Metabolomics Analysis Identifies Differential Metabolites as Biomarkers for Acute Myocardial Infarction.

Myocardial infarction (MI), including ST-segment elevation MI (STEMI) and non-ST-segment elevation MI (NSTEMI), is still a leading cause of death worldwide. Metabolomics technology was used to explore differential metabolites (DMs) as potential biomarkers for early diagnosis of STEMI and NSTEMI. In the study, 2531 metabolites, including 1925 DMs, were discovered. In the selected 27 DMs, 14 were successfully verified in a new cohort, and the AUC values were all above 0.8. There were 10 in STEMI group, namely L-aspartic acid, L-acetylcarnitine, acetylglycine, decanoylcarnitine, hydroxyphenyllactic acid, ferulic acid, itaconic acid, lauroylcarnitine, myristoylcarnitine, and cis-4-hydroxy-D-proline, and 5 in NSTEMI group, namely L-aspartic acid, arachidonic acid, palmitoleic acid, D-aspartic acid, and palmitelaidic acid. These 14 DMs may be developed as biomarkers for the early diagnosis of MI with high sensitivity and specificity. These findings have particularly important clinical significance for NSTEMI patients because these patients have no typical ECG changes.

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.

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.

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.

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 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.

Plasma Concentrations of Trace Elements Selenium and Cobalt During and After Coronary Artery Bypass Grafting Surgery.

Trace elements selenium (Se) and cobalt (Co) are essential in the human body, and a correlation between Se and cardiac surgery has been suggested. We investigated the plasma concentrations of Se and Co during and after coronary artery bypass grafting (CABG) surgery under cardiopulmonary bypass (CPB). From December 2019 to January 2020, preoperative plasma samples from isolated first-time CABG patients (n=20; 10 males, 10 females) were prospectively collected post-anesthesia and before CPB (T1), 45 min after CPB started (T2), 90 min after CPB started (T3), and postoperative days 1 (T4), and day 4 (T5). The plasma concentrations of Se and Co were measured. The Se concentration was significantly decreased at T2 (105.24±4.08 vs. 68.56±2.42 µg/L, p<0.001) and T3 (105.24±4.08 vs. 80.41±3.40 µg/L, p<0.001). The Co concentration was significantly decreased at T4 (0.35±0.19 vs. 0.26±0.13 µg/L, p<0.01) and T5 (0.35±0.19 vs. 0.23±0.11 µg/L, p<0.001). Five patients developed atrial fibrillation (AF); there was no other operative mortality or major morbidity. This is the first report of alterations of plasma Se and Co concentrations during and after CABG surgery. Our results may indicate that Se supplementation before or during CABG and Co supplementation after CABG may become necessary for patients undergoing CABG.

del Nido cardioplegia better preserves cardiac diastolic function but histidine-tryptophan-ketoglutarate is better for endothelial function.

OBJECTIVES: The effectiveness of myocardial protection of cardioplegia has been a matter of debate for decades. This study was designed to compare cardiac and endothelial protection of 3 clinically used cardioplegias: del Nido cardioplegia (DNC), histidine-tryptophan-ketoglutarate (HTK) and blood cardioplegia (BC) followed by HTK (BC + HTK) in a rat model of ischaemia/reperfusion (I/R). METHODS: Sixty male Wistar rats were subjected to either 120 min of global ischaemia at 4°C followed by 90 min of reperfusion (I/R) at 37°C or no I/R (control) in a Langendorff apparatus and were randomly allocated to 5 groups: control, I/R, DNC, HTK and BC + HTK. Cold cardioplegia solutions were administered at doses of 20 ml/kg for DNC and HTK or 10 ml/kg for BC followed by HTK. Haemodynamic parameters were continuously recorded using an intraventricular balloon. The endothelium-dependent relaxation to acetylcholine was measured in the left anterior descending artery using a myograph. Protein expression of cardiac troponin T (cTnT) and creatine kinase MB was determined by western blot. RESULTS: During reperfusion, HTK had higher left ventricular systolic pressure whereas DNC had lower left ventricular end-diastolic pressure, better left ventricular developed pressure and best +dp/dtmax and -dp/dtmax than the other 2 groups but the differences disappeared at the end of the reperfusion. HTK or BC + HTK preserves the acetylcholine-induced endothelium-dependent relaxation better than DNC (Emax = 48.2 ± 8.0% in DNC vs 75.0 ± 8.0% in HTK, P < 0.05; vs 96.9 ± 3.5% in BC + HTK, P < 0.001). The protein levels of cTnT and creatine kinase MB were downregulated in the 3 groups. CONCLUSIONS: All 3 cardioplegias prevented myocardial damage against I/R injury at the end of reperfusion. DNC demonstrated better preserved diastolic function of the left ventricle whereas HTK or BC + HTK showed better preserved coronary endothelial function. These findings may suggest that currently no 'perfect' cardioplegia exists and that exploration for the 'perfect' cardioplegia is needed.

Homocysteine alters vasoreactivity of human internal mammary artery by affecting the KCa channel family.

BACKGROUND: Hyperhomocysteinemia is an independent risk factor for atherosclerotic heart disease. We previously demonstrated that disruption of calcium-activated potassium (KCa) channel activity is involved in homocysteine-induced dilatory dysfunction of porcine coronary arteries. Recently we reported that the KCa channel family, including large-, intermediate-, and small-conductance KCa (BKCa, IKCa, and SKCa) subtypes, are abundantly expressed in human internal mammary artery (IMA). In this study, we further investigated whether homocysteine affects the expression and functionality of the KCa channel family in this commonly used graft for coronary artery bypass surgery (CABG). METHODS: Residual IMA segments obtained from patients undergoing CABG were studied in a myograph for the role of KCa subtypes in both vasorelaxation and vasoconstriction. The expression and distribution of KCa subtypes were detected by Western blot and immunohistochemistry. RESULTS: Both the BKCa channel activator NS1619 and the IKCa/SKCa channel activator NS309 evoked significant IMA relaxation. Homocysteine exposure suppressed NS1619-induced relaxation whereas showed no influence on NS309-induced response. Blockade of BKCa but not IKCa and SKCa subtypes significantly suppressed acetylcholine-induced relaxation and enhanced U46619-induced contraction. Homocysteine compromised the vasodilating activity of the BKCa subtype in IMA, associated with a lowered protein level of the BKCa ß1-subunit. Homocysteine potentiated the role of IKCa and SKCa subtypes in mediating endothelium-dependent relaxation without affecting the expression of these channels. CONCLUSIONS: Homocysteine reduces the expression of BKCa ß1-subunit and compromises the vasodilating activity of BKCa channels in IMA. Unlike BKCa, IKCa and SKCa subtypes are unessential for IMA vasoregulation, whereas the loss of BKCa functionality in hyperhomocysteinemia enhances the role of IKCa and SKCa subtypes in mediating endothelial dilator function. Targeting BKCa channels may form a strategy to improve the postoperative graft performance in CABG patients with hyperhomocysteinemia who receive IMA grafting.

Biomarkers and key pathways in atrial fibrillation associated with mitral valve disease identified by multi-omics study.

BACKGROUND: Mitral valve disease (MVD)-associated atrial fibrillation (AF) is one of the most common arrhythmias with an increased risk of thromboembolic events. This study aimed to identify the molecular mechanisms and possible biomarkers for chronic AF in MVD by using multi-omics methods. METHODS: This prospective study enrolled patients with MVD (n=100) undergoing mitral valve replacement surgery. The patients were allocated into chronic AF and sinus rhythm (SR) groups. Plasma samples were collected preoperatively. Proteomics was performed with isobaric tags for relative and absolute quantitation (iTRAQ) to identify differential proteins (DPs) between the two groups. The selected DPs were then validated in a new cohort of patients by enzyme-linked immunosorbent assay (ELISA). A gas chromatography-mass spectrometer was used in the metabolomics study to identify differential metabolites (DMs). Bioinformatics analyses were performed to analyze the results. RESULTS: Among the 447 plasma proteins and 322 metabolites detected, 57 proteins and 55 metabolites, including apolipoprotein A-I (ApoA-I), apolipoprotein A-II (ApoA-II), LIM domain only protein 7 (LMO7), and vitronectin (VN) were differentially expressed between AF and SR patients. Bioinformatics analyses identified enriched pathways related to AF, including peroxisome proliferator-activated receptor alpha (PPARα), the renin angiotensin aldosterone system (RAAS), galactose, biosynthesis of unsaturated fatty acids, and linoleic acid metabolism. CONCLUSIONS: Using integrated multi-omics technologies in MVD-associated AF patients, the present study, for the first time, revealed important signaling pathways, such as PPARα, as well as possible roles of other signaling pathways, including the RAAS and galactose metabolism to understand the molecular mechanism of MVD-associated AF. It also identified a large number of DPs and DMs. Some identified proteins and metabolites, such as ApoA-I, ApoA-II, LMO7, and VN, may be further developed as biomarkers for MVD-associated AF.

Mechanisms underlying the vasorelaxant effect of hydrogen sulfide on human saphenous vein.

Hydrogen sulfide (H2 S) represents the third and the youngest member of the gaseous transmitters family. The dominant effect of H2 S on isolated vessels is vasodilation. As the mechanism of H2 S-induced relaxation in human vessels remains unclear, the present study aimed to investigate the effects of H2 S donor, sodium hydrosulfide (NaHS), on isolated human saphenous vein (HSV) and to determine the mechanism of action. Our results showed that NaHS (1 µM-3 mM) induced a concentration-dependent relaxation of endothelium-intact HSV rings pre-contracted by phenylephrine. Pre-treatment with L-NAME, ODQ and KT5823 significantly inhibited NaHS-induced relaxation, while indomethacin induced partial inhibition. Among K+ channel blockers, the combination of apamin and TRAM-34 significantly affected the relaxation produced by NaHS, while iberiotoxin and glibenclamide only reduced maximal relaxation of HSV. NaHS partially relaxed endothelium-intact rings pre-contracted by high K+ , as well as phenylephrine-contracted rings in the presence of nifedipine. Additionally, the incubation of HSV rings with NaHS increased NO production. These results demonstrate that NaHS produces the concentration- and endothelium-dependent relaxation of isolated HSV. Vasorelaxation to NaHS probably involves activation of NO/cGMP/PKG pathway and partially prostacyclin. In addition, different K+ channels subtypes, especially SKCa and IKCa , as well as BKCa and KATP channels in high concentrations of NaHS, probably participate in the NaHS-induced vasorelaxation.

Differential expression profiles of circular RNAs in the rat hippocampus after deep hypothermic circulatory arrest.

Neurological dysfunction commonly occurs after cardiac surgery with deep hypothermic circulatory arrest (DHCA). The mechanisms underlying DHCA-associated brain injury remain poorly understood. This study determined the changes in expression profiles of circular RNAs (circRNAs) in the hippocampus in rats that underwent DHCA, with an attempt to explore the potential role of circRNAs in the brain injury associated with DHCA. Adult male Sprague Dawley rats were subjected to cardiopulmonary bypass with DHCA. Brain injury was evaluated by neurological severity scores and histological as well as transmission electron microscope examinations. The expression profiles of circRNAs in the hippocampal tissues were screened by microarray. Quantitative real-time PCR (RT-qPCR) was used to validate the reliability of the microarray results. Bioinformatic algorithms were applied to construct a competing endogenous RNA (ceRNA) network, and Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses were performed to explore the potential biological roles of the circRNAs. Out of 14 145 circRNAs screened, 56 were differentially expressed in the hippocampus between the DHCA and sham-operated rats, including 30 upregulated and 26 downregulated circRNAs. The expression changes of six selected circRNAs (upregulated: rno_circRNA_011190, rno_circRNA_012988, rno_circRNA_000544; downregulated: rno_circRNA_010393, rno_circRNA_012043, rno_circRNA_015149) were further confirmed by RT-qPCR. Bioinformatics analysis showed the enrichment of these confirmed circRNAs and their potential target mRNAs in several KEGG pathways including histidine metabolism, adipocytokine signaling, and cAMP signaling. By revealing the change expression profiles of circRNAs in the brain after DHCA, this study indicates possible involvements of these dysregulated circRNAs in brain injury and suggests a potential of targeting circRNAs for prevention and treatment of neurological dysfunction associated with DHCA.

Calcium-activated potassium channel family in coronary artery bypass grafts.

OBJECTIVES: We examined the expression, distribution, and contribution to vasodilatation of the calcium-activated potassium (KCa) channel family in the commonly used coronary artery bypass graft internal thoracic artery (ITA) and saphenous vein (SV) to understand the role of large conductance KCa (BKCa), intermediate-conductance KCa (IKCa), and small-conductance KCa (SKCa) channel subtypes in graft dilating properties determined by endothelium-smooth muscle interaction that is essential to the postoperative performance of the graft. METHODS: Real-time polymerase chain reaction and western blot were employed to detect the messenger RNA and protein level of KCa channel subtypes. Distribution of KCa channel subtypes was examined by immunohistochemistry. KCa subtype-mediated vasorelaxation was studied using wire myography. RESULTS: Both ITA and SV express all KCa channel subtypes with each subtype distributed in both endothelium and smooth muscle. ITA and SV do not differ in the overall expression level of each KCa channel subtype, corresponding to comparable relaxant responses to respective subtype activators. In ITA, BKCa is more abundantly expressed in smooth muscle than in endothelium, whereas SKCa exhibits more abundance in the endothelium. In comparison, SV shows even distribution of KCa channel subtypes in the 2 layers. The BKCa subtype in the KCa family plays a significant role in vasodilatation of ITA, whereas its contribution in SV is quite limited. CONCLUSIONS: KCa family is abundantly expressed in ITA and SV. There are differences between these 2 grafts in the abundance of KCa channel subtypes in the endothelium and the smooth muscle. The significance of the BKCa subtype in vasodilatation of ITA may suggest the potential of development of BKCa modulators for the prevention and treatment of ITA spasm during/after coronary artery bypass graft surgery.

Preoperative plasma biomarkers associated with atrial fibrillation after coronary artery bypass surgery.

OBJECTIVES: Postoperative atrial fibrillation (POAF) is a common complication in coronary artery bypass grafting (CABG) procedures. This prospective study aimed to investigate predisposition of proteins and metabolites correlated to POAF after CABG and related cellular pathways. METHODS: Preoperative plasma samples from patients undergoing CABG procedures were prospectively collected. After CABG, the patients were grouped to POAF or sinus rhythm (N = 170; n = 90 in the discovery set and n = 80 in the validation set). The plasma samples were analyzed using proteomics, metabolomics, and bioinformatics to identify the differential proteins and differential metabolites. The correlation between differential proteins and POAF was also investigated by multivariable regression analysis and receiver operator characteristic analysis. RESULTS: In the POAF(+) group, 29 differential proteins and 61 differential metabolites were identified compared with the POAF(-) group. The analysis of integrated omics revealed that preoperative alteration of peroxisome proliferators-activated receptor α and glutathione metabolism pathways increased the susceptibility of POAF after CABG. There was a correlation between plasma levels of apolipoprotein-C3, phospholipid transfer protein, glutathione peroxidase 3, cholesteryl ester transfer protein, and POAF. CONCLUSIONS: The present study for first time at multi-omics levels explored the mechanism of POAF and validated the results in a new cohort of patients, suggesting preexisting differential proteins and differential metabolites in the plasma of patients prone to POAF after CABG. Dysregulation of peroxisome proliferators-activated receptor α and glutathione metabolism pathways related to metabolic remodeling and redox imbalance-associated electrical remodeling may play a key role in the pathogenesis of POAF. Lower plasma phospholipid transfer protein, apolipoprotein-C3, higher cholesteryl ester transfer protein and glutathione peroxidase 3 levels are linked with POAF. These proteins/metabolites may be developed as biomarkers to predict POAF.

Novel mutations of TCTN3/LTBP2 with cellular function changes in congenital heart disease associated with polydactyly.

Congenital heart disease (CHD) associated with polydactyly involves various genes. We aimed to identify variations from genes related to complex CHD with polydactyly and to investigate the cellular functions related to the mutations. Blood was collected from a complex CHD case with polydactyly, and whole exome sequencing (WES) was performed. The CRISPR/Cas9 system was used to generate human pluripotent stem cell with mutations (hPSCs-Mut) that were differentiated into cardiomyocytes (hPSC-CMs-Mut) and analysed by transcriptomics on day 0, 9 and 13. Two heterozygous mutations, LTBP2 (c.2206G>A, p.Asp736Asn, RefSeq NM_000428.2) and TCTN3 (c.1268G>A, p.Gly423Glu, RefSeq NM_015631.5), were identified via WES but no TBX5 mutations were found. The stable cell lines of hPSCs-LTBP2mu /TCTN3mu were constructed and differentiated into hPSC-CMs-LTBP2mu /TCTN3mu . Compared to the wild type, LTBP2 mutation delayed the development of CMs. The TCTN3 mutation consistently presented lower rate and weaker force of the contraction of CMs. For gene expression pattern of persistent up-regulation, pathways in cardiac development and congenital heart disease were enriched in hPSCs-CM-LTBP2mu , compared with hPSCs-CM-WT. Thus, the heterozygous mutations in TCTN3 and LTBP2 affect contractility (rate and force) of cardiac myocytes and may affect the development of the heart. These findings provide new insights into the pathogenesis of complex CHD with polydactyly.

Protein biomarkers and risk scores in pulmonary arterial hypertension associated with ventricular septal defect: integration of multi-omics and validation.

The molecular mechanisms underlying pulmonary arterial hypertension (PAH) in congenital ventricular septal defects (VSD) are unclear. We aimed to reveal molecular pathways and potential biomarkers by multi-omics analysis in VSD-PAH. Plasma from 160 children, including 120 VSD patients with/without PAH and 40 healthy children was studied by integrated proteomics, metabolomics, and bioinformatics analyses. Proteomics identified 107 differential proteins (DPs) between patients with/without PAH including significantly increased adiponectin (ADIPO), dopamine ß-hydroxylase (DBH), alanyl membrane aminopeptidase (ANPEP), transferrin receptor 1, and glycoprotein Ib platelet α-subunit and decreased guanine nucleotide-binding protein Gs in VSD-PAH. Metabolomics discovered 191 differential metabolites between patients with/without PAH, including elevation of serotonin, taurine, creatine, sarcosine, and 2-oxobutanoate, and decrease of vanillylmandelic acid, 3,4-dihydroxymandelate, 15-keto-prostaglandin F2α, fructose 6-phosphate, l-glutamine, dehydroascorbate, hydroxypyruvate, threonine, l-cystine, and 1-aminocyclopropane-1-carboxylate. The DPs were validated in a new cohort of patients (n = 80). Integrated analyses identified key pathways, including cAMP, ECM receptor interaction, AMPK, hypoxia-inducible factor 1, PI3K-Akt signaling pathways, and amino acid metabolisms. Increased plasma protein levels of DBH, ADIPO, and ANPEP were found to be independently associated with the occurrence of PAH, with a new total risk score from these three proteins developed for clinical diagnosis. In this integrated multi-omics analysis in VSD-PAH patients, we have, for the first time, found that VSD-PAH patients present important differential proteins, metabolites, and key pathways. We have developed a total risk score (based on the plasma concentration of DBH, ANPEP, and ADIPO) as a predictor of development of PAH in CHD-VSD patients. Therefore, these proteins may be used as biomarkers, and the new total risk score has significant clinical implications in the diagnosis of PAH.