Acacetin alleviates autoimmune myocarditis by regulating CD4+ T cell mitochondrial respiration.

BACKGROUND: Myocarditis refers to an autoimmune inflammatory response of the myocardium with characterization of self-reactive CD4+ T cell activation, which lacks effective treatment and has a poor prognosis. Acacetin is a natural flavonoid product that has been reported to have anti-inflammatory effects. However, acacetin has not been investigated in myocarditis. METHODS: Oral acacetin treatment was administered in an experimental autoimmune myocarditis model established with myosin heavy chain-alpha peptide. Echocardiography, pathological staining, and RT-qPCR were used to detect cardiac function, myocardial injury, and inflammation levels. Flow cytometry was utilized to detect the effect of acacetin on CD4+ T cell function. RNA-seq, molecular docking, and microscale thermophoresis (MST) were employed to investigate potential mechanisms. Seahorse analysis, mitoSOX, JC-1, and mitotracker were utilized to detect the effect of acacetin on mitochondrial function. RESULTS: Acacetin attenuated cardiac injury and fibrosis as well as heart dysfunction, and reduced cardiac inflammatory cytokines and ratio of effector CD4+ T and Th17 cells. Acacetin inhibited CD4+ T cell activation, proliferation, and Th17 cell differentiation. Mechanistically, the effects of acacetin were related to reducing mitochondrial complex II activity thereby inhibiting mitochondrial respiration and mitochondrial reactive oxygen species in CD4+ T cells. CONCLUSION: Acacetin may be a valuable therapeutic drug in treating CD4+ T cell-mediated myocarditis.

Transient receptor potential vanilloid 4 (TRPV4) in neutrophils enhances myocardial ischemia/reperfusion injury.

The Ca2+-permeable TRPV4 cation channel is expressed in neutrophils and contributes to myocardial ischemia/reperfusion injury. Here we tested the hypotheses that TRPV4 promotes neutrophil activation and subsequently aggregates myocardial ischemia/reperfusion injury. TRPV4 protein was confirmed in neutrophils, and its function was assessed by the current and intracellular Ca2+ concentration elevations evoked by TRPV4 agonists. Furthermore, TRPV4 agonists dose-dependently promoted migration toward fMLP, reactive oxygen species production, and myeloperoxidase release, which were prevented by pretreatment with a selective TRPV4 antagonist, in neutrophils from TRPV4 knockout mice, Ca2+-free medium, or BAPTA-AM + Ca2+-free medium. Blockade of TRPV4 also inhibited the effects of commonly used neutrophil activators fMLP and PMA. Mechanically, TRPV4 regulated neutrophil activation, particularly reactive oxygen species production, by affecting PKCα, P38, and AKT via Ca2+ signaling. In addition, isolated hearts infused with neutrophils from wild-type mice showed additional myocardial ischemia/reperfusion injuries but not those infused with TRPV4 knockout. Our study reveals that TRPV4-mediated neutrophil activation enhances myocardial ischemia/reperfusion injury, and it might be a novel therapeutic target for myocardial ischemia/reperfusion injury and other neutrophil-mediated inflammatory diseases.

Inhibition of phosphoglycerate kinase 1 attenuates autoimmune myocarditis by reprogramming CD4+ T cell metabolism.

AIMS: CD4+ T cells are the major drivers of cardiac-specific autoimmunity in myocarditis, specifically Th1, Treg, and most significant Th17 cells. But the molecular mechanisms of their activation remain unclear. We aimed to elucidate the regulatory role of phosphoglycerate kinase 1 (PGK1) in CD4+ T cells and experimental autoimmune myocarditis (EAM). METHODS AND RESULTS: EAM was induced in BALB/c mice by subcutaneous injections with alpha myosin heavy chain peptide emulsified in complete Freund's adjuvant. Single-cell sequencing analysis found that glycolysis and PGK1 expression were elevated in cardiac CD4+ T and Th17 cells from myocarditis mice. Mice treated with PGK1 inhibitor NG52 showed less cardiac inflammation and fibrosis and better contractile function, as well as reduced cardiac infiltrating Th17 and Th1 cells and increased proportion of Treg. NG52 suppressed CD4+ T cell activation and differentiation of mice and myocarditis patients in vitro. Mechanistically, inhibition of PGK1 suppressed glycolytic activity and decreased pyruvate dehydrogenase kinase 1 (PDHK1) phosphorylation, thereby increasing reactive oxygen species (ROS) production in mitochondria and thus preventing Th17 cell differentiation. CONCLUSION: PGK1 may act as a key metabolic regulator of CD4+ T cell differentiation and regulates Th17 cell differentiation by regulating glycolysis and the PDHK1-ROS axis. Targeting PGK1 might be a promising strategy for the treatment of myocarditis.

Activation of transient receptor potential vanilloid 4 is involved in pressure overload-induced cardiac hypertrophy.

Previous studies, including our own, have demonstrated that transient receptor potential vanilloid 4 (TRPV4) is expressed in hearts and implicated in cardiac remodeling and dysfunction. However, the effects of TRPV4 on pressure overload-induced cardiac hypertrophy remain unclear. In this study, we found that TRPV4 expression was significantly increased in mouse hypertrophic hearts, human failing hearts, and neurohormone-induced hypertrophic cardiomyocytes. Deletion of TRPV4 attenuated transverse aortic constriction (TAC)-induced cardiac hypertrophy, cardiac dysfunction, fibrosis, inflammation, and the activation of NFκB - NOD - like receptor pyrin domain-containing protein 3 (NLRP3) in mice. Furthermore, the TRPV4 antagonist GSK2193874 (GSK3874) inhibited cardiac remodeling and dysfunction induced by TAC. In vitro, pretreatment with GSK3874 reduced the neurohormone-induced cardiomyocyte hypertrophy and intracellular Ca2+ concentration elevation. The specific TRPV4 agonist GSK1016790A (GSK790A) triggered Ca2+ influx and evoked the phosphorylation of Ca2+/calmodulin-dependent protein kinase II (CaMKII). But these effects were abolished by removing extracellular Ca2+ or GSK3874. More importantly, TAC or neurohormone stimulation-induced CaMKII phosphorylation was significantly blocked by TRPV4 inhibition. Finally, we show that CaMKII inhibition significantly prevented the phosphorylation of NFκB induced by GSK790A. Our results suggest that TRPV4 activation contributes to pressure overload-induced cardiac hypertrophy and dysfunction. This effect is associated with upregulated Ca2+/CaMKII mediated activation of NFκB-NLRP3. Thus, TRPV4 may represent a potential therapeutic drug target for cardiac hypertrophy and dysfunction after pressure overload.

Blockage of transient receptor potential vanilloid 4 prevents postoperative atrial fibrillation by inhibiting NLRP3-inflammasome in sterile pericarditis mice.

The incidence of atrial fibrillation (AF) increases after surgery and is associated with the activation of NLRP3-inflammation. Our previous studies have found that transient receptor potential vanilloid 4 (TRPV4) blockade reduces the susceptibility to AF, but its molecular mechanisms remains unclear. Therefore, we hypothesized that blockage of TRPV4 reduces the incidence of AF by inhibiting NLRP3-inflammasome in sterile pericarditis (SP) mice. In this study, we established SP mice by dusting talcum powder on atrial surfaces. We first confirmed that genetic or pharmacological TRPV4 inhibition reduced the susceptibility to AF in SP mice. We also found that the expression level of NLRP3-inflammasome and inflammatory cytokines significantly increased in the atria of SP mice, which further increased in application the TRPV4 agonist GSK1016790A (GSK101) and decreased in application the TRPV4 antagonist GSK2193874. More importantly, ERK inhibitor (U0126) or NF-κB inhibitor (Bay11-7082) could partially reverse GSK101-induced NLRP3-inflammasome up-regulation. Interestingly, U0126 can reversed GSK101-induced NF-κB phosphorylation, but Bay11-7082 cannot change GSK101-induced ERK phosphorylation. Finally, we shown that the activation of NLRP3-inflammasome and ERK/NF-κB signaling pathway significantly reduced in TRPV4-knockout SP mice. Collectively, our studies indicate that blockage of TRPV4 prevents AF in SP mice by inhibiting NLRP3-inflammasome through the ERK/NF-κB signaling pathway.

Identification of the distinctive role of DPT in dilated cardiomyopathy: a study based on bulk and single-cell transcriptomic analysis.

BACKGROUND: Dilated cardiomyopathy (DCM) is a common cause of heart failure. Cardiac remodeling is the main pathological change in DCM, yet the molecular mechanism is still unclear. Therefore, the present study aims to find potential crucial genes and regulators through bulk and single-cell transcriptomic analysis. METHODS: Three microarray datasets of DCM (GSE57338, GSE42955, GSE79962) were chosen from gene expression omnibus (GEO) to analyze the differentially expressed genes (DEGs). LASSO regression, SVM-RFE, and PPI network methods were then carried out to identify key genes. Another dataset (GSE116250) was used to validate these findings. To further identify DCM-associated specific cell types, transcription factors, and cell-cell interaction networks, GSEA, SCENIC, and CellPhoneDB were conducted on public datasets for single-cell RNA sequencing analysis of DCM (GSE109816 and GSE121893). Finally, reverse transcription-polymerase chain reaction (RT-PCR), western blot, and immunohistochemical were performed to validate DPT expression in fibroblasts and DCM. RESULTS: There were 281 DEGs between DCM and non-failing donors. CCL5 and DPT were identified to be key genes and both genes had a 0.844 area under the receiver operating curve (AUC) in the validation dataset. Further single-cell sequencing analysis revealed three main findings: (I) DPT was mainly expressed in fibroblasts and was significantly upregulated in DCM fibroblasts; (II) DPT+ fibroblasts were involved in the organization of the extracellular matrix (ECM) and collagen fibrils and were regulated by the transcription factor STAT3; and (III) DPT+ fibroblasts had high interactions with endothelial cells through including Ephrin-Eph, ACKR-CXCL, and JAG-NOTCH signal pathways. RT-PCR, western blot, and immunohistochemical confirmed that DPT was highly expressed and co-localized with Vimentin and p-STAT3 in DCM patients. STAT3 inhibitor S3I-201 reduced the expression of DPT in mouse cardiac fibroblasts. CONCLUSIONS: DPT could be used as a diagnostic marker and therapeutic target of DCM. DPT+ fibroblasts could be a novel regulator of the cardiac remodeling process in DCM.

Activation of transient receptor potential vanilloid 4 exacerbates myocardial ischemia-reperfusion injury via JNK-CaMKII phosphorylation pathway in isolated mice hearts.

Previous studies, including our own, have demonstrated that transient receptor potential vanilloid 4 (TRPV4) is involved in myocardial ischemia-reperfusion (IR) injury, yet its underlying molecular mechanism remains unclear. In this study, we isolated mice hearts for a Langendorff perfusion test and used HL-1 myocytes for in vitro assessments. We first confirmed that TRPV4 agonist (GSK101) enhanced myocardial IR injury, as demonstrated by the reduced recovery of cardiac function, larger myocardial infarct size, and more apoptotic cells. We also found that GSK101 could further increase the phosphorylation of JNK and CaMKII in isolated hearts during IR. Notably, GSK101 dose-dependently evoked the phosphorylation of JNK and CaMKII in isolated normal hearts. All above GSK101-induced effects could be significantly blocked by the pharmacological inhibition or genetic ablation of TRPV4. More importantly, JNK inhibition (with SP600125) or CaMKII inhibition (with KN93 or in transgenic AC3-I mice) could prevent GSK101-induced myocardial injury during IR. In HL-1 myocytes, GSK101 triggered Ca2+ influx and evoked the phosphorylation of JNK and CaMKII but these effects were abolished by removing extracellular Ca2+ or in the presence of a TRPV4 antagonist. Finally, we showed that in HL-1 myocytes and isolated hearts during IR, JNK inhibition significantly inhibited the phosphorylation of CaMKII induced by GSK101 but CaMKII inhibition had no effect on JNK activation induced by GSK101. Our data suggest that TRPV4 activation exacerbates myocardial IR injury via the JNK-CaMKII phosphorylation pathway.

Interleukin-6-Mediated-Ca2+ Handling Abnormalities Contributes to Atrial Fibrillation in Sterile Pericarditis Rats.

Pre-existing Ca2+ handling abnormalities constitute the arrhythmogenic substrate in patients developing postoperative atrial fibrillation (POAF), a common complication after cardiac surgery. Postoperative interleukin (IL)-6 levels are associated with atrial fibrosis in several animal models of POAF, contributing to atrial arrhythmias. Here, we hypothesize that IL-6-mediated-Ca2+ handling abnormalities contribute to atrial fibrillation (AF) in sterile pericarditis (SP) rats, an animal model of POAF. SP was induced in rats by dusting atria with sterile talcum powder. Anti-rat-IL-6 antibody (16.7 µg/kg) was administered intraperitoneally at 30 min after the recovery of anesthesia. In vivo electrophysiology, ex vivo optical mapping, western blots, and immunohistochemistry were performed to elucidate mechanisms of AF susceptibility. IL-6 neutralization ameliorated atrial inflammation and fibrosis, as well as AF susceptibility in vivo and the frequency of atrial ectopy and AF with a reentrant pattern in SP rats ex vivo. IL-6 neutralization reversed the prolongation and regional heterogeneity of Ca2+ transient duration, relieved alternans, reduced the incidence of discordant alternans, and prevented the reduction and regional heterogeneity of the recovery ratio of Ca2+ transient. In agreement, western blots showed that IL-6 neutralization reversed the reduction in the expression of ryanodine receptor 2 (RyR2) and phosphorylated phospholamban. Acute IL-6 administration to isolated rat hearts recapitulated partial Ca2+ handling phenotype in SP rats. In addition, intraperitoneal IL-6 administration to rats increased AF susceptibility, independent of fibrosis. Our results reveal that IL-6-mediated-Ca2+ handling abnormalities in SP rats, especially RyR2-dysfunction, independent of IL-6-induced-fibrosis, early contribute to the development of POAF by increasing propensity for arrhythmogenic alternans.

TRPV4 blockade suppresses atrial fibrillation in sterile pericarditis rats.

Atrial fibrillation (AF) commonly occurs after surgery and is associated with atrial remodeling. TRPV4 is functionally expressed in the heart, and its activation affects cardiac structure and functions. We hypothesized that TRPV4 blockade alleviates atrial remodeling and reduces AF induction in sterile pericarditis (SP) rats. TRPV4 antagonist GSK2193874 or vehicle was orally administered 1 day before pericardiotomy. AF susceptibility and atrial function were assessed using in vivo electrophysiology, ex vivo optical mapping, patch clamp, and molecular biology on day 3 after surgery. TRPV4 expression increased in the atria of SP rats and patients with AF. GSK2193874 significantly reduced AF vulnerability in vivo and the frequency of atrial ectopy and AF with a reentrant pattern ex vivo. Mechanistically, GSK2193874 reversed the abnormal action potential duration (APD) prolongation in atrial myocytes through the regulation of voltage-gated K+ currents (IK); reduced the activation of atrial fibroblasts by inhibiting P38, AKT, and STAT3 pathways; and alleviated the infiltration of immune cells. Our results reveal that TRPV4 blockade prevented abnormal changes in atrial myocyte electrophysiology and ameliorated atrial fibrosis and inflammation in SP rats; therefore, it might be a promising strategy to treat AF, particularly postoperative AF.

Colchicine prevents atrial fibrillation promotion by inhibiting IL-1ß-induced IL-6 release and atrial fibrosis in the rat sterile pericarditis model.

A few clinical trials have recently reported the potential effect of colchicine in preventing post-operative atrial fibrillation (POAF) and early atrial fibrillation (AF) recurrence after catheter pulmonary vein isolation. However, the molecular mechanisms through which colchicine inhibits AF remain unclear. We aim to assess the anti-AF effect of colchicine in the rat sterile pericarditis (SP) model and to investigate its molecular mechanisms. SP was induced in Sprague-Dawley rats by the epicardial application of sterile talc. Treatment with colchicine or vehicle began 1 d before pericardiotomy. AF was induced by transesophageal burst pacing on day 3 after surgery. Treatment with colchicine reduced the duration of AF and the probability of induction of AF in SP rats. The dose of 0.5 mg kg-1·day-1 had the best effect. Such treatment also reduced neutrophil infiltration, the mRNA expression of IL-6, TGF-ß, and TNF-α, atrial fibrosis, fibrosis related genes, and signal molecules (STAT3, P38, and AKT). Meanwhile, the release of IL-1ß (4-24 h) and IL-6 (4-72 h) in atria after surgery was significantly inhibited by colchicine. In cultured rat cardiac fibroblasts, colchicine treatment inhibited IL-1ß-induced expression of IL-6, which was accompanied by significantly decreased phosphorylation of P38, AKT, JNK, and NFκB. Interestingly, the supplementation of IL-6 abolished the anti-AF effect of colchicine in SP rats. Colchicine prevents AF in SP rats through the inhibition of IL-1ß-induced IL-6 release and subsequent atrial fibrosis. However, further studies are required to investigate whether colchicine inhibits POAF through other mechanisms.

Propofol Induces Cardioprotection Against Ischemia-Reperfusion Injury via Suppression of Transient Receptor Potential Vanilloid 4 Channel.

Ca2+ entry via the transient receptor potential vanilloid 4 (TRPV4) channel contributes to Ca2+ overload and triggers many pathophysiological conditions, including myocardial ischemia/reperfusion (I/R) injury. Propofol, a widely used intravenous anesthetic, attenuates myocardial I/R injury. However, the mechanism of propofol remains to be examined. The present study aims to test the hypothesis that propofol attenuates myocardial I/R injury through the suppression of TRPV4. We used a murine ex vivo model of myocardial I/R and in vitro cultured myocytes subjected to hypoxia/reoxygenation (H/R). Propofol or TRPV4 antagonist, HC-067047, attenuates myocardial I/R injury in isolated hearts. In addition, propofol, HC-067047, or TRPV4-siRNA attenuates H/R-induced intracellular Ca2+ concentration ([Ca2+]i) increase and cell viability reduction. On the contrary, TRPV4 agonist GSK1016790A exacerbates both ex vivo and in vitro myocardial injury. Pretreatment with propofol reverses the myocardial injury and intracellular Ca2+ overload induced by GSK1016790A at least in vitro. However, neither the combination of propofol and HC-067047 nor applying propofol to cells transfected with TRPV4-siRNA creates additional protective effects. In addition, propofol dose-dependently inhibits TRPV4-mediated Ca2+ entry induced by GSK1016790A and 4α-PDD. Propofol attenuates myocardial I/R injury partially through the suppression of TRPV4 channel and the subsequent inhibition of intracellular Ca2+ overload.

Blockade of Transient Receptor Potential Vanilloid 4 Enhances Antioxidation after Myocardial Ischemia/Reperfusion.

Antioxidative stress provides a cardioprotective effect during myocardial ischemia/reperfusion (I/R). Previous research has demonstrated that the blockade of transient receptor potential vanilloid 4 (TRPV4) attenuates myocardial I/R injury. However, the underlying mechanism remains unclear. The current study is aimed at investigating the antioxidative activity of TRPV4 inhibition and elucidating the underlying mechanisms in vitro and ex vivo. We found that the inhibiting TRPV4 by the selective TRPV4 blocker HC-067047 or specific TRPV4-siRNA significantly reduces reactive oxygen species (ROS) and methane dicarboxylic aldehyde (MDA) levels in H9C2 cells exposed to hypoxia/reoxygenation (H/R). Meanwhile, the activity of antioxidative enzymes, particularly superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px), is enhanced. Furthermore, after H/R, HC-067047 treatment increases the expression of P-Akt and the translocation of nuclear factor E2-related factor 2 (Nrf2) and related antioxidant response element (ARE) mainly including SOD, GSH-Px, and catalase (CAT). LY294002, an Akt inhibitor, suppresses HC-067047 and specific TRPV4-siRNA-induced Nrf2 expression and its nuclear accumulation. Nrf2 siRNA attenuates HC-067047 and specific TRPV4-siRNA-induced ARE expression. In addition, treatment with LY294002 or Nrf2 siRNA significantly attenuates the antioxidant and anti-injury effects of HC-067047 in vitro. Finally, in experiments on isolated rat hearts, we confirmed the antioxidative stress roles of TRPV4 inhibition during myocardial I/R and the application of exogenous H2O2. In conclusion, the inhibition of TRPV4 exerts cardioprotective effects through enhancing antioxidative enzyme activity and expressions via the Akt/Nrf2/ARE pathway.

Activation of transient receptor potential vanilloid 4 involves in hypoxia/reoxygenation injury in cardiomyocytes.

Transient receptor potential vanilloid 4 (TRPV4) is highly expressed in heart and vessels and can be activated during myocardial ischemia/reperfusion (I/R). Recently, we found that treatment with a selective TRPV4 antagonist HC-067047 significantly reduced infarct size, decreased troponin T levels and improved cardiac function in murine model myocardial I/R. This study was undertaken to investigate the mechanism underlying TRPV4-mediated myocardial I/R injury. To mimic myocardial I/R injury, we established a hypoxia/reoxygenation (H/R) model in H9C2 cells and neonatal rat ventricle myocytes (NRVMs) in vitro. TRPV4 mRNA and protein expression was confirmed in the H9C2 and NRVM, whereas functional TRPV4 activity was assessed from Ca2+ influx response to a TRPV4 agonist GSK1016790A. TRPV4 functional expression was significantly enhanced during H/R. Furthermore, H/R increased the intracellular Ca2+ concentration ([Ca2+]i) and induced cell injury, which were reversed by HC-067047 but was further aggravated by GSK1016790A. Moreover, HC-067047 treatment significantly alleviated the increase of reactive oxygen species (ROS) generation, the depolarization of mitochondrial membrane potential (Δψm) and the opening of mitochondrial permeability transition pore (mPTP) during H/R. On the contrary, GSK1016790A exacerbated those effects. Meanwhile, increase in [Ca2+]i and ROS induced by activation of TRPV4 was almost abolished when cells were cultured in Ca2+-free medium. In addition, ROS scavenger NAC obviously reversed activation of TRPV4-induced changes of Δψm and mPTP opening. Finally, we confirmed the direct roles of TRPV4 on cardiac injury and ROS generation in murine model myocardial I/R in vivo. In conclusion, activation of TRPV4 induces Ca2+ influx in cardiomyocytes, with subsequent ROS release, depolarizing of Δψm, opening mPTP, inducing injury and TRPV4 has key roles during I/R via these pathways.

Blockage of transient receptor potential vanilloid 4 alleviates myocardial ischemia/reperfusion injury in mice.

Transient receptor potential vanilloid 4 (TRPV4) is a Ca2+-permeable nonselective cation channel and can be activated during ischemia/reperfusion (I/R). This study tested whether blockade of TRPV4 can alleviate myocardial I/R injury in mice. TRPV4 expression began to increase at 1 h, reached statistically at 4 h, and peaked at 24-72 h. Treatment with the selective TRPV4 antagonist HC-067047 or TRPV4 knockout markedly ameliorated myocardial I/R injury as demonstrated by reduced infarct size, decreased troponin T levels and improved cardiac function at 24 h after reperfusion. Importantly, the therapeutic window for HC-067047 lasts for at least 12 h following reperfusion. Furthermore, treatment with HC-067047 reduced apoptosis, as evidenced by the decrease in TUNEL-positive myocytes, Bax/Bcl-2 ratio, and caspase-3 activation. Meanwhile, treatment with HC-067047 attenuated the decrease in the activation of reperfusion injury salvage kinase (RISK) pathway (phosphorylation of Akt, ERK1/2, and GSK-3ß), while the activation of survival activating factor enhancement (SAFE) pathway (phosphorylation of STAT3) remained unchanged. In addition, the anti-apoptotic effects of HC-067047 were abolished by the RISK pathway inhibitors. We conclude that blockade of TRPV4 reduces apoptosis via the activation of RISK pathway, and therefore might be a promising strategy to prevent myocardial I/R injury.

Signal Transducer and Activator of Transcription 3/MicroRNA-21 Feedback Loop Contributes to Atrial Fibrillation by Promoting Atrial Fibrosis in a Rat Sterile Pericarditis Model.

BACKGROUND: Postoperative atrial fibrillation is a frequent complication in cardiac surgery. The aberrant activation of signal transducer and activator of transcription 3 (STAT3) contributes to the pathogenesis of atrial fibrillation. MicroRNA-21 (miR-21) promotes atrial fibrosis. Recent studies support the existence of reciprocal regulation between STAT3 and miR-21. Here, we test the hypothesis that these 2 molecules might form a feedback loop that contributes to postoperative atrial fibrillation by promoting atrial fibrosis. METHODS AND RESULTS: A sterile pericarditis model was created using atrial surfaces dusted with sterile talcum powder in rats. The inflammatory cytokines interleukin (IL)-1ß, IL-6, transforming growth factor-ß, and tumor necrosis factor-α, along with STAT3 and miR-21, were highly upregulated in sterile pericarditis rats. The inhibition of STAT3 by S3I-201 resulted in miR-21 downregulation, which ameliorated atrial fibrosis and decreased the expression of the fibrosis-related genes, α-smooth muscle actin, collagen-1, and collagen-3; reduced the inhomogeneity of atrial conduction; and attenuated atrial fibrillation vulnerability. Meanwhile, treatment with antagomir-21 decreased STAT3 phosphorylation, alleviated atrial remodeling, abrogated sterile pericarditis-induced inhomogeneous conduction, and prevented atrial fibrillation promotion. The culturing of cardiac fibroblasts with IL-6 resulted in progressively augmented STAT3 phosphorylation and miR-21 levels. S3I-201 blocked IL-6 induced the expression of miR-21 and fibrosis-related genes in addition to cardiac fibroblast proliferation. Transfected antagomir-21 decreased the IL-6-induced cardiac fibroblast activation and STAT3 phosphorylation. The overexpression of miR-21 in cardiac fibroblasts caused the upregulation of STAT3 phosphorylation, enhanced fibrosis-related genes, and increased cell numbers. CONCLUSIONS: Our results have uncovered a novel reciprocal loop between STAT3 and miR-21 that is activated after heart surgery and can contribute to atrial fibrillation.

Lovastatin blocks Kv1.3 channel in human T cells: a new mechanism to explain its immunomodulatory properties.

Lovastatin is a member of Statins, which are beneficial in a lot of immunologic cardiovascular diseases and T cell-mediated autoimmune diseases. Kv1.3 channel plays important roles in the activation and proliferation of T cells, and have become attractive target for immune-related disorders. The present study was designed to examine the block effect of Lovastatin on Kv1.3 channel in human T cells, and to clarify its new immunomodulatory mechanism. We found that Lovastatin inhibited Kv1.3 currents in a concentration- and voltage-dependent manner, and the IC50 for peak, end of the pulse was 39.81 ± 5.11, 6.92 ± 0.95 µM, respectively. Lovastatin also accelerated the decay rate of current inactivation and negatively shifted the steady-state inactivation curves concentration-dependently, without affecting the activation curve. However, 30 µM Lovastatin had no apparent effect on KCa current in human T cells. Furthermore, Lovastatin inhibited Ca(2+) influx, T cell proliferation as well as IL-2 production. The activities of NFAT1 and NF-κB p65/50 were down-regulated by Lovastatin, too. At last, Mevalonate application only partially reversed the inhibition of Lovastatin on IL-2 secretion, and the siRNA against Kv1.3 also partially reduced this inhibitory effect of Lovastatin. In conclusion, Lovastatin can exert immunodulatory properties through the new mechanism of blocking Kv1.3 channel.

Interleukin-17A contributes to the development of post-operative atrial fibrillation by regulating inflammation and fibrosis in rats with sterile pericarditis.

Post-operative atrial fibrillation (AF) remains a common cause of morbidity. Increasing evidence indicates that inflammation and atrial fibrosis contribute to the pathogenesis of this condition. Interleukin (IL)-17A, a potent pro-inflammatory cytokine, has been implicated in the development of a number of cardiovascular diseases. However, its role in post-operative AF remains unknown. In the present study, sterile pericarditis (SP) was induced in rats by the epicardial application of sterile talc. AF was induced by transesophageal burst pacing. Western blot analysis was applied to quantify the expression of IL-17A. Quantitative PCR was used to detect the mRNA expression of IL-17A, IL-6, IL-1ß, transforming growth factor-ß1 (TGF-ß1), collagen type 1 (Col-1), collagen type 3 (Col-3) and α-smooth muscle actin (α-SMA). Gelatin zymography and reverse gelatin zymography were used to quantify the levels of matrix metalloproteinases (MMPs) and tissue inhibitors of MMPs (TIMPs). Histological analyses were performed to determine the extent of tissue inflammation and fibrosis. The rats with SP presented with a shorter refractoriness, a higher incidence and duration of AF, an enhanced susceptibility to developing AF, increased mRNA levels of AF-related pro-inflammatory cytokines (IL-6, IL-1ß and TGF-ß1), as well as marked atrial inflammation and fibrosis. The atrial IL-17A levels were elevated and correlated with the probability of developing AF. Treatment with anti-IL-17A monoclonal antibody decreased the levels of atrial IL-17A, prolonged refraction and markedly suppressed the development of AF. Simultaneously, inflammation and fibrosis were alleviated, which was further demonstrated by a decreased expression of AF-related pro-inflammatory cytokines, a downregulation in fibrosis-related mRNA expression (Col-1, Col-3 and α-SMA) and by the decreased activity of MMP-2/9 and TIMPs. Thus, the findings of our study indicate that IL-17A may play a pathogenic role in post-operative AF by inducing inflammation and fibrosis in rats with SP.