Functional dissection of human cardiac enhancers and noncoding de novo variants in congenital heart disease.

Rare coding mutations cause ∼45% of congenital heart disease (CHD). Noncoding mutations that perturb cis-regulatory elements (CREs) likely contribute to the remaining cases, but their identification has been problematic. Using a lentiviral massively parallel reporter assay (lentiMPRA) in human induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs), we functionally evaluated 6,590 noncoding de novo variants (ncDNVs) prioritized from the whole-genome sequencing of 750 CHD trios. A total of 403 ncDNVs substantially affected cardiac CRE activity. A majority increased enhancer activity, often at regions with undetectable reference sequence activity. Of ten DNVs tested by introduction into their native genomic context, four altered the expression of neighboring genes and iPSC-CM transcriptional state. To prioritize future DNVs for functional testing, we used the MPRA data to develop a regression model, EpiCard. Analysis of an independent CHD cohort by EpiCard found enrichment of DNVs. Together, we developed a scalable system to measure the effect of ncDNVs on CRE activity and deployed it to systematically assess the contribution of ncDNVs to CHD.

Clinical Profile and Risk Factors for Cardiac Death in Pediatric Patients With Primary Dilated Cardiomyopathy at a Tertiary Medical Center in China.

Aim: We sought to identify the clinical characteristics and risk factors for cardiac mortality in pediatric patients with primary dilated cardiomyopathy (DCM) in China. Methods: A total of 138 pediatric patients who were consecutively diagnosed with primary DCM from January 2011 to December 2020 were included. We assessed patients' clinical symptoms and performed laboratory examinations, electrocardiography, and echocardiography. Results: Of these patients, 79 (57%) had severe systolic dysfunction (left ventricular ejection fraction of < 30%), 79 (57.2%) developed DCM before 12 months of age, 62 (45%) were male, 121 (87.7%) presented with advanced heart failure (cardiac functional class III/IV), and 54 (39.1%) presented with arrhythmia. At a median follow-up of 12 months, the overall cardiac mortality rate was 33%, and 40 of 46 deaths occurred within 6 months following DCM diagnosis. A multivariate Cox regression analysis identified several independent cardiac death predictors, including an age of 12 months to 5 years [hazard ratio (HR) 2.799; 95% confidence interval (CI) 1.160-6.758; P = 0.022] or 10-15 years (HR 3.617; 95% CI 1.336-9.788; P = 0.011) at diagnosis, an elevated serum alanine aminotransferase (ALT) concentration (≥ 51.5 U/L) (HR 2.219; 95% CI 1.06-4.574; P = 0.031), and use of mechanical ventilation (HR 4.223; 95% CI 1.763-10.114; P = 0.001). Conclusion: The mortality rate of primary DCM without transplantation is high. Age, an elevated serum ALT concentration, and the need for mechanical ventilation predict mortality in patients with primary DCM, providing new insights into DCM risk stratification.

Next-Generation Sequencing Reveals Novel Genetic Variants for Dilated Cardiomyopathy in Pediatric Chinese Patients.

Dilated cardiomyopathy (DCM) is a myocardial disease characterized by bilateral or left ventricular cardiac dilation and systolic dysfunction that can lead to heart failure and sudden cardiac death in children. Many studies have focused on genetic variation in DCM-related genes in adult populations; however, the mutational landscape in pediatric DCM patients remains undetermined, especially in the Chinese population. We applied next-generation sequencing (NGS) technology to genetically analyze 46 pediatric DCM patients to reveal genotype-phenotype correlations. Our results indicated DCM-associated pathogenic mutations in 10 genes related to the structure or function of the sarcomere, desmosome, and cytoskeleton. We also identified 6 pathogenic mutations (5 novel) in the Titin (TTN) gene that resulted in truncated TTN variants in 6 (13%) out of 46 patients. Correlations between TTN mutations and clinical outcomes were assessed. Our data indicate that one-third of pediatric DCM cases are caused by genetic mutations. The role of TTN variants should not be underestimated in pediatric DCM and age-dependent pathogenic penetrance of these mutations should be considered for familial DCM cases. We argue that genetic testing of DCM cases is valuable for predicting disease severity, prognosis, and recurrence risk, and for screening first-degree relatives.

The Efficacy and Medium to Long-Term Follow-Up of Transcatheter Retrograde Closure of Perimembranous Ventricular Septal Defects via the Femoral Artery With Amplatzer Duct Occluder II in Children.

Objective: The aim of this study was to assess the efficacy and safety of transcatheter retrograde closure of perimembranous ventricular septal defect (pmVSD) via the femoral artery with an Amplatzer Duct Occluder II (ADO II) in children. Methods: The clinical and follow-up data of 102 children who had successfully undergone transcatheter retrograde closure of pmVSD via the femoral artery with Amplatzer Duct Occluder II (ADO II) from February 2012 to June 2019 in our center were retrospectively reviewed. Results: In 102 of 103 patients, the defects were successfully closed (99.0%). The average age was 4.03 ± 1.84 years and the mean weight was 17.50 ± 5.58 kg. The mean diameter of the defects was 2.58 ± 0.63 mm. Hundred of the occluders had a waist length of 4 mm. The complete closure rate was 89.2% 24 h after the procedure and 98% at the last follow-up. The follow-up period ranged from 6 to 92 months, with a median of 36 months. One child developed new mild aortic regurgitation (AR), and 9 patients developed new mild tricuspid regurgitation (TR). During follow-up, TR disappeared in 5 patients and decreased in 4 patients, and AR relieved in one patient. One patient developed intermittent complete left bundle branch block 4 days after the procedure and recovered sinus rhythm 2 days later. No serious complications occurred. Conclusion: Transcatheter retrograde closure of pmVSD via the femoral artery with ADO II in children is safe, feasible, and effective in selected patients.

Transcatheter Device Closure of Perimembranous and Intracristal Ventricular Septal Defects in Children: Medium- and Long-Term Results.

Background In children, the practice of transcatheter closure of intracristal ventricular septal defect (icVSD) has been limited. Currently, there is a lack of comparison between device closure of perimembranous ventricular septal defect (pmVSD) and icVSD, and long-term clinical outcomes are rare. Methods and Results This study included a total of 633 children (39 with icVSD and 594 with pmVSD), aged 18 months to 16 years, who underwent transcatheter closure of ventricular septal defect between January 2014 and December 2018. All patients were followed up until September 2020, with a median follow-up of 46 months in the pmVSD group and 52 months in the icVSD group. The procedural success rate was 96.3% and 84.6% in pmVSD and icVSD groups, respectively (P=0.002). The median of age, weight, procedure time, fluoroscopic time, and radiation dose were greater in the icVSD group compared with the pmVSD group. More eccentric ventricular septal defect occluders were used in the icVSD group. Most adverse events were minor without any intervention, with cardiac rhythm/conduction abnormalities being the most common. In the pmVSD group, 2 patients experienced complete atrioventricular block, with one implanting a permanent pacemaker and the other dying of cardiac arrest secondary to reversible complete atrioventricular block 40 days postprocedure. Complete left bundle-branch block occurred in 14 patients, and 12 cases were transient. In the icVSD group, no complete atrioventricular block or death occurred, and one patient developed transient complete left bundle-branch block. Conclusions In selected patients, transcatheter device closure of pmVSD and icVSD can be performed safely and successfully, with excellent medium- and long-term results in children.

The Phosphodiesterase-5 Inhibitor Vardenafil Improves the Activation of BMP Signaling in Response to Hydrogen Peroxide.

PURPOSE: The pleiotropic roles of phosphodiesterase-5 inhibitors (PDE5is) in cardiovascular diseases have attracted attention. The effect of vardenafil (a PDE5i) is partly mediated through reduced oxidative stress, but it is unclear whether vardenafil protects against hydrogen peroxide (H2O2)-induced endothelial cell injury, and the molecular mechanisms that are involved remain unknown. We determined the protective role of vardenafil on H2O2-induced endothelial cell injury in cultured human umbilical vein endothelial cells (HUVECs). METHODS AND RESULTS: Vardenafil decreased the number of TUNEL-positive cells, increased the Bcl2/Bax ratio, and ameliorated the numbers of BrdU-positive cells in H2O2-treated HUVECs. The bone morphogenetic protein receptor (BMPR)/p-Smad/MSX2 pathway was enhanced in response to H2O2, and vardenafil treatment could normalize this pathway. To determine whether the BMP pathway is involved, we blocked the BMP pathway using dorsomorphin, which abolished the protective effects of vardenafil. We found that vardenafil improved the H2O2-induced downregulation of BMP-binding endothelial regulator protein (BMPER), which possibly intersects with the BMP pathway in the regulation of endothelial cell injury in response to oxidative stress. CONCLUSIONS: We demonstrated for the first time that exogenous H2O2 activates BMPR expression and promotes Smad1/5/8 phosphorylation. Additionally, vardenafil can attenuate H2O2-induced endothelial cell injury in HUVECs. Vardenafil decreases apoptosis through an improved Bcl-2/Bax ratio and increases cell proliferation. Vardenafil protects against endothelial cell injury through ameliorating the intracellular oxidative stress level and BMPER expression. The protective role of vardenafil on H2O2-induced endothelial cell injury is mediated through BMPR/p-Smad/MSX2 in HUVECs.

Sonic hedgehog protein regulates fibroblast growth factor 8 expression in metanephric explant culture from BALB/c mice: Possible mechanisms associated with renal morphogenesis.

The sonic hedgehog (SHH) morphogen regulates cell differentiation and controls a number of genes during renal morphogenesis. To date, the effects of SHH on fibroblast growth factors (Fgfs) in embryonic kidney development remain unclear. In the present study, explants of BALB/c mouse embryonic kidney tissues were used to investigate the role of exogenous SHH on Fgf8 and Fgf10 expression levels ex vivo. Ureteric bud branches and epithelial metanephric derivatives were used to determine the renal morphogenesis with Dolichos biflorus agglutinin or hematoxylin­eosin staining. mRNA expression levels were determined using reverse transcription­quantitative polymerase chain reaction, while the protein expression levels were examined using immunohistochemistry and western blot analysis. During the initial stages of metanephric development, low levels of SHH, Fgf8, and Fgf10 expression were observed, which were found to increase significantly during more advanced stages of metanephric development. In addition, exogenous SHH protein treatment increased the number of ureteric bud branches and enhanced the formation of nephrons. Exogenous SHH reduced the Fgf8 mRNA and protein expression levels, whereas cyclopamine (an SHH­smoothened receptor inhibitor) interfered with SHH­mediated downregulation of Fgf8 expression. By contrast, exogenous SHH protein was not found to modulate Fgf10 mRNA and protein expression levels. In conclusion, these results indicate that the modulatory effects of SHH on BALB/c mouse metanephric explant cultures may involve the regulation of Fgf8 expression but not Fgf10 expression, which provides evidence for the functional role of Fgf proteins in renal morphogenesis.

High glucose induces the release of endothelin-1 through the inhibition of hydrogen sulfide production in HUVECs.

Hydrogen sulfide (H(2)S) has recently been identified as an endogenous gaseous signaling molecule. In the vascular system, the formation of H(2)S is catalyzed by cystathionine γ­lyase (CSE). Previous studies have demonstrated the protective effects of H(2)S on ischemic injury in various types of tissue. However,, little is known about the role of H(2)S in diabetes-associated vascular diseases. Thus, the aim of the present study was to examine the possible role of H(2)S in high glucose-induced vascular dysfunction, and to explore the underlying mechanisms. Human umbilical vein endothelial cells (HUVECs) were isolated from human umbilical veins. The levels of H(2)S following treatment with various levels of glucose were determined and the secretion of endothelin-1 (ET-1) was measured by ELISA. The mRNA and protein expression of CSE in the HUVECs was determined by real-time RT-PCR and western blot analysis, respectively. Treatment with high glucose (25 mmol/l) for 48 h significantly increased the secretion of ET-1 by HUVECs, with the concomitant suppression of H(2)S production and CSE protein expression. The increase in exogenous H(2)S levels through the administration of sodium hydrosulfide (NaHS) attenuated the high glucose-induced downregulation of CSE protein expression, and significantly inhibited the secretion of ET-1. These results suggest that the downregulation of CSE protein expression and the subsequent decrease in H(2)S production play a role in high glucose-induced vascular dysfunction possibly by increasing the secretion of ET-1 by endothelial cells.

Plasma 15-F2t-isoprostane in idiopathic pulmonary arterial hypertension.

BACKGROUND: 15-F2t-isoprostane (15-F2t-IsoP), a prostaglandin F2-like compound, is widely recognized as a biomarker of chronic heart failure. This study investigated the potential role and prognostic significance of plasma 15-F2t-IsoP in patients with idiopathic pulmonary arterial hypertension (IPAH). METHODS: Plasma 15-F2t-IsoP concentrations were determined in 80 consecutive IPAH patients at the time of their first right heart catheterization, and monitored for 30±12 months. The expression of 15-F2t-IsoP protein in autopsy lung samples was determined by immunohistochemical staining. RESULTS: Plasma 15-F2t-IsoP concentrations were significantly increased in patients with IPAH compared with healthy controls (91 pg/ml vs. 30 pg/ml, respectively; P<0.001). Patients with baseline 15-F2t-IsoP concentrations≥97 pg/ml had a significantly lower survival rate than those with lower baseline concentrations (P<0.001). During follow-up, 15-F2t-IsoP concentrations in survivors decreased, whereas concentrations in non-surviving patients increased further (P<0.05). Elevated concentrations of 15-F2t-IsoP were correlated with a severity of WHO functional class, lower 6-minute walking distance and mixed venous oxygen saturation, higher mean right atrial pressure and brain natriuretic peptide. Multivariate analysis revealed that the plasma 15-F2t-IsoP concentration was an independent factor associated with mortality. Histological studies showed that the expression of 15-F2t-IsoP was up-regulated in remodeled pulmonary vessels. CONCLUSIONS: An elevated plasma 15-F2t-IsoP concentration and a further increase during follow-up may be a risk factor for higher mortality in patients with IPAH.

Inhibition of hedgehog signaling by GANT58 induces apoptosis and shows synergistic antitumor activity with AKT inhibitor in acute T cell leukemia cells.

The hedgehog (Hh) signaling pathways have a crucial role in cell proliferation and survival, and the de-regulation of these pathways can lead to tumorigenesis. Here we investigated the expression and function of these pathways in acute T lymphocytic leukemia cells (T-ALL). Profiling of Hh pathway members revealed common expression of key Hh signaling effectors in all T-ALL cells. We found that T-ALL cells were insensitive to specific Smoothened (SMO) inhibition following the use of low concentrations of the SMO antagonist cyclopamine. In contrast, treatment with the novel GLI antagonist GANT58 reduced expression of the target gene Patched 1 as well as GLI family zinc finger 1 (GLI1) and preferentially decreased the viability of T-ALL cells. We also found perifosine, a novel AKT inhibitor, down-regulated GLI1 protein by dephosphorylation of AKT and GSK3ß dose-dependently and that pre-treatment with PD98059, a MEK/ERK pathway inhibitor, enhanced this down-regulation by 20%-30%. Then we questioned whether use of both GANT58 and AKT inhibitor together could confer a synergistic effect to decrease T-ALL cell viability. By applying the Chou-Talalay method, low concentration of GANT58 induced T-ALL cell death in a synergism fashion with perifosine or GSK690693 when used simultaneously. These findings indicate that the combined use of GANT58 and AKT inhibitor could help treat a broad range of malignant tumors in conjunction with existing cancer treatments.

The phosphodiesterase-5 inhibitor vardenafil reduces oxidative stress while reversing pulmonary arterial hypertension.

AIMS: Oxidative stress is implicated in the pathology of pulmonary arterial hypertension (PAH). Previously, we demonstrated that vardenafil, a phosphodiesterase-5 inhibitor, has potential as therapy for PAH, although the mechanism remained uncharacterized. Here, we aimed to determine baseline levels of oxidative stress in PAH and investigate whether vardenafil affects oxidative stress levels while improving PAH. METHODS AND RESULTS: Sprague-Dawley rats with monocrotaline-induced PAH were administered oral vardenafil 1 mg kg⁻¹ day⁻¹ for 21 days (n = 12). Treatment-naive patients (n = 15) with PAH were treated with vardenafil 5 mg twice daily for 3 months. Haemodynamic data and plasma levels of nitrate/nitrite and products of oxidative damage were determined in rats and patients. Histopathology, immunohistochemistry, and assessments of oxidative/anti-oxidative enzyme expression were performed in rat lung tissue. Compared with baseline (patients) or untreated controls (rats), vardenafil significantly reduced pulmonary vascular resistance and increased cardiac output (CO). In rats, vardenafil suppressed proliferation and enhanced apoptosis of pulmonary artery smooth muscle cells, attenuating small pulmonary artery remodelling, and right ventricular hypertrophy. Vardenafil significantly reduced levels of oxidative stress biomarkers, such as 8-iso-prostaglandin-F2α and 3-nitrotyrosine, and significantly increased nitric oxide (NO) levels in rats and patients. Furthermore, vardenafil significantly increased endothelial NO synthase expression and superoxide dismutase activity, and down-regulated nicotinamide adenine dinucleotide phosphate oxidase expression in rat lung tissue. CONCLUSION: Vardenafil reduces oxidative stress in rats and humans while improving PAH, warranting investigation of oxidative stress pathways as targets for PAH therapy.