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The primary pacemaking activity of the heart is determined by a spontaneous action potential (AP) within sinoatrial node (SAN) cells. This unique AP generation relies on two mechanisms: membrane clocks and calcium clocks. Nonhomologous arrhythmias are caused by several functional and structural changes in the myocardium. MicroRNAs (miRNAs) are essential regulators of gene expression in cardiomyocytes. These miRNAs play a vital role in regulating the stability of cardiac conduction and in the remodeling process that leads to arrhythmias. Although it remains unclear how miRNAs regulate the expression and function of ion channels in the heart, these regulatory mechanisms may support the development of emerging therapies. This study discusses the spread and generation of AP in the SAN as well as the regulation of miRNAs and individual ion channels. Arrhythmogenicity studies on ion channels will provide a research basis for miRNA modulation as a new therapeutic target.

Ca 2+ -activated K + channels are critical to cellular Ca 2+ homeostasis and excitability; they couple intracellular Ca 2+ and membrane voltage change. Of these, the small, 4–14 pS, conductance SK channels include three, KCNN1-3 encoded, SK1/KCa2.1, SK2/KCa2.2 and SK3/KCa2.3, channel subtypes with characteristic, EC 50 ∼ 10 nM, 40 pM, 1 nM, apamin sensitivities. All SK channels, particularly SK2 channels, are expressed in atrial, ventricular and conducting system cardiomyocytes. Pharmacological and genetic modification results have suggested that SK channel block or knockout prolonged action potential durations (APDs) and effective refractory periods (ERPs) particularly in atrial, but also in ventricular, and sinoatrial, atrioventricular node and Purkinje myocytes, correspondingly affect arrhythmic tendency. Additionally, mitochondrial SK channels may decrease mitochondrial Ca 2+ overload and reactive oxygen species generation. SK channels show low voltage but marked Ca 2+ dependences (EC 50 ∼ 300–500 nM) reflecting their α-subunit calmodulin (CaM) binding domains, through which they may be activated by voltage-gated or ryanodine-receptor Ca 2+ channel activity. SK function also depends upon complex trafficking and expression processes and associations with other ion channels or subunits from different SK subtypes. Atrial and ventricular clinical arrhythmogenesis may follow both increased or decreased SK expression through decreased or increased APD correspondingly accelerating and stabilizing re-entrant rotors or increasing incidences of triggered activity. This article is part of the theme issue ‘The heartbeat: its molecular basis and physiological mechanisms’.

Pulmonary hypertension (PH) associated with congenital heart disease is a progressive hemodynamic disease that can lead to increased pulmonary vascular resistance, vascular remodeling, and even right heart failure and death. LF3 is a novel inhibitor of the reporter gene activity of β-catenin/TCF4 interaction in the Wnt/β-catenin signal pathway. However, whether this action of LF3 can prevent PH development remains unclear. In this study, we investigated the therapeutic effect of LF3 in rat primary pulmonary artery smooth muscle cells (PASMCs) of the PH model. We found that LF3 inhibited the decrease in pulmonary artery acceleration time and ejection time by ultra-high-resolution ultrasound imaging and blocked the increase of pulmonary artery systolic pressure by using the BL420 biological function experimental system and right ventricular hypertrophy index by the electronic scales. Simultaneously, it prevented the increase of α-smooth muscle actin and fibronectin and the decrease of elastin in pulmonary arteries of rats in the PH group, as revealed by an immunohistochemical analysis. Moreover, cell proliferation and migration assays showed that LF3 significantly reduced the proliferation and migration of PASMCs. Western blotting and quantitative real-time polymerase chain reaction analyses revealed that LF3 suppressed the expression of proliferating cell nuclear antigens and Bcl-2 and increased the expression of Bax but did not alter the expressions of β-catenin and TCF4. Taken together, LF3 can reduce the migration and proliferation of PASMCs and induce their apoptosis to prevent the development of PH. It would be worthwhile to explore the potential use of LF3 in the treatment of PH.

Background The relationship between the ventricular rate (VR) during atrial fibrillation (AF) and skin sympathetic nerve activity (SKNA) remains unclear. Objective The purpose of this study was to test the hypothesis that SKNA bursts accelerate VR during AF. Methods We simultaneously recorded electrocardiogram and SKNA in 8 patients (median age 66.0 years [interquartile range {IQR} 59.0–77.0 years]; 4 men [50%]) with 30 paroxysmal AF episodes (all >10-minute long) and 12 patients (73.0 years [IQR 60.5–80.0 years]; 6 men [50%]) with persistent AF. The average amplitude of SKNA (aSKNA [μV]) during AF was analyzed in 1-minute windows and binned, showing 2 Gaussian distributions. We used the mean + 3SD of the first Gaussian distribution as the threshold that separates burst from baseline (nonburst) SKNA. All 1-minute aSKNA values above the threshold were detected, and the area between aSKNA and baseline of every 1 minute was calculated and added as burst area. Results VR was higher during SKNA bursts than during the nonburst period (103 beats/min [IQR 83–113 beats/min] vs 88 beats/min [IQR 76–101 beats/min], respectively; P = .003). In the highest quartile of the burst area during persistent AF, the scatterplot of maximal aSKNA and VR during each SKNA burst shows higher aSKNA and VR. The overall estimate of the correlation between maximal VR and aSKNA during bursts show a positive correlation in the highest quartile of the burst area (0.64; 95% confidence interval 0.54–0.74; P Conclusion SKNA bursts are associated with VR acceleration. These SKNA bursts may be new therapeutic targets for rate control during AF.

Background Existing methods for in vitro differentiation of human pluripotent stem cells (hPSCs) into sinoatrial node-like cells (SANLCs) require complex and undefined medium constituents. This might hinder the elucidation of the molecular mechanisms involved in cardiac subtype specification and prevent translational application. In our study, we aimed to establish a chemically defined differentiation methods to generate SANLCs effectively and stably. Methods We induced human embryonic stem cells (hESCs)/induced PSCs (hiPSCs) to pan-cardiomyocytes by temporal modulation of the WNT/β-catenin (WNT) signaling pathway with GSK3 inhibitor and WNT inhibitor. During cardiac mesoderm stage of the differentiation process, signaling of WNT, retinoid acid (RA), and fibroblast growth factor (FGF) was manipulated by three specific molecules. Moreover, metabolic selection was designed to improve the enrichment of SANLCs. Finally, RT-PCR, immunofluorescence, flow cytometry, and whole cell patch clamp were used to identify the SANLCs. Results WNT, RA, and FGF signaling promote the differentiation of hPSCs into SANLCs in a concentration- and time window-sensitive manner, respectively. Synergetic modulation of WNT, FGF, and RA signaling pathways enhance the pacemaker phenotype and improve the differentiation efficiency of SANLCs (up to 45%). Moreover, the purification based on lactate metabolism and glucose starvation further reached approximately 50% of SANLCs. Finally, the electrophysiological data demonstrate that cells differentiated with the proposed protocol produce a considerable number of SANLCs that display typical electrophysiological characteristics of pacemaker cells in vitro. Conclusion We provide an optimized and chemically defined protocol to generate SANLCs by combined modulation of WNT, RA, and FGF signaling pathways and metabolic selection by lactate enrichment and glucose starvation. This chemically defined method for generating SANLCs might provide a platform for disease modeling, drug discovery, predictive toxicology, and biological pacemaker construction.

Immunostaining of tissue sections from dogs with subcutaneous nerve stimulation.

Histology Staining Procedure for tissues from ambulatory dogs after subcutaneous nerve stimulation.

Background Extended-duration work shifts (EDWSs) might affect the health of physician residents, causing autonomic alteration. Skin sympathetic nerve activity (SKNA) recorded by noninvasive neuro-electrocardiography (neuECG) is used to estimate cardiac sympathetic tone. In this study, we aim to evaluate the impact of EDWSs on nocturnal SKNA assessed in resident doctors. Methods Twenty-four residents working EDWSs and 12 PhD students not working nightshift schedules were prospectively recruited. The neuECG was performed between 12 am and 6 am for 5 consecutive nights. SKNA was filtered from neuECG recorded signals. The questionnaires regarding work stress and sleep quality, blood pressure, and salivary alpha-amylase and cortisol levels were administered. Results The hours of weekly working and sleep opportunities were similar between residents and students, while residents reported more work stress and worse sleep quality. In residents, SKNA at 6 am (SKNA6am) was significantly higher than SKNA2am during the precall night, revealing a dipping pattern. However, the SKNA dipping disappeared during the on-call night and prominently flattened during the first postcall night, the full recovery of which was delayed until the second postcall nights. The morning blood pressure and salivary alpha-amylase and cortisol levels were similar between the precall and postcall days. In contrast, SKNA in students exhibited a constant dipping profile for all recorded nights. Conclusions In healthy young adults, SKNA presents a dip night. The SKNA dip is impaired by working a nightshift, with a delayed recovery. The neuECG might serve as a useful tool to detect subclinical autonomic disturbances in shiftworkers.

Background Subcutaneous nerve stimulation (ScNS) damages the stellate ganglion and improves rhythm control of atrial fibrillation (AF) in ambulatory dogs. Objective The purpose of this study was to test the hypothesis that thoracic ScNS can improve rate control in persistent AF. Methods We created persistent AF in 13 dogs and randomly assigned them to ScNS (n = 6) and sham control (n = 7) groups. 18F-2-Fluoro-2-deoxyglucose positron emission tomography/magnetic resonance imaging of the brain stem was performed at baseline and at the end of the study. Results The average stellate ganglion nerve activity reduced from 4.00 ± 1.68 μV after the induction of persistent AF to 1.72 ± 0.42 μV (P = .032) after ScNS. In contrast, the average stellate ganglion nerve activity increased from 3.01 ± 1.26 μV during AF to 5.52 ± 2.69 μV after sham stimulation (P = .023). The mean ventricular rate during persistent AF reduced from 149 ± 36 to 84 ± 16 beats/min (P = .011) in the ScNS group, but no changes were observed in the sham control group. The left ventricular ejection fraction remained unchanged in the ScNS group but reduced significantly in the sham control group. Immunostaining showed damaged ganglion cells in bilateral stellate ganglia and increased brain stem glial cell reaction in the ScNS group but not in the control group. The 18F-2-fluoro-2-deoxyglucose uptake in the pons and medulla was significantly (P = .011) higher in the ScNS group than the sham control group at the end of the study. Conclusion Thoracic ScNS causes neural remodeling in the brain stem and stellate ganglia, controls the ventricular rate, and preserves the left ventricular ejection fraction in ambulatory dogs with persistent AF.

Rationale: The ventricular conduction system (VCS) rapidly propagates electrical impulses through the working myocardium of the ventricles to coordinate chamber contraction. GWAS (Genome-wide association studies) have associated nucleotide polymorphisms, most are located within regulatory intergenic or intronic sequences, with variation in VCS function. Two highly correlated polymorphisms (r 2 >0.99) associated with VCS functional variation (rs13165478 and rs13185595) occur 5′ to the gene encoding the basic helix–loop–helix transcription factor HAND1 (heart- and neural crest derivatives-expressed protein 1). Objective: Here, we test the hypothesis that these polymorphisms influence HAND1 transcription thereby influencing VCS development and function. Methods and Results: We employed transgenic mouse models to identify an enhancer that is sufficient for left ventricle (LV) cis -regulatory activity. Two evolutionarily conserved GATA transcription factor cis -binding elements within this enhancer are bound by GATA4 and are necessary for cis -regulatory activity, as shown by in vitro DNA binding assays. CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9-mediated deletion of this enhancer dramatically reduces Hand1 expression solely within the LV but does not phenocopy previously published mouse models of cardiac Hand1 loss-of-function. Electrophysiological and morphological analyses reveals that mice homozygous for this deleted enhancer display a morphologically abnormal VCS and a conduction system phenotype consistent with right bundle branch block. Using 1000 Genomes Project data, we identify 3 additional single nucleotide polymorphisms (SNPs), located within the Hand1 LV enhancer, that compose a haplotype with rs13165478 and rs13185595. One of these SNPs, rs10054375, overlaps with a critical GATA cis -regulatory element within the Hand1 LV enhancer. This SNP, when tested in electrophoretic mobility shift assays, disrupts GATA4 DNA-binding. Modeling 2 of these SNPs in mice causes diminished Hand1 expression and mice present with abnormal VCS function. Conclusions: Together, these findings reveal that SNP rs10054375, which is located within a necessary and sufficient LV-specific Hand1 enhancer, exhibits reduces GATA DNA-binding in electrophoretic mobility shift assay, and this enhancer in total, is required for VCS development and function in mice and perhaps humans.

High output subcutaneous nerve stimulation (ScNS) remodels the stellate ganglia and suppresses cardiac arrhythmia.The purpose of this study was to test the hypothesis that long duration low output ScNS causes cardiac nerve sprouting and increases plasma norepinephrine concentration and the duration of paroxysmal atrial tachycardia (PAT) in ambulatory dogs.We prospectively randomized 22 dogs (11 males and 11 females) into 5 different output groups for 2 months of ScNS: 0 mA (sham) (n = 6), 0.25 mA (n = 4), 1.5 mA (n = 4), 2.5 mA (n = 4), and 3.5 mA (n = 4).As compared with baseline, the changes in the durations of PAT episodes per 48 hours were significantly different among different groups (sham, -5.0 ± 9.5 seconds; 0.25 mA, 95.5 ± 71.0 seconds; 1.5 mA, -99.3 ± 39.6 seconds; 2.5 mA, -155.3 ± 87.8 seconds; and 3.5 mA, -76.3 ± 44.8 seconds; P .001). The 3.5 mA group had a greater reduction in sinus heart rate than did the sham group (-29.8 ± 15.0 beats/min vs -14.5 ± 3.0 beats/min; P = .038). Immunohistochemical studies showed that the 0.25 mA group had a significantly increased while 2.5 mA and 3.5 mA stimulation had significantly reduced growth-associated protein 43 nerve densities in both atria and ventricles. The plasma norepinephrine concentrations in the 0.25 mA group was 5063.0 ± 4366.0 pg/mL, which was significantly higher than that in the other groups of dogs (739.3 ± 946.3; P = .009). There were no significant differences in the effects of simulation between males and females.In ambulatory dogs, low output ScNS causes cardiac nerve sprouting and increases plasma norepinephrine concentration and the duration of PAT episodes while high output ScNS is antiarrhythmic.

Long non-coding RNAs (lncRNAs) regulate diverse cellular processes, and their anomalous expression exert an essential role in the progression of many kinds of cancers, including colorectal carcinoma (CRC). The objective of this study was to investigate the role of lncRNA kcna3 and its underlying mechanism in CRC progression. The expression of lncRNA kcna3 in human CRC tissues and the adjacent non-tumor tissues was evaluated by RT-PCR. The correlations between lncRNA kcna3 expression levels and the overall survival (OS), as well as the clinicopathological features of CRC patients were analyzed. Gain-of-function and loss-of-function experiments were used to evaluate the effects of lncRNA kcna3 on the proliferation, apoptosis, migration, invasion and tumorigenesis of colon cancer SW620 cells. We found that lncRNA kcna3 was lowly expressed in CRC tissues, and its low expression was closely associated with patients' higher TNM grade and the higher occurrence rate of lymphatic metastasis and distant metastasis, as well as shorter OS. Enhanced expression of lncRNA kcna3 inhibited SW620 cells' proliferation, migration and invasion, and induced cell apoptosis in vitro, and repressed CRC tumor growth in vivo. Whereas knockdown of lncRNA kcna3 showed the opposite results. Mechanistically, up-regulation of lncRNA kcna3 decreased YAP1 protein expression and accelerated its degradation. The effects of lncRNA kcna3 overexpression on cell growth and tumorigenesis inhibition and apoptosis promotion were weakened when the expression of YAP1 was up-regulated. In conclusion, this study revealed that lncRNA kcna3 exerts a tumor-inhibit role in CRC progression through down-regulating YAP1 expression, indicating that lncRNA kcna3/YAP1 might be served as a new prognostic biomarker and therapeutic target for CRC.

To prepare a tissue-engineered pulmonary valved conduit (PVC) with good tensile strength and biocompatibility. Sixty adult porcine PVCs were used to determine the optimal decellularization time. Five juvenile porcine decellularized PVCs and five juvenile porcine crosslinked PVCs were subsequently prepared according to the optimized decellularization and crosslinking methods. All PVCs were implanted into juvenile sheep for 8 months and then were harvested for staining. With a low concentration of detergent (0.25% Triton X-100+0.25% sodium deoxycholate), the decellularization effect on porcine PVCs was complete by 24 hours, and there was minimal damage to the matrix. Gelatin embedding and 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) crosslinking improved the biomechanical properties of decellularized PVCs and reduced their immunogenicity. After implantation, the diameter and thickness of the PVCs in the decellularized and crosslinked groups increased significantly. In both groups, the conduits were unobstructed, with soft and smooth inner walls and without thrombosis, ulceration or neoplasia. The valves slightly degenerated with mild to moderate regurgitation. CD31-positive endothelial cells were visible on the inner surface of the conduits and valves. Scattered smooth muscle actin-positive cells were found in the middle layer of the conduit. The percentage of CD4- and CD68-positive cells and the calcium content were highest in decellularized porcine PVCs and lowest in ovine PVCs. The percentage of the matrix that was laminin-positive in decellularized and crosslinked porcine PVCs was lower than it was in ovine PVCs. Gelatin-embedded and EDC-crosslinked porcine PVCs can be "hosted" in sheep, with good biocompatibility, growth potential, and reduced calcification.

Objectives. To investigate the effects of cytotoxin-associated gene A- (CagA-) positive Helicobacter pylori on proliferation, invasion, autophagy, and expression of miR-125b-5p in colon cancer cells. Methods. Colon cancer cells were cocultured with H. pylori (CagA+) to analyze the effects of H. pylori on miR-125b-5p and autophagy. Colon cancer cells infected with H. pylori (CagA+) were mimicked by transfection of CagA plasmid. The effects of CagA on the proliferation, invasion, and autophagy of colon cancer cells were analyzed. Cell counting kit-8 (CCK-8), clone formation, and Transwell assays were used to detect cell viability, proliferation, and invasion ability, respectively. Proteins and miRNAs were detected by western blotting and qPCR, respectively. Results. H. pylori (CagA+) inhibited expression of miR-125b-5p and promoted autophagy in colon cancer cells. MiR-125 b-5p was underexpressed in colon cancer cells after CagA overexpression. CagA promoted colon cancer cell proliferation, invasion, and autophagy. Overexpression of miR-125b-5p inhibited the proliferation, invasion, and autophagy of colon cancer cells and reversed the effects of CagA. Conclusion. H. pylori (CagA+) infection may promote the development and invasion of colon cancer by inhibiting miR-125b-5p.

Objectives To investigate the effects of cytotoxin-associated gene A- (CagA-) positive Helicobacter pylori on proliferation, invasion, autophagy, and expression of miR-125b-5p in colon cancer cells. Methods Colon cancer cells were cocultured with H. pylori (CagA+) to analyze the effects of H. pylori on miR-125b-5p and autophagy. Colon cancer cells infected with H. pylori (CagA+) were mimicked by transfection of CagA plasmid. The effects of CagA on the proliferation, invasion, and autophagy of colon cancer cells were analyzed. Cell counting kit-8 (CCK-8), clone formation, and Transwell assays were used to detect cell viability, proliferation, and invasion ability, respectively. Proteins and miRNAs were detected by western blotting and qPCR, respectively. Results H. pylori (CagA+) inhibited expression of miR-125b-5p and promoted autophagy in colon cancer cells. MiR-125 b-5p was underexpressed in colon cancer cells after CagA overexpression. CagA promoted colon cancer cell proliferation, invasion, and autophagy. Overexpression of miR-125b-5p inhibited the proliferation, invasion, and autophagy of colon cancer cells and reversed the effects of CagA. Conclusion H. pylori (CagA+) infection may promote the development and invasion of colon cancer by inhibiting miR-125b-5p.

Aorto-cardiac fistula is a rare but potentially life-threatening condition. We herein report a rare case of chronic aorto-right ventricular fistula formation secondary to a stab penetrating injury to the heart and aorta occurred 15 years ago. The aorto-right ventricular fistula was not found until 15 years after the incident. The fistula had been repaired successfully to prevent further deterioration of cardiac function. Here, we report the clinical presentation, diagnosis and treatment strategies of the aorto-right ventricular fistula, and discuss the possible etiology of the development of the fistula after the penetrating injury.

Background Subcutaneous nerve stimulation (ScNS) delivered directly to large subcutaneous nerves can be either antiarrhythmic or proarrhythmic, depending on the stimulus output. Objective The purpose of this study was to perform a prospective randomized study in a canine model of persistent AF to test the hypothesis that high-output ScNS using blindly inserted subcutaneous electrodes can reduce ventricular rate (VR) during persistent atrial fibrillation (AF) whereas low-output ScNS would have opposite effects. Methods We prospectively randomized 16 male and 15 female dogs with sustained AF (>48 hours) induced by rapid atrial pacing into 3 groups (sham, 0.25 mA, 3.5 mA) for 4 weeks of ScNS (10 Hz, alternating 20-seconds ON and 60-seconds OFF). Results ScNS at 3.5 mA, but not 0.25 mA or sham, significantly reduced VR and stellate ganglion nerve activity (SGNA), leading to improvement of left ventricular ejection fraction (LVEF). No differences were found between the 0.25-mA and sham groups. Histologic studies showed a significant reduction of bilateral atrial fibrosis in the 3.5-mA group compared with sham controls. Only 3.5-mA ScNS had significant fibrosis in bilateral stellate ganglions. The growth-associated protein 43 (GAP43) staining of stellate ganglions indicated the suppression of GAP43 protein expression in the 3.5-mA group. There were no significant differences of nerve sprouting among all groups. There was no interaction between sex and ScNS effects on reduction of VR and SGNA, LVEF improvement, or results of histologic studies. Conclusion We conclude that 3.5-mA ScNS with blindly inserted electrodes can improve VR control, reduce atrial fibrosis, and partially improve LVEF in a canine model of persistent AF.

Background. Premature ventricular complexes (PVCs) exhibit circadian fluctuation. We determine if PVCs of different origin exhibit specific circadian patterns. Methods. We analyzed Holter recordings from patients with monomorphic PVCs who underwent catheter ablation. PVC circadian patterns were classified as fast-heart rate- (HR-) dependent (F-PVC), slow-HR-dependent (S-PVC), or HR-independent (I-PVC). PVC origins were determined intraprocedurally. Results. In a retrospective cohort of 407 patients, F-PVC and S-PVC typically exhibited diurnal and nocturnal predominance, respectively. Despite decreased circadian fluctuation, I-PVC generally had heavier nocturnal than diurnal burden. PVCs of left anterior fascicle origin were predominantly S-PVC, while those of posterior hemibranch origin were mostly F-PVC. PVCs originating from the aortic sinus of Valsalva (ASV) were predominantly I-PVC, while most PVCs arising from the left ventricular outflow tract (LVOT) were F-PVC. Using a diurnal/nocturnal PVC burden ratio of 0.92 as the cutoff value to distinguish LVOT from ASV origin achieved 97% sensitivity and, as further verification, an accuracy of 89% (16/18) in a prospective cohort of patients with PVCs originating from either ASV or LVOT. In contrast, PVCs originating from right ventricles, such as right ventricular outflow tract, did not show distinct circadian patterns. Conclusions. The circadian patterns exhibit origin specificity for PVCs arising from left ventricles. An analysis of Holter monitoring provides useful information on PVC localization in ablation procedure planning.

Background: The occurrence and development of atherosclerosis are closely related to shear stress on the endothelial cells. However, related molecular mechanisms of controlling mechanical signal transduction in endothelial cells remain largely unknown. Aim of the Study: To obtain this goal, here, a parallel-plate flow chamber was used to impose laminar shear stress (8.4 dyne/cm2 ) on endothelial cells for 0 minute (min), 10 min, 30 min, 60 min, and 120 min. Subjects and Methods: The RNA interference was employed to down-regulate scavenger receptor class B1 (SRB1) and LY294002 was performed to block the phosphorylation status of the phosphoinositide 3-kinase pathway. Then, the cell status was observed under the microscope, followed by detection of phosphorylated phosphoinositide 3-kinase (PI3K), p38, extracellular signal-regulated protein kinases 1 and 2 and the binding activities of activator protein 1, nuclear factorκB and endothelial nitric oxide synthase (eNOs) by western blot. The results showed that shear stress (8.4 dyne/cm2 ) for 60 min induced obvious cell morphologic changes. Meanwhile, PI3K phosphorylation and eNOS expression were significantly increased by shear stress while reversed by SRB1 si-RNA and LY294002. Additionally, shear stress caused the enhanced binding activity of nuclear transcription factor activator protein 1 (AP-1) to DNA from 10 to 120 min, while only at 30 and 60 min for NF-κB. After treatment with SRB1 si-RNA and LY294002 inhibitor, the binding activities of AP-1 and DNA was significantly decreased, however, there was no effect on NF-κB binding activity. Results: Therefore, SRB1 plays a crucial role in mediating shear stress-induced eNOS activation in endothelial cells via PI3K-AP1 pathway. These findings will provide an important understanding of shear stress-induced cell alterations in clinic detection

The ventricular conduction system (VCS), composed of the His bundle, the left and right bundle branches, and the Purkinje fiber network, rapidly propagates electrical impulses through the ventricles to coordinate chamber contraction. An ECG depicts VCS-mediated ventricular depolarization as the QRS interval. Disorders of the VCS may manifest as arrhythmias, and are associated with increased risk of sudden cardiac death and overall mortality. The genetic and developmental mechanisms underlying VCS dysfunction are poorly understood. Genome-wide association studies have identified multiple single nucleotide polymorphisms (SNPs) associated with VCS arrhythmias. A majority of these SNPs occur outside of coding domains, within intergenic or intronic regions. We hypothesize that such pathogenic SNPs may impact VCS development and function by altering the expression of critical genes. Two intergenic SNPs associated with QRS prolongation occur near the bHLH cardiac transcription factor HAND1 . We have identified a left ventricle (LV) enhancer, located between these two SNPs, that is necessary and sufficient for LV cis -regulatory activity. Two evolutionarily conserved GATA transcription factor consensus-binding sites within this enhancer are bound by GATA4 and necessary for cis -regulatory activity. CRISPR-mediated deletion of this enhancer dramatically reduced Hand1 expression solely within the LV. Mice homozygous for this deleted enhancer displayed dysregulated LV gene expression, morphologically abnormal His bundles and left bundle branches, and a VCS phenotype consistent with right bundle branch block. Genome-wide association analyses of human patients with QRS prolongation revealed additional, linked SNPs, one of which overlaps with a critical GATA cis -regulatory element within the LV-specific Hand1 enhancer. This SNP disrupts GATA4 binding. Ongoing studies of mice that genetically mimic these minor SNP variants will assess reduced Hand1 expression and impaired VCS function. We conclude that a LV-specific Hand1 enhancer is necessary for VCS development and a SNP associated with QRS prolongation directly influences GATA4 binding to a critical cis -regulatory element within this enhancer.

BACKGROUND. Simultaneous noninvasively recorded skin sympathetic nerve activity (SKNA) and electrocardiogram (neuECG) can be used to estimate cardiac sympathetic tone. We tested the hypothesis that large and prolonged SKNA bursts are associated with temporal clustering arrhythmias. METHODS. We recorded neuECG in 10 patients (69 ± 10 years old) with atrial fibrillation (AF) episodes and in 6 patients (50 ± 13 years old) with ventricular tachycardia (VT) or fibrillation (VF) episodes. Clustering was defined by an arrhythmic episode followed within 1 minute by spontaneous recurrences of the same arrhythmia. The neuECG signals were bandpass filtered between 500–1000 Hz to display SKNA. RESULTS. There were 22 AF clusters, including 231 AF episodes from 6 patients, and 9 VT/VF clusters, including 99 VT/VF episodes from 3 patients. A total duration of SKNA bursts associated with AF was longer than that during sinus rhythm (78.9 min/hour [interquartile range (IQR) 17.5–201.3] vs. 16.3 min/hour [IQR 14.5–18.5], P = 0.022). The burst amplitude associated with AF in clustering patients was significantly higher than that in nonclustering patients (1.54 μV [IQR 1.35–1.89], n = 114, vs. 1.20 μV [IQR 1.05–1.42], n = 21, P < 0.001). The SKNA bursts associated with VT/VF clusters lasted 9.3 ± 3.1 minutes, with peaks that averaged 1.13 ± 0.38 μV as compared with 0.79 ± 0.11 μV at baseline (P = 0.041). CONCLUSION. Large and sustained sympathetic nerve activities are associated with the temporal clustering of AF and VT/VF. FUNDING. This study was supported in part by NIH grants R42DA043391 (THE), R56 HL71140, TR002208-01, R01 HL139829 (PSC), a Charles Fisch Cardiovascular Research Award endowed by Suzanne B. Knoebel of the Krannert Institute of Cardiology (TK and THE), a Medtronic-Zipes Endowment, and the Indiana University Health-Indiana University School of Medicine Strategic Research Initiative (PSC).

Background The apamin-sensitive small-conductance calcium-activated K (SK) current IKAS modulates automaticity of the sinus node. IKAS blockade by apamin causes sinus bradycardia. Objective The purpose of this study was to test the hypothesis that IKAS modulates ventricular automaticity. Methods We tested the effects of apamin (100 nM) on ventricular escape rhythms in Langendorff-perfused rabbit ventricles with atrioventricular block (protocol 1) and on recorded transmembrane action potential of pseudotendons of superfused right ventricular endocardial preparations (protocol 2). Results All preparations exhibited spontaneous ventricular escape rhythms. In protocol 1, apamin decreased the atrial rate from 186.2 ± 18.0 bpm to 163.8 ± 18.7 bpm (N = 6; P = .006) but accelerated the ventricular escape rate from 51.5 ± 10.7 bpm to 98.2 ± 25.4 bpm (P = .031). Three preparations exhibited bursts of nonsustained ventricular tachycardia and pauses, resulting in repeated burst termination pattern. In protocol 2, apamin increased the ventricular escape rate from 70.2 ± 13.1 bpm to 110.1 ± 2.2 bpm (P = .035). Spontaneous phase 4 depolarization was recorded from the pseudotendons in 6 of 10 preparations at baseline and in 3 in the presence of apamin. There were no changes of phase 4 slope (18.37 ± 3.55 mV/s vs 18.93 ± 3.26 mV/s, N=3; P = .231, ), but the threshold of phase 0 activation (mV) reduced from –67.97 ± 1.53 to –75.26 ± 0.28 (P = .034). Addition of JTV-519, a ryanodine receptor 2 stabilizer, in 5 preparations reduced escape rate back to baseline. Conclusion Contrary to its bradycardic effect in the sinus node, IKAS blockade by apamin accelerates ventricular automaticity and causes repeated nonsustained ventricular tachycardia in normal ventricles. ryanodine receptor 2 blockade reversed the apamin effects on ventricular automaticity.

OBJECTIVES: To determine the effects of implanting a novel mosaic tissue-engineered porcine pulmonary artery valved conduit into the right ventricular outflow tract of sheep at a long-term follow-up. METHODS: The designed mosaic tissue-engineered porcine pulmonary artery valved conduits were implanted between the right ventricular outflow tract and distal pulmonary artery in sheep using the off-pump method. The sheep weight, conduit diameter, pulmonary valve annular diameter, left ventricular end-diastolic diameter, calcification and regurgitation of the pulmonary valve were measured preoperatively and at 6 and 12 months postoperatively. Macroscopic observation, ultrastructural analysis, endothelialization and detection of calcium content were performed after sacrificing the sheep at 12 months after surgery. RESULTS: The average sheep weight at 12 months after surgery was significantly higher than that preoperatively (P

Skin sympathetic nerve activity (SKNA) is useful for estimating sympathetic tone in humans.The purpose of this study was to test the hypotheses that (1) increased SKNA is associated with the onset and termination of paroxysmal atrial tachycardia (AT) and atrial fibrillation (AF) and (2) sinoatrial node response to SKNA is reduced in patients with more frequent AT or AF episodes.SKNA and electrocardiogram were recorded in 11 patients (4 men and 7 women; average age 66 ± 10 years), including 3 patients with AT (11 ± 18 episodes per patient) and 8 patients with AF (24 ± 26 episodes per patient).The average SKNA (aSKNA) 10 seconds before AT onset was 1.07 ± 0.10 μV and 10 seconds after termination was 1.27 ± 0.10 μV; both were significantly (P = .032 and P.0001) higher than that during sinus rhythm (0.97 ± 0.09 μV). The aSKNA 10 seconds before AF onset was 1.34 ± 0.07 μV and 10 seconds after termination was 1.31 ± 0.07 μV; both were significantly (P .0001) higher than that during sinus rhythm (1.04 ± 0.07 μV). The aSKNA before onset (P.0001) and after termination (P = .0011) was higher in AF than in AT. The sinus rate correlated (P.0001) with aSKNA in each patient (average r = 0.74; 95% confidence interval 0.65-0.84). The r value in each patient negatively correlated with the number of AT and AF episodes (r = -0.6493; 95% confidence interval -0.8990 to -0.08073; P = .0306).Increased SKNA was observed both at the onset and termination of AT and AF. Patients with more frequent AT and AF episodes had a weak correlation between sinus rate and aSKNA, suggesting sinoatrial node remodeling by tachycardia.

We report here the surgical treatment of a rare case of primary cardiac paraganglioma with an intratumoral coronary artery aneurysm.

To review the recent research progress of skeletal myoblasts for cardiac repair.The related literature about skeletal myoblasts for cardiac repair was reviewed, analyzed, and summarized.The results of animal experiments and clinical studies have shown that skeletal myoblasts been transplanted into the regional myocardial infarction area in different ways can improve cardiac function. But there are some challenges such as high loss rate of skeletal myoblasts and resulting in ventricular arrhythmias.Further studies can improve the safety and effectiveness of skeletal myoblasts for cardiac repair in the future.

To solve the shortage of hepatocytes for liver tissue engineering, to explore the possibility of proliferation of rat bone marrow mesenchymal stem cells (BMSCs) and the feasibility of differentiation of BMSCs into hepatocytes with a culture system containing cholestatic rat serum and hepatocyte growth factor (HGF) in vitro.Myeloid cells of femur and tibia were collected from the female healthy Wistar rats at the age of 6 weeks, the BMSCs were isolated, purified and identified. Normal and cholestatic rat serum were prepared from 40 healthy Wistar rats at the age of 12-14 weeks. The 3rd passage of BMSCs were harvested and added different cultures according to the following grouping: group A, DMEM plus 10%FBS; group B, hepatocyte growth medium (HGM) plus 5%FBS; group C, HGM plus 5% normal rat serum; group D, HGM plus 5% cholestatic rat serum; group E, HGM plus 5% cholestatic rat serum plus 25 microg/L HGF. The changes of cell morphology were observed, MTT assay was used to measure cell growth; the expression of alpha-fetoprotein (AFP) and cytokeratin 18 (CK18) were detected by immunocytochemistry; the glycogen deposit was examined by periodic acid-schiff (PAS) staining; and the urea content in culture supernatant was determined by glutamate dehydrogenase.Polygonal cells and binuclear cells were observed in groups D and E, while the shapes of cells in groups A, B, and C did not obviously change. The cell growth curve demonstrated that the speed of cells proliferation in group C was the fastest, the one in group B was the slowest; showing significant differences when compared with groups A, D, and E (P0.05). On the 7th day in groups D and E, the positive expressions of AFP and CK18 emerged, on the 14th day the positive expression of glycogen emerged. At the same period, the expression ratio was higher in group E than in group D (P0.05). The urea concentration increased gradually with induction time in groups D and E, the concentration was higher in group E than in group D (P0.05). No expressions of AFP, CK18, glycogen, and change of the urea concentration were observed in groups A, B, and C.Normal rat serum can obviously promote the growth of BMSCs; cholestatic rat serum which promote the growth of BMSCs can induce to differentiate into hepatocyte; and a combination of cholestatic serum and HGF can increase the differentiation ratio.

To observe the change of sino-atrial nodal tissue structure and ectopic pacing function after xenogenic sino-atrial nodal tissue transplanted into left ventricular wall, so as to provide new ideas for the treatment of sick sinus syndrome and severe atrioventricular block.Seventy healthy rabbits were selected, male or female, and weighing 1.5-2.0 kg. Of them, 42 were used as recipient animals and randomly divided into sham operation group, warm ischemia transplantation group, and cold ischemia transplantation group (n = 14), the other 28 were used as donors of warm ischemia and cold ischemia transplantation groups, which were sibling of the recipients. In recipients, a 6-mm-long and about 2-mm-deep incision was made in the vascular sparse area of left ventricular free wall near the apex. In sham operation group, the incision was sutured directly by 7-0 Prolene suture; in cold ischemia transplantation group, after the aortic roots cross-clamping, 4 degrees C cold crystalloid perfusion fluid infusion to cardiac arrest, then sinoatrial node were cut 5 mm x 3 mm for transplantation; in warm ischemia transplantation group, the same size of the sinus node tissue was captured for transplantation. After 1, 2, 3, and 4 weeks, 3 rabbits of each group were harvested to make bradycardia by stimulating bilateral vagus nerve and the cardiac electrical activity was observed; the transplanted sinus node histology and ultrastructural changes were observed.Thirty-six recipient rabbits survived (12 rabbits each group). At 1, 2, 3, and 4 weeks after bilateral vagus nerve stimulation, the cardiac electrical activity in each group was significantly slower, and showed sinus bradycardia. Four weeks after operation the heart rates of sham operation group, warm ischemia, and cold ischemia transplantation group were (81.17 +/- 5.67), (82.42 +/- 7.97), and (80.83 +/- 6.95) beats/minute, respectively; showing no significant difference among groups (P0.05). And no ectopic rhythm of ventricular pacing occurred. Sino-atrial nodal tissue survived in 6 of warm ischemic transplantation group and in 8 of cold ischemia transplantation group; showing no significant difference between two groups (P0.05). Two adjacent sinoatrial node cells, vacuole-like structure in the cytoplasm, a few scattered muscle microfilaments, and gap junctions between adjacent cells were found in transplanted sinus node.The allograft sinus node can survive, but can not play a role in ectopic pacing.

To locate sinoatrial node (SAN) in suckling pigs, to develop a reliable method for isolation, purification and cultivation of SAN cells and to observe the compatibility of SAN cells and Col I fiber scaffold.Five newborn purebred ChangBaiShan suckling pigs (male and female), aged less than 1-day-old and weighing 0.45-0.55 kg, were used. Multi-channels electrophysiological recorder was applied to detect the original site of atrial waves. Primary SAN cells harvested from that area were cultured by the conventional culture method and the purification culture method including differential velocity adherent technique and 5-BrdU treatment, respectively. Atrial myocytes isolated from the left atrium underwent purified culture. Cell morphology, time of cell attachment, time of unicellular pulsation, and pulsation frequency were observed using inverted microscope. The purified cultured SAN cells (5 x 10(5) cells/mL) were co-cultured with pre-wetted Col I fiber scaffold for 5 days, and then the cells were observed by HE staining and scanning electron microscope (SEM).The atrial waves occurred firstly at the area of SAN. The purified cultured SAN cells were spindle, triangular, and irregular in morphology, and the spindle cells comprised the greatest proportion. Atrial myocytes were not spindle-shaped, but primarily triangular and irregular. The proportion of spindle cells in the conventional cultured SAN cells was decreased from 73.0% +/- 2.9% in the purified cultured SAN cells, to 44.7% +/- 2.3% (P0.01), and the proportion of irregular cells increased from 7.0% +/- 1.7% in the purified cultured SAN cells to 36.1% +/- 2.6% (P0.01) . The proportion of the triangular cells in the purified and the conventional cultured SAN cells was 20.0% +/- 2.1% and 19.2% +/- 2.5%, respectively (P0.05). At 5 days after co-culture, HE staining displayed lots of SAN cells in Col I fiber scaffold, and SEM demonstrated conglobata adherence of the cells to the surface and lateral pore wall of scaffold, mutual connections of the cell processes, or attachment of cells to lateral pore wall of scaffold through pseudopodia.With accurate SAN location, the purification culture method containing differential velocity adherent technique and 5-BrdU treatment can increase the proportion of spindle cells and is a reliable method for the purification and cultivation of SAN cells. The SAN cells and Col I fiber scaffold have a good cellular compatibility.

To study the influence of ischemia-reperfusion on the expression of the hyperpolarization activated cyclic nucleotide gated cation channel 4 (HCN4) and to discuss the mechanism of functional disturbance of sinoatrial node tissue (SANT) after ischemia reperfusion injury (IRI).Eighty five healthy adult rabbits, weighing 2-3 kg, were randomly divided into 3 groups: control group [a suture passed under the root section of right coronary artery (RCA) without ligation, n=5], experimental group A (occluding the root section of RCA for 30 minutes, then loosening the root 2, 4, 8 and 16 hours, n=10), experimental group B (occluding the root section of RCA for 1 hour, then loosening the root 2, 4, 8 and 16 hours, n=10). At the end of the reperfusion, the SANT was cut off to do histopathological, transmission electron microscopical and immunohistochemical examinations and semi-quantitative analysis.The result of HE staining showed that patho-injure of sinoatrial node cell (SANC) happened in experimental groups A and B after 2 hours of reperfusion, the longer the reperfusion time was, the more serious patho-injure of SANC was after 4 and 8 hours of reperfusion, SANC reached peak of damage after 8 to 16 hours of reperfusion; patho-injure of SANC was more serious in experimental group B than in experimental group A at the same reperfusion time. Immunohistochemical staining showed that the expression of HCN4 located in cellular membrane and cytoplasm in the central area of SANC and gradually decreased from the center to borderline. The integral absorbance values of HCN4 expression in the control group (397.40 +/- 34.11) was significantly higher than those in the experimental group A (306.20 +/- 35.77, 216.60 +/- 18.59, 155.40 +/- 19.11 and 135.00 +/- 12.30) and in the experimental group B (253.70 +/- 35.66, 138.70 +/- 13.28, 79.10 +/- 9.60 and 69.20 +/- 8.42) after 2, 4, 8 and 16 hours of reperfusion (P0.05). With reperfusion time, the expression of HCN4 of SANC decreased, which was lowest after 8 hours of reperfusion; showing significant difference among 2, 4 and 8 hours after reperfusion (P0.05) and no significant difference between 8 and 16 hours after reperfusion (P0.05). At the same reperfusion time, the expression of HCN4 was higher in the experimental group A than in the experimental group B. The result of transmission electron microscope showed that ultramicrostructure of SANC was damaged after reperfusion in experimental groups A and B. The longer the reperfusion time was, the more serious ultramicrostructure damage of SANC was, and reached the peak of damage after 8 hours of reperfusion. Ultramicrostructure of SANC was not different between 8 and 16 hours of reperfusion. At the same reperfusion time, the ultramicrostructure damage of SANC was more serious in experimental group B than in experimental group A.IRI is harmful to the morphous and structure of SANC, and effects the expression of HCN4 of SANC, which is concerned with functional disturbance and arrhythmia.

OBJECTIVES: To determine the effects of implanting a novel mosaic tissue-engineered porcine pulmonary artery valved conduit into the right ventricular outflow tract of sheep at a long-term follow-up. METHODS: The designed mosaic tissue-engineered porcine pulmonary artery valved conduits were implanted between the right ventricular outflow tract and distal pulmonary artery in sheep using the off-pump method. The sheep weight, conduit diameter, pulmonary valve annular diameter, left ventricular end-diastolic diameter, calcification and regurgitation of the pulmonary valve were measured preoperatively and at 6 and 12 months postoperatively. Macroscopic observation, ultrastructural analysis, endothelialization and detection of calcium content were performed after sacrificing the sheep at 12 months after surgery. RESULTS: The average sheep weight at 12 months after surgery was significantly higher than that preoperatively (P