Shu-Lin Wu

CircRNA_000203 enhances the expression of fibrosis-associated genes by derepressing targets of miR-26b-5p, Col1a2 and CTGF, in cardiac fibroblasts

Circular RNAs (circRNAs) participate in regulating gene expression in diverse biological and pathological processes. The present study aimed to investigate the mechanism underlying the modulation of circRNA_000203 on expressions of fibrosis-associated genes in cardiac fibroblasts. CircRNA_000203 was shown upregulated in the diabetic mouse myocardium and in Ang-II-induced mouse cardiac fibroblasts. Enforced-expression of circRNA_000203 could increase expressions of Col1a2, Col3a1 and α-SMA in mouse cardiac fibroblasts. RNA pull-down and RT-qPCR assay indicated that circRNA_000203 could specifically sponge miR-26b-5p. Dual luciferase reporter assay revealed that miR-26b-5p interacted with 3′UTRs of Col1a2 and CTGF, and circ_000203 could block the interactions of miR-26b-5p and 3′UTRs of Col1a2 and CTGF. Transfection of miR-26b-5p could post-transcriptionaly inhibit expressions of Col1a2 and CTGF, accompanied with the suppressions of Col3a1 and α-SMA in cardiac fibroblasts. Additionally, over-expression of circRNA_000203 could eliminate the anti-fibrosis effect of miR-26b-5p in cardiac fibroblasts. Together, our results reveal that suppressing the function of miR-26b-5p contributes to the pro-fibrosis effect of circRNA_000203 in cardiac fibroblasts.

Involvement of Src in L-type Ca2+ channel depression induced by macrophage migration inhibitory factor in atrial myocytes.

Macrophage migration inhibitory factor (MIF) is a pleiotropic cytokine that controls inflammatory processes, and inflammation is known to play an important role in the pathogenesis of atrial fibrillation (AF). The present study sought to investigate whether MIF expression is responsible for the changes in L-type Ca2+ currents (I(Ca,L)) seen in AF. Whole-cell voltage-clamp recordings and biochemical assays were used to study the regulation and expression of I(Ca,L) in human atrial myocytes and in HL-1 cells. Basal I(Ca,L) was reduced in AF compared to sinus rhythm (SR) controls, mRNA and protein levels of the pore-forming alpha1C subunit of L-type Ca2+ channel (LCC alpha1C) were also decreased, while MIF expression levels were increased in AF. Levels of Src and activated Src (p-Src Y416) were higher in AF than in SR. Treatment of atrial myocytes from a patient with SR with human recombinant MIF (rMIF) (40 nM, 1 h) was found to depress I(Ca,L) amplitudes, while mouse rMIF (20 or 40 nM, 24 h) suppressed peak I(Ca,L) in HL-1 cells by approximately 69% and approximately 83% in a concentration-dependent manner. Mouse rMIF impaired the time-dependent recovery from inactivation of I(Ca,L) and down-regulated LCC alpha1C subunit levels. The depression of I(Ca,L) and decrease of LCC protein levels induced by rMIF were prevented by the Src inhibitors genistein and PP1. These results implicate MIF in the electrical remodeling that accompanies AF, probably by decreasing I(Ca,L) amplitudes through impairment of channel function, down-regulation of LCC alpha1C subunit levels, and the activation of c-Src kinases in atrial myocytes.

Silencing of desmoplakin decreases connexin43/Nav1.5 expression and sodium current in HL‑1 cardiomyocytes

Desmosomes and gap junctions are situated in the intercalated disks of cardiac muscle and maintain the integrity of mechanical coupling and electrical impulse conduction between cells. The desmosomal plakin protein, desmoplakin (DSP), also plays a crucial role in the stability of these interconnected components as well as gap junction connexin proteins. In addition to cell‑to‑cell junctions, other molecules, including voltage‑gated sodium channels (Nav1.5) are present in the intercalated disk and support the contraction of cardiac muscle. Mutations in genes encoding desmosome proteins may result in fatal arrhythmias, including arrhythmogenic right ventricular cardiomyopathy (ARVC). Therefore, the aim of the present study was to determine whether the presence of DSP is necessary for the normal function and localization of gap junction protein connexin43 (Cx43) and Nav1.5. To examine this hypothesis, RNA interference was utilized to knock down the expression of DSP in HL‑1 cells and the content, distribution and function of Cx43 and Nav1.5 was assessed. Western blotting and flow cytometry experiments revealed that Cx43 and Nav1.5 expression decreased following DSP silencing. In addition, immunofluorescence studies demonstrated that a loss of DSP expression led to an abnormal distribution of Cx43 and Nav1.5, while scrape‑loading dye/transfer revealed a decrease in dye transfer in DSP siRNA‑treated cells. The sodium current was also recorded by the whole‑cell patch clamp technique. The results indicated that DSP suppression decreased sodium current and slowed conduction velocity in cultured cells. The present study indicates that impaired mechanical coupling largely affects electrical synchrony, further uncovering the pathogenesis of ARVC.

ELECTROPHYSIOLOGICAL EFFECTS OF KETAMINE ON HUMAN ATRIAL MYOCYTES AT THERAPEUTICALLY RELEVANT CONCENTRATIONS

1 Ketamine is widely used for the induction of anaesthesia in high‐risk patients with cardiovascular instability or severe hypovolaemia. However, the ionic mechanisms involved in the effects of ketamine at therapeutically relevant concentrations in human cardiac myocytes are unclear. The present study was designed to investigate the effects of ketamine on L‐type Ca2+ (ICa), transient outward K+ (Ito), ultra‐rapid delayed rectifier K+ (IKur) and inward rectifier potassium (IK1) currents, as well as on action potentials, in human isolated atrial myocytes. 2 Atrial myocytes were isolated enzymatically from specimens of human atrial appendage obtained from patients undergoing coronary artery bypass grafting. The action potential and membrane currents were recorded in both current‐ and voltage‐clamp modes using the patch‐clamp technique. 3 Ketamine inhibited ICa with an IC50 of 1.8 µmol/L. In addition, 10 µmol/L ketamine decreased the ICa peak current at +10 mV from 5.1 ± 0.3 to 2.1 ± 0.4 pA/pF (P < 0.01), but did not change the threshold potential, peak current potential and reverse potential. 4 Ketamine had no effect on Ito, IKur or IK1, but it reversibly shortened the duration of the action potential in human atrial myocytes. 5 In conclusion, ketamine, at a clinically relevant concentration, shortens the action potential duration of the human atrial myocytes, probably by inhibiting ICa.

Mechanism of macrophage migration inhibitory factor-induced decrease of T-type Ca2+ channel current in atrium-derived cells

•  What is the central question of this study? Recent evidence indicates that T‐type calcium channels may play an important role in the pathogenesis of atrial fibrillation. We previously reported that macrophage migration inhibitory factor (MIF), a pro‐inflammatory cytokine, reduced L‐type calcium channel expression in atrial fibrillation. The role of MIF in the regulation of atrial T‐type calcium channels has not been previously investigated. •  What is the main finding and its importance? In the present study, we report that MIF decreases the T‐type calcium current in atrium‐derived myocytes through impairment of channel function and activation of c‐Src kinases, representing a potential pathogenic mechanism in atrial fibrillation.

Pharmacological effects of carvedilol on T-type calcium current in murine HL-1 cells

Carvedilol is widely used in the treatment of cardiovascular diseases including atrial fibrillation. T-type Ca(2+) channels have been recognized recently in the mechanisms underlying atrial arrhythmias. However, it is unclear whether carvedilol may affect the T-type Ca(2+) channel. The present study evaluated the pharmacological effects of carvedilol on T-type calcium current (I(Ca,T)) in the murine HL-1 cell line. I(Ca)(,T) was recorded by the whole-cell patch-clamp technique. Calcium transient was monitored by the fluorescent dye Fluo-4/AM and confocal laser scanning microscopy. Carvedilol reversibly inhibited I(Ca)(,T) in a concentration-dependent manner, with an IC50 of 2.1 microM. 3 microM carvedilol was found to decrease the peak I(Ca)(,T) amplitude at -20 mV from 20.1+/-1.8pA/pF to 10.9+/-2.1pA/pF. Carvedilol significantly shifted the steady-state inactivation curve of I(Ca)(,T) towards more negative potential by 12.8 mV, while the activation curve was not significantly altered. Carvedilol delayed recovery from inactivation of I(Ca)(,T), time constant (tau) was 112.4+/-3.5 ms in control and 270.1+/-4.7 ms in carvedilol. Carvedilol-induced inhibition rate in I(Ca)(,T) was enhanced with the increase in stimuli frequency, the inhibitory rate was 23.2+/-4.1% at 0.2 Hz and 47.2+/-0.6% at 2 Hz. Carvedilol still produced the significant decrease in the amplitude of I(Ca)(,T) in the presence of H-89, PKA inhibitor. Carvedilol significantly inhibited the amplitude of the calcium transient in a concentration-dependent manner. These findings indicate that carvedilol inhibits I(Ca)(,T) in atrial cells by mechanisms involving preferential interaction with the inactivated state of T-type Ca(2+) channel.

Myocyte-specific enhancer factor 2C: a novel target gene of miR-214-3p in suppressing angiotensin II-induced cardiomyocyte hypertrophy.

The role of microRNA-214-3p (miR-214-3p) in cardiac hypertrophy was not well illustrated. The present study aimed to investigate the expression and potential target of miR-214-3p in angiotensin II (Ang-II)-induced mouse cardiac hypertrophy. In mice with either Ang-II infusion or transverse aortic constriction (TAC) model, miR-214-3p expression was markedly decreased in the hypertrophic myocardium. Down-regulation of miR-214-3p was observed in the myocardium of patients with cardiac hypertrophy. Expression of miR-214-3p was upregulated in Ang-II-induced hypertrophic neonatal mouse ventricular cardiomyocytes. Cardiac hypertrophy was attenuated in Ang-II-infused mice by tail vein injection of miR-214-3p. Moreover, miR-214-3p inhibited the expression of atrial natriuretic peptide (ANP) and β-myosin heavy chain (MHC) in Ang-II-treated mouse cardiomyocytes in vitro. Myocyte-specific enhancer factor 2C (MEF2C), which was increased in Ang-II-induced hypertrophic mouse myocardium and cardiomyocytes, was identified as a target gene of miR-214-3p. Functionally, miR-214-3p mimic, consistent with MEF2C siRNA, inhibited cell size increase and protein expression of ANP and β-MHC in Ang-II-treated mouse cardiomyocytes. The NF-κB signal pathway was verified to mediate Ang-II-induced miR-214-3p expression in cardiomyocytes. Taken together, our results revealed that MEF2C is a novel target of miR-214-3p, and attenuation of miR-214-3p expression may contribute to MEF2Cexpressionin cardiac hypertrophy.

Role of tumour necrosis factor-a in the regulation of T-type calcium channel current in HL-1 cells.

Increasing evidence indicates that inflammation contributes to the initiation and perpetuation of atrial fibrillation (AF). Although tumour necrosis factor (TNF)‐α levels are increased in patients with AF, the role of TNF‐α in the pathogenesis of AF remains unclear. Besides L‐type Ca2+ currents (ICa,L), T‐type Ca2+ currents (ICa,T) also plays an important role in the pathogenesis of AF. This study was designed to use the whole‐cell voltage‐clamp technique and biochemical assays to explore if TNF‐α is involved in the pathogenesis of AF through regulating ICa,T in atrial myocytes. It was found that compared with sinus rhythm (SR) controls, T‐type calcium channel (TCC) subunit mRNA levels were decreased, while TNF‐α expression levels were increased, in human atrial tissue from patients with AF. In murine atrial myocyte HL‐1 cells, after culturing for 24 h, 12.5, 25 and 50 ng/mL TNF‐α significantly reduced the protein expression levels of the TCC α1G subunit in a concentration‐dependent manner. The peak current was reduced by the application of 12.5 or 25 ng/mL TNF‐α in a concentration‐dependent manner (from −15.08 ± 1.11 pA/pF in controls to −11.89 ± 0.83 pA/pF and −8.54 ± 1.55 pA/pF in 12.5 or 25 ng/mL TNF‐α group respectively). TNF‐α application also inhibited voltage‐dependent inactivation of ICa,T, shifted the inactivation curve to the left. These results suggest that TNF‐α is involved in the pathogenesis of AF, probably via decreasing ICa,T current density in atrium‐derived myocytes through impaired channel function and down‐regulation of channel protein expression. This pathway thus represents a potential pathogenic mechanism in AF.

A long-term, prospective, cohort study on the performance of right ventricular pacing leads: comparison of active-fixation with passive-fixation leads.

Active-fixation pacing leads allow the use of selective pacing sites. We evaluated their long-term performance versus passive-fixation leads in 199 newly implanted patients (n = 100 active and n = 99 passive). Postoperative pacing thresholds in the active group were higher than in the passive group (0.85 ± 0.31 V vs. 0.53 ± 0.21 V at baseline, P < 0.001). The active thresholds fell to 0.72 ± 0.23 V at 5 years with a significant drop at one month (0.68 ± 0.53 V, P = 0.003). The passive thresholds slightly increased to 0.72 ± 0.31 V at five years. Differences between groups were significant until three years (all P < 0.05). Active impedances were generally lower than passive impedances (600.44 ± 94.31Ω vs. 683.14 ± 110.98Ω at baseline), and both showed significant reductions at one month to 537.96 ± 147.43Ω in the active group, and after three months to 643.85 ± 82.40Ω in the passive group (both P < 0.01 vs. baseline). Impedance differences between groups were significant until four years (all P < 0.05). Adverse events included thresholds over 1 V, 5 of 6 active and 2 of 5 passive leads returned to below 1 V. One active left ventricular lead dislodged. One passive left subclavian lead insulation fracture occurred. Thus Active fixation pacing leads are stable in a five-year long-term follow up. There was no difference between active and passive leads in terms of electrical performance.

The enhancement of TXA2 receptors-mediated contractile response in intrarenal artery dysfunction in type 2 diabetic mice

&NA; Thromboxane A2 (TXA2) has been implicated in the pathogenesis of diabetic vascular complications, although the underlying mechanism remains unclear. The present study investigated the alterations in TXA2 receptor signal transduction in type 2 diabetic renal arteries. The contraction of renal arterial rings in control (db/m+) mice and type 2 diabetic (db/db) mice was measured by a Multi Myograph System. Intracellular calcium concentration ([Ca2+]i) in vascular smooth muscle cells was measured by Fluo‐4/AM dye and confocal laser scanning microscopy. Quantitative real‐time PCR and Western blot analysis were used to determine gene and protein expression levels, respectively. A stable TXA2 mimic U46619 caused markedly stronger dose‐dependent contractions in the renal arteries of db/db mice than in those of db/m+ mice. This response was completely blocked by a TXA2 receptor antagonist GR32191 and significantly inhibited by U73122. U46619‐induced vasoconstriction was increased in the presence of nifedipine in db/db mice compared with that in db/m+ mice, whereas the response to U46619 did not differ between the two groups in the presence of SKF96365. Sarcoplasmic reticulum Ca2+ release‐mediated and CaCl2‐induced contractions did not differ between the two groups. In db/db mice, store‐operated Ca2+(SOC) entry‐mediated contraction in the renal arteries and SOC entry‐mediated Ca2+ influx in smooth muscle cells were significantly increased. And the gene and protein expressions of TXA2 receptors, Orai1 and Stim1 were upregulated in the diabetic renal arteries. Therefore the enhancement of U46619‐induced contraction was mediated by the upregulation of TXA2 receptors and downstream signaling in the diabetic renal arteries.