Comprehensive analysis of RNA m6A methylation in pressure overload-induced cardiac hypertrophy.

AIM: To analyze and compare the mRNA N6-methyladenosine modifications in transverse aortic constriction induced mice hearts and normal mice hearts. MATERIALS AND METHODS: Colorimetric quantification was used to probe the changes in m6A modifications in the total RNA. The expression of m6A-related enzymes was analyzed via qRT-PCR and western blotting. RNA-seq and MeRIP-seq were performed to identify genes with differences in m6A modifications or expression in the transcriptome profile. RESULTS: Compared with the control group, the TAC group exhibited higher m6A methylation levels. FTO and WTAP were downregulated after TAC, while METTL3 was significantly downregulated at the protein level. MeRIP-seq revealed that 1179 m6A peaks were upmethylated and 733 m6A peaks were downmethylated, and biological analysis of these genes exhibited a strong relationship with heart function. CONCLUSION: Our findings provide novel information regarding m6A modification and gene expression changes in cardiac hypertrophy, which may be fundamental for further research.

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.

Association of sleep duration with risk of type 2 diabetes mellitus in a rural Chinese population: a nested case-control study.

PURPOSE: To investigate the association of sleep duration with type 2 diabetes mellitus (T2DM) in a rural Chinese population. METHODS: A 1:1 matched nested case-control study was performed based on a cohort that had been established in rural communities in Henan Province, China. T2DM patients and healthy controls (550 pairs) were included in this study. RESULTS: Abnormal sleep duration significantly increased the risk of T2DM with an approximate U-shaped association (sleep duration ≤ 6 h, OR = 1.742, 95% CI = 1.007-3.011, P = 0.047; sleep duration 8-9 h, OR = 1.462, 95% CI = 1.038-2.060, P = 0.030) compared with participants with a night sleep duration of 7-8 h, after adjusting for multiple confounders. When stratified by gender, only women were sensitive to shorter sleep duration (OR = 2.483, 95% CI = 1.149-5.366, P = 0.021). Abnormal sleep duration (too short or too long) had adverse effects on homeostasis model assessment (HOMA) and blood metabolites, and the effect was more noticeable in people with longer sleep durations. CONCLUSION: In a rural Chinese population, both too short and too long sleep duration increased the risk of T2DM. Especially women with less sleep duration have a higher risk of T2DM. Abnormal sleep also affects the HOMA index and metabolites; the relationship between HOMA-IR, total cholesterol, and LDL-Cholesterol with sleep duration was U-shaped, while fasting plasma glucose, body mass index, waist circumference, and triglyceride levels increased significantly only with longer sleep duration.

The Long Noncoding RNA Hepatocyte Nuclear Factor 4α Antisense RNA 1 Negatively Regulates Cytochrome P450 Enzymes in Huh7 Cells via Histone Modifications.

The maintenance of homeostasis of cytochromes P450 enzymes (P450s) under both physiologic and xenobiotic exposure conditions is ensured by the action of positive and negative regulators. In the current study, the hepatocyte nuclear factor 4α (HNF4A) antisense RNA 1 (HNF4A-AS1), an antisense long noncoding RNA of HNF4A, was found to be a negative regulator of the basal and rifampicin (RIF)-induced expression of nuclear receptors and downstream P450s. In Huh7 cells, knockdown of HNF4A-AS1 resulted in elevated expression of HNF4A, pregnane X receptor (PXR), and P450s (including CYP3A4) under both basal and RIF-induced conditions. Conversely, overexpression of HNF4A-AS1 led to decreased basal expression of constitutive androstane receptor, aryl hydrocarbon receptor, PXR, and all studied P450s. Of note, significantly diminished induction levels of PXR and CYP1A2, 2C8, 2C19, and 3A4 by RIF were also observed in HNF4A-AS1 plasmid-transfected Huh7 cells. Moreover, the negative feedback of HNF4A on HNF4A-AS1-mediated gene expression was validated using a loss-of-function experiment in this study. Strikingly, our data showed that increased enrichment levels of histone 3 lysine 4 trimethylation and HNF4A in the CYP3A4 promoter contribute to the elevated CYP3A4 expression after HNF4A-AS1 knockdown. Overall, the current study reveals that histone modifications contribute to the negative regulation of nuclear receptors and P450s by HNF4A-AS1 in basal and drug-induced levels. SIGNIFICANCE STATEMENT: Utilizing loss-of-function and gain-of-function experiments, the current study systematically investigated the negative regulation of HNF4A-AS1 on the expression of nuclear receptors (including HNF4A, constitutive androstane receptor, aryl hydrocarbon receptor, and pregnane X receptor) and P450s (including CYP1A2, 2E1, 2B6, 2D6, 2C8, 2C9, 2C19, and 3A4) in both basal and rifampicin-induced levels in Huh7 cells. Notably, this study is the first to reveal the contribution of histone modification to the HNF4A-AS1-mediated expression of CYP3A4 in Huh7 cells.

Glycogen synthase kinase-3ß inhibition alleviates activation of the NLRP3 inflammasome in myocardial infarction.

Inflammasome-promoted sterile inflammation following cardiac damage is critically implicated in heart dysfunction after myocardial infarction (MI). Glycogen synthase kinase-3 (GSK-3ß) is a prominent mediator of the inflammatory response, and high GSK-3 activity is associated with various heart diseases. We investigated the regulatory mechanisms of GSK-3ß in activation of the nod-like receptor family pyrin domain containing 3 (NLRP3) inflammasome in a rat model with successful induction of MI on days 2-28. An in vitro investigation was performed using newborn rat/human cardiomyocytes and fibroblast cultures under typical inflammasome stimulation and hypoxia treatment. GSK-3ß inhibition markedly improved myocardial dysfunction and prevented remodeling, with parallel reduction in the parameters of NLRP3 inflammasome activation after MI. GSK-3ß inhibition reduced NLRP3 inflammasome activation in cardiac fibroblasts, but not in cardiomyocytes. GSK-3ß's interaction with activating signal cointegrator (ASC) as well as GSK-3ß inhibition reduced ASC phosphorylation and oligomerization at the tissues and cellular levels. Taken together, these data show that GSK-3ß directly mediates NLRP3 inflammasome activation, causing cardiac dysfunction in MI.

Lamin A mutation impairs interaction with nucleoporin NUP155 and disrupts nucleocytoplasmic transport in atrial fibrillation.

Atrial fibrillation (AF) is the most common cardiac arrhythmia. Here, we show the identification and functional characterization of one AF-associated mutation p.Arg399Cys in lamin A/C. Co-immunoprecipitation and GST pull-down assays demonstrate that lamin A/C interacts with NUP155, which is a nucleoporin and causes AF when mutated. Lamin A/C mutation p.Arg399Cys impairs the interaction between lamin A/C and NUP155, and increases extractability of NUP155 from the nuclear envelope (NE). Mutation p.Arg399Cys leads to aggregation of lamin A/C in the nucleus, although it does not impair the integrity of NE upon cellular stress. Mutation p.Arg399Cys inhibits the export of HSP70 mRNA and the nuclear import of HSP70 protein. Electrophysiological studies show that mutation p.Arg399Cys decreases the peak cardiac sodium current by decreasing the cell surface expression level of cardiac sodium channel Nav 1.5, but does not affect IKr potassium current. In conclusion, our results indicate that lamin A/C mutation p.Arg399Cys weakens the interaction between nuclear lamina (lamin A/C) and the nuclear pore complex (NUP155), leading to the development of AF. The findings provide a novel molecular mechanism for the pathogenesis of AF.

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.

The HNF1α-Regulated LncRNA HNF1α-AS1 Is Involved in the Regulation of Cytochrome P450 Expression in Human Liver Tissues and Huh7 Cells.

Expression of cytochrome P450s (P450s) is regulated by epigenetic factors, such as DNA methylation, histone modifications, and noncoding RNAs through different mechanisms. Among these factors, long noncoding RNAs (lncRNAs) have been shown to play important roles in the regulation of gene expression; however, little is known about the effects of lncRNAs on the regulation of P450 expression. The aim of this study was to explore the role of lncRNAs in the regulation of P450 expression by using human liver tissues and hepatoma Huh7 cells. Through lncRNA microarray analysis and quantitative polymerase chain reaction in human liver tissues, we found that the lncRNA hepatocyte nuclear factor 1 alpha antisense 1 (HNF1α-AS1), an antisense RNA of HNF1α, is positively correlated with the mRNA expression of CYP2C8, 2C9, 2C19, 2D6, 2E1, and 3A4 as well as pregnane X receptor (PXR) and constitutive androstane receptor (CAR). Gain- and loss-of-function studies in Huh7 cells transfected with small interfering RNAs or overexpression plasmids showed that HNF1α not only regulated the expression of HNF1α-AS1 and P450s, but also regulated the expression of CAR, PXR, and aryl hydrocarbon receptor (AhR). In turn, HNF1α-AS1 regulated the expression of PXR and most P450s without affecting the expression of HNF1α, AhR, and CAR. Moreover, the rifampicin-induced expression of P450s was also affected by HNF1α and HNF1α-AS1. In summary, the results of this study suggested that HNF1α-AS1 is involved in the HNF1α-mediated regulation of P450s in the liver at both basal and drug-induced levels.

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.

CYP3A4*1G regulates CYP3A4 intron 10 enhancer and promoter activity in an allelic-dependent manner.

OBJECTIVE: CYP3A4*1G (G > A) in human CYP3A4 intron 10 is associated with therapeutic effects of CYP3A4-metabolized drugs. The aim of this study was to predict its function in the regulation of CYP3A4 expression. METHODS: Functional analysis of the CYP3A4*1G allele was performed using bioinformatic methods and enhancer or promoter reporter assays in HepG2 cells. RESULTS: Transcription regulatory elements like CAATboxes, TATA-boxes, Sp1, SMARCA3.01, and Box II-like sequence were present in the intron 10 of CYP3A4. SMARCA3.01 and Box II-like sequence were responsible for differential binding of transcription factors on the CYP3A4*1G allele. In CYP3A4*1G, the G allele enhanced expression of the CYP3A4 promoter in a position-dependent and orientation-dependent manner, however, the A allele enhanced expression of the CYP3A4 promoter in a position-independent and orientation-independent manner. In addition, the G allele and the A allele both displayed strong transcriptional activation, but the latter showed higher promoter activity than the former. Also, the A allele showed greater activity than the CYP3A4 promoter. CONCLUSION: These results in vitro suggest that CYP3A4*1G regulates CYP3A4 intron 10 enhancer and promoter activity in an allelic-dependent manner.

Implications of Bit1 and AIF overexpressions in esophageal squamous cell carcinoma.

Overwhelming evidence has demonstrated that Bit1 and AIF as mitochondrial proteins are implicated in the development and progression of a variety of tumors. However, their expressions and biological functions in esophageal squamous cell carcinoma (ESCC) remain to be delineated. In the present study, we found that Bit1, AIF, and Bcl-2 levels in ESCC tissues were significantly higher than those in normal esophageal epithelial tissues and dysplasia tissues (P < 0.05). Stepwise investigation demonstrated that Bit1 and Bcl-2 levels were both tightly associated with lymphatic metastasis and TNM staging (P < 0.05), and the levels of Bit1 mRNA as well as AIF and Bcl-2 proteins were both closely related to tumor differentiation (P < 0.05), but not related to the patients' age and gender (P > 0.05). Importantly, Bit1 mRNA and protein levels in ESCC with lymphatic metastasis and TNM staging in III and IV were markedly higher than that without lymphatic metastasis and TMN staging in I and II. Further analysis showed that expression of Bit1 protein was both positively correlated with expressions of AIF and Bcl-2 proteins (r = 0.408 and 0.405, respectively; P < 0.05). Correctively, our data cited earlier suggest that Bit1 plays pivotal roles in the development and progression of ESCC, and its biological functions in ESCC may be closely associated with AIF and Bcl-2 levels.

The long noncoding RNA HNF1A-AS1 with dual functions in the regulation of cytochrome P450 3A4.

Cytochrome P450 3A4 (CYP3A4) is the most important and abundant drug-metabolizing enzyme in the human liver. Inter-individual differences in the expression and activity of CYP3A4 affect clinical and precision medicine. Increasing evidence indicates that long noncoding RNAs (lncRNAs) play crucial roles in the regulation of CYP3A4 expression. Here, we showed that lncRNA hepatocyte nuclear factor 1 alpha-antisense 1 (HNF1A-AS1) exerted dual functions in regulating CYP3A4 expression in Huh7 and HepG2 cells. Mechanistically, HNF1A-AS1 served as an RNA scaffold to interact with both protein arginine methyltransferase 1 and pregnane X receptor (PXR), thereby facilitating their protein interactions and resulting in the transactivation of PXR and transcriptional alteration of CYP3A4 via histone modifications. Furthermore, HNF1A-AS1 bound to the HNF1A protein, a liver-specific transcription factor, thereby blocking its interaction with the E3 ubiquitin ligase tripartite motif containing 25, ultimately preventing HNF1A ubiquitination and protein degradation, further regulating the expression of CYP3A4. In summary, these results reveal the novel functions of HNF1A-AS1 as the transcriptional and post-translational regulator of CYP3A4; thus, HNF1A-AS1 may serve as a new indicator for establishing or predicting individual differences in CYP3A4 expression.

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.

Prenatal Lipopolysaccharide Exposure Alters Hepatic Drug-Metabolizing Enzyme Expression in Mouse Offspring via Histone Modifications.

Inflammation is a major regulator of drug-metabolizing enzymes (DMEs), therefore contributing to the interindividual variability of drug effects. However, whether prenatal inflammation affects DMEs expression in offspring remains obscure. This study investigated the effects of prenatal lipopolysaccharide (LPS) exposure on hepatic expression of inflammatory-related genes, nuclear receptors, and DMEs in offspring mice. Prenatal LPS exposure on gestational day (GD) 10 led to higher expression of NF-κB, Pxr, and Cyp2b10, while lower expression of Car, Ahr, Cyp3a11, and Ugt1a1 in postnatal day (PD) 30 offspring. However, multiple doses of LPS exposure on GD10-14 resulted in higher levels of inflammatory-related genes, Cyp1a2, and Cyp2b10, and lower levels of Pxr and Cyp3a11 in PD30 offspring liver. For PD60 offspring, decreased hepatic expression of NF-κB and IL-6, and increased expression of Pxr and Cyp3a11 were seen in single-dose LPS groups, whereas opposite results were observed in the multiple-dose LPS groups. Notably, enhanced H3K4me3 levels in the PXR response elements of the Cyp3a11 promoter were observed in the liver of PD60 offspring mice from dams treated with multiple doses of LPS during pregnancy. Overall, this study suggests that parental LPS exposure could persistently alter the hepatic expression of DMEs, and histone modifications may contribute to the long-term effects.

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.

An association study of HLA with levofloxacin-induced severe cutaneous adverse drug reactions in Han Chinese.

Levofloxacin-induced severe cutaneous adverse drug reactions (LEV-SCARs) remain unexplored. An association study of human leukocyte antigen (HLA) alleles with LEV-SCARs among 12 patients, 806 healthy subjects, and 100 levofloxacin-tolerant individuals was performed. The carrier frequencies of HLA-B∗13:01 (odds ratio [OR]: 4.50; 95% confidence interval [CI]: 1.15-17.65; p = 0.043), HLA-B∗13:02 (OR: 6.14; 95% CI: 1.73-21.76; p = 0.0072), and serotype B13 (OR: 17.73; 95% CI: 3.61-86.95; p = 4.85 × 10-5) in patients with LEV-SCARs were significantly higher than those of levofloxacin-tolerant individuals. Molecular docking analysis suggested that levofloxacin formed more stable binding models with HLA-B∗13:01 and HLA-B∗13:02 than with non-risk HLA-B∗46:01. Mass spectrometry revealed that nonapeptides bound to HLA-B∗13:02 shifted at several positions after exposure to levofloxacin. Prospective screening for serotype B13 (sensitivity: 83%, specificity: 78%) and alternative drug treatment for carriers may significantly decrease the incidence of LEV-SCARs.

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.