Shortened leukocyte telomere length as a potential biomarker for predicting the progression of atrial fibrillation from paroxysm to persistence in the short-term.

ABSTRACT: This study aimed to assess the role of leukocyte telomere length (LTL) in the development of atrial fibrillation (AF) among Chinese patients.This is a cross-sectional study. A total of 350 patients from June 2016 to December 2017 were retrospectively analyzed. These included 219 AF patients and 131 with sinus rhythm in the control group. Quantitative real-time PCR was used to measure relative LTL.The relative LTLs of all subjects (n = 350) ranged from 0.4 to 2.41 (0.98 ±â€Š0.29), showing a significant negative correlation (P < .001) with age. The AF-group had significantly shorter LTLs (0.93 ±â€Š0.26 vs 1.07 ±â€Š0.33, P < .001) and were older (61.50 ±â€Š6.49 vs 59.95 ±â€Š6.17, P = .028) than controls. LTLs among patients with persistent AF (PsAF), paroxysmal AF (PAF), and controls were significantly different (P < .001), with LTLs of PsAF patients being the shortest and controls being the longest. After adjusting for possible confounding factors, the PsAF group still showed significantly shorter LTLs than the PAF and control groups (P = .013 and P = .001, respectively). After an 18-month follow-up, 20 out of 119 PAF patients had progressed into PsAF and a relative LTL of ≤0.73 was an independent predictor for progression of PAF into PsAF.LTL was found to be shorter in patients with AF than in age-matched individuals with sinus rhythm and positively correlated with severity of AF. LTL shortening could be an independent risk factor for progression from paroxysmal AF to persistent AF in the short term.

Expression defect of the rare variant/Brugada mutation R1512W depends upon the SCN5A splice variant background and can be rescued by mexiletine and the common polymorphism H558R.

Background : Mutations in SCN5A that decrease Na current underlie arrhythmia syndromes such as the Brugada syndrome (BrS). SCN5A in humans has two splice variants, one lacking a glutamine at position 1077 (Q1077del) and one containing Q1077. We investigated the effect of splice variant background on loss-of-function and rescue for R1512W, a mutation reported to cause BrS. Methods and results : We made the mutation in both variants and expressed them in HEK-293 cells for voltage-clamp study. After 24 hours of transfection, the current expression level of R1512W was reduced by ~50% in both Q1077del and Q1077 compared to the wild-type (WT) channel, respectively. The activation and inactivation midpoint were not different between WT and mutant channels in both splice variant backgrounds. However, slower time constants of recovery and enhanced intermediate inactivation were observed for R1512W/Q1077 compared with WT-Q1077, while the recovery and intermediate inactivation parameters of R1512W/Q1077del were similar to WT-Q1077del. Furthermore, both mexiletine and the common polymorphism H558R restored peak sodium current (INa) amplitude of the mutant channel by increasing the cell surface expression of SCN5A. Conclusion : These findings provide further evidence that the splice variant affects the molecular phenotype with implications for the clinical phenotype, and they provide insight into the expression defect mechanisms and potential treatment in BrS.

The potential regulatory role of hsa_circ_0004104 in the persistency of atrial fibrillation by promoting cardiac fibrosis via TGF-ß pathway.

INTRODUCTION: The progression of paroxysmal AF (PAF) to persistent AF (PsAF) worsens the prognosis of AF, but its underlying mechanisms remain elusive. Recently, circular RNAs (circRNAs) were reported to be associated with cardiac fibrosis. In case of the vital role of cardiac fibrosis in AF persistency, we hypothesis that circRNAs may be potential regulators in the process of AF progression. MATERIALS AND METHODS: 6 persistent and 6 paroxysmal AF patients were enrolled as derivation cohort. Plasma circRNAs expressions were determined by microarray and validated by RT-PCR. Fibrosis level, manifested by serum TGF-ß, was determined by ELISA. Pathways and related non-coding RNAs involving in the progression of AF regulated were predicted by in silico analysis. RESULTS: PsAF patients showed a distinct circRNAs expression profile with 92 circRNAs significantly dysregulated (fold change ≥ 2, p < 0.05), compared with PAF patients. The validity of the expression patterns was subsequently validated by RT-PCR in another 60 AF patients (30 PsAF and PAF, respectively). In addition, all the 5 up and down regulated circRNAs were clustered in MAPK and TGF-beta signaling pathway by KEGG pathway analysis. Among the 5 circRNAs, hsa_circ_0004104 was consistently downregulated in PsAF group (0.6 ± 0.33 vs 1.46 ± 0.41, p < 0.001) and predicted to target several AF and/or cardiac fibrosis related miRNAs reported by previous studies. In addition, TGF-ß1 level was significantly higher in the PsAF group (5560.23 ± 1833.64 vs 2236.66 ± 914.89, p < 0.001), and hsa_circ_0004104 showed a significant negative correlation with TGF-ß1 level (r = - 0.797, p < 0.001). CONCLUSION: CircRNAs dysregulation plays vital roles in AF persistency. hsa_circ_0004104 could be a potential regulator and biomarker in AF persistency by promoting cardiac fibrosis via targeting MAPK and TGF-beta pathways.

p38/JNK Is Required for the Proliferation and Phenotype Changes of Vascular Smooth Muscle Cells Induced by L3MBTL4 in Essential Hypertension.

AIM: Hypertension is a complicated disorder with multifactorial etiology and high heritability. Our previous work has identified L3MBTL4 as a novel susceptibility gene for the development of essential hypertension, accompanied with activation of p38/JNK. Yet, little evidence has been reported whether p38/JNK contributed directly to L3MBTL4-induced vascular remodeling and exploring the potential mechanism of L3MBTL4 in vascular smooth muscle cells (VSMCs). METHODS: We evaluated the contribution of L3MBTL4 on proliferation, migration, and phenotype changes of VSMCs and further explored the critical role of p38 and JNK signaling pathway underlying. RESULTS: In L3MBTL4 transgenic rats, we found that the elevated blood pressure, increased left ventricular hypertrophy, and thickened vascular media layer were significantly relieved by both p38 and JNK inhibitors. Meanwhile, increased cell proliferation, advanced cell cycle progression, greater migratory capability, and synthetic phenotype were observed in L3MBTL4 overexpressed VSMCs, which could be blocked by either p38 or JNK inhibitor. CONCLUSIONS: Our findings pinpointed that p38 and JNK were required for the proliferation and phenotype changes of VSMCs induced by L3MBTL4 in hypertension. These novel findings yield new insights into the genetic and biological basis of hypertension and are fundamental for further studies to explore the intervention strategies targeting L3MBTL4 and p38/JNK to counteract the progression of hypertension.

Investigation of the Cellular Pharmacological Mechanism and Clinical Evidence of the Multi-Herbal Antiarrhythmic Chinese Medicine Xin Su Ning.

Xin Su Ning (XSN), a China patented and certified multi-herbal medicine, has been available in China since 2005 for treating cardiac ventricular arrhythmia including arrhythmia induced by ischemic heart diseases and viral myocarditis, without adverse reactions being reported. It is vitally important to discover pharmacologically how XSN as a multicomponent medicine exerts its clinical efficacy, and whether the therapeutic effect of XSN can be verified by standard clinical trial studies. In this paper we report our discoveries in a cellular electrophysiological study and in a three-armed, randomized, double-blind, placebo-controlled, parallel-group, multicenter trial. Conventional electrophysiological techniques were used to study the cellular antiarrhythmic mechanism of XSN. Data was then modeled with computational simulation of human action potential (AP) of the cardiac ventricular myocytes. The clinical trial was conducted with a total of 861 eligible participants randomly assigned in a ratio of 2:2:1 to receive XSN, mexiletine, or the placebo for 4 weeks. The primary and secondary endpoint was the change of premature ventricular contraction (PVC) counts and PVC-related symptoms, respectively. This trial was registered in the Chinese Clinical Trial Register Center (ChiCTR-TRC-14004180). We found that XSN prolonged AP duration of the ventricular myocytes in a dose-dependent, reversible manner and blocked potassium channels. Patients in XSN group exhibited significant total effective responses in the reduction of PVCs compared to those in the placebo group (65.85% vs. 27.27%, P < 0.0001). No severe adverse effects attributable to XSN were observed. In conclusion, XSN is an effective multicomponent antiarrhythmic medicine to treat PVC without adverse effect in patients, which is convincingly supported by its class I & III pharmacological antiarrhythmic mechanism of blocking hERG potassium channels and hNaV1.5 sodium channel reported in our earlier publication and prolongs AP duration both in ventricular myocytes and with computational simulation of human AP presented in this report.

Myofibroblast-Derived Exosomes Contribute to Development of a Susceptible Substrate for Atrial Fibrillation.

OBJECTIVE: Atrial fibrosis plays a critical role in atrial fibrillation (AF). A key event in the pathogenesis of fibrosis is the activation of fibroblasts (FBs) into myofibroblasts (MFBs). Paracrine factors released from MFBs lead to ion channel expression changes in cardiomyocytes (CMs). Downregulation of L-type calcium channel Cav1.2 expression is a hallmark of AF-associated ionic remodeling. However, whether exosome (Exo)-mediated crosstalk between MFBs and CMs regulates Cav1.2 expression remains unknown. METHODS: Atrial FBs and CMs were isolated and cultured from neonatal rats by enzymatic digestion. The activation of FBs into MFBs was induced by angiotensin II. Co-culture assay and in vitro Exo treatment were used to determine the effect of MFB-derived Exos on Cav1.2 expression. Confocal Ca2+ imaging was performed to examine the adrenergic stimulation-elicited Ca2+ influx signals. The levels of potential Cav1.2-inhibitory microRNAs (miRNAs) were measured by qRT-PCR. RESULTS: Untreated FBs expressed limited amounts of alpha smooth muscle actin (α-SMA), while angiotensin II induced a significant upregulation of α-SMA-expressing MFBs. Co-cultures of MFBs and CMs resulted in downregulation of Cav1.2 expression in CMs, which was largely abolished by pretreatment of MFBs with exosomal inhibitor GW4869. More importantly, treatment with MFB-derived Exos caused repression of Cav1.2 expression in CMs. Additionally, the adrenergic receptor agonist-elicited Ca2+ influx signals in CMs were remarkably attenuated by pretreatment with MFB-derived Exos, corresponding to the paralleled change in Cav1.2 expression. Finally, miR-21-3p, a potential Cav1.2-inhibitory miRNA, was enriched in MFB-derived Exos and upregulated in CMs in response to MFB-derived Exos. CONCLUSION: We uncover an Exo-mediated crosstalk between MFBs and CMs, contributing to increased vulnerability to AF by reducing the expression of Cav1.2 in CMs.

Disordered gut microbiota and alterations in metabolic patterns are associated with atrial fibrillation.

BACKGROUND: With the establishment of the heart-gut axis concept, accumulating studies suggest that the gut microbiome plays an important role in the pathogenesis of cardiovascular diseases. Yet, little evidence has been reported in characterizing the gut microbiota shift in atrial fibrillation. METHODS: We include the result of the global alterations that occur in the intestinal microbiota in a cohort of 50 patients with atrial fibrillation and 50 matched controls based on a strategy of metagenomic and metabolomic analyses. RESULTS: The alterations include a dramatic elevation in microbial diversity and a specific perturbation of gut microbiota composition. Overgrowth of Ruminococcus, Streptococcus, and Enterococcus, as well as reduction of Faecalibacterium, Alistipes, Oscillibacter, and Bilophila were detected in patients with atrial fibrillation. A gut microbial function imbalance and correlated metabolic pattern changes were observed with atrial fibrillation in both fecal and serum samples. The differential gut microbiome signatures could be used to identify patients with atrial fibrillation. CONCLUSIONS: Our findings characterize the disordered gut microbiota and microbial metabolite profiles in atrial fibrillation. Further research could determine whether intervention strategies targeting intestinal microbiome composition might be useful to counteract the progression of atrial fibrillation.

Study on the relationship between telomere length changes and recurrence of atrial fibrillation after radiofrequency catheter ablation.

INTRODUCTION: Advanced age is the foremost risk factor for atrial fibrillation (AF). Telomere length is a surrogate for biological aging, but the association between shortened leukocyte telomere length (LTL) and recurrence of AF (RAF) after ablation remains inconclusive. METHODS: In this prospective analysis, 282 patients underwent an initial catheter ablation for paroxysmal or persistent AF. The association between RAF and LTL was analyzed by univariate and multivariate Cox regression, as well as time-dependent receiver operating characteristic (ROC) analysis and Kaplan-Meier analysis. RESULTS: After a mean follow-up of 14.20 ± 5.04 months, RAF was documented in 78 of the 277 patients who completed the study (28.16%). In Cox proportional hazards models, LTL, age, diagnosis to ablation time (DTAT), N-terminal pronatriuretic peptide, and CHA2DS2-VASc score were significantly associated with RAF. After multivariable adjustment, LTL and DTAT were predicted as independent risk factors for RAF with hazard ratio (HR) of 3.17 (95% confidence interval [CI]: 1.23-8.15, P = 0.017) and 1.43 (95% CI: 1.10-1.86, P = 0.007), respectively. In addition, ROC analysis indicated the potential diagnostic value of LTL with an area under the curve of 0.64 (P < 0.001; sensitivity = 60.3%, specificity = 57.8%), and an optimum cut-off value of 1.040. LTL less than or equal to 1.040 was defined as shortened LTL, while LTL greater than 1.040 nonshortened LTL. Kaplan-Meier analysis showed RAF rate curve was separated significantly between two groups (21.2% vs 35.9%, log-rank test result P = 0.007). Patients with shortened LTL might have a higher risk for RAF with HR = 1.84 (P = 0.008). CONCLUSIONS: Shortened LTL is an independent risk factor for AF recurrence. Shortened LTL could be a potential biomarker in predicting RAF after ablation.

Dysbiotic gut microbes may contribute to hypertension by limiting vitamin D production.

BACKGROUND: Accumulating studies have suggested that gut microbiota (GM) dysbiosis and vitamin D3 deficiency each play an important role during the progression of hypertension (HTN). However, few studies have characterized the underlying interaction between GM shift and vitamin D3 deficiency in HTN patients. HYPOTHESIS: This study aimed to evaluate the possible crosstalk between GM dysbiosis and vitamin D deficiency in the pathogenesis of HTN. METHODS: In a cohort of 34 HTN patients and 15 healthy controls, we analyzed the fecal microbiota products, GM composition, and the interaction between GM and vitamin D3. RESULTS: Vitamin D3 was significantly decreased in feces of HTN patients (P = .006, vs controls) and was correlated with altered GM, including decreased Shannon index (R2 = 0.1296, P = .0111) and Pielou evenness (R2 = 0.1509, P = .0058). Moreover, vitamin D3 positively correlated with HTN-reduced bacterial genera, including Subdoligranulum (R2 = 0.181, P = .0023), Ruminiclostridium (R2 = 0.1217, P = .014), Intestinimonas (R2 = 0.2036, P = .0011), Pseudoflavonifractor (R2 = 0.1014, P = .0257), Paenibacillus (R2 = 0.089, P = .0373), and Marvinbryantia (R2 = 0.08173, P = .0464). Partial least squares structural equation modeling showed that 27.7% of the total effect of gut microbiome on HTN was mediated by limiting vitamin D production. Finally, receiver operating characteristic curve analysis revealed the predictive capacity of differential gut microbiome signatures and decreased vitamin D3 to distinguish HTN patients (AUC = 0.749, P = .006). CONCLUSIONS: Our findings suggest that the GM dysbiosis contributing to the development of HTN might be partially mediated by vitamin D3 deficiency. Future studies involving the underlying mechanism and intervention strategies targeting microbiome composition and vitamin D3 to counteract the progression of HTN are warranted.

Mexiletine rescues a mixed biophysical phenotype of the cardiac sodium channel arising from the SCN5A mutation, N406K, found in LQT3 patients.

INTRODUCTION: Individual mutations in the SCN5A-encoding cardiac sodium channel α-subunit usually cause a single cardiac arrhythmia disorder, some cause mixed biophysical or clinical phenotypes. Here we report an infant, female patient harboring a N406K mutation in SCN5A with a marked and mixed biophysical phenotype and assess pathogenic mechanisms. METHODS AND RESULTS: A patient suffered from recurrent seizures during sleep and torsades de pointes with a QTc of 530 ms. Mutational analysis identified a N406K mutation in SCN5A. The mutation was engineered by site-directed mutagenesis and heterologously expressed in HEK293 cells. After 48 hours incubation with and without mexiletine, macroscopic voltage-gated sodium current (INa) was measured with standard whole-cell patch clamp techniques. SCN5A-N406K elicited both a significantly decreased peak INa and a significantly increased late INa compared to wide-type (WT) channels. Furthermore, mexiletine both restored the decreased peak INa of the mutant channel and inhibited the increased late INa of the mutant channel. CONCLUSION: SCN5A-N406K channel displays both "gain-of-function" in late INa and "loss-of-function" in peak INa density contributing to a mixed biophysical phenotype. Moreover, our finding may provide the first example that mexiletine exerts a dual rescue of both "gain-of-function" and "loss-of-function" of the mutant sodium channel.

Arrhythmogenic Biophysical Phenotype for SCN5A Mutation S1787N Depends upon Splice Variant Background and Intracellular Acidosis.

BACKGROUND: SCN5A is a susceptibility gene for type 3 long QT syndrome, Brugada syndrome, and sudden infant death syndrome. INa dysfunction from mutated SCN5A can depend upon the splice variant background in which it is expressed and also upon environmental factors such as acidosis. S1787N was reported previously as a LQT3-associated mutation and has also been observed in 1 of 295 healthy white controls. Here, we determined the in vitro biophysical phenotype of SCN5A-S1787N in an effort to further assess its possible pathogenicity. METHODS AND RESULTS: We engineered S1787N in the two most common alternatively spliced SCN5A isoforms, the major isoform lacking a glutamine at position 1077 (Q1077del) and the minor isoform containing Q1077, and expressed these two engineered constructs in HEK293 cells for electrophysiological study. Macroscopic voltage-gated INa was measured 24 hours after transfection with standard whole-cell patch clamp techniques. We applied intracellular solutions with pH7.4 or pH6.7. S1787N in the Q1077 background had WT-like INa including peak INa density, activation and inactivation parameters, and late INa amplitude in both pH 7.4 and pH 6.7. However, with S1787N in the Q1077del background, the percentages of INa late/peak were increased by 2.1 fold in pH 7.4 and by 2.9 fold in pH 6.7 when compared to WT. CONCLUSION: The LQT3-like biophysical phenotype for S1787N depends on both the SCN5A splice variant and on the intracellular pH. These findings provide further evidence that the splice variant and environmental factors affect the molecular phenotype of cardiac SCN5A-encoded sodium channel (Nav1.5), has implications for the clinical phenotype, and may provide insight into acidosis-induced arrhythmia mechanisms.

Gene expression profile of increased heart rate in shensongyangxin-treated bradycardia rabbits.

Aims. The present study tries to investigate the gene expression profile of bradycardia rabbits' hearts after SSYX (SSYX, a traditional Chinese medicine) treatment. Methods. Eighteen adult rabbits were randomly assigned in three groups: sham, model, and SSYX treatment groups. Heart rate was recorded in rabbits and total RNA was isolated from hearts. Gene expression profiling was conducted and quantitative real-time reverse transcription-polymerase chain reaction (RT-PCR) was performed to confirm the gene expression results. Patch clamp using human induced pluripotent stem cell-derived cardiomyocytes was applied to record the calcium current in the presence of SSYX. Results. The mean RR interval reduced after six weeks due to the injury of the sinoatrial node in the model group. This effect was partially reversed by 4-week SSYX treatment. cDNA microarray demonstrated that genes related with pacemaker current, calcium ion homeostasis, and signaling were altered by SSYX treatment. Results from patch clamp demonstrated that SSYX reduced the calcium current which is consistent with gene expression results. Conclusion. The present study shows mRNA remodeling of bradycardia and demonstrates that SSYX is effective in treating bradycardia by reversing altered gene expression in bradycardia models. Reduced calcium current by SSYX also confirmed the gene expression results.

Comparison of read-through effects of aminoglycosides and PTC124 on rescuing nonsense mutations of HERG gene associated with long QT syndrome.

Aminoglycosides promote the readthrough of premature stop codons introduced by nonsense mutations to produce full-length proteins in genetic disease models. The read-through effects of different aminoglycosides and PTC124 on HERG gene have yet to be adequately elucidated. The wild-type (WT) or mutant genes were transiently transfected in HEK293 cells. The read-through effect was examined by adding drugs into culture medium for 24 h. Western blot analysis and patch clamping were performed to evaluate the expression and function of the genes. The mRNA levels were determined using qPCR. The results showed that G418 and PTC124 significantly increased the protein expression of R1014X mutant in a dose-dependent manner and produced a full-length protein. The maximal protein levels after G418, gentamicin or PTC124 treatment were 39.1±2.4, 18.6±0.3 or 10.3±1.0%, respectively, of the WT level. Tobramycin did not exhibit a read-through effect. The mRNA levels, however, did not differ between WT and mutant gene. The tail current densities of R1014X channels at 40 mV were 22.57±2.26 pA/pF for G418, 16.21±1.49 pA/pF for gentamicin and 9.62±0.73 pA/pF for PTC124. The leftward shift of the activation curve was corrected only by G418 and gentamicin. The read-through effects of W927X, R863X and E698X revealed that as the mutation site approached the N-terminal, the rescue efficiency was decreased. The above results suggest that aminoglycosides and PTC124 induced different effects on rescue nonsense mutations of the HERG gene. The mutation site was a significant factor in determining the pharmacological rescue efficiency.

Digenic inheritance novel mutations in SCN5a and SNTA1 increase late I(Na) contributing to LQT syndrome.

SCN5A and SNTA1 are reported susceptible genes for long QT syndrome (LQTS). This study was designed to elucidate a plausible pathogenic arrhythmia mechanism for the combined novel mutations R800L-SCN5A and A261V-SNTA1 on cardiac sodium channels. A Caucasian family with syncope and marginally prolonged QT interval was screened for LQTS-susceptibility genes and found to harbor the R800L mutation in SCN5A and A261V mutation in SNTA1, and those with both mutations had the strongest clinical phenotype. The mutations were engineered into the most common splice variant of human SCN5A and SNTA1 cDNA, respectively, and sodium current (INa) was characterized in human embryonic kidney 293 cells cotransfected with neuronal nitric oxide synthase (nNOS) and the cardiac isoform of the plasma membrane Ca-ATPase (PMCA4b). Peak INa densities were unchanged for wild-type (WT) and for mutant channels containing R800L-SCN5A, A261V-SNTA1, or R800L-SCN5A plus A261V-SNTA1. However, late INa for either single mutant was moderately increased two- to threefold compared with WT. The combined mutations of R800L-SCN5A plus A261V-SNTA1 significantly enhanced the INa late/peak ratio by 5.6-fold compared with WT. The time constants of current decay of combined mutant channel were markedly increased. The gain-of-function effect could be blocked by the N(G)-monomethyl-l-arginine, a nNOS inhibitor. We conclude that novel mutations in SCN5A and SNTA1 jointly exert a nNOS-dependent gain-of-function on SCN5A channels, which may consequently prolong the action potential duration and lead to LQTS phenotype.