Zbtb16 increases susceptibility of atrial fibrillation in type 2 diabetic mice via Txnip-Trx2 signaling.

Atrial fibrillation (AF) is the most prevalent sustained cardiac arrhythmia, and recent epidemiological studies suggested type 2 diabetes mellitus (T2DM) is an independent risk factor for the development of AF. Zinc finger and BTB (broad-complex, tram-track and bric-a-brac) domain containing 16 (Zbtb16) serve as transcriptional factors to regulate many biological processes. However, the potential effects of Zbtb16 in AF under T2DM condition remain unclear. Here, we reported that db/db mice displayed higher AF vulnerability and Zbtb16 was identified as the most significantly enriched gene by RNA sequencing (RNA-seq) analysis in atrium. In addition, thioredoxin interacting protein (Txnip) was distinguished as the key downstream gene of Zbtb16 by Cleavage Under Targets and Tagmentation (CUT&Tag) assay. Mechanistically, increased Txnip combined with thioredoxin 2 (Trx2) in mitochondrion induced excess reactive oxygen species (ROS) release, calcium/calmodulin-dependent protein kinase II (CaMKII) overactivation, and spontaneous Ca2+ waves (SCWs) occurrence, which could be inhibited through atrial-specific knockdown (KD) of Zbtb16 or Txnip by adeno-associated virus 9 (AAV9) or Mito-TEMPO treatment. High glucose (HG)-treated HL-1 cells were used to mimic the setting of diabetic in vitro. Zbtb16-Txnip-Trx2 signaling-induced excess ROS release and CaMKII activation were also verified in HL-1 cells under HG condition. Furthermore, atrial-specific Zbtb16 or Txnip-KD reduced incidence and duration of AF in db/db mice. Altogether, we demonstrated that interrupting Zbtb16-Txnip-Trx2 signaling in atrium could decrease AF susceptibility via reducing ROS release and CaMKII activation in the setting of T2DM.

Ubiquitin C-terminal hydrolase L1 promotes expression of programmed cell death-ligand 1 in non-small-cell lung cancer cells.

Programmed cell death-ligand 1 (PD-L1) expressed on cancer cells can cause immune escape of non-small-cell lung cancer (NSCLC). Elucidation of the regulatory mechanisms of the PD-L1 expression is a prerequisite for establishing new tumor immunotherapy strategies. Ubiquitin C-terminal hydrolase L1 (UCHL1) is a regulator of cellular signaling transduction that is aberrantly expressed in NSCLC. However, it is not known whether UCHL1 regulates the expression of PD-L1 in NSCLC cells. In the present study, we found that UCHL1 promotes the expression of PD-L1 in NSCLC cell lines. In addition, UCHL1 expressed in NSCLC cells inhibited activation of Jurkat cells through upregulation of PD-L1 expression in in vitro experiments. Moreover, UCHL1 upregulates PD-L1 expression through facilitating activation of the AKT-P65 signaling pathway. In conclusion, these results indicated that UCHL1 promoted PD-L1 expression in NSCLC cells. This finding implied that inhibition of UCHL1 might suppress immune escape of NSCLC through downregulation of PD-L1 expression in NSCLC cells.

Ntsr1 contributes to pulmonary hypertension by enhancing endoplasmic reticulum stress via JAK2-STAT3-Thbs1 signaling.

Pulmonary hypertension (PH) is a severe clinical syndrome with pulmonary vascular remodeling and poor long-term prognosis. Neurotensin receptor 1 (Ntsr1), serve as one of the G protein-coupled receptors (GPCRs), implicates in various biological processes, but the potential effects of Ntsr1 in PH development are unclear. The Sugen/Hypoxia (SuHx) or monocrotaline (MCT) induced rat PH model was used in our study and the PH rats showed aggravated pulmonary artery remodeling and increased right ventricular systolic pressure (RVSP). Our results revealed that Ntsr1 induced endoplasmic reticulum (ER) stress response via ATF6 activation contributed to the development of PH. Moreover, RNA-sequencing (RNA-seq) and phosphoproteomics were performed and the Ntsr1-JAK2-STAT3-thrombospondin 1 (Thbs1)-ATF6 signaling was distinguished as the key pathway. In vitro, pulmonary artery smooth muscle cells (PASMCs) under hypoxia condition showed enhanced proliferation and migration properties, which could be inhibited by Ntsr1 knockdown, JAK2 inhibitor (Fedratinib) treatment, STAT3 inhibitior (Stattic) treatment, Thbs1 knockdown or ATF6 knockdown. In addition, adeno-associated virus 1 (AAV1) were used to knockdown the expression of Ntsr1, Thbs1 or ATF6 in rats and reversed the phenotype of PH. In summary, our results reveal that Ntsr1-JAK2-STAT3-Thbs1 pathway can induce enhanced ER stress via ATF6 activation and increased PASMC proliferation and migration capacities, which can be mechanism of the pulmonary artery remodeling and PH. Targeting Ntsr1 might be a novel therapeutic strategy to ameliorate PH.

Dimethyl Fumarate Ameliorates Doxorubicin-Induced Cardiotoxicity By Activating the Nrf2 Pathway.

Doxorubicin (DOX) is limited in clinical application because of its cardiotoxicity. Oxidative stress and apoptosis are crucial in DOX-induced cardiac injury. Dimethyl fumarate (DMF) is an FDA-approved oral drug with powerful effects to reduce oxidative stress and apoptosis through the Nrf2 pathway. This study was aimed to determine whether DMF can protect against DOX-induced cardiac injury. We used both neonatal rat cardiomyocytes (NRCMs) in vitro and DOX-induced cardiac toxicity in vivo to explore the effects of DMF. The results showed that DMF significantly improved cell viability and morphology in NRCMs. In addition, DMF alleviated DOX-induced cardiac injury in rats, as evidenced by decreased CK-MB, LDH levels, improved survival rates, cardiac function, and pathological changes. Moreover, DMF significantly inhibited cardiac oxidative stress by reducing MDA levels and increasing GSH, SOD, and GSH-px levels. And DMF also inhibited DOX-induced cardiac apoptosis by modulating Bax, Bcl-2 and cleaved caspase-3 expression. Moreover, DMF exerted its protective effects against DOX by promoting Nrf2 nuclear translocation, which activated its downstream antioxidant gene Hmox1. Silencing of Nrf2 attenuated the protective effects of DMF in NRCMs as manifested by increased intracellular oxidative stress, elevated apoptosis levels, and decreased cell viability. In addition, DMF showed no protective effects on the viability of DOX-treated tumor cells, which suggested that DMF does not interfere with the antitumor effect of DOX in vitro. In conclusion, our data confirmed that DMF alleviated DOX-induced cardiotoxicity by regulating oxidative stress and apoptosis through the Nrf2 pathway. DMF may serve as a new candidate to alleviate DOX-related cardiotoxicity in the future.

Loss of Camk2n1 aggravates cardiac remodeling and malignant ventricular arrhythmia after myocardial infarction in mice via NLRP3 inflammasome activation.

AIMS: Inflammation response and subsequent ventricular remodeling are critically involved in the development of ventricular arrhythmia post myocardial infarction (MI). However, as the vital endogenous inhibitor of calcium/calmodulin-dependent protein kinase II (CaMKII), the effects of CaMKII inhibitor 1 (Camk2n1) on the process of arrhythmia substrate generation following MI remains unclear. In this study, we investigated the role of Camk2n1 in ventricular arrhythmia post-MI and the underlying mechanisms. METHODS AND RESULTS: Camk2n1 was mainly expressed in cardiomyocytes and inhibited the phosphorylation of CaMKIIδ in the infarcted border zone. Compared to wild type (WT) littermates mice, Camk2n1 knockout mice (Camk2n1-/-) manifested exacerbated cardiac dysfunction, larger fibrosis area, higher incidence of premature ventricular contractions (PVCs) and higher vulnerability to ventricular tachycardia (VT) or ventricular fibrillation (VF) after MI. The results of RNA sequencing (RNA-seq) identified that excessive activation of NLRP3 inflammasome was responsible for aggravated inflammation response which led to adverse cardiac remodeling in Camk2n1-/- mice subjected to MI. More importantly, both in vivo and in vitro experiments verified that aggravated NLRP3 inflammasome activation occurred via CaMKIIδ-p38/JNK pathway in Camk2n1-/- mice. CONCLUSIONS: Collectively, our results highlight the importance of Camk2n1 in alleviating ventricular remodeling and malignant ventricular arrhythmia post-MI by reducing cardiomyocytes inflammation activation via CaMKIIδ-p38/JNK-NLRP3 inflammasome pathway, targeting Camk2n1 might serve as a novel therapeutic strategy after MI.

Activin receptor-like kinase 4 haplodeficiency alleviates the cardiac inflammation and pacing-induced ventricular arrhythmias after myocardial infarction.

BACKGROUND: Inflammation process is an important determinant for subsequent changes in cardiac function and remodeling after acute myocardial infarction (MI). Recent studies have implicated that ALK4 haplodeficiency improves cardiac function after MI. However, it remains unknown if the beneficial effects are partly attributed to ALK4 haplodeficiency-induced modulation on inflammatory response in the inflammatory phase of MI. In this research, we aimed to explore the mechanism of ALK4 haplodeficiency in the inflammatory stage of MI. METHODS: ALK4, CD16, and CD14 were detected in peripheral blood mononuclear cells (PBMCs) isolated from MI patients and healthy volunteers. ALK4 haplodeficiency (ALK4+/-) mice and wild-type (WT) littermates were randomly divided into the sham group and the MI group. Inflammation cytokines and chemokines were measured. Echocardiography and intracardiac electrophysiological recordings were performed on the 3rd day and the 7th day after MI operation. ALK4 expression and inflammation cytokines were also detected in LPS- or IL-4-stimulated bone marrow-derived macrophages (BMDM) from the ALK4+/- mice and WT littermates. RESULTS: ALK4 gene expression in circulating monocytes of MI patients was higher than that in those of healthy volunteers. Cardiac inflammation and vulnerability of ventricular arrhythmia after acute myocardial injury are significantly alleviated in ALK4+/- mice as compared to WT littermates. On the 3rd day post-MI, the level of M1 macrophages were decreased in ALK4+/- mice as compared to WT littermates, while the level of M2 macrophages were increased on the 7th day post-MI. BMDM isolated from ALK4+/- mice displayed reduced secretion of pro-inflammation cytokines after stimulation by LPS in hypoxic condition and increased secretion of anti-inflammation cytokines after stimulation by IL-4. As a result, the haplodeficiency of ALK4 might be responsible for reduced inflammation response in the post-MI stage. CONCLUSIONS: ALK4 haplodeficiency reduces cardiac inflammation, improves cardiac function, and finally reduces the vulnerability of ventricular arrhythmia in the inflammatory stage after MI.

Renal Denervation by Noninvasive Stereotactic Radiotherapy Induces Persistent Reduction of Sympathetic Activity in a Hypertensive Swine Model.

Background We have previously reported the feasibility of noninvasive stereotactic body radiotherapy (SBRT) as a novel approach for renal denervation. Methods and Results Herein, from a translational point of view, we assessed the antihypertensive effect and chronological evolution of SBRT-induced renal nerve injury within 6 months in a hypertensive swine model. Hypertension was induced in swine by subcutaneous implantation of deoxycorticosterone acetate pellets in combination with a high-salt diet. A single dose of 25 Gy with SBRT was delivered for renal denervation in 9 swine within 3.4±1.0 minutes. Blood pressure levels at baseline and 1 and 6 months post-SBRT were comparable to control (n=5), whereas renal norepinephrine was significantly lower at 6 months (P<0.05). Abdominal computed tomography, performed before euthanasia and renal function assessment, remained normal. Standard semiquantitative histological assessment showed that compared with control (1.4±0.4), renal nerve injury was greater at 1 month post-SBRT (2.3±0.3) and peaked at 6 months post-SBRT (3.2±0.8) (P<0.05), along with a higher proportion of active caspase-3-positive nerves (P<0.05). Moreover, SBRT resulted in continuous dysfunction of renal sympathetic nerves and low level of nerve regeneration in 6 months by immunohistochemistry analysis. Conclusions SBRT delivering 25 Gy for renal denervation was safe and related to sustained reduction of sympathetic activity by aggravating nerve damage and inhibiting nerve regeneration up to 6 months; however, its translation to clinical trial should be cautious because of the negative blood pressure response in the deoxycorticosterone acetate-salt hypertensive swine model.

Macrophages facilitate post myocardial infarction arrhythmias: roles of gap junction and KCa3.1.

Effective therapeutic targets against post-myocardial infarction (MI) arrhythmias remain to be discovered. We aimed to investigate the role of macrophages in post-MI arrhythmias. Methods: Mononuclear cell accumulation, macrophage polarization from M0 to M1 subset, and gap junction formation were analyzed in MI patients and MI mice by flow cytometry, immunofluorescence and patch clamping. Differentially expressed genes were identified by RNA sequencing. Macrophages and cardiomyocytes were cocultured in vitro, and the effects of gap junction and KCa3.1 on electrophysiological properties were assessed by patch clamping. The effects of KCa3.1 inhibition on post-MI arrhythmias were assessed by intracardiac stimulation and ambulatory electrocardiograms in vivo. Results: Percentage of pro-inflammatory mononuclear cells were significantly elevated in patients with post-MI arrhythmias compared with MI patients without arrhythmias and healthy controls (p<0.001). Macrophages formed gap junction with cardiomyocytes in MI border zones of MI patient and mice, and pro-inflammatory macrophages were significantly increased 3 days post-MI (p<0.001). RNA sequencing identified Kcnn4 as the most differentially expressed gene encoding ion channel, and the upregulation is mainly attributed to macrophage accumulation and polarization into pro-inflammatory subset. In vitro coculture experiments demonstrated that connection with M0 macrophages via gap junction slightly shortened the action potential durations (APDs) of cardiomyocytes. However, the APD90 of cardiomyocytes connected with M1 macrophages were significantly prolonged (p<0.001), which were effectively attenuated by gap junction inhibition (p=0.002), KCa3.1 inhibition (p=0.008), KCa3.1 silencing (p<0.001) and store-operated Ca2+ channel inhibition (p=0.005). In vivo results demonstrated that KCa3.1 inhibition significantly decreased the QTc durations (p=0.031), intracardiac stimulation-induced ventricular arrhythmia durations (p=0.050) and incidence of premature ventricular contractions (p=0.030) in MI mice. Conclusion: Macrophage polarization leads to APD heterogeneity and post-MI arrhythmias via gap junction and KCa3.1 activation. The results provide evidences of a novel mechanism of post-MI heterogeneous repolarization and arrhythmias, rendering macrophages and KCa3.1 to be potential therapeutic targets.