METTL3-m6A-Rubicon axis inhibits autophagy in nonalcoholic fatty liver disease.

N6-methyladenosine (m6A) mRNA modification plays critical roles in various biological events and is involved in multiple complex diseases. However, the role of m6A modification in autophagy in nonalcoholic fatty liver disease (NAFLD) remains largely unknown. Here, we report that m6A modification was increased in livers of NAFLD mouse models and in free fatty acid (FFA)-treated hepatocytes, and the abnormal m6A modification was attributed to the upregulation of methyltransferase like 3 (METTL3) induced by lipotoxicity. Knockdown of METTL3 promoted hepatic autophagic flux and clearance of lipid droplets (LDs), while overexpression of METTL3 inhibited these processes. Mechanistically, METTL3 directly bound to Rubicon mRNA and mediated the m6A modification, while YTH N6-methyladenosine RNA binding protein 1 (YTHDF1), as a partner of METTL3, interacted with the m6A-marked Rubicon mRNA and promoted its stability. Subsequently, RUBICON inhibited autophagosome-lysosome fusion and further blocked clearance of LDs. Taken together, our results showed a critical role of METTL3 and YTHDF1 in regulating lipid metabolism via the autophagy pathway and provided a novel insight into m6A mRNA methylation in NAFLD.

Lipotoxicity-induced circGlis3 impairs beta cell function and is transmitted by exosomes to promote islet endothelial cell dysfunction.

AIMS/HYPOTHESIS: Lipotoxicity constitutes the major driving force for type 2 diabetes. Circular RNAs (circRNAs) play important roles in regulating beta cell function and exosomes are essential mediators of intercellular communication. The role of exosomal circRNAs in type 2 diabetes remains largely unknown. We aimed to examine whether lipotoxicity induces dysregulation of circRNAs in beta cell-derived exosomes and to determine the contribution of exosomal circRNAs to the development of type 2 diabetes. METHODS: Exosomes were extracted from MIN6 cells treated with palmitate or BSA, and RNA sequencing was performed. CircGlis3 (Gli-similar 3) expression level was validated by qPCR. The impact of circGlis3 on beta cell function and the deleterious effects of exosomal circGlis3 on islet endothelial cells (islet ECs) were investigated in vitro and in vivo in human and mouse models by gain or loss of function assays. The molecular mechanism of circGlis3 was explored by RNA pull-down and immunoprecipitation assays. RESULTS: Beta cell-derived exosomal circGlis3 was significantly upregulated under lipotoxic conditions, and exosomal circGlis3 levels were also elevated in the serum of mouse models of diabetes and participants with type 2 diabetes. CircGlis3 participated in lipotoxicity-induced beta cell dysfunction in vitro and in vivo. Moreover, beta cell-derived exosomal circGlis3 could be transferred to islet ECs and reduce the cell viability, cell migration and angiogenesis of islet ECs. Mechanistically, circGlis3 promoted the degradation of glucocorticoid modulatory element-binding protein 1 (GMEB1) by facilitating the interaction between GMEB1 and mindbomb E3 ubiquitin protein ligase 2 (MIB2), thus suppressing the phosphorylation of heat shock protein 27 (HSP27). CONCLUSIONS/INTERPRETATION: Our study points to the involvement of circGlis3 in diabetes development, and exosomal circGlis3 transfer as a communication mode between beta cells and islet ECs, suggesting that circGlis3 might be a potential biomarker and therapeutic target for type 2 diabetes. DATA AVAILABILITY: The RNA-sequencing data have been deposited in the NCBI Sequence Read Archive (SRA) database, with accession number PRJNA689673. Mass spectrometry data are available via ProteomeXchange with identifier PXD024693.

Myoglobin modulates the Hippo pathway to promote cardiomyocyte differentiation.

The endogenous mechanisms that propagate cardiomyocyte differentiation and prevent de-differentiation remain unclear. While the expression of the heme protein myoglobin increases by over 50% during cardiomyocyte differentiation, a role for myoglobin in regulating cardiomyocyte differentiation has not been tested. Here, we show that deletion of myoglobin in cardiomyocyte models decreases the gene expression of differentiation markers and stimulates cellular proliferation, consistent with cardiomyocyte de-differentiation. Mechanistically, the heme prosthetic group of myoglobin catalyzes the oxidation of the Hippo pathway kinase LATS1, resulting in phosphorylation and inactivation of yes-associated protein (YAP). In vivo, myoglobin-deficient zebrafish hearts show YAP dephosphorylation and accelerated cardiac regeneration after apical injury. Similarly, myoglobin knockdown in neonatal murine hearts shows increased YAP dephosphorylation and cardiomyocyte cycling. These data demonstrate a novel role for myoglobin as an endogenous driver of cardiomyocyte differentiation and highlight myoglobin as a potential target to enhance cardiac development and improve cardiac repair and regeneration.

Reduced acetylation of TFAM promotes bioenergetic dysfunction in the failing heart.

General control of amino acid synthesis 5-like 1 (GCN5L1) was previously identified as a key regulator of protein lysine acetylation in mitochondria. Subsequent studies demonstrated that GCN5L1 regulates the acetylation status and activity of mitochondrial fuel substrate metabolism enzymes. However, the role of GCN5L1 in response to chronic hemodynamic stress is largely unknown. Here, we show that cardiomyocyte-specific GCN5L1 knockout mice (cGCN5L1 KO) display exacerbated heart failure progression following transaortic constriction (TAC). Mitochondrial DNA and protein levels were decreased in cGCN5L1 KO hearts after TAC, and isolated neonatal cardiomyocytes with reduced GCN5L1 expression had lower bioenergetic output in response to hypertrophic stress. Loss of GCN5L1 expression led to a decrease in the acetylation status of mitochondrial transcription factor A (TFAM) after TAC in vivo, which was linked to a reduction in mtDNA levels in vitro. Together, these data suggest that GCN5L1 may protect from hemodynamic stress by maintaining mitochondrial bioenergetic output.

Serum albumin and prognosis in elderly patients with nonischemic dilated cardiomyopathy.

AIMS: Hypoalbuminemia was extensively used to diagnose malnutrition in older adults. Malnutrition was associated with mortality in elderly patients with cardiovascular diseases. The relationship between hypoalbuminemia and clinical outcomes in elderly patients with nonischemic dilated cardiomyopathy (NIDCM) remains unknown. METHODS: A total of 1058 consecutive patients with NIDCM (age ≥60 years) were retrospectively enrolled from January 2010 to December 2019. Univariate and multivariate analyses were performed to assess the association of hypoalbuminemia with clinical outcomes. RESULTS: Patients with hypoalbuminemia were older (69.29 ±â€Š6.67 vs. 67.61 ±â€Š5.90 years, P  < 0.001) and had higher prevalence of in-hospital and long-term death than those without (6.9 vs. 1.7%, 50.7 vs. 35.2%, P  < 0.001). Logistic regression analysis showed that hypoalbuminemia was significantly related to in-hospital death [odds ratio (OR): 4.334, 95% confidence interval (CI): 2.185-8.597, P  < 0.001]. Kaplan-Meier survival analysis showed that patients with hypoalbuminemia had worse prognosis than those with nonhypoalbuminemia (log-rank χ2 28.96, P  < 0.001). After adjusting for age, serum creatinine, HDL-C, AST/ALT hypoalbuminemia, LVEF and diabetes, hypoalbuminemia remained an independent predictor for long-term death (hazard ratio 1.322, 95% CI 0.046-1.670, P  = 0.019). CONCLUSION: Hypoalbuminemia was associated with increased risk of in-hospital and long-term mortality in elderly patients with NIDCM.

Myocardial brain-derived neurotrophic factor regulates cardiac bioenergetics through the transcription factor Yin Yang 1.

AIMS: Brain-derived neurotrophic factor (BDNF) is markedly decreased in heart failure patients. Both BDNF and its receptor, tropomyosin-related kinase receptor (TrkB), are expressed in cardiomyocytes; however, the role of myocardial BDNF signalling in cardiac pathophysiology is poorly understood. Here, we investigated the role of BDNF/TrkB signalling in cardiac stress response to exercise and pathological stress. METHODS AND RESULTS: We found that myocardial BDNF expression was increased in mice with swimming exercise but decreased in a mouse heart failure model and human failing hearts. Cardiac-specific TrkB knockout (cTrkB KO) mice displayed a blunted adaptive cardiac response to exercise, with attenuated upregulation of transcription factor networks controlling mitochondrial biogenesis/metabolism, including peroxisome proliferator-activated receptor gamma coactivator 1 alpha (PGC-1α). In response to pathological stress (transaortic constriction, TAC), cTrkB KO mice showed an exacerbated heart failure progression. The downregulation of PGC-1α in cTrkB KO mice exposed to exercise or TAC resulted in decreased cardiac energetics. We further unravelled that BDNF induces PGC-1α upregulation and bioenergetics through a novel signalling pathway, the pleiotropic transcription factor Yin Yang 1. CONCLUSION: Taken together, our findings suggest that myocardial BDNF plays a critical role in regulating cellular energetics in the cardiac stress response.

Predicting response to immunotherapy in gastric cancer via assessing perineural invasion-mediated inflammation in tumor microenvironment.

BACKGROUND: The perineural invasion (PNI)-mediated inflammation of the tumor microenvironment (TME) varies among gastric cancer (GC) patients and exhibits a close relationship with prognosis and immunotherapy. Assessing the neuroinflammation of TME is important in predicting the response to immunotherapy in GC patients. METHODS: Fifteen independent cohorts were enrolled in this study. An inflammatory score was developed and validated in GC. Based on PNI-related prognostic inflammatory signatures, patients were divided into Clusters A and B using unsupervised clustering. The characteristics of clusters and the potential regulatory mechanism of key genes were verified by RT-PCR, western-blot, immunohistochemistry and immunofluorescence in cell and tumor tissue samples.The neuroinflammation infiltration (NII) scoring system was developed based on principal component analysis (PCA) and visualized in a nomogram together with other clinical characteristics. RESULTS: Inflammatory scores were higher in GC patients with PNI compared with those without PNI (P < 0.001). NII.clusterB patients with PNI had abundant immune cell infiltration in the TME but worse prognosis compared with patients in the NII.clusterA patients with PNI and non-PNI subgroups. Higher immune checkpoint expression was noted in NII.clusterB-PNI. VCAM1 is a specific signature of NII.clusterB-PNI, which regulates PD-L1 expression by affecting the phosphorylation of STAT3 in GC cells. Patients with PNI and high NII scores may benefit from immunotherapy. Patients with low nomogram scores had a better prognosis than those with high nomogram scores. CONCLUSIONS: Inflammation mediated by PNI is one of the results of tumor-nerve crosstalk, but its impact on the tumor immune microenvironment is complex. Assessing the inflammation features of PNI is a potential method in predicting the response of immunotherapy effectively.

Coupling Lipid Labeling and Click Chemistry Enables Isolation of Extracellular Vesicles for Noninvasive Detection of Oncogenic Gene Alterations.

Well-preserved molecular cargo in circulating extracellular vesicles (EVs) offers an ideal material for detecting oncogenic gene alterations in cancer patients, providing a noninvasive diagnostic solution for detection of disease status and monitoring treatment response. Therefore, technologies that conveniently isolate EVs with sufficient efficiency are desperately needed. Here, a lipid labeling and click chemistry-based EV capture platform ("Click Beads"), which is ideal for EV message ribonucleic acid (mRNA) assays due to its efficient, convenient, and rapid purification of EVs, enabling downstream molecular quantification using reverse transcription digital polymerase chain reaction (RT-dPCR) is described and demonstrated. Ewing sarcoma protein (EWS) gene rearrangements and kirsten rat sarcoma viral oncogene homolog (KRAS) gene mutation status are detected and quantified using EVs isolated by Click Beads and matched with those identified in biopsy specimens from Ewing sarcoma or pancreatic cancer patients. Moreover, the quantification of gene alterations can be used for monitoring treatment responses and disease progression.

Circ-Tulp4 promotes ß-cell adaptation to lipotoxicity by regulating soat1 expression.

This study aimed to identify circular RNAs differentially expressed in the islets of type 2 diabetes (T2DM) models and clarify their roles in the control of ß-cell functions. Circular RNAs dysregulated in the islets of diabetic db/db mice were identified by high-throughput RNA sequencing. Then, the expression level of the selected circular RNA circ-Tulp4 was confirmed by real-time PCR in the islets of diabetic models and Min6 cells. MTS, EdU, western blot, flow cytometric analysis, and luciferase assay were performed to investigate the impact of circ-Tulp4 on ß-cell functions. This study identified thousands of circular RNAs in mouse pancreatic islets. The circ-Tulp4 level significantly decreased in the diabetic models and altered in the Min6 cells under lipotoxic condition. The modulation of circ-Tulp4 level in Min6 cells regulated cell proliferation. Furthermore, an interaction was demonstrated between circ-Tulp4 and miR-7222-3p, which suppressed the expression of cholesterol esterification-related gene, sterol O-acyltransferase 1 (SOAT1). The accumulation of soat1 activated cyclin D1 expression, thus promoting cell cycle progression. These findings showed that circ-Tulp4 regulated ß-cell proliferation via miR-7222-3p/soat1/cyclin D1 signaling. Our research suggested that circ-Tulp4 might be a potential therapeutic intervention for T2DM. Besides, soat1 might be important for ß-cell adaptation to lipotoxicity.