three times consecutively following Chinese Society of Hypertension guidelines. We assessed the BP phenotype based on five criteria: completeness of readings, percentage of identical BP readings, odd BP readings, end-digit preference and trends in BP from the first to the third reading. The proportion of participants with identical readings were 2.0% and 3.1% for systolic (SBP) and diastolic blood pressure (DBP), respectively. Among 26,898 BP values, 0.3% ended in an odd number. Among observers, the prevalence of identical readings varied from 0% to 5.3% for SBP and from 0% to 6.8% for DBP. Compared with the expected frequency of 20%, those ending in 0 had a lower frequency (17.2%; p < 0.001), whereas those ending in 8 had a higher frequency (22.4%; p < 0.001). From the first to the third measurement, SBP and DBP decreased (p < 0.001) by 0.87 and 0.55 mmHg, respectively. In conclusion, the procedures set up in the Gaoyou study produced a high-quality BP phenotype.