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Pre-excitation syndrome can either mimic or mask myocardial infarction, making the diagnosis of acute myocardial infarction difficult. Herein, we report the case of a male patient with Wolf-Parkinson-White (WPW) syndrome who presented to our emergency department with severe chest pain. Non-ST-elevation myocardial infarction was suspected because of cardiac enzyme elevation and abnormal ST-T changes identified through electrocardiography. The patient underwent percutaneous coronary intervention; a left anterior descending artery stenotic lesion was dilated, and drug-eluting stents were implanted. One month later, he underwent successful radiofrequency catheter ablation for his accessory pathway and tachycardia. We present the series of electrocardiographic ST-T abnormalities to raise awareness of the value of diagnosing myocardial injury early in patients with WPW syndrome.

Vascular endothelial growth factor (VEGF), a pivotal activator of angiogenesis and calcium (Ca2+) signaling in endothelial cells, was shown to increase collagen production in atrial fibroblasts. In this study, we evaluated whether VEGF may regulate Ca2+ homeostasis in atrial fibroblasts and contribute to its profibrogenesis. Migration, and proliferation analyses, patch-clamp assay, Ca2+ fluorescence imaging, and western blotting were performed using VEGF-treated (300 pg/mL or 1000 pg/mL) human atrial fibroblasts with or without coadministration of Ethylene glycol tetra-acetic acid (EGTA, 1 mmol/L), or KN93 (a Ca2+/calmodulin-dependent protein kinase II [CaMKII] inhibitor, 10 μmol/L). VEGF (1000 pg/mL) increased migration, myofibroblast differentiation, pro-collagen type I, pro-collagen type III production, and phosphorylated VEGF receptor 1 expression of fibroblasts. VEGF (1000 pg/mL) increased the nonselective cation current (INSC) of transient receptor potential (TRP) channels and potassium current of intermediate-conductance Ca2+-activated K+ (KCa3.1) channels thereby upregulating Ca2+ entry. VEGF upregulated phosphorylated ERK expression. An ERK inhibitor (PD98059, 50 μmol/L) attenuated VEGF-activated INSC of TRP channels. The presence of EGTA attenuated the profibrotic effects of VEGF on pro-collagen type I, pro-collagen type III production, myofibroblast differentiation, and migratory capabilities of fibroblasts. VEGF upregulated the expression of phosphorylated CaMKII in fibroblasts, which was attenuated by EGTA. In addition, KN93 reduced VEGF-increased pro-collagen type I, pro-collagen type III production, myofibroblast differentiation, and the migratory capabilities of fibroblasts. In conclusion, we found that VEGF increases atrial fibroblast activity through CaMKII signaling by enhancing Ca2+ entry. Our findings provide benchside evidence leading to a potential novel strategy targeting atrial myopathy and arrhythmofibrosis. Vascular endothelial growth factor increases the profibrotic activity of atrial fibroblasts by activating the currents through transient receptor potential channels and intermediate-conductance calcium-activated K+ (KCa3.1) channels and by enhancing Ca2+ entry-induced phosphorylated Ca2+/calmodulin-dependent protein kinase II (CaMKII) signaling. These findings suggest a novel strategy targeting atrial myopathy and arrhythmofibrosis.

Aims Macrophage migration inhibitory factor (MIF), a pleiotropic inflammatory cytokine, is highly expressed in patients with atrial fibrillation (AF). Inflammation increases the risk of AF and is primarily triggered by pulmonary vein (PV) arrhythmogenesis. This study investigated whether MIF can modulate the electrical activity of the PV and examined the underlying mechanisms of MIF. Methods and results A conventional microelectrode, a whole-cell patch clamp, western blotting, and immunofluorescent confocal microscopy were used to investigate electrical activity, calcium (Ca2+) regulation, protein expression, ionic currents, and cytosolic reactive oxygen species (ROS) in rabbit PV tissue and isolated single cardiomyocytes with and without MIF incubation (100 ng/mL, treated for 6 h). The MIF (100 ng/mL)-treated PV tissue (n = 8) demonstrated a faster beating rate (1.8 ± 0.2 vs. 2.6 ± 0.1 Hz, P < 0.05), higher incidence of triggered activity (12.5 vs. 100%, P < 0.05), and premature atrial beat (0 vs. 100%, P < 0.05) than the control PV tissue (n = 8). Compared with the control PV cardiomyocytes, MIF-treated single PV cardiomyocytes had larger Ca2+ transients (0.6 ± 0.1 vs. 1.0 ± 0.1, ΔF/F0, P < 0.05), sarcoplasmic reticulum Ca2+ content (0.9 ± 0.20 vs. 1.7 ± 0.3 mM of cytosol, P < 0.05), and cytosolic ROS (146.8 ± 5.3 vs. 163.7 ± 3.8, ΔF/F0, P < 0.05). Moreover, MIF-treated PV cardiomyocytes exhibited larger late sodium currents (INa-Late), L-type Ca2+ currents, and Na+/Ca2+ exchanger currents than the control PV cardiomyocytes. KN93 [a selective calcium/calmodulin-dependent protein kinase II (CaMKII) blocker, 1 μM], ranolazine (an INa-Late inhibitor, 10 μM), and N-(mercaptopropionyl) glycine (ROS inhibitor, 10 mM) reduced the beating rates and the incidence of triggered activity and premature captures in the MIF-treated PV tissue. Conclusion Macrophage migration inhibitory factor increased PV arrhythmogenesis through Na+ and Ca2+ dysregulation through the ROS activation of CaMKII signalling, which may contribute to the genesis of AF during inflammation. Anti-CaMKII treatment may reverse PV arrhythmogenesis. Our results clearly reveal a key link between MIF and AF and offer a viable therapeutic target for AF treatment.

Galectin-3 (Gal-3) is a biomarker of atrial fibrillation (AF) that mediates atrial inflammation. CD98 is the membrane surface receptor for Gal-3. Nevertheless, the role of the Gal-3/CD98 axis in atrial arrhythmogenesis is unclear. In this study, we investigated the effects of Gal-3/CD98 signalling on atrial pathogenesis.Whole cell patch clamp and western blotting were used to analyse calcium/potassium homeostasis and calcium-related signalling in Gal-3-administrated HL-1 atrial cardiomyocytes with/without CD98 neutralized antibodies. Telemetry electrocardiographic recording, Masson's trichrome staining and immunohistochemistry staining of atrium were obtained from mice having received tail-vein injections with Gal-3.Gal-3-treated HL-1 myocytes had a shorter action potential duration, smaller L-type calcium current, increased sarcoplasmic reticulum (SR) calcium content, NaGal-3 recombinant protein administration increases atrial fibrosis and arrhythmogenesis through CD98 signalling. Targeting Gal-3/CD98 axis might be a novel therapeutic strategy for patients with AF and high Gal-3 levels.

BACKGROUND Inhibition of histone deacetylases (HDACs) attenuates cardiac fibrosis. In this study, we evaluated whether the inhibition of class I HDACs can attenuate angiotensin II (ANG II)-induced fibrogenesis and mitochondrial malfunction through its effects on reactive oxygen species (ROS) and calcium dysregulation in human cardiac fibroblasts (CFs). METHODS Seahorse XF24 extracellular flux analyser, fluorescence staining, Western blotting, HDAC activity assays and Transwell migration assay were used to study mitochondrial respiration, adenosine triphosphate (ATP) production, mitochondrial calcium uptake and ROS, HDAC expression and activity and fibroblast activity in CFs without (control) or with ANG II (100 nM) and/or MS-275 (HDAC class 1 inhibitor, 10 μM) for 24 h. RESULTS ANG II increased HDAC activity without changing protein expression in CFs. Compared with controls, ANG II-treated CFs had greater migration activity, higher ATP production, maximal respiration and spare capacity with higher mitochondrial Ca2+ uptake and ROS generation, which was attenuated by the administration of MS-275. ANG II activated CFs by increasing mitochondrial calcium content and ATP production, which may be caused by increased HDAC activity. Inhibition of HDAC1 attenuated the effects of ANG II by reducing mitochondrial ROS generation and calcium overload. CONCLUSIONS Modulating mitochondrial function by regulation of HDAC may be a novel strategy for controlling CF activity.

Fructose is a main dietary sugar involved in the excess sugar intake-mediated progression of cardiovascular diseases and cardiac arrhythmias. Chronic intake of fructose has been the focus on the possible contributor to the metabolic diseases and cardiac inflammation. Recently, the small intestine was identified to be a major organ in fructose metabolism. The overconsumption of fructose induces dysbiosis of the gut microbiota, which, in turn, increases intestinal permeability and activates host inflammation. Endotoxins and metabolites of the gut microbiota, such as lipopolysaccharide, trimethylamine N-oxide, and short-chain fatty acids, also influence the host inflammation and cardiac biofunctions. Thus, high-fructose diets cause heart–gut axis disorders that promote cardiac arrhythmia. Understanding how gut microbiota dysbiosis-mediated inflammation influences the pathogenesis of cardiac arrhythmia may provide mechanisms for cardiac arrhythmogenesis. This narrative review updates our current understanding of the roles of excessive intake of fructose on the heart-gut axis and proposes potential strategies for inflammation-associated cardiac vascular diseases.

Background: Atrial fibrosis plays an important role in the genesis of heart failure and atrial fibrillation. The left atrium (LA) exhibits a higher level of fibrosis than the right atrium (RA) in heart failure and atrial arrhythmia. However, the mechanism for the high fibrogenic potential of the LA fibroblasts remains unclear. Calcium (Ca2+) signaling contributes to the pro-fibrotic activities of fibroblasts. This study investigated whether differences in Ca2+ homeostasis contribute to differential fibrogenesis in LA and RA fibroblasts. Methods: Ca2+ imaging, a patch clamp assay and Western blotting were performed in isolated rat LA and RA fibroblasts. Results: The LA fibroblasts exhibited a higher Ca2+ entry and gadolinium-sensitive current compared with the RA fibroblasts. The LA fibroblasts exhibited greater pro-collagen type I, type III, phosphorylated Ca2+/calmodulin-dependent protein kinase II (CaMKII), phosphorylated phospholipase C (PLC), stromal interaction molecule 1 (STIM1) and transient receptor potential canonical (TRPC) 3 protein expression compared with RA fibroblasts. In the presence of 1 mmol/L ethylene glycol tetra-acetic acid (EGTA, Ca2+ chelator), the LA fibroblasts had similar pro-collagen type I, type III and phosphorylated CaMKII expression compared with RA fibroblasts. Moreover, in the presence of KN93 (a CaMKII inhibitor, 10 μmol/L), the LA fibroblasts had similar pro-collagen type I and type III compared with RA fibroblasts. Conclusion: The discrepancy of phosphorylated PLC signaling and gadolinium-sensitive Ca2+ channels in LA and RA fibroblasts induces different levels of Ca2+ influx, phosphorylated CaMKII expression and collagen production.

Cardiac fibrosis plays a vital role in the pathogenesis of heart failure. Fibroblast activity is enhanced by increases in store-operated Ca2+ entry (SOCE) and calcium release-activated calcium channel protein 1 (Orai1) levels. Lithium regulates SOCE; however, whether therapeutic concentrations of lithium can be used to inhibit cardiac fibrogenesis is unknown. Migration and proliferation assays, Western blotting, real-time reverse-transcription polymerase chain reaction analysis, and calcium fluorescence imaging were performed in human cardiac fibroblasts treated with or without LiCl at 1.0 mM (i.e., therapeutic peak level) or 0.1 mM (i.e., therapeutic trough level) for 24 h. Results showed that LiCl (0.1 mM, but not 1.0 mM) inhibited the migration and collagen synthesis ability of cardiac fibroblasts. Additionally, thapsigargin-induced SOCE was reduced in fibroblasts treated with LiCl (0.1 mM). The expression level of Orai1 was lower in LiCl (0.1 mM)-treated fibroblasts relative to the fibroblasts without LiCl treatment. Fibroblasts treated with a combination of LiCl (0.1 mM) and 2-APB (10 μM, an Orai1 inhibitor) demonstrated similar migration and collagen synthesis abilities as those in LiCl (0.1 mM)-treated fibroblasts. Altogether, lithium at therapeutic trough levels reduced the migration and collagen synthesis abilities of human cardiac fibroblasts by inhibiting SOCE and Orai1 expression.

Background Calcific aortic valve disease is associated with ageing and high mortality. However, no effective pharmacological treatment has been developed. Vascular endothelial growth factor (VEGF) and its receptor are overexpressed in the calcified aortic valve tissue. However, the role of VEGF in calcific aortic valve disease pathogenesis and its underlying mechanisms remain unclear. Materials and methods Runt-related transcription factor 2 expression and calcium-related signalling were investigated in porcine valvular interstitial cells with or without human VEGF-A recombinant protein (VEGF165 , 1-100 ng/mL) treatment and/or calmodulin-dependent kinase II (CaMKII) inhibitor (KN93, 10 µmol/L) and inositol triphosphate receptor inhibitor (2-aminoethyldiphenyl borate, 30 µmol/L) for 5 days. Results VEGF165 -treated cells had higher Runt-related transcription factor 2 expression and CaMKII/ adenosine 3',5'-monophosphate response element-binding protein (CREB) signalling activation than did control cells. KN93 reduced Runt-related transcription factor 2 expression and CREB phosphorylation in VEGF165 -treated cells. The 2-aminoethyldiphenyl borate also reduced Runt-related transcription factor 2 expression in VICs treated with VEGF165 . Conclusion VEGF upregulated Runt-related transcription factor 2 expression in VICs by activating the IP3R/CaMKII/CREB signalling pathway.

Background Rivaroxaban, a widely used factor Xa inhibitor in reducing stroke in atrial fibrillation (AF) patients has multiple biological effects with activation of protease-activated receptor (PAR) signaling. Atrial fibrosis plays a critical role in the pathophysiology of AF. In this study, we evaluated whether rivaroxaban regulates atrial fibroblast activity and its underlying mechanisms. Methods and results Migration, proliferation analyses, nitric oxide (NO) production assay, calcium fluorescence imaging, and western blots were conducted in human atrial fibroblasts with or without rivaroxaban (100 nmol/L or 300 nmol/L) and co-administration of L-NAME (L-NG-nitro arginine methyl ester, 100 μmol/L), EGTA (Ethylene glycol tetra-acetic acid, 1 mmol/L), thrombin (0.5 U/mL), PAR1 agonist peptide (TFLLR-NH2, 100 μmol/L), PAR1 inhibitor (SCH79797, 0.5 μmol/L) and PAR2 inhibitor (GB83, 10 μmol/L). Atrial fibrosis was examined in isoproterenol (100 mg/kg, subcutaneous injection)-treated rats with or without rivaroxaban (10 mg/kg/day orally for 14 consecutive days). Rivaroxaban reduced the migration, pro-collagen type I production, and proliferation of atrial fibroblasts. Rivaroxaban decreased phosphorylated endothelial NO synthase (eNOS) (Thr 495, an inhibitory phosphorylated site of eNOS), and calcium (Ca2+) entry, and increased NO production. Moreover, L-NAME blocked the effects of rivaroxaban on fibroblast collagen and NO production. In the presence of EGTA, the migratory capability was similarly decreased in atrial fibroblasts with and without treatment with rivaroxaban (100 nmol/L), which suggests that rivaroxaban decreases migratory capability of atrial fibroblasts by inhibiting Ca2+ entry. Additionally, rivaroxaban significantly attenuated the effects of thrombin, and TFLLR-NH2 on migratory, proliferative, and pro-collagen type I production capability in atrial fibroblasts. SCH79797 or GB83 decreased pro-collagen type I production, migration, and proliferation capability in fibroblasts, but combined SCH79797 or GB83 with and without rivaroxaban had similar fibroblast activity. Moreover, rivaroxaban significantly decreased atrial fibrosis in isoproterenol-treated rats. Conclusions Rivaroxaban (100–300 nmol/L) regulates atrial fibroblast activity and atrial fibrosis by increasing NO production and decreasing Ca2+ entry through inhibition of PAR signaling.

Rivaroxaban, a direct factor Xa inhibitor, is widely used to reduce the chance of stroke in patients with atrial fibrillation (AF). It is not clear why the prothrombin time (PT) of the international normalized ratio (INR) fails to correlate with treatment using rivaroxaban in patients with AF. In this study, patient characteristics, the rivaroxaban dosage, AF type, drug history, biochemical properties, and hematological profiles were assessed in patients treated with rivaroxaban. In 69 patients with AF receiving rivaroxaban, 27 (39.1%) patients had a normal INR (≤1.1, group 1), 27 (39.1%) patients had a slightly prolonged INR (1.1∼1.5, group 2), and 15 (21.7%) patients had a significantly prolonged INR (>1.5, group 3). Group 1 patients had a higher incidence of a stroke history than did patients in group 2 ( P = .026) and group 3 ( P = .032). We scored patients with a persistent AF pattern (1 point), paroxysmal AF pattern (0 point), renal function (ie, the creatinine clearance rate in mL/min/1.73 m2 of >60 as 0 points, of 30∼60 as 1 point, and of

Atrial fibrillation (AF) is a common form of arrhythmia with serious public health impacts, but its underlying mechanisms are not yet fully understood. Vascular endothelial growth factor (VEGF) is highly expressed in the atrium of patients with AF, but whether VEGF affects AF pathogenesis remains unclear. Pulmonary veins (PVs) are important sources for the genesis of atrial tachycardia or AF. Therefore, this study assessed the effects of VEGF on PV electrophysiological properties and evaluated its underlying mechanisms. Conventional microelectrodes and whole-cell patch clamps were performed using isolated rabbit PV preparations or single isolated PV cardiomyocytes before and after VEGF or VEGF receptor (VEGFR), Akt, NOS inhibitor administration. We found that VEGF (0.1, 1, and 10 ng/mL) reduced the PV beating rate in a dose-dependent manner. Furthermore, VEGF (10 ng/mL) reduced late diastolic depolarization and diastolic tension. Isoproterenol increased PV beating and burst firing, which was attenuated by VEGF (1 ng/mL). In the presence of VEGFR-1 inhibition (ZM306416 at 10 μM) and L-NAME (100 μM), VEGF (1 ng/mL) did not alter PV spontaneous activity. In isolated PV cardiomyocytes, VEGF (1 ng/mL) decreased L-type calcium, sodium/calcium exchanger, and late sodium currents. In conclusion, we found that VEGF reduces PV arrhythmogenesis by modulating sodium/calcium homeostasis through VEGFR-1/NOS signaling pathway.

Vascular endothelial growth factor (VEGF) inhibitors, including monoclonal antibodies and tyrosine kinase inhibitors (TKIs), are important as anticancer treatments through curbing tumour angiogenesis and growth. VEGF inhibitors have significant cardiovascular effects. By blocking VEGF receptors, ligands, or signal pathways, VEGF inhibitors disturb the balance between vasodilation and vasoconstriction, undermine endothelial cell integrity, and activate cardiomyocyte apoptosis. VEGF inhibitors increase risks of hypertension, heart failure, thromboembolism and arrhythmia. Genetic and geographic studies showed that genetic polymorphisms likely play significant predictive or prognostic roles in cardiovascular toxicity associated with VEGF inhibitors. This review updates current understandings of VEGF inhibitors on cardiovascular toxicity, explores potential mechanisms, and clarifies whether genetic or ethnic factors contribute to their adverse effects. Key Messages VEGF inhibitors disturb the balance between vasodilation and vasoconstriction, undermine endothelial cell integrity and activate cardiomyocyte apoptosis. VEGF inhibitors increase risks of hypertension, heart failure, thromboembolism and arrhythmia. Genetic and geographic studies showed that genetic polymorphisms likely play significant predictive or prognostic roles in cardiovascular toxicity associated with VEGF inhibitors.

Background A Pitx2c deficiency increases the risk of atrial fibrillation (AF). Atrial structural remodelling with fibrosis blocks electrical conduction and leads to arrhythmogenesis. A Pitx2c deficiency enhances profibrotic transforming growth factor (TGF)-β expression and calcium dysregulation, suggesting that Pitx2c may play a role in atrial fibrosis. The purposes of this study were to evaluate whether a Pitx2c deficiency modulates cardiac fibroblast activity and study the underlying mechanisms. Materials and methods A migration assay, proliferation analysis, Western blot analysis and calcium fluorescence imaging were conducted in Pitx2c-knockdown human atrial fibroblasts (HAFs) using short hairpin (sh)RNA or small interfering (si)RNA. Results Compared to control HAFs, Pitx2c-knockdown HAFs had a greater migration but a similar proliferative ability. Pitx2c-knockdown HAFs had a higher calcium influx with enhanced phosphorylation of calmodulin kinase II (CaMKII), α-smooth muscle actin and matrix metalloproteinase-2. In the presence of a CaMKII inhibitor (KN-93, 0.5 μmol/L), control and Pitx2c-knockdown HAFs exhibited similar migratory abilities. Conclusion These findings suggest that downregulation of Pitx2c may regulate atrial fibrosis through modulating calcium homeostasis, which may contribute to its role in anti-atrial fibrosis, and Pitx2c downregulation may change the atrial electrophysiology and AF occurrence through modulating fibroblast activity.

Diabetes mellitus (DM) has significant effects on cardiac calcium (Ca2+) and sodium (Na+) regulation. Clinical studies have shown that empagliflozin (Jardiance&trade, ) has cardiovascular benefits, however the mechanisms have not been fully elucidated. This study aimed to investigate whether empagliflozin modulates cardiac electrical activity as well as Ca2+/Na+ homeostasis in DM cardiomyopathy. Electrocardiography, echocardiography, whole-cell patch-clamp, confocal microscopic examinations, and Western blot, were performed in the ventricular myocytes of control and streptozotocin-induced DM rats, with or without empagliflozin (10 mg/kg for 4 weeks). The results showed that the control and empagliflozin-treated DM rats had smaller left ventricular end-diastolic diameters and shorter QT intervals than the DM rats. In addition, the prolonged action potential duration in the DM rats was attenuated in the empagliflozin-treated DM rats. Moreover, the DM rats had smaller sarcoplasmic reticular Ca2+ contents, intracellular Ca2+ transients, L-type Ca2+, reverse mode Na+-Ca2+exchanger currents, lower protein expressions of sarcoplasmic reticulum ATPase, ryanodine receptor 2 (RyR2), but higher protein expressions of phosphorylated RyR2 at serine 2808 than the control and empagliflozin-treated DM rats. The incidence and frequency of Ca2+ sparks, cytosolic and mitochondrial reactive oxygen species, and late Na+ current and Na+/hydrogen-exchanger currents were greater in the DM rats than in the control and empagliflozin-treated DM rats. Empagliflozin significantly changed Ca2+ regulation, late Na+ and Na+/hydrogen-exchanger currents and electrophysiological characteristics in DM cardiomyopathy, which may contribute to its cardioprotective benefits in DM patients.

// Shih-Yu Huang 1,2 , Yao-Chang Chen 3 , Yu-Hsun Kao 1,4 , Ming-Hsiung Hsieh 5,6 , Yung-Kuo Lin 5,6 , Cheng-Chih Chung 1,5 , Ting-I Lee 7,8 , Wen-Chin Tsai 9 , Shih-Ann Chen 10 and Yi-Jen Chen 1,5 1 Graduate Institute of Clinical Medicine, College of Medicine, Taipei Medical University, Taipei, Taiwan 2 Division of Cardiology, Department of Internal Medicine, Cathay General Hospital, Taipei, Taiwan 3 Department of Biomedical Engineering, National Defense Medical Center, Taipei, Taiwan 4 Department of Medical Education and Research, Wan Fang Hospital, Taipei Medical University, Taipei, Taiwan 5 Division of Cardiovascular Medicine, Department of Internal Medicine, Wan Fang Hospital, Taipei Medical University, Taipei, Taiwan 6 Division of Cardiology, Department of Internal Medicine, School of Medicine, College of Medicine, Taipei Medical University, Taipei, Taiwan 7 Department of General Medicine, School of Medicine, College of Medicine, Taipei Medical University, Taipei, Taiwan 8 Division of Endocrinology and Metabolism, Department of Internal Medicine, Wan Fang Hospital, Taipei Medical University, Taipei, Taiwan 9 Division of Cardiology, Tzu-Chi General Hospital, Institute of Medical Sciences, Tzu-Chi University, Hualien, Taiwan 10 Division of Cardiology and Cardiovascular Research Center, Veterans General Hospital-Taipei, Taipei, Taiwan Correspondence to: Yi-Jen Chen, email: // Keywords : atrial fibrillation, calcium regulation, fibroblast growth factor-23, pulmonary vein, late sodium current, Pathology Section Received : July 25, 2016 Accepted : September 29, 2016 Published : October 04, 2016 Abstract Fibroblast growth factor 23 (FGF23), elevated in chronic renal failure, increases atrial arrhythmogenesis and dysregulates calcium homeostasis. Late sodium currents (I Na-Late ) critically induces ectopic activity of pulmoanry vein (the most important atrial fibrillation trigger). This study was to investigate whether FGF23 activates the I Na-Late leading to calcium dysregulation and increases PV arrhythmogenesis. Patch clamp, western blot, and confocal microscopy were used to evaluate the electrical activities, calcium homeostasis, and mitochondrial reactive oxygen species (ROS) in PV cardiomyocytes with or without FGF23 (0.1 or 1 ng/mL) incubation for 4~6 h. Compared to the control, FGF23 (1 ng/mL, but not 0.1 ng/mL)-treated PV cardiomyocytes had a faster beating rate. FGF23 (1 ng/mL)-treated PV cardiomyocytes had larger I Na-Late , calcium transients, and mitochondrial ROS than controls. However, ranolazine (an inhibitor of I Na-Late ) attenuated FGF23 (1 ng/mL)-increased beating rates, calcium transients and mitochondrial ROS. FGF23 (1 ng/mL)-treated PV cardiomyocytes exhibited larger phosphorylation of calcium/calmodulin-dependent protein kinase II (CaMKII). Chelerythrine chloride (an inhibitor of protein kinase C) decreased I Na-Late in FGF23 (1 ng/mL)-treated PV cardiomyocytes. However, KN93 (a selective CaMKII blocker) decreased I Na-Late in control and FGF23 (1 ng/mL)-treated PV cardiomyocytes to a similar extent. In conclusion, FGF23 increased PV arrhythmogenesis through sodium and calcium dysregulation by acting protein kinase C signaling.

The extracellular matrix (ECM) and transforming growth factor-β (TGF)-β are important in cardiac fibrosis, however, the effects of the ECM on TGF‑β signaling remain to be fully elucidated. The aims of the present study were to evaluate the role of collagen in TGF‑β signaling and examine the underlying mechanisms. In the present study, western blot analysis was used to examine TGF‑β signaling in HL‑1 cells treated with and without (control) type I collagen (10 µg/ml), which was co‑administered with either an anti‑β1 integrin antibody (10 µg/ml) or a stretch‑activated channel inhibitor (gadolinium; 50 µM). Cell proliferation and adhesion assays were used to investigate the roles of integrin, mechanical stretch and mitogen‑activated protein kinases (MAPKs) on cell proliferation and adhesion. The type I collagen (10 µg/ml)‑treated HL‑1 cells were incubated with or without anti‑β1 integrin antibody (10 µg/ml), gadolinium (50 µM) or inhibitors of p38 (SB203580; 3 µM), extracellular signal‑regulated kinase (ERK; PD98059; 50 µM) and c‑Jun N‑terminal kinase (JNK; SP600125; 50 µM). Compared with the control cells, the collagen‑treated HL‑1 cells had lower expression levels of type I and type II TGF‑β receptors (TGFβRI and TGFβRII), with an increase in phosphorylated focal adhesion kinase (FAK), p38 and ERK1/2, and a decrease in JNK. Incubation with the anti‑β1 integrin antibody reversed the collagen‑induced downregulation of the expression of TGFβRII and phosphorylated FAK. Gadolinium downregulated the expression levels of TGFβRI and small mothers against decapentaplegic (Smad)2/3, and decreased the levels of phosphorylated p38, ERK1/2 and JNK. In addition, gadolinium reversed the collagen‑induced activation of p38 and ERK1/2. In the presence of gadolinium and anti‑β1 integrin antibody, collagen regulated the expression levels of TGFβRI, TGFβRII and Smad2/3, but did not alter the phosphorylation of p38, ERK1/2 or JNK. In addition, collagen increased cell proliferation and adhesion, and this collagen‑induced cell proliferation was inhibited by the anti‑β1 integrin antibody and ERK inhibitor. Taken together, the data obtained suggested that collagen differentially regulated the expression levels of TGFβRI and TGFβRII, and modulated the phosphorylation of MAPKs through integrin‑ or stretch‑dependent and ‑independent signaling pathways.

a Graduate Institute of Clinical Medicine, College of Medicine, Taipei Medical University, Taipei, Taiwan b Division of Cardiology, Chi-Mei Medical Center, Tainan, Taiwan c School of Medicine, Chung Shan Medical University, Taichung, Taiwan d Department of Medical Education and Research, Wan Fang Hospital, Taipei Medical University, Taipei, Taiwan e Department of Biomedical Engineering, National Defense Medical Center, Taipei, Taiwan f Division of Cardiovascular Medicine, Department of Internal Medicine, Wan Fang Hospital, Taipei Medical University, Taipei, Taiwan

Autophagy serves a role in preserving cellular homeostasis. Diabetes mellitus (DM) impairs cardiac autophagy and is associated with an accumulation of cytotoxic proteins that may provoke apoptosis and damage cardiomyocytes. Histone deacetylase (HDAC) inhibitors attenuate cardiac fibrosis and inflammation, and improve cardiomyopathy resulting from DM. However, the effect of HDAC inhibition on autophagy in DM cardiomyopathy has not been investigated. The purpose of the present study was to evaluate whether HDAC inhibition modulates cardiac autophagy and to investigate the potential mechanisms in type 2 DM (T2DM) hearts. Electrocardiography was used to evaluate cardiac function and western blotting was used to evaluate protein expression in autophagy, the serine/threonine protein kinase mTOR (mTOR) signaling pathway, poly adenosine diphosphate ribose polymerase 1 (PARP1), insulin signaling, advanced glycosylation end product‑specific receptor (RAGE), and proinflammatory cytokines in control rats and in rats treated with a high‑fat diet (60% fat) and low‑dose streptozotocin (35 mg/kg) in order to induce T2DM, with or without an HDAC inhibitor (MPT0E014; 50 mg/kg/rat daily for 7 days). Compared with the control rats, T2DM and T2DM rats treated with MPT0E014 exhibited elevated blood glucose levels and similar body weights. However, T2DM rats treated with MPT0E014 and control rats had a smaller left ventricular end‑diastolic diameter compared with the T2DM rats. The control and T2DM rats treated with MPT0E014 had greater protein expression of cardiac phosphorylated (p)‑5' adenosine monophosphate‑activated protein kinase α 2, light chain 3‑II, Beclin‑1, glucose transporter 4, p‑protein kinase B, and insulin receptor substrate‑1 (Ser 307) compared with T2DM rats. In addition, control and T2DM rats treated with MPT0E014 had decreased cardiac protein expression of cleaved PARP1, p‑mTOR‑S2448, p‑P70S6K‑Thr‑389, RAGE, tumor necrosis factor‑α, and interleukin‑6 compared with T2DM rats. The present study demonstrated that MPT0E014 may improve cardiac function in T2DM rats by modulating myocardial autophagy, inflammation and insulin signaling.

Background Androgen deficiency produces heart failure, which can be ameliorated by testosterone supplementation. Cardiac fibrosis plays a critical role in the pathophysiology of heart failure. This study aimed to evaluate whether testosterone can attenuate cardiac fibroblast activity through modulating transforming growth factor (TGF)-β and angiotensin (Ang) II signaling. Methods Migration, proliferation, myofibroblast differentiation, collagen production, and transcription signaling were evaluated in adult male rat (weighing 300–350g) cardiac fibroblasts with and without incubation with testosterone (10nM) and co-administration of TGF-β1 (10ng/ml) or Ang II (100nM) by cell migration analysis, proliferation assay, soluble collagen measurement, zymographic analysis, immunofluorescence microscopy, real-time PCR and Western blot. Results Compared to those without testosterone, testosterone-treated fibroblasts exhibited less collagen production. Testosterone-treated fibroblasts also had less migration, proliferation, myofibroblast differentiation, and collagen production in the presence of TGF-β1, or had less collagen production with Ang II. Testosterone-treated fibroblasts had decreased phosphorylated Akt, mammalian target of rapamycin, and 4E binding protein-1 irrespective of TGF-β1 treatment and had increased matrix metalloproteinase (MMP)-2 in the presence of TGF-β1 treatment, and had decreased phosphorylated P38 and Smad 2/3 levels in the presence of Ang II. Cardiac fibroblasts with and without testosterone had similar mRNA and protein expressions of total Akt and total Smad 2/3 irrespective of TGF-β1 or Ang II treatment. Conclusion Physiological level of testosterone attenuated Akt and Smad 2/3 phosphorylation mediated by TGF-β1 and angiotensin II respectively, which can result in decreased cardiac fibroblast activation and potentially contribute to beneficial effects in heart failure.

Chronic heart failure (HF) is a major health problem throughout the world. Gender has significant effects on the pathophysiology of HF. Low levels of free testosterone are independently associated with increased chronic HF symptoms and mortality. Cardiac fibrosis plays a pivotal role in structural remodeling in HF. Transforming growth factor (TGF)-β, angiotensin II and oxidative stress contribute to the activity/extent of cardiac fibrosis. Androgen deficiency can up-regulate TGF-β expression under angiotensin II stimulation in vivo. In this review, we summarized the potential mechanisms accounting for the effects of androgen on cardiac fibrosis through regulating fibrocytes activity under TGF, which can explain wound healing and cardiac fibrosis in male with acute myocardial injury and chronic HF.

Aim Atrial fibrosis plays a pivotal role in the pathophysiology of heart failure (HF). The left atrium (LA) experiences greater fibrosis than the right atrium (RA) during HF. It is not clear whether LA cardiac fibroblasts contain distinctive activities that predispose LA to fibrosis. Methods LA and RA fibrosis were evaluated in healthy and isoproterenol induced HF Sprague-Dawley rats. Rat LA and RA primary isolated fibroblasts were subjected to proliferation assay, oxidative stress assay, cell migration analysis, collagen measurement, cytokine array and western blot. Results Healthy rat LA and RA had a similar extent of collagen deposition. HF significantly increased fibrosis to a greater severity in LA than in RA. Compared to isolated RA fibroblasts, the in vitro experiments showed that isolated LA fibroblasts had higher oxidative stress and exhibited higher collagen, transforming growth factor-β1, connective tissue growth factor production, and less vascular endothelial growth factor (VEGF) production, but had similar migration, myofibroblast differentiation and proliferation activities. VEGF significantly increased the collagen production ability of LA fibroblasts, but not RA fibroblasts. LA fibroblasts had more phosphorylated ERK1/2 and P38 expression. ERK inhibitor (PD98059, 50 μmol/L) significantly attenuated collagen production, increased VEGF production in RA fibroblasts but not in LA fibroblasts. P38 inhibitor (SB203580, 30 μmol/L) significantly attenuated collagen production in LA fibroblasts but not in RA fibroblasts. P38 inhibitor also significantly increased VEGF production in RA and LA fibroblasts. Conclusions Differences in profibrotic activity between LA and RA fibroblasts may be caused by different responses to mitogen-activated protein kinase signaling. This article is protected by copyright. All rights reserved.

Background The calcific aortic valve (AV) disease is a common disease with the unclear mechanism, and optimal pharmacological treatment remains unavailable. Epigenetic modulation by histone acetyltransferase (HAT) plays a critical role in osteogenic transdifferentiation and atherosclerosis. The purposes of this study were to investigate whether HAT contributes to the pathophysiology of AV calcification and assess the therapeutic potential of HAT inhibition. Methods Porcine valvular interstitial cells (VICs) were treated with osteogenic medium (10ng/mL of tumor necrosis factor-α and 4mmol/L of high phosphate) for 7days. We analyzed the RNA and protein expression of myofibroblastic (α-SMA, vimentin, collagen 1A1, collagen 3, Egr-1, MMP2, MMP9) and osteoblastic markers (osteocalcin and alkaline phosphatase) in VICs, and studied the effects of a p300 inhibitor (C646, 10μmol/L) on calcification (Alizarin Red S staining), osteogenesis, HAT activity, the mitogen-activated protein kinase (MAPK) and Akt pathway, and Klotho expression on VICs. Results Osteogenic medium treated VICs had higher expressions of osteocalcin, alkaline phosphatase and acetylated lysine-9 of histone H 3 (ac-H3K9) than control cells. C646 attenuated osteogenesis of VICs with simultaneous inhibition of the HAT activity of p300. There was neither significant increase of p300 protein nor p300 transcript during the osteogenesis process. Additionally, osteogenic medium treated VICs decreased the expression of Klotho, which is attenuated by C646. Conclusions Activated HAT activity of p300 modulates AV calcification through osteogenic transdifferentiation of VICs with Klotho modulation. P300 inhibition is a potential therapeutic target for AV calcification.

Cardiac fibrosis plays a critical role in the pathophysiology of cardiovascular disease. It has been observed that curcumin has several cardiovascular effects. The purpose of this study was to evaluate whether curcumin can attenuate cardiac fibroblasts activity.We evaluated the migration, proliferation, collagen production, and transcription signaling in rat cardiac fibroblasts isolated from Sprague-Dawley rats (males, weighing 300-350 g) that were or were not incubated with curcumin (25 μM) and the co-administration of transforming growth factor (TGF)- β1 (10 ng/ml) or angiotensin (Ang) II (100 nM) by a cell migration analysis, proliferation assay, and Western blot analysis. Compared to those without curcumin, curcumin-treated cardiac fibroblasts exhibited lower migratory, proliferative abilities and collagen production at the baseline and after the co-administration of TGF-β1 or Ang II. Curcumin-treated cardiac fibroblasts had increased matrix metalloproteinase (MMP)-2 activity in the presence of Ang II treatment. Curcumin-treated cardiac fibroblasts down-regulated phosphorylated protein kinase B (Akt) and phosphorylated Smad2/3 expression irrespective of TGF-β1 treatment. Curcumin also decreased phosphorylated extracellular signal-regulated kinase (ERK)1/2 levels in the presence of Ang II.Curcumin attenuated Akt, Smad2/3, and ERK1/2 phosphorylation which were mediated by TGF-β1 and angiotensin II. This resulted in decreased cardiac fibroblast activation and supports the assertion that curcumin is an effective antifibrotic agent which can be used to treat heart failure.Angiotensin; Curcumin; Fibroblasts; Heart failure; Transforming growth factor.