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.

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.