Variants of the promoter of MYH6 gene in congenital isolated and sporadic patent ductus arteriosus: case-control study and cellular functional analyses.

Patent ductus arteriosus (PDA) is a common form of congenital heart disease. The MYH6 gene has important effects on cardiovascular growth and development, but the effect of variants in the MYH6 gene promoter on ductus arteriosus is unknown. DNA was extracted from blood samples of 721 subjects (428 patients with isolated and sporadic PDA and 293 healthy controls) and analyzed by sequencing for MYH6 gene promoter region variants. Cellular function experiments with three cell lines (HEK-293, HL-1, and H9C2 cells) and bioinformatics analyses were performed to verify their effects on gene expression. In the MYH6 gene promoter, 11 variants were identified. Four variants were found only in patients with PDA and 2 of them (g.3434G>C and g.4524C>T) were novel. Electrophoretic mobility shift assay showed that the transcription factors bound by the promoter variants were significantly altered in comparison to the wild-type in all three cell lines. Dual luciferase reporter showed that all the 4 variants reduced the transcriptional activity of the MYH6 gene promoter (P < 0.05). Prediction of transcription factors bound by the variants indicated that these variants alter the transcription factor binding sites. These pathological alterations most likely affect the contraction of the smooth muscle of ductus arteriosus, leading to PDA. This study is the first to focus on variants at the promoter region of the MYH6 gene in PDA patients with cellular function tests. Therefore, this study provides new insights to understand the genetic basis and facilitates further studies on the mechanism of PDA formation.

Metabolomics and Biomarkers for Paroxysmal and Persistent Atrial Fibrillation.

BACKGROUND: Atrial fibrillation (AF) is the most common type of arrhythmia worldwide and is associated with serious complications. This study investigated the metabolic biomarkers associated with AF and the differences in metabolomics and associated metabolic biomarkers between paroxysmal AF (AFPA) and persistent AF. METHODS AND RESULTS: Plasma samples were prospectively collected from patients with AF and patients in sinus rhythm with negative coronary angiography. The patients were divided into 3 groups: AFPA, persistent AF, and sinus rhythm (N=54). Metabolomics (n=36) using ultra-high-performance liquid chromatography mass spectrometry was used to detect differential metabolites that were validated in a new cohort (n=18). The validated metabolites from the validation phase were further analyzed by receiver operating characteristic. Among the 36 differential metabolites detected by omics assay, 4 were successfully validated with area under the curve >0.8 (P<0.05). Bioinformatics analysis confirmed the enrichment pathways of unsaturated fatty acid biosynthesis, glyoxylate and dicarboxylate metabolism, and carbon metabolism. Arachidonic acid was a potential biomarker of AFPA, glycolic acid and L-serine were biomarkers of AFPA and persistent AF, and palmitelaidic acid was a biomarker of AFPA. CONCLUSIONS: In this metabolomics study, we detected 36 differential metabolites in AF, and 4 were validated with high sensitivity and specificity. These differential metabolites are potential biomarkers for diagnosis and monitoring of disease course. This study therefore provides new insights into the precision diagnosis and management of AF.

Homocysteine impairs the anticontractile/vasorelaxing activity of perivascular adipose tissue surrounding human internal mammary artery.

OBJECTIVES: Perivascular adipose tissue (PVAT) surrounding human internal mammary artery (IMA) possesses anticontractile property. Its function under pathological conditions is barely studied. We previously reported that homocysteine impairs the vasodilator function of IMA through endothelium and smooth muscle-dependent mechanisms. This study investigated the effect of homocysteine on the function of PVAT and the associated mechanisms. METHODS: Residual IMA tissues were collected from patients undergoing coronary artery bypass grafting. Vasoreactivity was studied using myograph. Adiponectin was measured by ELISA. Expressions of adiponectin receptors (AdipoRs), eNOS and p-eNOS were determined by RT-qPCR and Western blot. RESULTS: Exposure to homocysteine augmented the contractile responses of PVAT-intact IMA to U46619 and potassium chloride, regardless with or without endothelium. Such augmentation was also observed in skeletonized IMA with transferred, homocysteine-exposed PVAT. Homocysteine attenuated the relaxant response of PVAT-intact while endothelium-denuded vessels to acetylcholine. Homocysteine lowered adiponectin content in the PVAT, downregulated the expression of AdipoR1 and AdipoR2 as well as eNOS and p-eNOS in skeletonized IMA. The relaxant response of skeletonized IMA to AdipoR agonist AdipoRon was blunted by homocysteine or eNOS inhibitor, and homocysteine significantly attenuated the inhibitory effect of eNOS inhibitor on AdipoRon-induced relaxation. CONCLUSIONS: Homocysteine impairs the anticontractile/vasorelaxing activity of PVAT surrounding the IMA through inhibiting adiponectin/AdipoR/eNOS/nitric oxide signalling pathway.

Association between serum gamma-glutamyltransferase and early-onset coronary artery disease: a retrospective case-control study.

BACKGROUND: Serum gamma-glutamyltransferase (GGT) activity has been proposed as a promising predictor of atherosclerosis-related complications and a prognostic marker for cardiovascular diseases. The objective of this study was to investigate the potential correlation between serum levels of GGT and early-onset coronary artery disease (EOCAD). METHODS: A retrospective, hospital-based case-control study was conducted, which included 860 patients with EOCAD and gender- and age-matched controls. Serum levels of GGT were measured using the reference measurement procedure on an automatic biochemistry analyser. RESULTS: The serum GGT levels of patients with EOCAD (34.90 ± 31.44 U/L) were significantly higher than those of the control group (21.57 ± 16.44 U/L, p < .001). Elevated serum levels of GGT were found to be an independent risk factor for EOCAD, with an odds ratio (OR) of 1.021 (95% confidence interval (CI): 1.014-1.029). Additionally, for every quartile increase in serum GGT levels, the risk of developing EOCAD increased by 1.6-fold. Moreover, serum GGT levels were significantly associated with disease severity, with lower GGT levels observed in patients without significant vascular disease (31.74 ± 24.06 U/L) compared to those with two-vessel disease (33.06 ± 25.00 U/L, p = .002) and three-vessel disease (37.75 ± 36.76 U/L, p = .001). CONCLUSIONS: The results of this study suggest that elevated serum GGT levels are associated with the development of EOCAD, and GGT may be implicated in the pathogenesis of the disease. Further large-scale prospective studies are needed to explore the potential relationship between serum GGT levels and the dynamic development of EOCAD.

Tetralogy of Fallot: variants of MYH6 gene promoter and cellular functional analyses.

BACKGROUND: Tetralogy of Fallot (TOF) is a common form of congenital heart disease. The MYH6 gene has important effects on cardiovascular growth and development. METHODS: In 608 subjects, including 315 TOF patients, we investigated the MYH6 gene promoter variants and verified the effect on gene expression by using cellular functional experiments with three cell lines (HEK-293, HL-1, and H9C2 cells) and bioinformatics analysis. RESULTS: In the MYH6 gene promoter, 12 variants were identified from 608 subjects. Five variants were found only in patients with TOF and two of them (g.3384G>T and g.4518T>C) were novel. Electrophoretic mobility shift assay with three cell lines (HEK-293, HL-1, and H9C2) showed significant changes in the transcription factors bound by the promoter variants compared to the wild-type. Dual luciferase reporter showed that four of the five variants reduced the transcriptional activity of the MYH6 gene promoter (p < 0.05). CONCLUSIONS: This study is the first to test the cellular function of variants in the promoter region of the MYH6 gene in patients with TOF, which provides new insights into the genetic basis of TOF and provides a basis for further study of the mechanism of TOF formation. IMPACT: DNA from 608 human subjects was sequenced for MYH6 gene promoter region variants with five variants found only in TOF patients and two were novel. EMSA and dual luciferase reporter experiments in three cell lines found these variants pathological. Prediction by JASPAR database indicated that these variants alter the transcription factor binding sites. The study, for the first time, confirmed that there are variants at the MYH6 gene promoter region and these variants alter the cellular function. The variants found in this study suggest the possible pathological role in the formation of TOF.

Identification and Functional Verification of CITED2 Gene Promoter Region in Patients with Patent Ductus Arteriosus.

Patent ductus arteriosus (PDA) is a common congenital heart disease. CITED2 plays an important role in the development of the heart, and genetic variants in its coding region are significantly associated with cardiac malformations. However, the role of variants in the promoter region of CITED2 in the development of PDA remains unclear. We extracted the peripheral blood of 646 subjects (including 353 PDA patients and 293 unrelated healthy controls) for sequencing. We identified 13 promoter variants of the CITED2 gene (including 2 novel heterozygous variants). Of the 13 variants, 10 were found only in PDA patients. In mouse cardiomyocytes (HL-1) and rat cardiac myocytes (RCM), the transcriptional activity of the CITED2 gene promoter was significantly changed by the variants (p < 0.05). The results of the experiments of electrophoretic mobility indicated that these variants may affect the transcription of the CITED2 gene by influencing the binding ability of transcription factors. These results, combined with the JASPAR database analysis, showed that the destruction/production of transcription factor binding sites due to the variants in the promoter region of the CITED2 gene may directly or indirectly affect the binding ability of transcription factors. Our results suggest for the first time that variants at the CITED2 promoter region may cause low expression of CITED2 protein related to the formation of PDA.

Lysine crotonylation of SERCA2a correlates to cardiac dysfunction and arrhythmia in Sirt1 cardiac-specific knockout mice.

Protein post-translational modifications (PTMs) are important regulators of protein functions and produce proteome complexity. SIRT1 has NAD+-dependent deacylation of acyl-lysine residues. The present study aimed to explore the correlation between lysine crotonylation (Kcr) on cardiac function and rhythm in Sirt1 cardiac-specific knockout (ScKO) mice and related mechanism. Quantitative proteomics and bioinformatics analysis of Kcr were performed in the heart tissue of ScKO mice established with a tamoxifen-inducible Cre-loxP system. The expression and enzyme activity of crotonylated protein were assessed by western blot, co-immunoprecipitation, and cell biology experiment. Echocardiography and electrophysiology were performed to investigate the influence of decrotonylation on cardiac function and rhythm in ScKO mice. The Kcr of SERCA2a was significantly increased on Lys120 (1.973 folds). The activity of SERCA2a decreased due to lower binding energy of crotonylated SERCA2a and ATP. Changes in expression of PPAR-related proteins suggest abnormal energy metabolism in the heart. ScKO mice had cardiac hypertrophy, impaired cardiac function, and abnormal ultrastructure and electrophysiological activities. We conclude that knockout of SIRT1 alters the ultrastructure of cardiac myocytes, induces cardiac hypertrophy and dysfunction, causes arrhythmia, and changes energy metabolism by regulating Kcr of SERCA2a. These findings provide new insight into the role of PTMs in heart diseases.

Inhibition of the Activating Transcription Factor 6 Branch of Endoplasmic Reticulum Stress Ameliorates Brain Injury after Deep Hypothermic Circulatory Arrest.

Neurological dysfunction is a common complication of deep hypothermic circulatory arrest (DHCA). Endoplasmic reticulum (ER) stress plays a role in neuronal ischemia-reperfusion injury; however, it is unknown whether it contributes to DHCA-induced brain injury. Here, we aimed to investigate the role of ER stress in a rat DHCA model and cell hypothermic oxygen-glucose deprivation reoxygenation (OGD/R) model. ER stress and apoptosis-related protein expression were identified using Western blot analysis. Cell counting assay-8 and flow cytometry were used to determine cell viability and apoptosis, respectively. Brain injury was evaluated using modified neurological severity scores, whereas brain injury markers were detected through histological examinations and immunoassays. We observed significant ER stress molecule upregulation in the DHCA rat hippocampus and in hypothermic OGD/R PC-12 cells. In vivo and in vitro experiments showed that ER stress or activating transcription factor 6 (ATF6) inhibition alleviated rat DHCA-induced brain injury, increased cell viability, and decreased apoptosis accompanied by C/EBP homologous protein (CHOP). ER stress is involved in DHCA-induced brain injury, and the inhibition of the ATF6 branch of ER stress may ameliorate this injury by inhibiting CHOP-mediated apoptosis. This study establishes a scientific foundation for identifying new therapeutic targets for perioperative brain protection in clinical DHCA.

Genetic Variants of ISL1 Gene Promoter Identified from Congenital Tetralogy of Fallot Patients Alter Cellular Function Forming Disease Basis.

Tetralogy of Fallot (TOF) is the most common cyanotic congenital heart disease in newborns. ISL1 is a master transcription factor in second heart field development, whereas the roles of ISL1 gene promoter variants in TOF patients have not been genetically investigated. Total DNA extraction from 601 human subjects, including 308 TOF patients and 293 healthy controls, and Sanger sequencing were performed. Four variants (including one novel heterozygous variant) within the ISL1 gene promoter were only found in TOF patients. Functional analysis of DNA sequence variants was performed by using the dual-luciferase reporter assay and demonstrated that three of the four variants significantly decreased the transcriptional activity of ISL1 gene promoter in HL-1 cells (p < 0.05). Further, the online JASPAR database and electrophoretic mobility shift assay showed that the three variants affected the binding of transcription factors and altered ISL1 expression levels. In conclusion, the current study for the first time demonstrated that the variants identified from the ISL1 gene promoter region are likely involved in the development of TOF by affecting the transcriptional activity and altering the ISL1 expression level. Therefore, these findings may provide new insights into the molecular etiology and potential therapeutic strategy of TOF.

Soluble epoxide hydrolase and TRPC3 channels jointly contribute to homocysteine-induced cardiac hypertrophy: Interrelation and regulation by C/EBPß.

OBJECTIVES: Studies in certain cardiac hypertrophy models suggested the individual role of soluble epoxide hydrolase (sEH) and canonical transient receptor potential 3 (TRPC3) channels, however, whether they jointly mediate hypertrophic process remains unexplored. Hyperhomocysteinemia promotes cardiac hypertrophy while the involvement of sEH and TRPC3 channels remains unknown. This study aimed to explore the role of, and interrelation between sEH and TRPC3 channels in homocysteine-induced cardiac hypertrophy. METHODS: Rats were fed methionine-enriched diet to induce hyperhomocysteinemia. H9c2 cells and neonatal rat cardiomyocytes were incubated with homocysteine. Cardiac hypertrophy was evaluated by echocardiography, histological examination, immunofluorescence imaging, and expressions of hypertrophic markers. Epoxyeicosatrienoic acids (EETs) were determined by ELISA. TRPC3 current was recorded by patch-clamp. Gene promotor activity was measured using dual-luciferase reporter assay. RESULTS: Inhibition of sEH by 1-trifluoromethoxyphenyl-3-(1-propionylpiperidin-4-yl) urea (TPPU) reduced ventricular mass, lowered the expression of hypertrophic markers, decreased interstitial collagen deposition, and improved cardiac function in hyperhomocysteinemic rats, associated with restoration of EETs levels in myocardium. TPPU or knockdown of sEH suppressed TRPC3 transcription and translation as well as TRPC3 current that were enhanced by homocysteine. Exogenous 11,12-EET inhibited homocysteine-induced TRPC3 expression and cellular hypertrophy. Silencing C/EBPß attenuated, while overexpressing C/EBPß promoted homocysteine-induced hypertrophy and expressions of sEH and TRPC3, resulting respectively from inhibition or activation of sEH and TRPC3 gene promoters. CONCLUSIONS: sEH and TRPC3 channels jointly contribute to homocysteine-induced cardiac hypertrophy. Homocysteine transcriptionally activates sEH and TRPC3 genes through a common regulatory element C/EBPß. sEH activation leads to an upregulation of TRPC3 channels via a 11,12-EET-dependent manner.

Hematological parameters and early-onset coronary artery disease: a retrospective case-control study based on 3366 participants.

Background: Thrombosis and inflammation are crucial elements in the pathogenesis of cardiovascular disease. Hematological parameters elucidate information involving the inflammatory and blood coagulation processes. Objectives: The current study explored the association of hematological parameters with EOCAD to identify specific risk factors. Design: A single-center retrospective case-control study was conducted with 1693 coronary artery disease patients and 1693 controls. Methods: Hematological parameters were examined through an automated analyzer. Results: The basophil percentage was significantly reduced in EOCAD (0.43 ± 0.26, p < 0.001) and MI (0.33 ± 0.24, p < 0.001) groups compared with controls (0.54 ± 0.28). The eosinophil percentage was also significantly lower in EOCAD (2.21 ± 1.71, p < 0.001) and MI (1.71 ± 2.44, p < 0.001) groups compared with controls (2.41 ± 1.75). The lymphocyte percentage in patients of EOCAD and MI and controls was 31.65 ± 7.93, 25.48 ± 9.43, and 34.82 ± 7.28, respectively. A significant difference was observed among the groups (p < 0.001). Except for the mean corpuscular hemoglobin (MCH), other red blood cell (RBC) parameters significantly differed between EOCAD patients and controls. The red blood cell distribution width (RDW), hematocrit (HCT), RBC count, mean corpuscular hemoglobin concentration (MCHC), mean corpuscular volume (MCV), and hemoglobin level were associated with EOCAD prevalence after adjusting for baseline differences. Platelet volume distribution width (PDW) also correlated with EOCAD prevalence (ORadjust = 1.087, 95% CI: 1.044-1.131). Conclusions: Hematological parameters are closely associated with EOCAD. Moreover, leukocyte parameters correlated with the presence and severity of the disease. In addition, erythrocyte parameters were associated with the disease presence but not with the disease severity. Among the platelet parameters, only PDW was related to the disease presence.

Antispastic Effect of Fasudil and Cocktail of Fasudil and Nitroglycerin in Internal Thoracic Artery.

BACKGROUND: Spasm of arterial grafts in coronary artery bypass grafting is a clinical problem and can occasionally be lethal. Perioperative spasm in the internal thoracic artery (ITA) and coronary arteries occurs in 0.43% of patients. This study aimed to investigate the antispastic effect of a RhoA/Rho-kinase (Rho-associated coiled-coil-containing protein kinase [ROCK]) inhibitor (fasudil) with and without nitroglycerin in combination in the ITA. METHODS: Isolated human ITA rings taken from 68 patients who were undergoing coronary bypass were studied in a myograph. Cumulative concentration-relaxation curves for fasudil (-9 to -3.5 log M) were established in the ITA, which was precontracted with potassium chloride or U46619. The inhibitory effect of fasudil (-6.3 or -5.3 log M) or fasudil in combination with nitroglycerin were also tested. The ROCK2 protein was measured by Western blot. RESULTS: Fasudil caused similar relaxation in ITA rings contracted by potassium chloride or U46619. Pretreatment with -5.3 log M fasudil significantly depressed contraction induced by potassium chloride (P = .004 vs control; P = .017 vs -6.3 log M) and U46619 (P = .010 vs control; P = .041 vs. -6.3 log M). Fasudil in combination with nitroglycerin had more effect and more rapid and sustained relaxation than either vasodilator alone. Fasudil caused a decrease of ROCK2 protein content (P = .014). CONCLUSIONS: Fasudil fully relaxes some vasoconstrictor-induced contraction and decreases ROCK2 protein content in the ITA. The combination of fasudil and nitroglycerin has a superior effect than either vasodilator alone. The new cocktail solution composed of fasudil and nitroglycerin (pH 7.4) has effective antispastic action and may prove to be a new antispastic method for arterial conduits during coronary bypass surgery.

Risk factors for development of acute renal failure in 5077 coronary artery bypass grafting patients in the current era.

BACKGROUND: Acute renal failure (ARF) is one of the major complications after coronary artery bypass grafting (CABG) surgery. The risk factors are changing along with the technical evolution. The aim of this study was to identify the risk factors for ARF requiring dialysis after CABG surgery in the current era. METHODS: Between April 2012 and November 2019, 5077 consecutive patients who underwent CABG were analyzed retrospectively. The patients were divided into ARF group and non-ARF group according to whether ARF occurred and dialysis was required after operation. Univariate analysis was performed to find possible factors associated with ARF. Any variables that had trends to be associated with ARF were included in stepwise multiple logistic regression analysis. RESULTS: Of the 5077 patients who underwent CABG, 53 (1.04%) developed ARF requiring dialysis whereas 5024 (98.96%) were in non-ARF group. Cardiopulmonary bypass (CPB) time (odds ratio [OR], 1.009; 95% confidence interval [CI], 1.003-1.016; p = .006), insertion of intra-aortic balloon pump (IABP; OR, 19.294; 95% CI, 5.49-67.808; p = .000), and low ejection fraction (EF; OR, 0.943; 95% CI, 0.894-0.994; p = .030) were independent risk factors for development of ARF requiring dialysis in patients undergoing CABG surgery. CONCLUSION: Our study identified prolonged CPB time, insertion of IABP, and low EF as independent risk factors for developing ARF requiring dialysis after CABG. The results suggest that shortening of CPB time and protection of cardiac function are important factors to prevent ARF and that special care should be taken to protect the renal function when the patient need insertion of IABP.

Pathophysiological Role of Variants of the Promoter Region of CITED2 Gene in Sporadic Tetralogy of Fallot Patients with Cellular Function Verification.

Tetralogy of Fallot (TOF) is a common congenital heart malformation. Genetic variants in the CITED2 coding region are known to be significantly associated with cardiac malformation, but the role of variants in the CITED2 promoter region in the development of TOF remains unclear. In this study, we investigated CITED2 promoter variants in the DNA of 605 subjects, including 312 TOF patients and 293 unrelated healthy controls, by Sanger sequencing. We identified nine CITED2 gene promoter variants (including one novel heterozygous variant). Six were found only in patients with TOF and none in the control group. The transcriptional activity of the CITED2 gene promoter in mouse cardiomyocyte (HL-1) cells was significantly altered by the six variants (p < 0.05). The results of the electrophoretic mobility change assay and JASPAR database analysis showed that these variants generated or destroyed a series of possible transcription factor binding sites, resulting in changes in the CITED2 protein expression. We conclude that CITED2 promoter variants in TOF patients affect transcriptional activity and may be involved in the occurrence and progression of TOF. These findings may provide new insights into molecular pathogenesis and potential therapeutic insights in patients with TOF.

Genetic Variants of CITED2 Gene Promoter in Human Atrial Septal Defects: Case-Control Study and Cellular Functional Verification.

Atrial septal defect (ASD) is one of the most common forms of congenital heart disease (CHD). Genetic variants in the coding region of the CITED2 gene are known to be significantly correlated with CHD, but the role of variants in the promoter region of CITED2 is unknown. We investigated variants in the promoter of the CITED2 gene in 625 subjects (332 ASD and 293 healthy controls) through Sanger sequencing. Four variants in the CITED2 gene promoter were found only in eight ASD patients with zero occurrence in the control subjects (one case of g.4078A>C(rs1165649373), one case of g.4240C>A(rs1235857801), four cases of g.4935C>T(rs111470468), two cases of g.5027C>T(rs112831934)). Cellular functional analysis showed that these four variants significantly changed the transcriptional activity of the CITED2 gene promoter in HEK-293 and HL-1 cells. Electrophoretic mobility change assay results and JASPAR database analysis demonstrated that these variants created or destroyed a series of possible transcription factor binding sites, resulting in changes in the expression of CITED2 protein. We conclude that the variants of CITED2 promoter in ASD patients affect the transcriptional activity and are likely involved in the occurrence and development of ASD. These findings provide new perspectives on the pathogenesis and potential therapeutic insights of ASD.

Identification and functional analysis of variants of MYH6 gene promoter in isolated ventricular septal defects.

BACKGROUND: Ventricular septal defect is the most common form of congenital heart diseases. MYH6 gene has a critical effect on the growth and development of the heart but the variants in the promoter of MYH6 is unknown. PATIENTS AND METHODS: In 604 of the subjects (311 isolated and sporadic ventricular septal defect patients and 293 healthy controls), DNA was extracted from blood samples and MYH6 gene promoter region variants were analyzed by sequencing. Further functional verification was performed by cellular experiments using dual luciferase reporter gene analysis, electrophoretic mobility shift assays, and bioinformatics analysis. RESULTS: Nine variants were identified in the MYH6 gene promoter and two of those variants [g.4085G>C(rs1222539675) and g.4716G>A(rs377648095)] were only found in the ventricular septal defect patients. Cellular function experiments showed that these two variants reduced the transcriptional activity of the MYH6 gene promoter (p < 0.001). Further analysis with online JASPAR database suggests that these variants may alter a set of putative transcription factor binding sites that possibly lead to changes in myosin subunit expression and ventricular septal defect formation. CONCLUSIONS: Our study for the first time identifies variants in the promoter region of the MYH6 gene in Chinese patients with isolated and sporadic ventricular septal defect. These variants significantly reduced MYH6 gene expression and affected transcription factor binding sites and therefore are pathogenic. The present study provides new insights in the role of the MYH6 gene promoter region to better understand the genetic basis of VSD formation.

Identification and functional analysis of genetic variants of ISL1 gene promoter in human atrial septal defects.

BACKGROUND: Atrial septal defect (ASD) is a common type of congenital heart disease. A gene promoter plays pivotal role in the disease development. This study was designed to investigate the pathological role of variants of the ISL1 gene promoter region in ASD patients. METHODS: Total DNA extracted from 625 subjects, including 332 ASD patients and 293 healthy controls, was sequenced to identify variants in the promoter region of ISL1 gene. Further functional analyses of the variants were performed with dual luciferase reporter assay and electrophoretic mobility shift assay (EMSA). All possible binding sites of transcription factor affected by the identified variants were predicted using the JASPAR database. RESULTS: Four variants in the ISL1 gene promoter were found only in patients with ASD by sequencing. Three of the four variants [g.4923 G > C (rs541081886), g.5079 A > G (rs1371835943) and g.5309 G > A (rs116222082)] significantly decreased the transcriptional activities compared with the wild-type ISL1 gene promoter (p < 0.05). The EMSA revealed that these variants [g.4923 G > C (rs541081886), g.5079 A > G (rs1371835943) and g.5309 G > A (rs116222082)] in the ISL1 gene promoter affected the number and affinity of binding sites of transcription factors. Further analysis with the online JASPAR database demonstrated that a cluster of putative binding sites for transcription factors may be altered by these variants. CONCLUSIONS: These sequence variants identified from the promoter region of ISL1 gene in ASD patients are probably involved in the development of ASD by affecting the transcriptional activity and altering ISL1 levels. Therefore, these findings may provide new insights into the molecular etiology and potential therapeutic strategy of ASD.

Targeting IRE1α-JNK-c-Jun/AP-1-sEH Signaling Pathway Improves Myocardial and Coronary Endothelial Function Following Global Myocardial Ischemia/Reperfusion.

Objectives: Endoplasmic reticulum (ER) stress and soluble epoxide hydrolase (sEH) upregulation/activation have been implicated in myocardial ischemia/reperfusion (I/R) injury. We previously reported that ER stress mediates angiotensin II-induced sEH upregulation in coronary endothelium, whether and how ER stress regulates sEH expression to affect postischemic cardiac function remain unexplored. This study aimed to unravel the signaling linkage between ER stress and sEH in an ex vivo model of myocardial I/R injury. Methods: Hearts from male Wistar-Kyoto rats were mounted on a Langendorff apparatus and randomly allocated to 7 groups, including control, I/R (30-min ischemia and 60-min reperfusion), and I/R groups pretreated with one of the following inhibitors: 4-PBA (targeting: ER stress), GSK2850163 (IRE1α), SP600125 (JNK), SR11302 (AP-1), and DCU (sEH). The inhibitor was administered for 15 min before ischemia with a peristaltic pump. Hemodynamic parameters including left ventricular systolic pressure (LVSP), left ventricular end-diastolic pressure (LVEDP), and maximal velocity of contraction (+dp/dtmax) and relaxation (-dp/dtmax) of the left ventricle were continuously recorded using an intraventricular balloon. Endothelial dilator function of the left anterior descending artery was studied in a wire myograph upon completion of reperfusion. The expression of ER stress molecules, JNK, c-Jun, and sEH was determined by western-blot. Results: I/R decreased LVSP (105.5±6.4 vs. 146.9±13.4 mmHg), and increased LVEDP (71.4±3.0 vs. 6.0±2.7 mmHg), with a resultant decreased LVDP (34.1±9.2 vs. 140.9±13.1 mmHg). I/R attenuated +dp/dtmax (651.7±142.1 vs. 2806.6±480.6 mmHg/s) and -dp/dtmax (-580.0±109.6 vs. -2118.0±244.9 mmHg/s) (all ps<0.001). The I/R-induced cardiac dysfunction could be alleviated by 4-PBA (LVSP 119.5±15.6 mmHg, p<0.01; LVEDP 21.2±4.2 mmHg, LVDP 98.3±12.0 mmHg, +dp/dtmax 2166.7±208.4 mmHg/s, and -dp/dtmax -1350.9±99.8 mmHg/s, all ps<0.001), GSK2850163 (LVSP 113.4±10.9 mmHg, p<0.01; LVEDP 37.1±3.1 mmHg, LVDP 76.3±13.9 mmHg, +dp/dtmax 1586.5±263.3 mmHg/s, -dp/dtmax -1127.7±159.9 mmHg/s, all ps<0.001), SP600125 (LVSP 113.9±5.6 mmHg, LVDP 40.5±3.3 mmHg, +dp/dtmax 970.1±89.8 mmHg/s, all ps<0.01), SR11302 (LVSP 97.9±7.5 mmHg, p<0.01; LVEDP 52.7±8.6mmHg, p<0.001; LVDP 45.2±9.8mmHg, p<0.05; +dp/dtmax 1231.5±196.6 mmHg/s, p<0.01; -dp/dtmax -658.3±68.9 mmHg/s, p<0.05), or DCU (LVSP 109.9±4.1 mmHg, p<0.01; LVEDP 11.7±1.8 mmHg, LVDP 98.2±4.9 mmHg, +dp/dtmax 1869.8±121.9 mmHg/s, and -dp/dtmax -1492.3±30.8 mmHg/s, all ps<0.001). The relaxant response of the coronary artery to acetylcholine was decreased after I/R in terms of both magnitude and sensitivity (p<0.001). All inhibitors improved acetylcholine-induced relaxation. Global I/R increased sEH expression and induced ER stress in both myocardium and coronary artery. Inhibition of ER stress or IRE1α downregulated I/R-induced sEH expression and inhibited JNK and c-Jun phosphorylation. Both JNK and AP-1 inhibitors lowered sEH level in myocardium and coronary artery in I/R-injured hearts. Conclusions: This study deciphered the molecular linkage between ER stress and sEH regulation in global I/R insult by uncovering a novel signaling axis of IRE1α-JNK-c-Jun/AP-1-sEH, which provided basis for future research on the therapeutic potential of targeting the IRE1α-JNK-c-Jun/AP-1-sEH axis for ischemic myocardial injury.

Identification of novel rare copy number variants associated with sporadic tetralogy of Fallot and clinical implications.

Tetralogy of Fallot (TOF) is the most common cyanotic congenital heart disease. Highly penetrant copy number variants (CNVs) and genes related to the etiology of TOF likely exist with differences among populations. We aimed to identify CNV contributions to sporadic TOF cases in Han Chinese. Genomic DNA was extracted from peripheral blood in 605 subjects (303 sporadic TOF and 302 unaffected Han Chinese [Control] from cardiac centers in China) and analyzed by genome-wide association study (GWAS). The GWAS results were compared with existing Database of Genetic Variants. These CNVs were further validated by qPCR. Bioinformatics analyses were performed with protein-protein interaction (PPI) network and KEGG pathway enrichment. Across all chromosomes 119 novel "TOF-specific CNVs" were identified with prevalence of CNVs of 21.5% in chromosomes 1-20 and 37.0% including Chr21/22. In chromosomes 1-20, CNVs on 11q25 (encompasses genes ACAD8, B3GAT1, GLB1L2, GLB1L3, IGSF9B, JAM3, LOC100128239, LOC283177, MIR4697, MIR4697HG, NCAPD3, OPCML, SPATA19, THYN1, and VPS26B) and 14q32.33 (encompasses genes THYN1, OPCML, and NCAPD3) encompass genes most likely to be associated with TOF. Specific CNVs found on the chromosome 21 (6.3%) and 22(11.9%) were also identified in details. PPI network analysis identified the genes covering the specific CNVs related to TOF and the signaling pathways. This study for first time identified novel TOF-specific CNVs in the Han Chinese with higher frequency than in Caucasians and with 11q25 and 14q32.33 not reported in TOF of Caucasians. These novel CNVs identify new candidate genes for TOF and provide new insights into genetic basis of TOF.