Lei Ruan

FOXP3 demethylation as a means of identifying quantitative defects in regulatory T cells in acute coronary syndrome

OBJECTIVE The contribution of regulatory T cells (Tregs) to the pathogenesis of acute coronary syndrome (ACS) remains poorly understood. One core obstacle is the lack of Treg-specific markers. A highly conserved CpG enriched element in forkhead box P3 intron 1 (FOXP3 i l) is unmethylated only in Tregs, and measuring the unmethylation of FOXP3 i l can be used to identify the role of Tregs in clinical diseases. This study investigated whether analyzing the demethylation status of FOXP3 i 1 is a more reliable means than using Treg-specific surface markers in ACS. METHODS AND RESULTS We evaluated circulating Tregs percentages on different levels including cell frequencies (CD4(+)CD25(hi)FOXP3(+)Tregs and CD4(+)CD25(hi)CD45(+)naïve Tregs) or FOXP3 mRNA, FOXP3 i 1 demethylation status and related cytokine secretion in 89 patients with ACS and 35 controls. FOXP3 i 1 demethylation assay showed that the amount of Tregs in ACS patients was significantly reduced than that in controls (p = 0.0005). However, flow cytometry analysis did not identify any reduction of CD4(+)CD25(hi)FOXP3(+)Tregs in ACS patients. Notably, younger patients had higher percentage of CD4(+)CD25(hi)FOXP3(+)Tregs but decreased percentage of CD4(+)CD25(hi)CD45(+)naïve Tregs than either controls or older patients. Furthermore, a DNA hypomethylation agent increased the amount of CD4(+)CD25(hi)FOXP3(+)Tregs and Tregs related cytokine IL-10 and suppressed the production of pro-inflammatory cytokine interferon-γ by inducing FOXP3 i 1 demethylation in vitro. CONCLUSIONS A quantitative defect of Tregs, suggestive of decreased peripheral tolerance, could be a potential hallmark of ACS disease. Targeting FOXP3 i l demethylation might elevate the inhibitory activity of Tregs in ACS.

Kv1.3 channels as a potential target for immunomodulation of CD4+CD28null T cells in patients with acute coronary syndrome

Modulation of CD4(+)CD28null T cells through K+ channels could provide potential novel targets for the treatment acute coronary syndrome (ACS). However, the surface phenotype and K+ channel expression of CD4(+)CD28null T cells in patients with ACS is unclear. The aim of this study was to investigate the surface phenotype and K+ channel expression of CD4(+)CD28null T cells in patients with ACS. We found that more than 80% of CD4(+)CD28null T cells in patients with ACS showed a CD45RA(-)CD45RO(+)CCR7- surface phenotype. CD4(+)CD28(null) T expressed small numbers of the voltage-gated Kv1.3 and intermediate-conductance Ca2+-activated K+ channel KCa3.1 when quiescent, but increased Kv1.3 expression 4-fold with little change in KCa3.1 levels upon activation. Consistent with their channel phenotypes, the production of interferon-γ and perforin in CD4(+)CD28null T cells was suppressed by the specific Kv1.3 blocker 5-(4-phenoxybutoxy)psoralen PAP-1. Therefore, selective targeting of Kv1.3 in CD4(+)CD28null T cells may hold potential therapeutic promise for ACS.

Pharmacological enhancement of cardiac gap junction coupling prevents arrhythmias in canine LQT2 model.

Gap junctions contribute to the transmural heterogeneity of repolarization in the normal heart and under conditions of prolonged QT interval in the diseased heart. This study examined whether enhancing of gap junction coupling can reduce transmural dispersion of repolarization (TDR) and prevent torsade de pointes (TdP) in a canine LQT2 model. Canine left ventricular wedge preparations were perfused with delayed rectifier potassium current (IKr) blocker d-sotalol to mimic LQT2 and the antiarrhythmic peptide 10 (AAP10) was used as a gap junction coupling enhancer. As compared with the control group, the LQT2 group had significantly augmented TDR and higher incidence of TdP associated with increased nonphosphorylated connexin 43 (Cx43). AAP10 prevented augmentation of TDR and induction of TdP while rescuing Cx43 phosphorylation. There was no significant change in the quantity and spatial distribution of Cx43. These data indicate that gap junction enhancer AAP10 can prevent augmentation of TDR and suppress TdP by preventing dephosphorylation of Cx43 in a LQT2 model.

Ability of ambulatory ECG-based T-wave alternans to modify risk assessment of cardiac events: a systematic review

BackgroundExercise-based spectral T-wave alternans (TWA) has been proposed as a noninvasive tool-identifying patients at risk of sudden cardiac death (SCD) and cardiac mortality. Prior studies have indicated that ambulatory electrocardiogram (AECG)-based TWA is an important alternative platform to exercise for risk stratification of cardiac events. This study sought to review data regarding 24-hour AECG-based TWA and to discuss its potential role in risk stratification of fatal cardiac events across a series of patient risk profiles.MethodsProspective clinical studies of the predictive value of AECG-based TWA obtained with daily activity published between January 1990 and November 2014 were retrieved. Major endpoints included composite endpoint of SCD, cardiac mortality, and severe arrhythmic events.ResultsData were accumulated from 5 studies involving a total of 1,588 patients, including 317 positive and 1,271 negative TWA results. Compared with the negative group, positive group showed increased rates of SCD (hazard ratio [HR]: 7.49, 95% confidence interval [CI]: 2.65 to 21.15), cardiac mortality (HR: 4.75, 95% CI: 0.42 to 53.55), and composite endpoint (SCD, cardiac mortality, and severe arrhythmic events, HR: 5.94, 95% CI: 1.80 to 19.63). For the 4 studies evaluating TWA measured using the modified moving average method, the HR associated with a positive versus negative TWA result was 9.51 (95% CI: 4.99 to 18.11) for the composite endpoint.ConclusionsThe positive group of AECG-based TWA has a nearly six-fold risk of severe outcomes compared with the negative group. Therefore, AECG-based TWA provides an accurate means of predicting fatal cardiac events.

Effect of the Kv1.3 voltage-gated potassium channel blocker PAP-1 on the initiation and progress of atherosclerosis in a rat model

AbstractAcute coronary syndrome is a serious medical emergency. It occurs when an atherosclerotic plaque ruptures, leading to thrombus formation within a coronary artery. Previous studies have shown that T cells are involved in the initiation and progression of acute coronary syndrome. CD4+CD28null T lymphocytes increase in atherosclerotic plaque, and voltage-gated potassium channel Kv1.3 blockers can suppress the function of these cells in vitro by preventing exocytosis of their cytoplasmic granules. The purpose of this study was to investigate the effect of PAP-1, a small molecule voltage-gated potassium channel Kv1.3 blocker, on the development of atherosclerosis (AS) in a rat model and the potential mechanism for this effect. Plasma lipids, interferonγ, CRP, CD4+CD28null T cells, and perforin were increased and unstable atherosclerotic plaques developed in the rat model of AS. Blockade of the Kv1.3 potassium channel via PAP-1 administration decreased perforin levels and prevented plaque formation but had no effect on the other changes seen in this AS model. These findings suggest that the small molecule, voltage-gated potassium channel Kv1.3 blocker PAP-1 can suppress the development of AS in a rat model, most likely by inhibiting the exocytosis of cytoplasmic granules from CD4+CD28null T cells.

Increasing gap junction coupling suppresses ibutilide-induced torsades de pointes.

Drug-induced torsades de pointes (TdP) is common with class III antiarrhythmic drugs. Increased transmural dispersion of repolarization (TDR) contributes significantly to the development of TdP. Gap junctions play an important role in maintaining TDR in long QT syndrome. The present study examined the effect of a gap junction enhancer, antiarrhythmic peptide 10 (AAP10), on ibutilide-induced TdP. Coronary-perfused rabbit ventricular wedge preparations were used to evaluate the effect of AAP10 on ibutilide-induced arrhythmia. Transmural electrocardiograms and action potentials were recorded simultaneously. Early afterdepolarizations (EADs), R-on-T extrasystole, TdP and changes in Tpeak-end (Tp-e) and the Tp-e/QT ratio were observed. Changes in the levels of non-phosphorylated connexin 43 (Cx43) were measured by immunoblotting. Compared with those in the control group, the QT interval, Tp-e/QT and incidence rates of EAD and TdP increased with augmented dephosphorylation in the ventricular wedge preparations perfused with ibutilide under conditions of hypokalemia and hypomagnesemia. In the presence of AAP10, the incidence rates of EAD and TdP were reduced and the Tp-e/QT ratio decreased, with a parallel reduction in the level of non-phosphorylated Cx43. The results indicate that AAP10 suppressed ibutilide-induced TdP under conditions of hypokalemia and hypomagnesemia by decreasing TDR. AAP10 reduced TDR, possibly by preventing the dephosphorylation of Cx43 and thereby increasing myocardial cell gap junction coupling.

Antiarrhythmic Peptide AAP10 Prevents Arrhythmias Induced by Protein Kinase C Activation in Rabbit Left Ventricular Wedges

As the mechanisms underlying PKC activation induced arrhythmias are not yet fully verified, we investigated the role of gap junctions in arrhythmias induced by PKC activation.Arterially-perfused rabbit left ventricular preparations were randomly assigned to perfusion with phorbol ester (PMA) or in combination with AAP10. Transmural ECG as well as action potentials from both endocardium and epicardium were simultaneously recorded throughout all experiments. Changes in connexin43 (Cx43) and nonphosphorylated Cx43 on S368 were measured by Western blot analysis.In the PMA group, the QT interval was shortened, the interval from the peak to the end of the electrocardiographic T wave (Tp-e) and induced nonsustained ventricular tachycardia (VT) were increased, and the expressions of Cx43 and nonphosphorylated Cx43 on S368 were decreased compared with the control group. Compared with the PMA group, without significant changes in the QT interval and the expression of nonphosphorylated connexin43 on S368, Tp-e and induced VT decreased and the expression of Cx43 increased in the AAP10 group.AAP10 can prevent PMA-induced rabbit ventricular arrhythmias by attenuating the increase of Tp-e and the decrease of expression of Cx43. These data suggest that increasing gap junction coupling prevents arrhythmias induced by protein kinase C activation.

Functional prediction of miR-3144-5p in human cardiac myocytes based on transcriptome sequencing and bioinformatics

Background: RAN guanine nucleotide release factor (RANGRF) encoding protein MOG1 plays an important role in cardiac arrhythmia, so we intended to investigate the regulatory miRNA of RANGRF and explore its potential regulatory mechanism in arrhythmogenesis. Methods: Based on bioinformatic analysis, miR-3144-5p was predicted to be a regulatory miRNA of RANGRF, which were then validated through a dual-luciferase reporter plasmid assay. Subsequently, the expression level of miR-3144-5p in human cardiac myocytes (HCMs) was detected, followed by cell transfection with miR-3144-5p mimics. Transcriptome sequencing was then performed in HCMs with or without transfection. The sequencing results were subjected to bioinformatic analyses, including differentially expressed gene (DEG) analysis, functional enrichment analysis, protein–protein interaction (PPI) network analysis, miRNA-target gene analysis, and miRNA-transcription factor (TF)-target gene coregulatory network analysis. Results: There really existed a regulatory relation between miR-3144-5p and RANGRF. The expression level of miR-3144-5p was low in HCMs. After cell transfection, miR-3144-5p expression level significantly increased in HCMs. Bioinformatic analyses of the transcriptome sequencing results identified 300 upregulated and 271 downregulated DEGs between miR-3144-5p mimic and control group. The upregulated genes ISL1 and neuregulin 1 (NRG1) were significantly enriched in cardiac muscle cell myoblast differentiation (GO:0060379). CCL21 was one of the hub genes in the PPI network and also a target gene of miR-3144-5p. Moreover, the TF of v-Myc avian myelocytomatosis viral oncogene neuroblastoma-derived homolog (MYCN) was involved in the miR-3144-5p-TF-target gene coregulatory network and interacted with the target genes of miR-3144-5p. Conclusion: ISL1, NRG1, CCL21, and MYCN were differentially expressed in the miR-3144-5p mimic group, suggesting that miR-3144-5p overexpression plays a role in HCMs by regulating these genes and TF. This study may provide new insight into the mechanisms behind the progression of cardiac arrhythmia.

Fluconazole-induced long QT syndrome via impaired human ether-a-go-go-related gene (hERG) protein trafficking in rabbits

Aims hERG protein trafficking deficiency has long been known in drug-induced long QT syndrome (LQTS). However, validated evidence from in vivo data kept scanty. Our goal was to investigate the proarrhythmic action of fluconazole and its underlying mechanism in an animal model. Methods and results Twenty female Japanese long-eared white rabbits were randomly distributed into a control group and a fluconazole group for a chronic 2-week treatment. The control group was treated with 0.5% sodium carboxymethylcellulose (CMCNa), and the fluconazole group was treated with fluconazole. Electrocardiograms (ECGs) were recorded during the experimental period. Isolated arterially perfused left ventricular wedge preparations from the rabbits were made 2 weeks after treatment, and the arrhythmia events, the transmural ECG, and action potential from both the endocardium and epicardium were recorded. The changes in hERG protein expression were measured by western blot. The fluconazole group showed a longer QT interval 1 week after treatment (P < 0.05) and a higher arrhythmia occurrence 2 weeks after treatment (P < 0.05) than the control group. The fluconazole group also showed a longer transmural dispersion of repolarization and a higher occurrence of life-threatening torsades de pointes in arterially perfused left ventricular preparations. Furthermore, western blot analysis showed that the density of mature hERG protein was lower in the fluconazole group than that in the control group. Conclusion Fluconazole can prolong the QT interval and possess proarrhythmic activity due to its inhibition of hERG protein trafficking in our experimental model. These findings may impact the clinical potential of fluconazole in humans.

Speckle tracking echocardiography assessment of global and regional contraction dysfunction in the mice model of pressure overload.

SummarySpeckle tracking echocardiography (STE) has been applied to the evaluation of cardiac contraction dysfunction. However, there were few studies on alteration of global and regional STE parameters in the process of myocardial hypertrophy and heart failure. In this study, STE was applied to evaluate the global and regional cardiac function under heart failure and hypertrophy in the mice model of pressure overload. Adult mice were subjected to mild or severe aortic banding with a 25 Gauge (G) or 27 G needle. After surgery, STE and conventional echocardiography were used in the sham group (n=10), mild trans-aortic banding (TAB) group (n=14) and severe TAB group (n=10) for 8 weeks. The results showed that the mice subjected to severe TAB showed a significant change in fractional shortening (FS), left ventricular (LV) mass, and left ventricular end diastolic diameter (LVEDD) (P<0.05 for each). Meanwhile, there were no significant differences in FS and LVEDD between the sham group and mild TAB group during the experimental procedures (P>0.05 for both). STE analysis revealed that longitudinal strain (LS) was significantly decreased in the severe TAB group as compared with the sham and mild TAB groups (P<0.05 for both) from the postoperative week 1. LS in the mild TAB group was reduced as compared to the sham group (P<0.05). Radial strain (RS) and circumferential strain (CS) were significantly decreased in the severe TAB group as compared to the sham group and the mild TAB group (P<0.05 for both) from the postoperative week 1 (P<0.05 for both). Compared to the sham group, CS in the mild TAB group maintained unchanged during the test period, and RS was reduced only on the postoperative week 6 (P<0.05). Finally, regional contraction dysfunction was analyzed in both hypertrophic and failing myocardium in longitudinal and radial directions. It was found that LS was largest in the apex region and RS was smallest in the apex region in the healthy and hypertrophic myocardium. It was also found that compared to the sham group, only base longitudinal strain in the mild TAB group was decreased. Each of regional strain in the severe TAB group was uniformly depressed in radial and longitudinal directions. It is concluded that STE has provided a non-invasive and highly feasible way to explore the global and regional contraction dysfunction in hypertrophic and heart failure myocardium in the murine model of pressure overload.Speckle tracking echocardiography (STE) has been applied to the evaluation of cardiac contraction dysfunction. However, there were few studies on alteration of global and regional STE parameters in the process of myocardial hypertrophy and heart failure. In this study, STE was applied to evaluate the global and regional cardiac function under heart failure and hypertrophy in the mice model of pressure overload. Adult mice were subjected to mild or severe aortic banding with a 25 Gauge (G) or 27 G needle. After surgery, STE and conventional echocardiography were used in the sham group (n=10), mild trans-aortic banding (TAB) group (n=14) and severe TAB group (n=10) for 8 weeks. The results showed that the mice subjected to severe TAB showed a significant change in fractional shortening (FS), left ventricular (LV) mass, and left ventricular end diastolic diameter (LVEDD) (P<0.05 for each). Meanwhile, there were no significant differences in FS and LVEDD between the sham group and mild TAB group during the experimental procedures (P>0.05 for both). STE analysis revealed that longitudinal strain (LS) was significantly decreased in the severe TAB group as compared with the sham and mild TAB groups (P<0.05 for both) from the postoperative week 1. LS in the mild TAB group was reduced as compared to the sham group (P<0.05). Radial strain (RS) and circumferential strain (CS) were significantly decreased in the severe TAB group as compared to the sham group and the mild TAB group (P<0.05 for both) from the postoperative week 1 (P<0.05 for both). Compared to the sham group, CS in the mild TAB group maintained unchanged during the test period, and RS was reduced only on the postoperative week 6 (P<0.05). Finally, regional contraction dysfunction was analyzed in both hypertrophic and failing myocardium in longitudinal and radial directions. It was found that LS was largest in the apex region and RS was smallest in the apex region in the healthy and hypertrophic myocardium. It was also found that compared to the sham group, only base longitudinal strain in the mild TAB group was decreased. Each of regional strain in the severe TAB group was uniformly depressed in radial and longitudinal directions. It is concluded that STE has provided a non-invasive and highly feasible way to explore the global and regional contraction dysfunction in hypertrophic and heart failure myocardium in the murine model of pressure overload.