Empagliflozin suppressed cardiac fibrogenesis through sodium-hydrogen exchanger inhibition and modulation of the calcium homeostasis.

BACKGROUND: The novel sodium-glucose co-transporter 2 inhibitor (SGLT2i) potentially ameliorates heart failure and reduces cardiac arrhythmia. Cardiac fibrosis plays a pivotal role in the pathophysiology of HF and atrial myopathy, but the effect of SGLT2i on fibrogenesis remains to be elucidated. This study investigated whether SGLT2i directly modulates fibroblast activities and its underlying mechanisms. METHODS AND RESULTS: Migration, proliferation analyses, intracellular pH assay, intracellular inositol triphosphate (IP3) assay, Ca2+ fluorescence imaging, and Western blotting were applied to human atrial fibroblasts. Empagliflozin (an SGLT2i, 1, or 5 µmol/L) reduced migration capability and collagen type I, and III production. Compared with control cells, empagliflozin (1 µmol/L)- treated atrial fibroblasts exhibited lower endoplasmic reticulum (ER) Ca2+ leakage, Ca2+ entry, inositol trisphosphate (IP3), lower expression of phosphorylated phospholipase C (PLC), and lower intracellular pH. In the presence of cariporide (an Na+-H+ exchanger (NHE) inhibitor, 10 µmol/L), control and empagliflozin (1 µmol/L)-treated atrial fibroblasts revealed similar intracellular pH, ER Ca2+ leakage, Ca2+ entry, phosphorylated PLC, pro-collagen type I, type III protein expression, and migration capability. Moreover, empagliflozin (10 mg/kg/day orally for 28 consecutive days) significantly increased left ventricle systolic function, ß-hydroxybutyrate and decreased atrial fibrosis, in isoproterenol (100 mg/kg, subcutaneous injection)-induced HF rats. CONCLUSIONS: By inhibiting NHE, empagliflozin decreases the expression of phosphorylated PLC and IP3 production, thereby reducing ER Ca2+ release, extracellular Ca2+ entry and the profibrotic activities of atrial fibroblasts.

Silicon Oxy-Nitride for the Low Refractive Index Layers in the Mirror Coatings of the Cryogenic Laser Interferometer Gravitational Waves Detector.

The low refractive index layers in the mirror coatings of the room-temperature laser interferometer gravitational waves detectors are silica deposited by the ion beam sputter method. However, the silica film suffers from the cryogenic mechanical loss peak, hindering its application for the next generation detector operated at cryogenics. New low refractive index materials need to be explored. We study amorphous silicon oxy-nitride (SiON) films deposited using the method of plasma-enhanced chemical vapor deposition. By changing the N_{2}O/SiH_{4} flow rate ratio, we can tune the refractive index of the SiON smoothly from nitridelike to silicalike of ∼1.48 at 1064 nm, 1550 nm, and 1950 nm. Thermal anneal reduced the refractive index down to ∼1.46 and effectively reduced the absorption and cryogenic mechanical loss; the reductions correlated with the N─H bond concentration decrease. Extinction coefficients of the SiONs at the three wavelengths are reduced down to 5×10^{-6}∼3×10^{-7} by annealing. Cryogenic mechanical losses at 10 K and 20 K (for ET and KAGRA) of the annealed SiONs are significantly lower than the annealed ion beam sputter silica. They are comparable at 120 K (for LIGO-Voyager). Absorption from the vibrational modes of the N─H terminal-hydride structures dominates over the absorption from other terminal hydrides, the Urbach tail, and the silicon dangling bond states in SiON at the three wavelengths.

Ketogenic Diet Regulates Cardiac Remodeling and Calcium Homeostasis in Diabetic Rat Cardiomyopathy.

A ketogenic diet (KD) might alleviate patients with diabetic cardiomyopathy. However, the underlying mechanism remains unclear. Myocardial function and arrhythmogenesis are closely linked to calcium (Ca2+) homeostasis. We investigated the effects of a KD on Ca2+ homeostasis and electrophysiology in diabetic cardiomyopathy. Male Wistar rats were created to have diabetes mellitus (DM) using streptozotocin (65 mg/kg, intraperitoneally), and subsequently treated for 6 weeks with either a normal diet (ND) or a KD. Our electrophysiological and Western blot analyses assessed myocardial Ca2+ homeostasis in ventricular preparations in vivo. Unlike those on the KD, DM rats treated with an ND exhibited a prolonged QTc interval and action potential duration. Compared to the control and DM rats on the KD, DM rats treated with an ND also showed lower intracellular Ca2+ transients, sarcoplasmic reticular Ca2+ content, sodium (Na+)-Ca2+ exchanger currents (reverse mode), L-type Ca2+ contents, sarcoplasmic reticulum ATPase contents, Cav1.2 contents. Furthermore, these rats exhibited elevated ratios of phosphorylated to total proteins across multiple Ca2+ handling proteins, including ryanodine receptor 2 (RyR2) at serine 2808, phospholamban (PLB)-Ser16, and calmodulin-dependent protein kinase II (CaMKII). Additionally, DM rats treated with an ND demonstrated a higher frequency and incidence of Ca2+ leak, cytosolic reactive oxygen species, Na+/hydrogen-exchanger currents, and late Na+ currents than the control and DM rats on the KD. KD treatment may attenuate the effects of DM-dysregulated Na+ and Ca2+ homeostasis, contributing to its cardioprotection in DM.

Class I HDAC modulates angiotensin II-induced fibroblast migration and mitochondrial overactivity.

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.

Effect of antidiabetic drugs on the risk of atrial fibrillation: mechanistic insights from clinical evidence and translational studies.

Diabetes mellitus (DM) is an independent risk factor for atrial fibrillation (AF), which is the most common sustained arrhythmia and is associated with substantial morbidity and mortality. Advanced glycation end product and its receptor activation, cardiac energy dysmetabolism, structural and electrical remodeling, and autonomic dysfunction are implicated in AF pathophysiology in diabetic hearts. Antidiabetic drugs have been demonstrated to possess therapeutic potential for AF. However, clinical investigations of AF in patients with DM have been scant and inconclusive. This article provides a comprehensive review of research findings on the association between DM and AF and critically analyzes the effect of different pharmacological classes of antidiabetic drugs on AF.

Lithium Treatment Improves Cardiac Dysfunction in Rats Deprived of Rapid Eye Movement Sleep.

Rapid eye movement (REM) sleep deprivation triggers mania and induces cardiac fibrosis. Beyond neuroprotection, lithium has cardioprotective potential and antifibrotic activity. This study investigated whether lithium improved REM sleep deprivation-induced cardiac dysfunction and evaluated the potential mechanisms. Transthoracic echocardiography, histopathological analysis, and Western blot analysis were performed in control and REM sleep-deprived rats with or without lithium treatment (LiCl of 1 mmol/kg/day administered by oral gavage for 4 weeks) in vivo and in isolated ventricular preparations. The results revealed that REM sleep-deprived rats exhibited impaired contractility and greater fibrosis than control and lithium-treated REM sleep-deprived rats. Western blot analysis showed that REM sleep-deprived hearts had higher expression levels of transforming growth factor beta (TGF-ß), phosphorylated Smad 2/3, and alpha-smooth muscle actin than lithium-treated REM sleep-deprived and control hearts. Moreover, lithium-treated REM sleep-deprived hearts had lower expression of angiotensin II type 1 receptor, phosphorylated nuclear factor kappa B p65, calcium release-activated calcium channel protein 1, transient receptor potential canonical (TRPC) 1, and TRPC3 than REM sleep-deprived hearts. The findings suggest that lithium attenuates REM sleep deprivation-induced cardiac fibrogenesis and dysfunction possibly through the downregulation of TGF-ß, angiotensin II, and Ca2+ signaling.

Role of Endothelin-1 in Right Atrial Arrhythmogenesis in Rabbits with Monocrotaline-Induced Pulmonary Arterial Hypertension.

Atrial arrhythmias are considered prominent phenomena in pulmonary arterial hypertension (PAH) resulting from atrial electrical and structural remodeling. Endothelin (ET)-1 levels correlate with PAH severity and are associated with atrial remodeling and arrhythmia. In this study, hemodynamic measurement, western blot analysis, and histopathology were performed in the control and monocrotaline (MCT, 60 mg/kg)-induced PAH rabbits. Conventional microelectrodes were used to simultaneously record the electrical activity in the isolated sinoatrial node (SAN) and right atrium (RA) tissue preparations before and after ET-1 (10 nM) or BQ-485 (an ET-A receptor antagonist, 100 nM) perfusion. MCT-treated rabbits showed an increased relative wall thickness in the pulmonary arterioles, mean cell width, cross-sectional area of RV myocytes, and higher right ventricular systolic pressure, which were deemed to have PAH. Compared to the control, the spontaneous beating rate of SAN-RA preparations was faster in the MCT-induced PAH group, which can be slowed down by ET-1. MCT-induced PAH rabbits had a higher incidence of sinoatrial conduction blocks, and ET-1 can induce atrial premature beats or short runs of intra-atrial reentrant tachycardia. BQ 485 administration can mitigate ET-1-induced RA arrhythmogenesis in MCT-induced PAH. The RA specimens from MCT-induced PAH rabbits had a smaller connexin 43 and larger ROCK1 and phosphorylated Akt than the control, and similar PKG and Akt to the control. In conclusion, ET-1 acts as a trigger factor to interact with the arrhythmogenic substrate to initiate and maintain atrial arrhythmias in PAH. ET-1/ET-A receptor/ROCK signaling may be a target for therapeutic interventions to treat PAH-induced atrial arrhythmias.

Calcium dysregulation increases right ventricular outflow tract arrhythmogenesis in rabbit model of chronic kidney disease.

Chronic kidney disease (CKD) increases the risk of arrhythmia. The right ventricular outflow tract (RVOT) is a crucial site of ventricular tachycardia (VT) origination. We hypothesize that CKD increases RVOT arrhythmogenesis through its effects on calcium dysregulation. We analysed measurements obtained using conventional microelectrodes, patch clamp, confocal microscopy, western blotting, immunohistochemical examination and lipid peroxidation for both control and CKD (induced by 150 mg/kg neomycin and 500 mg/kg cefazolin daily) rabbit RVOT tissues or cardiomyocytes. The RVOT of CKD rabbits exhibited a short action potential duration, high incidence of tachypacing (20 Hz)-induced sustained VT, and long duration of isoproterenol and tachypacing-induced sustained and non-sustained VT. Tachypacing-induced sustained and non-sustained VT in isoproterenol-treated CKD RVOT tissues were attenuated by KB-R7943 and partially inhibited by KN93 and H89. The CKD RVOT myocytes had high levels of phosphorylated CaMKII and PKA, and an increased expression of tyrosine hydroxylase-positive neural density. The CKD RVOT myocytes exhibited large levels of Ito , IKr , NCX and L-type calcium currents, calcium leak and malondialdehyde but low sodium current, SERCA2a activity and SR calcium content. The RVOT in CKD with oxidative stress and autonomic neuron hyperactivity exhibited calcium handling abnormalities, which contributed to the induction of VT.

Vascular endothelial growth factor on Runt-related transcript factor-2 in aortic valve cells.

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.

Physiological testosterone attenuates profibrotic activities of rat cardiac fibroblasts through modulation of nitric oxide and calcium homeostasis.

Testosterone deficiency is associated with poor prognosis among patients with chronic heart failure (HF). Physiological testosterone improves the exercise capacity of patients with HF. In this study, we evaluated whether treatment with physiological testosterone contributes to anti-fibrogenesis by modifying calcium homeostasis in cardiac fibroblasts and we studied the underlying mechanisms. Nitric oxide (NO) analyses, calcium (Ca2+) fluorescence, and Western blotting were performed in primary isolated rat cardiac fibroblasts with or without (control cells) testosterone (10, 100, 1,000 nmol/L) treatment for 48 hours. Physiological testosterone (10 nmol/L) increased NO production and phosphorylation at the inhibitory site of the inositol trisphosphate (IP3) receptor, thereby reducing Ca2+ entry, phosphorylated Ca2+/calmodulin-dependent protein kinase II (CaMKII) expression, type I and type III pro-collagen production. Non-physiological testosterone-treated fibroblasts exhibited similar NO and collagen production capabilities as compared to control (testosterone deficient) fibroblasts. These effects were blocked by co-treatment with NO inhibitor (L-NG-nitro arginine methyl ester [L-NAME], 100 µmol/L). In the presence of the IP3 receptor inhibitor (2-aminoethyl diphenylborinate [2-APB], 50 µmol/L), testosterone-deficient and physiological testosterone-treated fibroblasts exhibited similar phosphorylated CaMKII expression. When treated with 2-APB or CaMKII inhibitor (KN93, 10 µmol/L), testosterone-deficient and physiological testosterone-treated fibroblasts exhibited similar type I, and type III collagen production. In conclusion, physiological testosterone activates NO production, and attenuates the IP3 receptor/Ca2+ entry/CaMKII signaling pathway, thereby inhibiting the collagen production capability of cardiac fibroblasts.

Lithium Reduces Migration and Collagen Synthesis Activity in Human Cardiac Fibroblasts by Inhibiting Store-Operated Ca2+ Entry.

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.

Fibroblast Growth Factor 23 Stimulates Cardiac Fibroblast Activity through Phospholipase C-Mediated Calcium Signaling.

Fibroblast growth factor (FGF)-23 induces hypertrophy and calcium (Ca2+) dysregulation in cardiomyocytes, leading to cardiac arrhythmia and heart failure. However, knowledge regarding the effects of FGF-23 on cardiac fibrogenesis remains limited. This study investigated whether FGF-23 modulates cardiac fibroblast activity and explored its underlying mechanisms. We performed MTS analysis, 5-ethynyl-2'-deoxyuridine assay, and wound-healing assay in cultured human atrial fibroblasts without and with FGF-23 (1, 5 and 25 ng/mL for 48 h) to analyze cell proliferation and migration. We found that FGF-23 (25 ng/mL, but not 1 or 5 ng/mL) increased proliferative and migratory abilities of human atrial fibroblasts. Compared to control cells, FGF-23 (25 ng/mL)-treated fibroblasts had a significantly higher Ca2+ entry and intracellular inositol 1,4,5-trisphosphate (IP3) level (assessed by fura-2 ratiometric Ca2+ imaging and enzyme-linked immunosorbent assay). Western blot analysis showed that FGF-23 (25 ng/mL)-treated cardiac fibroblasts had higher expression levels of calcium release-activated calcium channel protein 1 (Orai1) and transient receptor potential canonical (TRPC) 1 channel, but similar expression levels of α-smooth muscle actin, collagen type IA1, collagen type Ⅲ, stromal interaction molecule 1, TRPC 3, TRPC6 and phosphorylated-calcium/calmodulin-dependent protein kinase II when compared with control fibroblasts. In the presence of ethylene glycol tetra-acetic acid (a free Ca2+ chelator, 1 mM) or U73122 (an inhibitor of phospholipase C, 1 µM), control and FGF-23-treated fibroblasts exhibited similar proliferative and migratory abilities. Moreover, polymerase chain reaction analysis revealed that atrial fibroblasts abundantly expressed FGF receptor 1 but lacked expressions of FGF receptors 2-4. FGF-23 significantly increased the phosphorylation of FGF receptor 1. Treatment with PD166866 (an antagonist of FGF receptor 1, 1 µM) attenuated the effects of FGF-23 on cardiac fibroblast activity. In conclusion, FGF-23 may activate FGF receptor 1 and subsequently phospholipase C/IP3 signaling pathway, leading to an upregulation of Orai1 and/or TRPC1-mediated Ca2+ entry and thus enhancing human atrial fibroblast activity.

Empagliflozin and Liraglutide Differentially Modulate Cardiac Metabolism in Diabetic Cardiomyopathy in Rats.

Glucagon-like peptide 1 receptor agonists (GLP-1RAs) and sodium-glucose cotransporter-2 inhibitors (SGLT2is) are antihyperglycemic agents with cardioprotective properties against diabetic cardiomyopathy (DCM). However, the distinctive mechanisms underlying GLP-1RAs and SGLT2is in DCM are not fully elucidated. The purpose of this study was to investigate the impacts of GLP1RAs and/or SGLT2is on myocardial energy metabolism, cardiac function, and apoptosis signaling in DCM. Biochemistry and echocardiograms were studied before and after treatment with empagliflozin (10 mg/kg/day, oral gavage), and/or liraglutide (200 µg/kg every 12 h, subcutaneously) for 4 weeks in male Wistar rats with streptozotocin (65 mg/kg intraperitoneally)-induced diabetes. Cardiac fibrosis, apoptosis, and protein expression of metabolic and inflammatory signaling molecules were evaluated by histopathology and Western blotting in ventricular cardiomyocytes of different groups. Empagliflozin and liraglutide normalized myocardial dysfunction in diabetic rats. Upregulation of phosphorylated-acetyl coenzyme A carboxylase, carnitine palmitoyltransferase 1ß, cluster of differentiation 36, and peroxisome proliferator-activated receptor-gamma coactivator, and downregulation of glucose transporter 4, the ratio of phosphorylated adenosine monophosphate-activated protein kinase α2 to adenosine monophosphate-activated protein kinase α2, and the ratio of phosphorylated protein kinase B to protein kinase B in diabetic cardiomyocytes were restored by treatment with empagliflozin or liraglutide. Nucleotide-binding oligomerization domain, leucine-rich repeat and pyrin domain-containing 3, interleukin-1ß, tumor necrosis factor-α, and cleaved caspase-1 were significantly downregulated in empagliflozin-treated and liraglutide-treated diabetic rats. Both empagliflozin-treated and liraglutide-treated diabetic rats exhibited attenuated myocardial fibrosis and apoptosis. Empagliflozin modulated fatty acid and glucose metabolism, while liraglutide regulated inflammation and apoptosis in DCM. The better effects of combined treatment with GLP-1RAs and SGLT2is may lead to a potential strategy targeting DCM.

Vascular endothelial growth factor enhances profibrotic activities through modulation of calcium homeostasis in human atrial fibroblasts.

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.

Clinical Risk Factors for Therapeutic Lithium-Associated Electrocardiographic Changes in Patients With Bipolar Disorder.

PURPOSE/BACKGROUND: Lithium, a common medication used in bipolar disorder treatment, can exert an inhibitory effect on sodium and potassium channels and potentially cause cardiac electrical conduction disturbance and corrected QT (QTc) prolongation. This study aimed to examine whether lithium at therapeutic levels can change electrocardiographic parameters in different groups of patients with bipolar disorder and to identify the potential clinical risk factors. METHODS/PROCEDURES: Standard 12-lead electrocardiogram data before and after lithium treatment in bipolar disorder patients after at least 2-week dropout of psychotropic medications were analyzed. FINDINGS/RESULTS: A total of 39 patients with bipolar disorder receiving lithium treatment were enrolled. Nineteen patients (48.7%) exhibited increased from P wave beginning to QRS complex beginning intervals after lithium treatment (mean serum level, 0.653 ± 0.247 mmol/L). Twenty-four patients (61.5%) exhibited increased a combination of Q, R, and S waves complex durations and increased QTc intervals. Twenty-three patients (59.0%) exhibited increased corrected JT (JTc) intervals. The patient group with increased QTc or JTc intervals exhibited a higher mean systolic blood pressure than did the patient group without increased QTc (134.7 ± 19.2 mm Hg vs 115.7 ± 11.8 mm Hg, P = 0.020) or JTc intervals (134.4 ± 19.6 mm Hg vs 117.6 ± 13.3 mm Hg, P = 0.054), respectively. Biochemical and hemodynamic parameters were comparable between patients with and without increased a combination of Q, R, and S waves complex durations or from P wave beginning to QRS complex beginning intervals. IMPLICATIONS/CONCLUSIONS: Elevated systolic blood pressure may be the risk factor for the ventricular conduction delay in bipolar disorder patients receiving lithium at therapeutic levels.

Klotho modulates electrical activity and calcium homeostasis in pulmonary vein cardiomyocytes via PI3K/Akt signalling.

AIMS: Klotho, a potential antiageing protein has remarkable cardiovascular effects, which is lower in the patients with chronic kidney disease (CKD). Chronic kidney disease increases the risk of atrial fibrillation, majorly triggered by pulmonary vein (PV) arrhythmogenesis. This study investigated whether klotho protein can modulate PV electrical activity and the underlying potential mechanisms. METHODS AND RESULTS: A conventional microelectrode and whole-cell patch clamp were used to investigate the action potentials and ionic currents in isolated rabbit PV tissue preparations and single cardiomyocytes before and after klotho administration. Phosphoinositide 3-kinase (PI3K)/Akt signalling was studied using western blotting. Klotho significantly reduced PV spontaneous beating rates in PV tissue preparations at 1.0 and 3.0 ng/mL (but not at 0.1 and 0.3 ng/mL). In the presence of the Akt inhibitor (10 µM), klotho (1.0 and 3.0 ng/mL) did not change PV electrical activities. Klotho (1.0 ng/mL) significantly decreased the late sodium current (INa-Late) and L-type calcium current (ICa-L), similar to the Akt inhibitor (10 µM). Western blots demonstrated that klotho (1.0 ng/mL)-treated PV cardiomyocytes had less phosphorylation of Akt (Ser473) compared with klotho-untreated cardiomyocytes. Compared with control PVs, klotho at relatively lower concentrations (0.1 and 0.3 ng/mL) significantly reduced beating rates and decreased the amplitudes of delay afterdepolarizations in CKD PVs. CONCLUSION: Klotho modulated PV electrical activity by inhibiting PI3K/Akt signalling, which may provide a novel insight into CKD-induced arrhythmogenesis.

Therapeutic potential of vitamin D in AGE/RAGE-related cardiovascular diseases.

Cardiovascular diseases (CVDs) are among the leading threats to human health. The advanced glycation end product (AGE) and receptor for AGE (RAGE) signaling pathway regulates the pathogenesis of CVDs, through its effects on arterial stiffness, atherosclerosis, mitochondrial dysfunction, oxidative stress, calcium homeostasis, and cytoskeletal function. Targeting the AGE/RAGE pathway is a potential therapeutic strategy for ameliorating CVDs. Vitamin D has several beneficial effects on the cardiovascular system. Experimental findings have shown that vitamin D regulates AGE/RAGE signaling and its downstream effects. This article provides a comprehensive review of the mechanistic insights into AGE/RAGE involvement in CVDs and the modulation of the AGE/RAGE signaling pathways by vitamin D.

Sodium hydrosulphide restores tumour necrosis factor-α-induced mitochondrial dysfunction and metabolic dysregulation in HL-1 cells.

Tumour necrosis factor (TNF)-α induces cardiac metabolic disorder and mitochondrial dysfunction. Hydrogen sulphide (H2 S) contains anti-inflammatory and biological effects in cardiomyocytes. This study investigated whether H2 S modulates TNF-α-dysregulated mitochondrial function and metabolism in cardiomyocytes. HL-1 cells were incubated with TNF-α (25 ng/mL) with or without sodium hydrosulphide (NaHS, 0.1 mmol/L) for 24 hours. Cardiac peroxisome proliferator-activated receptor (PPAR) isoforms, pro-inflammatory cytokines, receptor for advanced glycation end products (RAGE) and fatty acid metabolism were evaluated through Western blotting. The mitochondrial oxygen consumption rate and adenosine triphosphate (ATP) production were investigated using Seahorse XF24 extracellular flux analyzer and bioluminescence assay. Fluorescence intensity using 2', 7'-dichlorodihydrofluorescein diacetate was used to evaluate mitochondrial oxidative stress. NaHS attenuated the impaired basal and maximal respiration, ATP production and ATP synthesis and enhanced mitochondrial oxidative stress in TNF-α-treated HL-1 cells. TNF-α-treated HL-1 cells exhibited lower expression of PPAR-α, PPAR-δ, phosphorylated 5' adenosine monophosphate-activated protein kinase-α2, phosphorylated acetyl CoA carboxylase, carnitine palmitoyltransferase-1, PPAR-γ coactivator 1-α and diacylglycerol acyltransferase 1 protein, but higher expression of PPAR-γ, interleukin-6 and RAGE protein than control or combined NaHS and TNF-α-treated HL-1 cells. NaHS modulates the effects of TNF-α on mitochondria and the cardiometabolic system, suggesting its therapeutic potential for inflammation-induced cardiac dysfunction.

Pitx2c inhibition increases atrial fibroblast activity: Implications in atrial arrhythmogenesis.

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)-ß 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.

Empagliflozin Attenuates Myocardial Sodium and Calcium Dysregulation and Reverses Cardiac Remodeling in Streptozotocin-Induced Diabetic Rats.

Diabetes mellitus (DM) has significant effects on cardiac calcium (Ca2+) and sodium (Na⁺) regulation. Clinical studies have shown that empagliflozin (Jardiance™) 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.