Intermittent Hypoxia Prevents Myocardial Mitochondrial Ca2+ Overload and Cell Death during Ischemia/Reperfusion: The Role of Reactive Oxygen Species.

It has been documented that reactive oxygen species (ROS) contribute to oxidative stress, leading to diseases such as ischemic heart disease. Recently, increasing evidence has indicated that short-term intermittent hypoxia (IH), similar to ischemia preconditioning, could yield cardioprotection. However, the underlying mechanism for the IH-induced cardioprotective effect remains unclear. The aim of this study was to determine whether IH exposure can enhance antioxidant capacity, which contributes to cardioprotection against oxidative stress and ischemia/reperfusion (I/R) injury in cardiomyocytes. Primary rat neonatal cardiomyocytes were cultured in IH condition with an oscillating O2 concentration between 20% and 5% every 30 min. An MTT assay was conducted to examine the cell viability. Annexin V-FITC and SYTOX green fluorescent intensity and caspase 3 activity were detected to analyze the cell death. Fluorescent images for DCFDA, Fura-2, Rhod-2, and TMRM were acquired to analyze the ROS, cytosol Ca2+, mitochondrial Ca2+, and mitochondrial membrane potential, respectively. RT-PCR, immunocytofluorescence staining, and antioxidant activity assay were conducted to detect the expression of antioxidant enzymes. Our results show that IH induced slight increases of O2-· and protected cardiomyocytes against H2O2- and I/R-induced cell death. Moreover, H2O2-induced Ca2+ imbalance and mitochondrial membrane depolarization were attenuated by IH, which also reduced the I/R-induced Ca2+ overload. Furthermore, treatment with IH increased the expression of Cu/Zn SOD and Mn SOD, the total antioxidant capacity, and the activity of catalase. Blockade of the IH-increased ROS production abolished the protective effects of IH on the Ca2+ homeostasis and antioxidant defense capacity. Taken together, our findings suggest that IH protected the cardiomyocytes against H2O2- and I/R-induced oxidative stress and cell death through maintaining Ca2+ homeostasis as well as the mitochondrial membrane potential, and upregulation of antioxidant enzymes.

Combining Induced Pluripotent Stem Cells and Genome Editing Technologies for Clinical Applications.

In this review, we introduce current developments in induced pluripotent stem cells (iPSCs), site-specific nuclease (SSN)-mediated genome editing tools, and the combined application of these two novel technologies in biomedical research and therapeutic trials. The sustainable pluripotent property of iPSCs in vitro not only provides unlimited cell sources for basic research but also benefits precision medicines for human diseases. In addition, rapidly evolving SSN tools efficiently tailor genetic manipulations for exploring gene functions and can be utilized to correct genetic defects of congenital diseases in the near future. Combining iPSC and SSN technologies will create new reliable human disease models with isogenic backgrounds in vitro and provide new solutions for cell replacement and precise therapies.

Baicalein Ameliorates Pulmonary Arterial Hypertension Caused by Monocrotaline through Downregulation of ET-1 and ETAR in Pneumonectomized Rats.

Baicalein (BE) extracted from Scutellaria baicalensis Georgi is able to alleviate various cardiovascular and inflammatory diseases. However, the effects of BE on pulmonary arterial hypertension (PAH) remain unknown. Therefore, the present study aimed to examine whether BE ameliorates pneumonectomy and monocrotaline-induced PAH in rats and further investigate the underlying molecular mechanisms. Administration of BE greatly attenuated the development of PAH as evidenced by an improvement of its characteristic features, including elevation of right ventricular systolic pressure, right ventricular hypertrophy, and pulmonary vascular remodeling. Moreover, the increased protein expression of endothelin-1 (ET-1) and ETA receptor (ETAR), superoxide overproduction, and activation of Akt/ERK1/2/GSK3[Formula: see text]/[Formula: see text]-catenin pathway that occurred in the lungs of PAH rats were markedly reversed by BE treatment. Compared with the untreated PAH rats, higher expression of endothelial nitric oxide synthase (eNOS), but lower levels of inducible nitric oxide synthase and vWF were observed in BE-treated PAH rats. Collectively, treatment with BE remarkably attenuates the pathogenesis of PAH, and the protection of BE may be associated with suppressing Akt/Erk1/2/GSK3[Formula: see text]/[Formula: see text]-catenin/ET-1/ETAR signaling and preventing endothelial dysfunction. These results suggest that BE is a potential agent for treatment of PAH.

Intermittent Hypoxia Inhibits Na+-H+ Exchange-Mediated Acid Extrusion Via Intracellular Na+ Accumulation in Cardiomyocytes.

BACKGROUND/AIMS: Intermittent hypoxia (IH) has been shown to exert preconditioning-like cardioprotective effects. It also has been reported that IH preserves intracellular pH (pHi) during ischemia and protects cardiomyocytes against ischemic reperfusion injury. However, the exact mechanism is still unclear. METHODS: In this study, we used proton indicator BCECF-AM to analyze the rate of pHi recovery from acidosis in the IH model of rat neonatal cardiomyocytes. Neonatal cardiomyocytes were first treated with repetitive hypoxia-normoxia cycles for 1-4 days. Cells were then acid loaded with NH4Cl, and the rate of pHi recovery from acidosis was measured. RESULTS: We found that the pHi recovery rate from acidosis was much slower in the IH group than in the room air (RA) group. When we treated cardiomyocytes with Na+-H+ exchange (NHE) inhibitors (Amiloride and HOE642) or Na+-free Tyrode solution during the recovery, there was no difference between RA and IH groups. We also found intracellular Na+ concentration ([Na+]i) significantly increased after IH exposure for 4 days. However, the phenomenon could be abolished by pretreatment with ROS inhibitors (SOD and phenanathroline), intracellular calcium chelator or Na+-Ca2+ exchange (NCX) inhibitor. Furthermore, the pHi recovery rate from acidosis became faster in the IH group than in the RA group when inhibition of NCX activity. CONCLUSIONS: These results suggest that IH would induce the elevation of ROS production. ROS then activates Ca2+-efflux mode of NCX and results in intracellular Na+ accumulation. The rise of [Na+]i further inhibits the activity of NHE-mediated acid extrusion and retards the rate of pHi recovery from acidosis during IH.

Pulseless electrical activity in acute massive pulmonary embolism during thrombolytic therapy.

We report a case of acute pulmonary embolism with hemodynamic instability diagnosed by a computed tomography pulmonary angiogram. The patient developed pulseless electrical activity during systemic thrombolytic therapy with recombinant tissue plasminogen activator. Successful return of spontaneous circulation was achieved after immediate cardiopulmonary resuscitation with chest compressions for 6 min. His electrocardiogram (ECG) on arrival in the emergency department displayed sinus tachycardia, an S wave in lead I, a Q wave in lead III, incomplete right bundle branch block (RBBB), T-wave inversion (TWI) in leads V1-V3, ST elevation in leads aVR and V1, and ST depression in leads I, II, III, aVF, and V4-V6. These characteristic ECG changes might have prognostic value for clinical deterioration. He recovered after treatment. After discharge, the ECG showed resolution of TWI in leads V1-V3 and incomplete RBBB, suggesting recovery from right ventricular dysfunction, which was confirmed by an echocardiogram on follow in the outpatient department.

Angiotensin converting enzyme DD genotype is associated with acute coronary syndrome severity and sudden cardiac death in Taiwan: a case-control emergency room study.

BACKGROUND: Angiotensin converting enzyme (ACE) gene insertion/deletion (I/D) polymorphisms have been associated with acute coronary syndrome (ACS); however, several controversial results have also been found in different studied populations. This hospital-based, emergency room, case-control study in Taiwan retrospectively investigated 111 ACS patients, and 195 non-coronary subjects as a control group, to study the effects of ACE I/D polymorphism in the most urgent ACS patients. ACE I/D polymorphisms were determined by polymerase chain reaction-based assays and their associations with ACS risk, severity, and sudden cardiac death were determined. RESULTS: The ACE DD genotype was associated with ACS incidence. The DD genotype was associated with a significant 4-fold higher risk of ACS in multivariate analysis (odds ratio (OR) = 4.295; 95% confidence interval (CI): 1.436-12.851, p = 0.009), and a 3.35-fold higher risk of acute myocardial infarction. DD genotype carriers also had more than 3-fold higher risks of stenosis in all the three coronary arteries, left anterior descending artery infarction, and anterior wall infarction. In addition, the DD genotype was also associated with a higher risk of sudden cardiac death (OR = 6.484, 95% CI: 1.036-40.598, p = 0.046). CONCLUSIONS: This study demonstrated that the ACE DD genotype is an independent risk factor for ACS, and in particular, for acute myocardial infarction. In addition, the ACE DD genotype is also associated with greater ACS severity and a higher risk of sudden cardiac death. ACE genotyping is recommended for patients with a history of ACS, and more intensive preventive care is suggested for patients with the DD genotype.

The toxic effect of Amiodarone on valve formation in the developing heart of zebrafish embryos.

BACKGROUND: Amiodarone is a class D drug given to treat arrhythmia, including pregnant women, but its effects on the developing heart have not been studied. Although some studies have suggested that this drug is safe for fetuses, they have been conducted on mothers with fetuses at or beyond six months of gestational age. RESULTS: The occurrence of valve defect was positively proportional to Amiodarone concentrations over 9 µM, but not lower than 6 µM. Ectopic overexpression of versican was observed at the atrioventricular canal of the Amiodarone-treated embryos at 15 µM (EC(50)). VE-cadherin (cdh5), normally downregulated at the endocardial cushion, was also ectopically overexpressed in the Amiodarone-treated embryos. Knockdown of either versican or cdh5 in the Amiodarone-treated embryos could rescue the valve defect caused by Amiodarone. CONCLUSIONS: By inducing versican ectopical overexpression, leading, in turn, to cdh5 ectopical overexpression, Amiodarone treatment causes failure of cardiac valve formation in zebrafish embryos.

Inhibitory effect of n-butylidenephthalide on neointimal hyperplasia in balloon injured rat carotid artery.

This investigation was designed to determine the inhibitory effects and mechanisms of n-butylidenephthalide (BP) from Angelica sinensis on smooth muscle cell (SMC) proliferation in vitro and in balloon injured rat carotid artery. Treatment of cultured rat aorta SMC-derived A7r5 cells with 25-100 µg/mL BP significantly inhibited the proliferation and arrested the cell cycle in G(0)/G(1) phase. BP induced the expression and migration of Nur77 from the nucleus to the cytoplasm. Among signal pathways, JNK and p38 MAPK were phosphorylated after BP treatment. In vivo, the neointimal area of common carotid artery 2 weeks after balloon injury reduced significantly in Sprague-Dawley rats treated with 150-300 mg/kg BP compared with the control. The proliferative activity indicated by immunohistochemical detection of Ki-67 positive cells in the neointima was significantly decreased in the 60-300 mg/kg BP treatment groups. The apoptotic activity indicated by cleaved caspase-3 positive cells and Nur77 positive cells in the neointima was significantly increased in rats treated with 60-300 mg/kg BP. This study demonstrated BP inhibited neointimal hyperplasia in balloon injured rat carotid artery due to its dual effects of proliferative inhibition and apoptotic induction on SMCs. Up-regulation of Nur77 gene may partly explain the antihyperplasia activity of BP on the neointima.

Plasma C-reactive protein and 5-lipoxygenase-activating protein gene promoter poly-A polymorphism in patients with coronary artery disease.

OBJECTIVES: The purpose of this study was to explore the possible associations of the 5-lipoxygenase-activating protein (FLAP) poly-A genotype, plasma high-sensitivity C-reactive protein (hsCRP) and the extent score of coronary artery disease (CAD). METHODS: The 17A/21A genotypes and plasma hsCRP levels were determined in 555 Chinese patients, 424 with and 131 without CAD. The luciferase reporter assay was performed to explore the functional significance of promoter poly-A polymorphism. RESULTS: CAD patients showed significantly higher plasma hsCRP (p = 0.007) than non-CAD subjects, but no differences in the 17A allele carriers and frequency. The extent score of CAD was significantly correlated with plasma hsCRP (p = 0.03). Furthermore, the 17A allele carriers showed significantly higher hsCRP than the 21A homozygotes (p = 0.02). Multiple linear regression analysis documented an impact of the poly-A genotype on plasma hsCRP (p = 0.03). In vitro, the 17A construct was found to have greater promoter activity than the 21A construct (p = 0.02). CONCLUSIONS: The present study demonstrated a significant correlation of FLAP gene promoter 17A allele carriers with higher plasma hsCRP levels in patients with CAD. This association might be related to the increased transcriptional activity of the FLAP gene and the resulting pro-inflammatory effect on the 5-lipoxygenase pathway.

Plasma adiponectin, T94G gene polymorphism and PAI-1 in patients with and without hypertension.

BACKGROUND: Reduced adiponectin level has been associated with metabolic syndrome, type 2 diabetes, coronary artery disease and gene polymorphisms, but the interrelationships of T94G genotype, plasma adiponectin and plasminogen activator inhibitor-1 (PAI-1) are less understood. PATIENTS AND METHODS: The T94G genotypes and plasma levels of adiponectin, and PAI-1 were determined in 568 Chinese patients, 212 with and 356 without hypertension, to study the possible associations of T94G genotype, plasma adiponectin, PAI-1 and blood pressure. RESULTS: Hypertensive patients showed significantly lower plasma adiponectin (9.7 +/- 11.1 vs. 11.5 +/- 10.0 microg/ml, p = 0.04) and higher PAI-1 (p < 0.001) levels but not significantly greater adiponectin TT genotype percentage (38.7 vs. 33.5%) and T allele frequency (0.620 vs. 0.585) than normotensive subjects. Plasma adiponectin was inversely related to PAI-1 activity (r = -0.09, p = 0.03) and antigen (r = -0.202, p < 0.001). Furthermore, the TT genotypic group showed significantly lower plasma adiponectin level (10.4 +/- 10.5 vs. 13.4 +/- 10.8 mug/ml, p = 0.03) and higher plasma PAI-1 activity (17.0 +/- 9.7 vs. 13.5 +/- 7.6 IU/ml, p = 0.003) and antigen (32.3 +/- 22.7 vs. 25.9 +/- 14.7 ng/ml, p = 0.01) than the GG genotypic group. Multiple linear regression analysis in all study subjects, in men and in normotensives documented an impact of adiponectin T94G genotype on plasma levels of adiponectin (p = 0.007, 0.003 and 0.03) and PAI-1 activity (p = 0.02, 0.03 and 0.04) and antigen (p = 0.03, 0.007 and 0.04) after adjustment for potential confounding factors. CONCLUSIONS: The present study demonstrated a significant correlation of the TT genotype with lower plasma adiponectin and higher plasma PAI-1 levels in a Chinese population. The contribution of this genotype seemed greater in men and normotensives. It suggested the adiponectin gene T94G polymorphism might affect the regulation of circulating adiponectin and PAI-1.

Association of interleukin-6 gene G-174C polymorphism and plasma plasminogen activator inhibitor-1 level in Chinese patients with and without hypertension.

BACKGROUND: The interleukin-6 (IL-6) gene promoter G-174C polymorphism has been associated with insulin resistance, hypertension, and coronary artery disease; however, its relationship with plasma PAI-1 level has not yet been studied. METHODS: The G-174C genotypes and plasma PAI-1 antigen and activity were determined in 424 Chinese subjects, 207 with hypertension and 217 without, to study the possible effects of IL-6 genotypes on the regulation of PAI-1 and blood pressure. RESULTS: Hypertensive patients showed significantly greater percentage of IL-6 GG genotype (51.7% v 33.2%, P < .001) and G allele frequency (71.7% v 59%, P < .001) than normotensive subjects. The GG genotypic group had significantly higher plasma PAI-1 activity (16.1 +/- 9.8 v 12.3 +/- 7.5 IU/mL, P = .03) and antigen (32.4 +/- 23.2 v 23.2 +/- 13.5 ng/mL, P = .01) than the CC genotypic group, with intermediate values in the GC genotypic group (15.9 +/- 9.0 IU/mL and 29.1 +/- 17.5 ng/mL). Multiple linear regression analysis in all study subjects and in normotensive subjects documented an independent dominant effect of IL-6 G-174C gene polymorphism on plasma levels of PAI-1 activity (P = .02 and .01) and antigen (P = .02 and .03) after log transformation and adjustment for confounding factors. CONCLUSIONS: The present study showed a positive association of the IL-6 GG genotype with hypertension and with elevated plasma PAI-1 level in normotensive individuals in a Chinese population in Taiwan. Our findings suggest that the IL-6 gene promoter G-174C polymorphism may affect the regulation of PAI-1 and blood pressure through an inflammatory mechanism.

Association of PAI-1 gene promoter 4g/5g polymorphism with plasma PAI-1 activity in Chinese patients with and without hypertension.

BACKGROUND: The PAI-1 gene promoter 4G/5G polymorphism was found to be associated with plasma PAI-1 activity in white but not yet in Chinese patients. Hypertension might exaggerate the 4G/5G genotype effect on plasma PAI-1. METHODS: The 4G/5G genotypes and plasma PAI-1 levels were determined in 565 Chinese, 211 with and 354 without hypertension to study the genotype effect and the mode of gene-environment interaction. RESULTS: Hypertensive patients showed significantly higher plasma PAI-1 activity (18.2 +/- 10.0 v 14.6 +/- 8.8 IU/mL, P <.001) than normotensive subjects, and also higher body mass index (BMI) and plasma triglyceride (TG), but had neither significant difference in the 4G allele frequency (0.531 v 0.549) nor in the 4G/4G genotype percentage (24.6% v 26.5%). The 4G/4G genotypic group had higher plasma PAI-1 activity (17.6 +/- 10.2 v 14.5 +/- 7.3 IU/mL, P =.027) than the 5G/5G genotypic groups, but the statistic significance was present in women (18.1 +/- 10.0 v 14.8 +/- 6.9 IU/mL, P =.025) and not in men (17.2 +/- 10.5 v 14.3 +/- 7.7 IU/mL, P =.39) after log transformation. Multiple regression analysis of all cases documented the independent effect of BMI (P =.000), plasma TG (P =.000), age (P =.006), gender (P =.046), and the PAI-1 genotype (4G/4G v 5G/5G, P =.012) on plasma PAI-1 activity. However, the significant association of 4G/4G genotypes with higher plasma PAI-1 activity was present in women (P =.004) but not in men. There was a significant difference (P =.04) on the plasma TG-PAI-1 activity correlations between the 4G/4G (r = 0.521) and 5G/5G (r = 0.117) genotypic groups of hypertensive patients. CONCLUSIONS: The present study showed that the 4G/4G genotype was associated with elevated plasma PAI-1 activity in Chinese patients with and without hypertension. The contribution of the PAI-1 genotype seemed larger in women. In hypertensives carrying the 4G/4G genotype, higher TG was correlated with higher PAI-1, suggesting a possible contribution of gene-environmental interaction to their high risk for atherothrombotic disease.

Carotid wall thickening, left ventricular hypertrophy, and insulin resistance in patients with hypertension.

BACKGROUND: Insulin resistance was associated with hypertension and coronary artery disease, and might play a role in the development of carotid atherosclerosis and cardiac hypertrophy. METHODS: The relationship of insulin resistance, estimated by steady-state plasma glucose (SSPG) with the B-mode ultrasound-measured carotid intima-medial thickness (IMT) and the M-mode echocardiographically determined left ventricular mass (LVM), was examined in 82 Chinese patients with hypertension. RESULTS: Insulin-resistant patients with obesity, glucose intolerance, dyslipidemia, and hypofibrinolysis show no significantly greater LVM index and carotid IMT than nonresistant individuals. Patients with either left ventricular hypertrophy or carotid wall thickening did not present significantly higher SSPG than those without these abnormalities. Neither carotid IMT nor LVM was associated with SSPG on multiple regression analysis. CONCLUSIONS: The study suggested that insulin resistance was not an important contributor for carotid thickening and cardiac hypertrophy in Chinese patients with hypertension.