The caspase-1 inhibitor VX765 upregulates connexin 43 expression and improves cell-cell communication after myocardial infarction via suppressing the IL-1ß/p38 MAPK pathway.

Connexin 43 (Cx43) is the most important protein in the gap junction channel between cardiomyocytes. Abnormalities of Cx43 change the conduction velocity and direction of cardiomyocytes, leading to reentry and conduction block of the myocardium, thereby causing arrhythmia. It has been shown that IL-1ß reduces the expression of Cx43 in astrocytes and cardiomyocytes in vitro. However, whether caspase-1 and IL-1ß affect connexin 43 after myocardial infarction (MI) is uncertain. In this study we investigated the effects of VX765, a caspase-1 inhibitor, on the expression of Cx43 and cell-to-cell communication after MI. Rats were treated with VX765 (16 mg/kg, i.v.) 1 h before the left anterior descending artery (LAD) ligation, and then once daily for 7 days. The ischemic heart was collected for histochemical analysis and Western blot analysis. We showed that VX765 treatment significantly decreased the infarct area, and alleviated cardiac dysfunction and remodeling by suppressing the NLRP3 inflammasome/caspase-1/IL-1ß expression in the heart after MI. In addition, VX765 treatment markedly raised Cx43 levels in the heart after MI. In vitro experiments were conducted in rat cardiac myocytes (RCMs) stimulated with the supernatant from LPS/ATP-treated rat cardiac fibroblasts (RCFs). Pretreatment of the RCFs with VX765 (25 µM) reversed the downregulation of Cx43 expression in RCMs and significantly improved intercellular communication detected using a scrape-loading/dye transfer assay. We revealed that VX765 suppressed the activation of p38 MAPK signaling in the heart tissue after MI as well as in RCMs stimulated with the supernatant from LPS/ATP-treated RCFs. Taken together, these data show that the caspase-1 inhibitor VX765 upregulates Cx43 expression and improves cell-to-cell communication in rat heart after MI via suppressing the IL-1ß/p38 MAPK pathway.

RNAi silencing of the 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR) gene inhibits vitellogenesis in Chinese mitten crab Eriocheir sinensis.

The sesquiterpenoid methyl farnesoate (MF), a de-epoxide form of insect juvenile hormone III (JH III), plays an essential role in regulating many crucial physiological processes in crustaceans including vitellogenesis and reproduction. 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR) is an important rate-limiting enzyme in the mevalonate pathway, which is critical for the synthesis of JH III and MF. In the present study, a full-length cDNA encoding HMGR (EsHMGR) in Eriocheir sinensis was isolated and characterised. Sequence analysis of EsHMGR revealed that it belongs to Class I HMGR family proteins with HMG-CoA-binding and NADPH-binding domains, both important for HMGR activity. In addition to its ubiquitous tissue expression, expression of EsHMGR was highly specific to the ovary, the main site of Vg synthesis. During ovarian development, EsHMGR expression in ovary displayed a stage-specific pattern, and was correlated with expression of vitellogenin (EsVg) in hepatopancreas, which suggests that EsHMGR possibly involved in vitellogenesis. To further investigate the functional role of EsHMGR in vitellogenin biosynthesis in E. sinensis, RNA interference-mediated gene silencing was carried out both in vitro and in vivo. Quantitative PCR results showed that injection of EsHMGR double-stranded RNA (dsRNA) led to a significant decrease in EsVg expression levels in ovary and hepatopancreas both in vitro and in vivo. Taken together, the results suggest that EsHMGR is involved in vitellogenin biosynthesis in female E. sinensis, which may provide a new resource for HMGR enzymes participating in reproduction in crustaceans.

Synthesis of Aluminophosphate Molecular Sieves in Alkaline Media.

Unlike conventional aluminosilicate zeolites synthesized in alkaline media, aluminophosphate molecular sieves (AlPOs) have always been prepared under acidic conditions in the past three decades; this has been regarded as one of essential factors for synthesis, except for the case of silica-substituted analogues (SAPOs). For the first time, we demonstrate herein a simple and generalized route for synthesizing various types of aluminophosphate molecular sieves in alkaline media. A series of aluminophosphate sieves and their analogues have been prepared with different quaternary ammonium cations as structure-directing agents in this manner. The above successes have extended the systematic media from acidic or neutral to alkaline for the preparation of a series of aluminophosphate molecular sieves, which possibly open an alternative route for the synthesis of aluminophosphate molecular sieves.

Digoxin improves steatohepatitis with differential involvement of liver cell subsets in mice through inhibition of PKM2 transactivation.

The cardiac glycoside digoxin was identified as a potent suppressor of pyruvate kinase isoform 2-hypoxia-inducible factor-α (PKM2-HIF-1α) pathway activation in liver injury mouse models via intraperitoneal injection. We have assessed the therapeutic effects of digoxin to reduce nonalcoholic steatohepatitis (NASH) by the clinically relevant oral route in mice and analyzed the cellular basis for this effect with differential involvement of liver cell subsets. C57BL/6J male mice were placed on a high-fat diet (HFD) for 10 wk and started concurrently with the gavage of digoxin (2.5, 0.5, 0.125 mg/kg twice a week) for 5 wk. Digoxin significantly reduced HFD-induced hepatic damage, steatosis, and liver inflammation across a wide dosage range. The lowest dose of digoxin (0.125 mg/kg) showed significant protective effects against liver injury and sterile inflammation. Consistently, digoxin attenuated HIF-1α sustained NLRP3 inflammasome activation in macrophages. We have reported for the first time that PKM2 is upregulated in hepatocytes with hepatic steatosis, and digoxin directly improved hepatocyte mitochondrial dysfunction and steatosis. Mechanistically, digoxin directly bound to PKM2 and inhibited PKM2 targeting HIF-1α transactivation without affecting PKM2 enzyme activation. Thus, oral digoxin showed potential to therapeutically inhibit liver injury in NASH through the regulation of PKM2-HIF-1α pathway activation with involvement of multiple cell types. Because of the large clinical experience with oral digoxin, this may have significant clinical applicability in human NASH.NEW & NOTEWORTHY This study is the first to assess the therapeutic efficacy of oral digoxin on nonalcoholic steatohepatitis (NASH) in a high-fat diet (HFD) mouse model and to determine the divergent of cell type-specific effects. Oral digoxin reduced liver damage, steatosis, and inflammation in HFD mice. Digoxin attenuated hypoxia-inducible factor (HIF)-1α axis-sustained inflammasome activity in macrophages and hepatic oxidative stress response in hepatocytes via the regulation of PKM2-HIF-1α axis pathway activation. Oral digoxin may have significant clinical applicability in human NASH.

CHID1 positively regulates RLR antiviral signaling by targeting the RIG-I/VISA signalosome.

Retinoic acid-inducible gene-I (RIG-I) belongs to the RIGI-like receptors (RLRs), a class of primary pattern recognition receptors. It senses viral double-strand RNA in the cytoplasm and delivers the activated signal to its adaptor virus-induced signaling adapter (VISA), which then recruits the downstream TNF receptor-associated factors and kinases, triggering a downstream signal cascade that leads to the production of proinflammatory cytokines and antiviral interferons (IFNs). However, the mechanism of RIG-I-mediated antiviral signaling is not fully understood. Here, we demonstrate that chitinase domain-containing 1 (CHID1), a member of the chitinase family, positively regulates the RLR antiviral signaling pathway by targeting the RIG-I/VISA signalosome. CHID1 overexpression enhances the activation of nuclear factor κB (NF-кB) and interferon regulatory factor 3 (IRF3) triggered by Sendai virus (SeV) by promoting the polyubiquitination of RIG-I and VISA, thereby potentiating IFN-ß production. CHID1 knockdown in human 239T cells inhibits SeV-induced activation of IRF3 and NF-κB and the induction of IFN-ß. These results indicate that CHID1 positively regulates RLR antiviral signal, revealing the novel mechanism of the RIG-I antiviral signaling pathway.

FKBP8 inhibits virus-induced RLR-VISA signaling.

The mitochondrial antiviral signal protein mitochondrial antiviral signaling protein, also known as virus-induced signaling adaptor (VISA), plays a key role in regulating host innate immune signaling pathways. This study identifies FK506 binding protein 8 (FKBP8) as a candidate interacting protein of VISA through the yeast two-hybrid technique. The interaction of FKBP8 with VISA, retinoic acid inducible protein 1 (RIG-I), and IFN regulatory factor 3 (IRF3) was confirmed during viral infection in mammalian cells by coimmunoprecipitation. Overexpression of FKBP8 using a eukaryotic expression plasmid significantly attenuated Sendai virus-induced activation of the promoter interferons ß (IFN-ß), and transcription factors nuclear factor κ-light chain enhancer of activated B cells (NF-κB) and IFN-stimulated response element (ISRE). Overexpression of FKBP8 also decreased dimer-IRF3 activity, but enhanced virus replication. Conversely, knockdown of FKBP8 expression by RNA interference showed opposite effects. Further studies indicated that FKBP8 acts as a negative interacting partner to regulate RLR-VISA signaling by acting on VISA and TANK binding kinase 1 (TBK1). Additionally, FKBP8 played a negative role on virus-induced signaling by inhibiting the formation of TBK1-IRF3 and VISA-TRAF3 complexes. Notably, FKBP8 also promoted the degradation of TBK1, RIG-I, and TRAF3 resulting from FKBP8 reinforced Sendai virus-induced endogenous polyubiquitination of RIG-I, TBK1, and TNF receptor-associated factor 3 (TRAF3). Therefore, a novel function of FKBP8 in innate immunity antiviral signaling regulation was revealed in this study.

Three distinct 3-methylcytidine (m3C) methyltransferases modify tRNA and mRNA in mice and humans.

Chemical RNA modifications are central features of epitranscriptomics, highlighted by the discovery of modified ribonucleosides in mRNA and exemplified by the critical roles of RNA modifications in normal physiology and disease. Despite a resurgent interest in these modifications, the biochemistry of 3-methylcytidine (m3C) formation in mammalian RNAs is still poorly understood. However, the recent discovery of trm141 as the second gene responsible for m3C presence in RNA in fission yeast raises the possibility that multiple enzymes are involved in m3C formation in mammals as well. Here, we report the discovery and characterization of three distinct m3C-contributing enzymes in mice and humans. We found that methyltransferase-like (METTL) 2 and 6 contribute m3C in specific tRNAs and that METTL8 only contributes m3C to mRNA. MS analysis revealed that there is an ∼30-40% and ∼10-15% reduction, respectively, in METTL2 and -6 null-mutant cells, of m3C in total tRNA, and primer extension analysis located METTL2-modified m3C at position 32 of tRNAThr isoacceptors and tRNAArg(CCU) We also noted that METTL6 interacts with seryl-tRNA synthetase in an RNA-dependent manner, suggesting a role for METTL6 in modifying serine tRNA isoacceptors. METTL8, however, modified only mRNA, as determined by biochemical and genetic analyses in Mettl8 null-mutant mice and two human METTL8 mutant cell lines. Our findings provide the first evidence of the existence of m3C modification in mRNA, and the discovery of METTL8 as an mRNA m3C writer enzyme opens the door to future studies of other m3C epitranscriptomic reader and eraser functions.

Insights of the Crystallization Process of Molecular Sieve AlPO4-5 Prepared by Solvent-Free Synthesis.

Crystallization of AlPO4-5 with AFI structure under solvent-free conditions has been investigated. Attention was mainly focused on the characterization of the intermediate phases formed at the early stages during the crystallization. The development in the long-range ordering of the solid phases as a function of crystallization time was monitored by XRD, SEM, IR, UV-Raman, and MAS NMR techniques. Particularly, the UV-Raman spectroscopy was employed to obtain the information on the formation process of the framework. J-HMQC (27)Al/(31)P double-resonance NMR experiments were used to identify the P-O-Al bonded species in the intermediate phases. For the first time the P-O-Al bonded species in the intermediate phases can be correctly described through using this advanced NMR technique. The crystallization under solvent-free conditions appears to follow the pathway: The initial amorphous raw material is converted to an intermediate phase which has four-/six-membered ring species, then gradually transformed into crystalline AlPO4-5. This observation is not consistent with the common idea that the intermediate phase is the semicrystalline intermediates with a three-dimensional structure.

Highly Efficient Heterogeneous Hydroformylation over Rh-Metalated Porous Organic Polymers: Synergistic Effect of High Ligand Concentration and Flexible Framework.

A series of diphosphine ligand constructed porous polymers with stable and flexible frameworks have been successfully synthesized under the solvothermal conditions from polymerizing the corresponding vinyl-functionalized diphosphine monomers. These insoluble porous polymers can be swollen by a wide range of organic solvents, showing similar behavior to those of soluble analogues. Rather than just as immobilizing homogeneous catalysts, these porous polymers supported with Rh species demonstrate even better catalytic performance in the hydroformylations than the analogue homogeneous catalysts. The sample extraordinary performance could be attributed to the combination of high ligand concentration and flexible framework of the porous polymers. Meanwhile, they can be easily separated and recycled from the reaction systems without losing any activity and selectivity. This excellent catalytic performance and easy recycling heterogeneous catalyst property make them be very attractive. These diphosphine ligand constructed porous polymers may provide new platforms for the hydroformylation of olefins in the future.

The protease inhibitor atazanavir blocks hERG K(+) channels expressed in HEK293 cells and obstructs hERG protein transport to cell membrane.

AIM: Atazanavir (ATV) is a HIV-1 protease inhibitor for the treatment of AIDS patients, which is recently reported to provoke excessive prolongation of the QT interval and torsades de pointes (TdP). In order to elucidate its arrhythmogenic mechanisms, we investigated the effects of ATV on the hERG K(+) channels expressed in HEK293 cells. METHODS: hERG K(+) currents were detected using whole-cell patch clamp recording in HEK293 cells transfected with EGFP-hERG plasmids. The expression of hERG protein was measured with Western blotting. Two mutants (Y652A and F656C) were constructed in the S6 domain within the inner helices of hERG K(+) channels that were responsible for binding of various drugs. The trafficking of hERG protein was studied with confocal microscopy. RESULTS: Application of ATV (0.01-30 µmol/L) concentration-dependently decreased hERG K(+) currents with an IC50 of 5.7±1.8 µmol/L. ATV (10 µmol/L) did not affect the activation and steady-state inactivation of hERG K(+) currents. Compared with the wild type hERG K(+) channels, both Y652A and F656C mutants significantly reduced the inhibition of ATV on hERG K(+) currents. Overnight treatment with ATV (0.1-30 µmol/L) concentration-dependently reduced the amount of fully glycosylated 155 kDa hERG protein without significantly affecting the core-glycosylated 135 kDa hERG protein in the cells expressing the WT-hERG protein. Confocal microscopy studies confirmed that overnight treatment with ATV obstructed the trafficking of hERG protein to the cell membrane. CONCLUSION: ATV directly blocks hERG K(+) channels via binding to the residues Y652 and F656 in the S6 domain, and indirectly obstructs the transport of the hERG protein to the cell membrane.

Solvent-free syntheses of hierarchically porous aluminophosphate-based zeolites with AEL and AFI structures.

Development of sustainable routes for synthesizing aluminophosphate-based zeolites are very important because of their wide applications. As a typical sustainable route, solvent-free synthesis of zeolites not only decreases polluted wastes but also increases product yields. Systematic solvent-free syntheses of hierarchically porous aluminophosphate-based zeolites with AEL and AFI structures is presented. XRD patterns and SEM images show that these samples have high crystallinity. N2 sorption isotherm tests show that these samples are hierarchically porous, and their surface areas are comparable with those of corresponding zeolites from hydrothermal route. Chosen as an example, catalytic oxidation of ethylbenzene with O2 shows that cobalt substituted APO-11 from the solvent-free route (S-CoAPO-11) is more active than conventional CoAPO-11 from hydrothermal route owing to the sample hierarchical porosity.

Blockage of hERG current and the disruption of trafficking as induced by roxithromycin.

Roxithromycin is an oral macrolide antibiotic agent that has been repeatedly reported to provoke excessive prolongation of the Q-T interval and torsades de pointes in clinical settings. To investigate the mechanisms underlying the arrhythmogenic side effects of roxithromycin, we studied the molecular mechanisms of roxithromycin on human ether-à-go-go-related gene (hERG) K(+) channels expressed in human embryonic kidney (HEK293) cells. Roxithromycin was found to inhibit wild-type (WT) hERG currents in a concentration-dependent manner with a half-maximum block concentration (IC50) of 55.8 ± 9.1 µmol/L. S6 residue hERG mutants (Y652A and F656C) showed reduced levels of hERG current blockage attributable to roxithromycin. Roxithromycin also inhibited the trafficking of hERG protein to the cell membrane, as confirmed by Western blot analysis and confocal microscopy. These findings indicate that roxithromycin may cause acquired long-QT syndrome via direct inhibition of hERG current and by disruption of hERG protein trafficking. Mutations in drug-binding sites (Y652A or F656C) of the hERG channel were found to attenuate hERG current blockage by roxithromycin, but did not significantly alter the disruption of trafficking.

Epigenetic Regulation of Macrophage Polarization in Cardiovascular Diseases.

Cardiovascular diseases (CVDs) are the leading cause of hospitalization and death worldwide, especially in developing countries. The increased prevalence rate and mortality due to CVDs, despite the development of several approaches for prevention and treatment, are alarming trends in global health. Chronic inflammation and macrophage infiltration are key regulators of the initiation and progression of CVDs. Recent data suggest that epigenetic modifications, such as DNA methylation, posttranslational histone modifications, and RNA modifications, regulate cell development, DNA damage repair, apoptosis, immunity, calcium signaling, and aging in cardiomyocytes; and are involved in macrophage polarization and contribute significantly to cardiac disease development. Cardiac macrophages not only trigger damaging inflammatory responses during atherosclerotic plaque formation, myocardial injury, and heart failure but are also involved in tissue repair, remodeling, and regeneration. In this review, we summarize the key epigenetic modifications that influence macrophage polarization and contribute to the pathophysiology of CVDs, and highlight their potential for the development of advanced epigenetic therapies.

ALKBH5 inhibitors as a potential treatment strategy in heart failure-inferences from gene expression profiling.

Heart Failure (HF) is a complex clinical syndrome in which the heart is unable to provide enough blood flow to meet metabolic needs and lacks efficient venous return. HF is a major risk factor for morbidity and mortality with cardiovascular diseases globally. Despite enormous research, the molecular markers relevant to disease prognosis and management remain not well understood. Here, we analyzed the whole transcriptomes of 18 failing hearts and 15 non-failing hearts (predominantly of Caucasian origin), by applying the standard in silico tools. The analyses revealed novel gene-markers including ALKBH5 of mRNA demethylation and KMT2E of histone modification processes, significantly over-expressed in the HF compared with the non-failing hearts (FDR < 0.05). To validate the over-expression of ALKBH5, we determined the global m6A level in hypoxic H9c2 cells using a dot blot assay. The global m6A level was found markedly lower in the hypoxic H9c2 cells than in the control cells. Additionally, the expression of ALKBH5 in the H9c2 cells was quantified by the qPCR and found to be 1.18 times higher at 12 h (p < 0.05), and 1.67 times higher at 24 h of hypoxia (p < 0.01) compared with the control cells, indicating a likely role of ALKBH5 in the failing cardiac cells. Furthermore, we identified several compounds through the virtual screening of 11,272 drug-like molecules of the ZINC15 database to inhibit the ALKBH5 in a molecular docking process. Collectively, the study revealed novel markers potentially involved in the pathophysiology of HF and suggested plausible therapeutic molecules for the management of the disease.

Dummy molecularly imprinted polymers as the coating of stir bar for sorptive extraction of bisphenol A in tap water.

A unique stir bar coated with dummy molecularly imprinted polymers for bisphenol A was prepared by sol-gel technique. The scanning electron microscopic image of the coating presented a homogeneous surface with a thickness of about 57 ± 2.5 µm. The Fourier transform infrared spectrum of the coating proved the incorporating of dummy molecularly imprinted polymers with the sol-gel network. When used to extract bisphenol A from aqueous solution containing bisphenol A and its three analogs (4-tert-butylphenol, 4,4'-dihydroxybiphenyl, and 3,3',5,5'-tetrabromo-bisphenol A). Dummy molecularly imprinted polymers-coated stir bar showed better selectivity than the bars coated with polydimethylsiloxane or non-imprinted polymers. The extraction conditions including stirring speed, pH, and extraction time were optimized. After back extraction with methanol, the extracts were analyzed by high-performance liquid chromatography-fluorescence detection. The linear range was 0.0228-0.456 ng/mL with correlation coefficient of 0.9994 and the detection limit was about 5.70 × 10(-3) ng/mL based on three times ratio of signal-to-noise. The method was applied to the determination of trace bisphenol A in tap water.

Potential therapeutic strategies for myocardial infarction: the role of Toll-like receptors.

Myocardial infarction (MI) is a life-threatening condition among patients with cardiovascular diseases. MI increases the risk of stroke and heart failure and is a leading cause of morbidity and mortality worldwide. Several genetic and epigenetic factors contribute to the development of MI, suggesting that further understanding of the pathomechanism of MI might help in the early management and treatment of this disease. Toll-like receptors (TLRs) are well-known members of the pattern recognition receptor (PRR) family and contribute to both adaptive and innate immunity. Collectively, studies suggest that TLRs have a cardioprotective effect. However, prolonged TLR activation in the response to signals generated by damage-associated molecular patterns (DAMPs) results in the release of inflammatory cytokines and contributes to the development and exacerbation of myocardial inflammation, MI, ischemia-reperfusion injury, myocarditis, and heart failure. The objective of this review is to discuss and summarize the association of TLRs with MI, highlighting their therapeutic potential for the development of advanced TLR-targeted therapies for MI.

GSK-3ß-mediated activation of NLRP3 inflammasome leads to pyroptosis and apoptosis of rat cardiomyocytes and fibroblasts.

We previously demonstrated that GSK-3ß mediates NLRP3 inflammasome activation and IL-1ß production in cardiac fibroblasts (CFs) after myocardial infarction (MI). In this study, we show how GSK-3ß-mediated activation of the NLRP3 inflammasome/caspase-1/IL-1ß pathway leads to apoptosis and pyroptosis of cardiomyocytes (CMs) and CFs. Administration of lipopolysaccharide (LPS)/ATP to primary newborn rat cardiac fibroblasts (RCFs) led to increase in proteins of NLRP3, apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC), caspase-1, IL-1ß, and IL-18. Additionally, the expression of caspase-3 and N-terminal fragments of gasdermin D (N-GSDMD) and the Bax/Bcl-2 ratio increased. Administration of the GSK-3ß inhibitor SB216763 reduced the levels of apoptosis- and pyroptosis-related proteins regulated by NLRP3 inflammasome activation in RCFs. Next, we transferred the culture supernatant of LPS/ATP-treated RCFs to in vitro primary newborn rat cardiomyocytes (RCMs). The results showed that SB216763 attenuate the upregulation of the ratios of Bax/Bcl-2 and the expression of caspase-3 and N-GSDMD in RCMs. Direct stimulation of RCMs and H9c2 cells with recombinant rat IL-1ß increased the p-GSK-3ß/GSK-3ß and Bax/Bcl-2 ratios and the expression of caspase-3 and N-GSDMD, while both SB216763 and TLR1 (an IL-1ß receptor inhibitor) markedly reduced these effects, as assessed using propidium iodide positive staining and the lactate dehydrogenase release assay. The caspase-11 inhibitor wedelolactone decreased the expression level of N-GSDMD but did not alter the p-GSK-3ß/GSK-3ß ratio. Lastly, we established a Sprague-Dawley rat MI model to confirm that SB216763 diminished the increase in caspase-3 and N-GSDMD expression and the Bax/Bcl-2 ratio in the ischemic area. These data demonstrate that GSK-3ß regulates apoptosis and pyroptosis of RCMs and RCFs due to NLRP3 inflammasome activation in RCFs.

Glycogen synthase kinase 3 beta inhibitor SB216763 improves Kir2.1 expression after myocardia infraction in rats.

BACKGROUND: Abnormal ion channel currents caused by myocardial electrical remodeling is one of the main causes of malignant arrhythmias. Glycogen synthase kinase 3ß (GSK-3ß) is the main therapeutic target following ischemia as it regulates nerve cell channels. However, few studies have investigated its role in myocardial electrical remodeling. The present study aimed to investigate the role of GSK-3ß in a rat myocardial infarction (MI)-induced electrical remodeling and potential effects on cardiac ionic channels including KCNJ2/Kir2.1/IK1. METHODS: Ligation of the left anterior descending artery in rats was performed to establish a MI model. The rats were randomly divided into three groups, the sham, MI, and MI + SB group. The animals in the latter group were administered SB216763 (GSK-3ß inhibitor) at a dose of 0.6 mg·kg-1·day-1. The ventricular function was assessed by echocardiography, electrocardiography, and histological analysis 7 days post-surgery. Serum was collected to measure lactate dehydrogenase and cardiac troponin I levels, and the mRNA and protein levels of the KCNJ2/Kir2.1/IK1 channel in the heart tissues were assessed. H9c2 cells were cultured to examine the effects of SB216763 on the protein expression of Kir2.1 channel under hypoxic conditions. RESULTS: The results revealed that SB216763 ameliorated acute cardiac injury and improved myocardial dysfunction. Moreover, SB216763 increased the mRNA and protein expression of Kir2.1 during MI. Furthermore, SB216763 treatment abrogated the decreased expression of Kir2.1 in H9c2 cells under hypoxic conditions. CONCLUSIONS: GSK-3ß inhibition upregulates Kir2.1 expression in a rat model of MI.

Ticagrelor reduces doxorubicin-induced pyroptosis of rat cardiomyocytes by targeting GSK-3ß/caspase-1.

Doxorubicin (Dox) is a widely used clinical drug whose cardiotoxicity cannot be ignored. Pyroptosis (inflammatory cell death) has gradually gained attention in the context of Dox-induced cardiotoxicity. In addition to the inhibition of platelet activation by ticagrelor, little is known about its other pharmacological effects. Glycogen synthase kinase 3ß (GSK-3ß) has been shown to contribute to the pathological process of pyroptosis, but whether it is related to the potential role of ticagrelor is unclear. In this study, we investigated the effects of ticagrelor on Dox-induced pyroptosis in cardiomyocytes. Rats were treated with ticagrelor (7.5 mg/kg, i.g.) 1 h before intravenous injection of Dox (2.5 mg/kg), once every 3 days, six times in total. Hearts were collected for histochemical analysis and western blot detection 8 weeks after the last administration. Ticagrelor was shown to significantly improve cardiac function by inhibiting GSK-3ß/caspase-1/GSDMD activation. In vitro experiments were conducted using rat cardiac myocytes (RCMs) and rat embryonic cardiac-derived H9c2 cells. Pretreatment with ticagrelor (10 µm) significantly inhibited Dox (1 µm)-induced hypertrophy and reversed the upregulation of GSDMD-NT expression. We showed that ticagrelor suppressed the activation of Akt caused by Dox in the heart tissue as well as in RCMs/H9c2 cells caused by Dox. When GSK-3ß expression was absent in H9c2 cells, the inhibitory effect of ticagrelor on Dox-induced caspase-1/GSDMD activation was weakened. These data showed that ticagrelor reduced Dox-induced pyroptosis in rat cardiomyocytes by targeting GSK-3ß/caspase-1.

GSK3ß mediates the spatiotemporal dynamics of NLRP3 inflammasome activation.

Subcellular machinery of NLRP3 is essential for inflammasome assembly and activation. However, the stepwise process and mechanistic basis of NLRP3 engagement with organelles remain unclear. Herein, we demonstrated glycogen synthase kinase 3ß (GSK3ß) as a molecular determinant for the spatiotemporal dynamics of NLRP3 inflammasome activation. Using live cell multispectral time-lapse tracking acquisition, we observed that upon stimuli NLRP3 was transiently associated with mitochondria and subsequently recruited to the Golgi network (TGN) where it was retained for inflammasome assembly. This occurred in relation to the temporal contact of mitochondria to Golgi apparatus. NLRP3 stimuli initiate GSK3ß activation with subsequent binding to NLRP3, facilitating NLRP3 recruitment to mitochondria and transition to TGN. GSK3ß activation also phosphorylates phosphatidylinositol 4-kinase 2 Α (PI4k2A) in TGN to promote sustained NLRP3 oligomerization. Our study has identified the interplay between GSK3ß signaling and the organelles dynamics of NLRP3 required for inflammasome activation and opens new avenues for therapeutic intervention.