Analyses of lncRNA and mRNA profiles in recurrent atrial fibrillation after catheter ablation.

BACKGROUND: Atrial fibrillation (AF) is the most common cardiac arrhythmia worldwide. Catheter ablation has become a crucial treatment for AF. However, there is a possibility of atrial fibrillation recurrence after catheter ablation. Our study sought to elucidate the role of lncRNA‒mRNA regulatory networks in late AF recurrence after catheter ablation. METHODS: We conducted RNA sequencing to profile the transcriptomes of 5 samples from the presence of recurrence after AF ablation (P-RAF) and 5 samples from the absence of recurrence after AF ablation (A-RAF). Differentially expressed genes (DEGs) and long noncoding RNAs (DE-lncRNAs) were analyzed using the DESeq2 R package. The functional correlations of the DEGs were assessed through Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses. A protein‒protein interaction (PPI) network was constructed using STRING and Cytoscape. We also established a lncRNA‒mRNA regulatory network between DE-lncRNAs and DEGs using BEDTools v2.1.2 software and the Pearson correlation coefficient method. To validate the high-throughput sequencing results of the hub genes, we conducted quantitative real-time polymerase chain reaction (qRT‒PCR) experiments. RESULTS: A total of 28,528 mRNAs and 42,333 lncRNAs were detected. A total of 96 DEGs and 203 DE-lncRNAs were identified between the two groups. GO analysis revealed that the DEGs were enriched in the biological processes (BPs) of "regulation of immune response" and "regulation of immune system process", the cellular components (CCs) of "extracellular matrix" and "cell‒cell junction", and the molecular functions (MFs) of "signaling adaptor activity" and "protein-macromolecule adaptor activity". According to the KEGG analysis, the DEGs were associated with the "PI3K-Akt signaling pathway" and "MAPK signaling pathway." Nine hub genes (MMP9, IGF2, FGFR1, HSPG2, GZMB, PEG10, GNLY, COL6A1, and KCNE3) were identified through the PPI network. lncRNA-TMEM51-AS1-201 was identified as a core regulator in the lncRNA‒mRNA regulatory network, suggesting its potential impact on the recurrence of AF after catheter ablation through the regulation of COL6A1, FGFR1, HSPG2, and IGF2. CONCLUSIONS: The recurrence of atrial fibrillation after catheter ablation may be associated with immune responses and fibrosis, with the extracellular matrix playing a crucial role. TMEM51-AS1-201 has been identified as a potential key target for AF recurrence after catheter ablation.

Gene modules and genes associated with postoperative atrial fibrillation: weighted gene co-expression network analysis and circRNA-miRNA-mRNA regulatory network analysis.

Background: Atrial fibrillation (AF) is the most common complication in patients undergoing cardiac surgery. However, the pathogenesis of postoperative AF (POAF) is elusive, and research related to this topic is sparse. Our study aimed to identify key gene modules and genes and to conduct a circular RNA (circRNA)-microRNA (miRNA)-messenger RNA (mRNA) regulatory network analysis of POAF on the basis of bioinformatic analysis. Methods: The GSE143924 and GSE97455 data sets from the Gene Expression Omnibus (GEO) database were analyzed. Weighted gene co-expression network analysis (WGCNA) was used to identify the key gene modules and genes related to POAF. A circRNA-miRNA-mRNA regulatory network was also built according to differential expression analysis. Functional enrichment analysis was further performed according to Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis. Results: WGCNA identified 2 key gene modules and 44 key genes that were significantly related to POAF. Functional enrichment analysis of these key genes implicated the following important biological processes (BPs): endosomal transport, protein kinase B signaling, and transcription regulation. The circRNA-miRNA-mRNA regulatory network suggested that KLF10 may take critical part in POAF. Moreover, 2 novel circRNAs, hsa_circRNA_001654 and hsa_circRNA_005899, and 2 miRNAs, hsa-miR-19b-3p and hsa-miR-30a-5p, which related with KLF10, were involved in the network. Conclusions: Our study provides foundational expression profiles following POAF based on WGCNA. The circRNA-miRNA-mRNA network offers insights into the BPs and underlying mechanisms of POAF.

Association between systemic inflammatory response index and left ventricular remodeling and systolic dysfunction in atrial fibrillation patients.

BACKGROUND: Cardiac remodeling and dysfunction can be caused by atrial fibrillation (AF). The aim of this research is to investigate the relationship between the systemic inflammatory response index (SIRI) and left ventricular (LV) remodeling and systolic function in individuals with AF. METHODS: 416 patients with AF who were admitted to the Second Department of Cardiology in the East Ward of the Qingdao Municipal Hospital between January 2020 and May 2022 were included in the present retrospective research. The relationship between SIRI and various cardiac parameters was analyzed. The patients' left atrial (LA) enlargement and left ventricular (LV) hypertrophy and systolic dysfunction were evaluated. SIRI was calculated by the formula: neutrophil × monocyte/lymphocyte. RESULTS: SIRI significantly correlated with LV end-diastolic diameter (LVDd), LV posterior wall thickness at end-diastole (LVPWTd), interventricular septal thickness at end-diastole (IVSTd), LV mass index (LVMI), LV ejection fraction (LVEF), LA diameter (LAD), C-reactive protein (CRP), and N-terminal pro-B-type natriuretic peptide (NT-proBNP) in patients with AF. In multivariate linear regression analyses, SIRI was discovered to be significantly related to LVMI (ln-transformed) (p = 0.025), LVEF (ln-transformed) (p = 0.005), and LAD (ln-transformed) (p = 0.007). In multivariate logistic regression, the highest quartile of SIRI (SIRI > 1.62) was significantly associated with LV hypertrophy (p = 0.026), impaired LV systolic function (p = 0.002), and LA enlargement (p = 0.025). CONCLUSIONS: SIRI was significantly associated with LV remodeling and systolic function impairment in patients with AF. SIRI may serve as a reliable and convenient inflammatory biomarker for detecting impaired cardiac structure and systolic function in patients with AF.

Loss of α7nAChR enhances endothelial-to-mesenchymal transition after myocardial infarction via NF-κB activation.

The myocardial fibrosis in response to myocardial infarction (MI) is closely related to the dysbalance of endothelial-to-mesenchymal transition (EndMT). Although numerous reports indicate that α7 nicotinic acetylcholine receptor (α7nAChR) activates the cholinergic anti-inflammatory pathway (CAP) to regulate the magnitude of inflammatory responses, the role of α7nAChR in myocardial fibrosis, as well as the underlying mechanisms, have not been elucidated. In this study, we evaluated cardiac function, fibrosis, and EndMT signaling using a mouse model of MI and interleukin (IL)-1ß-induced human cardiac microvascular endothelial cells (HCMECs). In vivo, α7nAChR deletion increased cardiac dysfunction, exacerbated the cardiac inflammatory response, and NF-κB activation, and enhanced EndMT, as shown by higher expression levels of fibroblast markers (FSP-1, α-SMA, collagen I, Snail) and decreased levels of the FGFR1, glucocorticoid receptor (GR) and endothelial marker (CD31) compared to wild-type mice. In vitro, the pharmacological activation of α7nAChR with PNU282987 significantly inhibited IL-1ß-induced EndMT, as shown by a reduced transition to the fibroblast-like phenotype and the expression of fibrotic markers. Moreover, the IL-1ß-mediated activation of NF-κB pathway was suppressed by PNU282987. This anti-EndMT effect of α7nAChR was associated with regulation of Snail. Furthermore, Western blot analysis further revealed that the GR antagonist RU38486 could partially counteract the effect of PNU282987 on NF-κB expression. In conclusion, our results show that α7nAChR is involved in cardiac fibrosis by inhibiting EndMT, providing a novel approach to the treatment of MI.

LncRNA LINC00961 regulates endothelial­mesenchymal transition via the PTEN­PI3K­AKT pathway.

The long noncoding RNA LINC00961 plays a crucial role in cancer and cardiovascular diseases. In the present study, the role and underlying mechanism of LINC00961 in endothelial­mesenchymal transition (EndMT) induced by transforming growth factor beta (TGF­ß), was investigated. Human cardiac microvascular endothelial cells were transfected with LV­LINC00961 or short hairpin LINC00961 plasmids to overexpress or knock down LINC00961 in the cells, respectively. The cells were then exposed to TGF­ß in serum­free medium for 48 h to induce EndMT. Flow cytometric analysis, Cell Counting Kit­8 assay and immunofluorescence staining were performed to examine the cell apoptosis rate, assess cell viability, and identify CD31+/α­SMA+ double­positive cells, respectively. Western blotting and reverse transcription­ quantitative polymerase chain reaction were used to evaluate protein and mRNA expression, respectively. Injury to endothelial cells and EndMT was induced by TGF­ß in a time­dependent manner. LINC00961 overexpression promoted injury and EndMT, whereas LINC00961 knockdown had the opposite effects. Knockdown of LINC00961 attenuated EndMT and injury to endothelial cells induced by TGF­ß via the PTEN­PI3K­AKT pathway. Inhibition of LINC00961 expression may prevent the occurrence of EndMT­related cardiovascular diseases, such as myocardial fibrosis and heart failure. Therefore, LINC00961 shows potential as a therapeutic target for cardiovascular diseases.

Relationship between Plasma Trimethylamine N-Oxide Levels and Renal Dysfunction in Patients with Hypertension.

INTRODUCTION: Trimethylamine N-oxide (TMAO) is a metabolite produced by gut bacteria. Although increased TMAO levels have been linked to hypertension (HTN) and chronic kidney disease (CKD) with poor prognosis, no clinical studies have directly addressed the relationship between them. In this study, we investigated the relationship between TMAO and renal dysfunction in hypertensive patients. METHODS: We included healthy controls (n = 50), hypertensive patients (n = 46), and hypertensive patients with renal dysfunction (n = 143). Their blood pressure values were taken as the highest measured blood pressure. Renal function was evaluated using the estimated glomerular filtration rate. Plasma TMAO levels were measured using high-performance liquid chromatography tandem mass spectrometry. RESULTS: We found significant differences in plasma TMAO levels among the 3 groups (p < 0.01). The plasma TMAO of patients with HTN was significantly higher than that of healthy people, and the plasma TMAO of patients with HTN complicated by renal dysfunction was significantly higher than either of the other groups. Patients in the highest TMAO quartile were at a higher risk of developing CKD stage 5 than those in the lowest quartile. In the receiver operating characteristic curve, the area under the curve of TMAO combined with ß 2-macroglobulin for predicting renal dysfunction in patients with HTN was 0.85 (95% confidence interval 0.80-0.90). CONCLUSION: An elevated TMAO level reflects higher levels of HTN and more severe renal dysfunction. TMAO, combined with ß 2-macroglobulin levels, may assist in diagnosing CKD in hypertensive patients. Plasma TMAO has predictive value for early kidney disease in hypertensive patients.

Metformin Attenuates Hypoxia-induced Endothelial Cell Injury by Activating the AMP-Activated Protein Kinase Pathway.

ABSTRACT: Metformin reduces the incidence of cardiovascular diseases, and potential underlying mechanisms of action have been suggested. Here, we investigated the role of metformin in endothelial cell injury and endothelial-mesenchymal transition (EndMT) induced by hypoxia. All experiments were performed in human cardiac microvascular endothelial cells (HCMECs). HCMECs were exposed to hypoxic conditions for 24, 48, 72, and 96 hours, and we assessed the cell viability by cell counting kit 8; metformin (2, 5, 10, and 20 mmol/L) was added to the cells after exposure to the hypoxic conditions for 48 hours. The cells were randomly divided into the control group, hypoxia group, hypoxia + metformin group, hypoxia + control small interfering RNA group, hypoxia + small interfering Prkaa1 (siPrkaa1) group, and hypoxia + siPrkaa1 + metformin group. Flow cytometry and cell counting kit 8 were used to monitor apoptosis and assess cell viability. Immunofluorescence staining was used to identify the CD31+/alpha smooth muscle actin+ double-positive cells. Quantitative real-time-PCR and Western blot were used for mRNA and protein expression analyses, respectively. Hypoxia contributed to endothelial injuries and EndMT of HCMECs in a time-dependent manner, which was mainly manifested as decreases in cell viability, increases in apoptotic rate, and changes in expression of apoptosis-related and EndMT-related mRNAs and proteins. Furthermore, metformin could attenuate the injuries and EndMT caused by hypoxia. After metformin treatment, phosphorylated-AMPK (pAMPK) and p-endothelial nitric oxide synthase expression increased, whereas p-mammalian target of rapamycin expression decreased. However, results obtained after transfection with siPrkaa1 were in contrast to the results of metformin treatment. In conclusion, metformin can attenuate endothelial injuries and suppress EndMT of HCMECs under hypoxic conditions because of its ability to activate the AMPK pathway, increase p-AMPK/AMP-activated protein kinase, and inhibit mammalian target of rapamycin.

Protective effects of acetylcholine on hypoxia-induced endothelial-to-mesenchymal transition in human cardiac microvascular endothelial cells.

Endothelial-to-mesenchymal transition (EndMT) has been reported as a key factor in myocardial fibrosis. Acetylcholine (ACh), a neurotransmitter of the vagus nerve, has been confirmed to exert cardio-protective properties with unclear mechanisms. In this study, the specific markers of cell injury, EndMT, inflammation, and autophagy were measured. We found that treatment with ACh prevented hypoxia-induced cell viability reduction and apoptosis in human cardiac microvascular endothelial cells (HCMECs). Additionally, our results indicate that pre-treatment with ACh significantly suppresses hypoxia-induced EndMT and NF-κB activation in HCMECs. ACh also reduced hypoxia-inducible factor (HIF)-1ɑ protein levels under hypoxia. Knock down of HIF-1ɑ enhanced the inhibitory effect of ACh on NF-κB activation. The NF-κB-specific small molecule inhibitor BAY 11-7082, prostaglandin E2, and LY294002 prevented hypoxia-induced EndMT. Moreover, our data show that hypoxia triggers autophagy in HCMECs, and ACh significantly upregulates autophagy activity. Pre-treatment of HCMECs with 3-methyladenine or chloroquine partially reversed ACh-induced EndMT inhibition. These results suggest that ACh may confer protection against hypoxia-induced EndMT through the inhibition of NF-κB and the induction of autophagy.

Klotho improves diabetic cardiomyopathy by suppressing the NLRP3 inflammasome pathway.

AIMS: NLRP3 inflammasome activation is essential for the development and prognosis of diabetic cardiomyopathy (DCM). The anti-aging protein Klotho is suggested to modulate tissue inflammatory responses. The aim of the present study was to examine the protective effects of Klotho on DCM. MAIN METHODS: A streptozotocin-induced diabetes mouse model was established to assess the effects of Klotho in vivo, which was administered for 12 weeks. The characteristics of type 1 DCM were evaluated by general status, echocardiography, and histopathology. The expression of associated factors was determined by RT-qPCR and western blotting. Parallel experiments to determine the molecular mechanism through which Klotho prevents DCM were performed using H9C2 cells exposed to high glucose (35 mM). KEY FINDINGS: Diabetes-induced increases in serum creatine kinase-muscle/brain and lactate dehydrogenase levels, cardiac fibrosis, cardiomyocyte apoptosis, and cardiac dysfunction were ameliorated by Klotho. Additionally, Klotho suppressed TXNIP expression, NLRP3 inflammasome activation, and expression of the inflammatory cytokines tumor necrosis factor ɑ, interleukin-1ß, and interleukin-18 in vivo. In high glucose-cultured cardiomyocytes, Klotho and N-acetylcysteine significantly downregulated intracellular reactive oxygen species generation and TXNIP/NLRP3 inflammasome activation. Pretreatment of H9C2 cells with NLRP3 siRNA or Klotho prevented high glucose-induced inflammation and apoptosis in H9C2 cells. SIGNIFICANCE: Our results demonstrate that the protective effect of Klotho on diabetes-induced cardiac injury is associated with inhibition of the NLRP3 inflammasome pathway, suggesting its therapeutic potential for DCM.

Vaspin prevents myocardial injury in rats model of diabetic cardiomyopathy by enhancing autophagy and inhibiting inflammation.

NLRP3 inflammasome activation plays an important role in diabetic cardiomyopathy (DCM). It is known that autophagy is related to the activation of inflammasomes during oxidative stress. Visceral adipose tissue-derived serine protease inhibitor (Vaspin), is an adipocytokine that has been shown to exert a protective effect on autophagic activity, but whether and how Vaspin improves myocardial damage in DCM remain unclear. In this study, we explored the role of Vaspin in DCM using a streptozotocin (STZ)-induced diabetes model. Cardiac function, cardiomyocyte apoptosis, myocardial tissue morphology, and mitochondrial morphology in diabetic rats were improved after eight weeks of Vaspin treatment. Vaspin treatment augmented autophagy activation in diabetic rat hearts. Moreover, the activation of NLRP3 inflammasome was inhibited by Vaspin, followed by a decrease in the cleavage of caspase-1 and maturation of IL-1ß and TNF-ɑ. In vitro studies found that the mitochondrial reactive oxygen species (ROS) generation as well as the depolarization of the mitochondrial membrane in H9C2cells induced by high glucose were attenuated by Vaspin. This inhibitory effect of Vaspin on NLRP3 inflammasome activation was due to the protection of autophagy activity and was abolished after the treatment of autophagy inhibitor (3-MA). These results demonstrate that Vaspin alleviates STZ-induced myocardial injury and renders a cardioprotective effect by suppressing NLRP3 inflammasome activation and promoting autophagy.

Vaspin Prevents Tumor Necrosis Factor-α-Induced Apoptosis in Cardiomyocytes by Promoting Autophagy.

Visceral adipose tissue-derived serine protease inhibitor (Vaspin) is an adipocytokine that has been shown to exert anti-inflammatory effects and inhibits apoptosis under diabetic conditions. This study was designed to investigate the impact of vaspin on autophagy in tumor necrosis factor (TNF)-α-induced injury in cardiomyocytes and its cardioprotective effects in the pathogenesis of diabetic cardiomyopathy (DCM). H9C2 cells were treated with TNF-α with or without vaspin in vitro. Tumor necrosis factor-α treatment inhibited autophagy and promoted apoptosis in H9C2 cells after stimulating for 24 hours. Pretreatment with vaspin significantly mitigated apoptosis induced by TNF-α partly because of augment effects of vaspin on autophagy as demonstrated by a higher ratio of LC3-II/LC3-I, higher expression of Beclin-1, and increased autophagosomes formation. Furthermore, the AKT agonist IGF-1 significantly reversed the effect of vaspin on autophagy. In vivo DCM model was also developed by treating rats with streptozotocin followed by intraperitoneal injection with vaspin. In DCM rats, upregulation of vaspin reversed cardiac dysfunction, as identified by increased left ventricular ejection fractions and fractional shortening levels, a higher Em/Am ratio, and lower levels of TNF-α, lactate dehydrogenase, creatine kinase, and creatine kinase-myocardial isoenzyme. In conclusion, vaspin attenuated the TNF-α-induced apoptosis by promoting autophagy probably through inhibiting the PI3K/AKT/mTOR pathway and further ameliorated the cardiac dysfunction in DCM rats.

Predictive value of exercise-induced atrial natriuretic peptide secretion for the presence of left atrial low-voltage areas in patients with persistent atrial fibrillation.

Objectives Left atrial (LA) low-voltage areas (LVAs) are a strong predictor of atrial fibrillation (AF) recurrence after pulmonary vein isolation (PVI). However, a non-invasive method for evaluating LA-LAVs has not been established yet. The objective of our study was to assess the predictive value of the plasma atrial natriuretic peptide (ANP) level for the presence of LA-LVAs in patients with persistent AF (PeAF). Methods Seventy-two PeAF patients underwent an exercise stress test preprocedurally. LA voltage maps were created after PVI. Demographic, clinical and echocardiographic data were recorded. Plasma levels of ANP at baseline (ANP0) and increase induced by exercise (ΔANP) were also measured. Results Compared with patients without LA-LVAs, patients with LA-LVAs had a longer history of AF, higher CHADS2 score and higher ANP0 and lower ΔANP. LA-LVAs extent correlated with duration of AF history, CHADS2 score and ΔANP (R = -0.76, P < 0.01). Only ΔANP independently predicted the presence of LA-LVAs (OR =1.63, P = 0.02). Derived from the ROC curve, ΔANP <55 pg/mL predicted the presence of LA-LVAs with high accuracy (AUC =0.78; 95% CI =0.57-0.87, P < 0.01). Conclusions Exercise-induced secretion of ANP may be used to predict the presence of LA-LVAs in patients with PeAF before catheter ablation.

Autophagy attenuates endothelial-to-mesenchymal transition by promoting Snail degradation in human cardiac microvascular endothelial cells.

Endothelial-to-mesenchymal transition (EndMT) mainly exists in cardiovascular development and disease progression, and is well known to contribute to cardiac fibrosis. Recent studies indicated that autophagy also participates in the regulation of cardiac fibrosis. However, the precise role of autophagy in cardiac fibrosis and the underlying molecular mechanism remain unclear. The present study aimed to explore the role of autophagy in EndMT, reveal the underlying molecular mechanism, and seek new therapy for cardiac fibrosis. In the present study, we found that EndMT and autophagy were induced simultaneously by hypoxia in human cardiac microvascular endothelial cells (HCMECs). Rapamycin, an autophagy enhancer, attenuated EndMT with promoting angiogenesis, while 3-methyladenine (3-MA) and chloroquine (CQ), agents that inhibit autophagy, accelerated the progression accompanied by the decrease in counts of tube formation under hypoxia conditions. Interestingly, intervening autophagy by rapamycin, 3-MA, or CQ did not affect hypoxia-induced autocrine TGFß signaling, but changed the expression of Snail protein without alterations in the expression of Snail mRNA. Furthermore, the colocalization of LC3 and Snail indicated that autophagy might mediate Snail degradation under hypoxia conditions in HCMECs. Interaction of p62, the substrate of autophagy, with Snail by co-immunoprecipitation especially in hypoxia-incubated cells confirmed the hypothesis. In conclusion, autophagy serves as a cytoprotective mechanism against EndMT to promote angiogenesis by degrading Snail under hypoxia conditions, suggesting that autophagy targetted therapeutic strategies may be applicable for cardiac fibrosis by EndMT.

RUNX3 modulates hypoxia-induced endothelial-to-mesenchymal transition of human cardiac microvascular endothelial cells.

Endothelial-mesenchymal transition (EndMT) is an essential mechanism in the cardiovascular system, for both cardiovascular development and cardiovascular diseases (CVDs). Recent studies indicate that runt-related transcription factor 3 (RUNX3) contributes to EndMT and endothelial cell dysfunction. However, the underlying molecular mechanism remains unknown. The present study was designed to investigate the role of RUNX3 in EndMT and endothelial cell function, and to elucidate the underlying molecular mechanism. Human cardiac microvascular endothelial cells (HCMECs) were incubated in strictly controlled hypoxic conditions (1% O2). HCMECs were cultured under normoxic conditions (21% O2), and then moved to a strictly controlled hypoxic environment (1% O2). Under this hypoxic condition, the cells were transfected with the lentiviral vector containing RUNX3 or an empty lentiviral vector for 8 h. After the cells were cultured under hypoxic conditions for 4 days, CD31 and α-smooth muscle actin colocalization were assessed by immunofluorescence microscopy. Transwell migration and tube formation assays were used to examine the migration and angiogenesis ability. RT-qPCR and western blotting were used to determine the expression of molecules involved in EndMT. Hypoxia induced the transition of HCMECs to mesenchymal cells and markedly promoted tube formation and cell migration. Transforming growth factor-ß (TGF-ß) and Notch signaling were activated during the hypoxia-induced EndMT of HCMECs. RUNX3 knockdown attenuated EndMT of HCMECs, promoted angiogenic phenotype, and reduced endothelial cell migration. In conclusion, our results showed that RUNX3 knockdown attenuated hypoxia-induced EndMT and reversed endothelial cell functions. RUNX3 is a common downstream target of TGF-ß and Notch signaling, and may be a novel therapeutic target for treating CVD mediated by EndMT.

Fibrillatory wave amplitude on transesophageal ECG as a marker of left atrial low-voltage areas in patients with persistent atrial fibrillation.

BACKGROUND: Low-voltage areas (LVAs) are frequently observed in patients with persistent atrial fibrillation (PeAF) and may represent adverse atrial remodeling. However, noninvasive method of evaluating LAVs is not well established. METHODS: In a cohort of 68 patients with PeAF, endocardial voltage maps of left atrium (LA) were created during sinus rhythm after pulmonary vein isolation (PVI). LVAs were defined as areas with electrogram amplitudes <0.5 mV. LA-LVAs were correlated with clinical, echocardiographic, surface, and transesophageal electrocardiography (TE-ECG) variables. RESULTS: LA voltage mapping revealed any degree of LA-LVAs in 50 (73.5%) patients. Patients with LA-LVAs were older, had a longer history of AF, and lower fibrillatory wave (F wave) amplitude on TE-ECG (0.27 ± 0.06 vs 0.39 ± 0.08 mv, p < .01) as compared to patients without LA-LVAs. The extent of LA-LVAs was weakly correlated with age (R = 0.36, p = .03) and AF duration (R = 0.26, p = .02), but significantly correlated with F-wave amplitude on TE-ECG (R = -0.57, p < .01). Only F-wave amplitude on TE-ECG was found as independent predictor for the presence of LA-LVAs (OR = 1.53, 95% CI = 1.09-2.96, p = .03). A receiver operating characteristic (ROC) curve identified an F-wave amplitude of 0.29 mV (AUC = 0.788; sensitivity = 68.4%; specificity = 73.2%) on TE-ECG as the optimal cutoff value for predicting LA-LVAs. CONCLUSIONS: As a noninvasive investigation, F-wave amplitude on TE-ECG may be used as an indicator for the presence of LA-LVAs.

[Knockdown of RUNX3 inhibits hypoxia-induced endothelial-to-mesenchymal transition of human cardiac microvascular endothelial cells].

Objective To investigate the effects of Runt-related transcription factor 3 (RUNX3) knockdown on hypoxia-induced endothelial-to-mesenchymal transition (EndoMT) of human cardiac microvascular endothelial cells (HCMECs), and elucidate the underlying molecular mechanism. Methods HCMECs were cultured in hypoxic conditions and infected with RUNX3-RNAi lentivirus to knock-down the expression of RUNX3. Reverse transcription PCR was performed to detect the mRNA expressions of RUNX3 and EndoMT related genes such as CD31, vascular endothelial cadherin (VE-cadherin), α-smooth muscle actin (α-SMA) and fibroblast-specific protein-1 (FSP-1); Western blotting was used to determine the protein expressions of RUNX3, CD31, α-SMA and another molecules involved in EndoMT; and immunofluorescence cytochemistry was applied to observe the colocalization of CD31 and α-SMA. Results Hypoxia induced the transition of HCMECs to mesenchymal cells. Hypoxia up-regulated the expression of TGF-ß2, Smad2/3, phosphorylation of Smad2/3 (p-Smad2/3), Notch-1, Hes1, and Hey1; knockdown of RUNX3 down-regulated the levels of Smad2/3, p-Smad2/3, Hes1, and Hey1 to different extents, and raised the levels of TGF-ß2 and Notch-1. Conclusion Knockdown of RUNX3 in HCMECs attenuates hypoxia-induced EndoMT via partially inhibiting TGF-ß and Notch signaling pathway.

Protective effects of Notch1 signaling activation against high glucose-induced myocardial cell injury: Analysis of its mechanisms of action.

Notch1 plays an important role in cardiomyocyte apoptosis and cardiac fibrosis. However, the effects of Notch1 on diabetic cardiomyopathy (DCM) and its mechanisms of action remain unclear. In the present study, we sought to investigate the role of Notch1 in, and its effects on high glucose (HG)­induced myocardial cell apoptosis and myocardial fibrosis. H9c2 cells exposed to HG were used to establish an in vitro model of myocardial injury. The H9c2 cells were cultured with normal glucose (NG; 5.5 mmol/L­ NG), and were then epxosed to HG (33 mmol/L­ HG), a γ­secretase inhibitor (DAPT), and were transfected with a lentiviral vector containing the Notch1 intracellular domain (N1ICD; lentivirus­N1ICD). At 72 h following exposure to HG, DAPT or transfection with lentivirus­N1ICD, myocardial cell viability was assessed using a Cell Counting kit­8 (CCK­8) assay. Cell apoptosis was measured using Annexin V/propidium iodide (PI) double staining and flow cytometry. The mRNA expression levels of hairy/enhancer of split­1 (Hes­1) and hairy/enhancer-of-split related with YRPW motif­1 (Hey­1) were measured by quantitative PCR (qPCR), while the protein expression of N1ICD, Bax, Bcl­2, transforming growth factor­ß1 (TGF­ß1) and connective tissue growth factor (CTGF), and the levels of phosphorylated (p-)AKT, total (t-)AKT, p­phosphoinositide 3-kinase (PI3K) and t­AKT were measured by western blot analysis. Our results revealed that exposure to HG induced apoptosis and upregulated TGF­ß1 and CTGF expression in the H9c2 cardiomyocytes. Furthermore, the Notch1 and PI3K/AKT signaling pathways were activated following transfection with lentivirus­N1ICD, and this activation enhanced myocardial cell viability, prevented cardiomyocyte apoptosis and decreased TGF­ß1 and CTGF expression. On the whole, our data demonstrate that the overexpression of Notch1 prevents HG­induced cardiomyocyte apoptosis and decreases CTGF expression in H9c2 cells exposed to HG. Thus, Notch1 may be used to prevent the development of DCM and to inhibit cardiac fibrosis. The findings of our study may prove to be of use in the development of novel therapeutic strategies for DCM.

[Research progress of Tbx3 in cardiac biological pacemaker].

The early cardiac biological pacemaker studies were mostly around HCN channel, and how to build a biological pacemaker through the enhanced If current. In recent years, however, people found that the genes of Tbx3 could play an important role in the development of cardiac conduction system, especially in processes of the maturity of the sinoatrial node and maintenance of its function. And the Tbx3 can further optimize the biological pacemaker. Therefore, it could be a new therapeutic focus in biological pacemaker and treatment of cardiac conduction system disease. This paper summarizes some of the latest research progress of the Tbx3 in biological pacemaker in recent years. We hope that this review could provide theoretical basis for the clinical applications of Tbx3.

Effect of NRG-1/ErbB signaling intervention on the differentiation of bone marrow stromal cells into sinus node-like cells.

The neuregulin-1 (NRG-1)/ErbB signaling pathway is a crucial regulator of cardiac development and plays an important role in the formation of the cardiac special conduction system. To establish a rat bone marrow stromal cell (BMSC) cardiomyocyte (CM)-like differentiation model, BMSCs were treated with 5-azacytidine and fibroblast growth factor basic (FGF-basic) for 24 hours and then cocultured with neonatal rat CMs in a Transwell culture system. The feasibility of regulating the differentiation of BMSCs into sinoatrial node cells by manipulating the NRG-1/ErbB pathway was investigated. Three weeks after induction, reverse transcription-polymerase chain reaction analysis revealed that inhibition of NRG-1/ErbB signaling (using AG1478) greatly enhanced the expression of HCN4, Tbx3, and Tbx2. Additionally, Tbx3 protein levels were higher than in the control group and even produced distinct nodal-type action potentials. The expression of Nkx2.5 in the NRG-1 group (treated with exogenous NRG-1) was higher than the other 2 groups. The expression of phospho-Akt was also increased in the NRG-1 group but decreased in the AG1478 group. Together, these data demonstrate that inhibiting the endogenous NRG-1/ErbB signaling pathway when rat BMSCs differentiate into CMs can greatly enhance the pacemaker phenotype. Akt signaling may be one of the underlying molecular mechanisms responsible for these results.

Gene transfer of human neuregulin-1 attenuates ventricular remodeling in diabetic cardiomyopathy rats.

Neuregulin-1 (NRG-1) is a cardioactive growth factor released from endothelial cells. However, the effect of NRG-1 on ventricular remodeling in diabetic cardiomyopathy (DCM) remains unclear. The aim of the present study was to investigate the pathophysiological role of NRG-1 in a rat model of DCM. Rat cardiac microvascular endothelial cells (CMECs) were transfected with human NRG-1 (hNRG-1) lentivirus. The hNRG-1 medium was utilized to culture rat cardiomyocytes. The cardiomyocytes were counted with a hemacytometer to determine the proliferation index and Annexin V/propidium iodide double staining was employed to examine the apoptotic rate. A rat model of DCM was induced by an intraperitoneal injection of streptozotocin. The hNRG-1 lentivirus was injected into the myocardium of the DCM model rats. Four weeks after the lentiviral injection, cardiac catheterization was performed to evaluate the cardiac function. Apoptotic cells were determined by terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) staining. Left ventricular sections were stained with Masson's trichrome to investigate the myocardial collagen content. The expression levels of related genes and proteins were analyzed. The results indicated that hNRG-1 conditioned medium stimulated the proliferation and counteracted the apoptosis of cardiomyocytes in vitro. In the rats with DCM, gene transfer of hNRG-1 to the myocardium improved heart function, as indicated by invasive hemodynamic measurements. In addition, hNRG-1 reduced the number of apoptotic cells, decreased the expression of bax and increased the expression of bcl-2 in the myocardium of the DCM model rats. Myocardial fibrosis and type I and III pro-collagen mRNA levels in the myocardium were significantly reduced by hNRG-1. hNRG-1 also increased the expression of phospho-Akt and phospho-eNOS in the myocardium. In conclusion, the gene transfer of hNRG-1 ameliorates cardiac dysfunction in diabetes. Although further studies are required, NRG-1 appears to protect cardiomyocytes against apoptosis and to reduce the extent of myocardial interstitial fibrosis.