Long-term reliability of the phospholamban (PLN) p.(Arg14del) risk model in predicting major ventricular arrhythmia: a landmark study.

AIMS: Recently, a genetic variant-specific prediction model for phospholamban (PLN) p.(Arg14del)-positive individuals was developed to predict individual major ventricular arrhythmia (VA) risk to support decision-making for primary prevention implantable cardioverter defibrillator (ICD) implantation. This model predicts major VA risk from baseline data, but iterative evaluation of major VA risk may be warranted considering that the risk factors for major VA are progressive. Our aim is to evaluate the diagnostic performance of the PLN p.(Arg14del) risk model at 3-year follow-up. METHODS AND RESULTS: We performed a landmark analysis 3 years after presentation and selected only patients with no prior major VA. Data were collected of 268 PLN p.(Arg14del)-positive subjects, aged 43.5 ± 16.3 years, 38.9% male. After the 3 years landmark, subjects had a mean follow-up of 4.0 years (± 3.5 years) and 28 (10%) subjects experienced major VA with an annual event rate of 2.6% [95% confidence interval (CI) 1.6-3.6], defined as sustained VA, appropriate ICD intervention, or (aborted) sudden cardiac death. The PLN p.(Arg14del) risk score yielded good discrimination in the 3 years landmark cohort with a C-statistic of 0.83 (95% CI 0.79-0.87) and calibration slope of 0.97. CONCLUSION: The PLN p.(Arg14del) risk model has sustained good model performance up to 3 years follow-up in PLN p.(Arg14del)-positive subjects with no history of major VA. It may therefore be used to support decision-making for primary prevention ICD implantation not merely at presentation but also up to at least 3 years of follow-up.

Increased DNA methylation contributes to the early ripening of pear fruits during domestication and improvement.

BACKGROUND: DNA methylation is an essential epigenetic modification. However, its contribution to trait changes and diversity in the domestication of perennial fruit trees remains unknown. RESULTS: Here, we investigate the variation in DNA methylation during pear domestication and improvement using whole-genome bisulfite sequencing in 41 pear accessions. Contrary to the significant decrease during rice domestication, we detect a global increase in DNA methylation during pear domestication and improvement. We find this specific increase in pear is significantly correlated with the downregulation of Demeter-like1 (DML1, encoding DNA demethylase) due to human selection. We identify a total of 5591 differentially methylated regions (DMRs). Methylation in the CG and CHG contexts undergoes co-evolution during pear domestication and improvement. DMRs have higher genetic diversity than selection sweep regions, especially in the introns. Approximately 97% of DMRs are not associated with any SNPs, and these DMRs are associated with starch and sucrose metabolism and phenylpropanoid biosynthesis. We also perform correlation analysis between DNA methylation and gene expression. We find genes close to the hypermethylated DMRs that are significantly associated with fruit ripening. We further verify the function of a hyper-DMR-associated gene, CAMTA2, and demonstrate that overexpression of CAMTA2 in tomato and pear callus inhibits fruit ripening. CONCLUSIONS: Our study describes a specific pattern of DNA methylation in the domestication and improvement of a perennial pear tree and suggests that increased DNA methylation plays an essential role in the early ripening of pear fruits.

NLRC4-mediated pyroptosis was involved in coagulation disorders of acute pancreatitis.

BACKGROUND: Acute pancreatitis (AP) is a potentially lethal acute disease highly involved in coagulation disorders. Pyroptosis has been reported to exacerbate coagulation disorders, yet this implication has not been illustrated completely in AP. METHODS: RNA sequencing data of peripheral blood of AP patients were downloaded from the Gene Expression Omnibus database. Gene set variation analysis and single sample gene set enrichment analysis were used to calculate the enrichment score of coagulation-related signatures and pyroptosis. Spearman and Pearson correlation analysis was used for correlation analysis. Peripheral blood samples and related clinical parameters were collected from patients with AP and healthy individuals. A severe AP (SAP) model of mice was established using caerulein and lipopolysaccharide. Enzyme-linked immunosorbent assay, chemiluminescence immunoassay and immunohistochemical analysis were employed to detect the level of coagulation indicators and pyroptosis markers in serum and pancreas tissues. Additionally, we evaluated the effect of pyroptosis inhibition and NLRC4 silence on the function of human umbilical vein endothelial cells (HUVECs). RESULTS: Coagulation disorders were significantly positively correlated to the severity of AP, and they could be a predictor for AP severity. Further analyses indicated that six genes-DOCK9, GATA3, FCER1G, NLRC4, C1QB and C1QC-may be involved in coagulation disorders of AP. Among them, NLRC4 was positively related to pyroptosis that had a positive association with most coagulation-related signatures. Data from patients showed that NLRC4 and other pyroptosis markers, including IL-1ß, IL-18, caspase1 and GSDMD, were significant correlation to AP severity. In addition, NLRC4 was positively associated with coagulation indicators in AP patients. Data from mice showed that NLRC4 was increased in the pancreas tissues of SAP mice. Treatment with a pyroptosis inhibitor effectively alleviated SAP and coagulation disorders in mice. Finally, inhibiting pyroptosis or silencing NLRC4 could relieve endothelial dysfunction in HUVECs. CONCLUSIONS: NLRC4-mediated pyroptosis damages the function of endothelial cells and thereby exacerbates coagulation disorders of AP. Inhibiting pyroptosis could improve coagulation function and alleviate AP.

Protein subcellular relocalization and function of duplicated flagellar calcium binding protein genes in honey bee trypanosomatid parasite.

The honey bee trypanosomatid parasite, Lotmaria passim, contains two genes that encode the flagellar calcium binding protein (FCaBP) through tandem duplication in its genome. FCaBPs localize in the flagellum and entire body membrane of L. passim through specific N-terminal sorting sequences. This finding suggests that this is an example of protein subcellular relocalization resulting from gene duplication, altering the intracellular localization of FCaBP. However, this phenomenon may not have occurred in Leishmania, as one or both of the duplicated genes have become pseudogenes. Multiple copies of the FCaBP gene are present in several Trypanosoma species and Leptomonas pyrrhocoris, indicating rapid evolution of this gene in trypanosomatid parasites. The N-terminal flagellar sorting sequence of L. passim FCaBP1 is in close proximity to the BBSome complex, while that of Trypanosoma brucei FCaBP does not direct GFP to the flagellum in L. passim. Deletion of the two FCaBP genes in L. passim affected growth and impaired flagellar morphogenesis and motility, but it did not impact host infection. Therefore, FCaBP represents a duplicated gene with a rapid evolutionary history that is essential for flagellar structure and function in a trypanosomatid parasite.

Identification of allograft inflammatory factor-1 suppressing the progression and indicating good prognosis of osteosarcoma.

BACKGROUND: Osteosarcoma is one of the most common cancers worldwide. Intense efforts have been made to elucidate the pathogeny, but the mechanisms of osteosarcoma are still not well understood. We aimed to investigate the potential biomarker, allograft inflammatory factor-1 (AIF1), affecting the progression and prognosis of osteosarcoma. METHODS: Three microarray datasets were downloaded from GEO datasets and one was obtained from the TCGA dataset. The differentially expressed genes (DEGs) were identified. GO and KEGG functional enrichment analyses of overlapped DEGs were performed. The PPI network of overlapped DEGs was constructed by STRING and visualized with Cytoscape. Overall survival (OS) and Metastasis free survival (MFS) were analyzed from GSE21257. Finally, the effect of the most relevant core gene affecting the progression of osteosarcoma was examined in vitro. RESULTS: One hundred twenty six DEGs were identified, consisting of 65 upregulated and 61 downregulated genes. Only AIF1 was significantly associated with OS and MFS. It was found that AIF1 could be enriched into the NF-κB signaling pathway. GSEA and ssGSEA analyses showed that AIF1 was associated with the immune invasion of tumors. Cell experiments showed that AIF1 was underexpressed in osteosarcoma cell lines, while the malignant propriety was attenuated after overexpressing the expression of AIF1. Moreover, AIF1 also affects the expression of the NF-κB pathway. CONCLUSION: In conclusion, DEGs and hub genes identified in the present study help us understand the molecular mechanisms underlying the carcinogenesis and progression of osteosarcoma, and provide candidate targets for diagnosis and treatment of osteosarcoma.

Maternal RNA transcription in Dlk1-Dio3 domain is critical for proper development of the mouse placental vasculature.

The placenta is a unique organ for ensuring normal embryonic growth in the uterine. Here, we found that maternal RNA transcription in Dlk1-Dio3 imprinted domain is essential for placentation. PolyA signals were inserted into Gtl2 to establish a mouse model to prevent the expression of maternal RNAs in the domain. The maternal allele knock-in (MKI) and homozygous (HOMO) placentas showed an expanded junctional zone, reduced labyrinth and poor vasculature impacting both fetal and maternal blood spaces. The MKI and HOMO models displayed dysregulated gene expression in the Dlk1-Dio3 domain. In situ hybridization detected Dlk1, Gtl2, Rtl1, miR-127 and Rian dysregulated in the labyrinth vasculature. MKI and HOMO induced Dlk1 to lose imprinting, and DNA methylation changes of IG-DMR and Gtl2-DMR, leading to abnormal gene expression, while the above changes didn't occur in paternal allele knock-in placentas. These findings demonstrate that maternal RNAs in the Dlk1-Dio3 domain are involved in placental vasculature, regulating gene expression, imprinting status and DNA methylation.

NLRC4 methylation and its response to intravenous immunoglobulin therapy in Kawasaki disease: a case control study.

BACKGROUND: Kawasaki disease (KD) is a systemic vasculitis accompanied by many systemic physiological and biochemical changes. Elucidating its molecular mechanisms is crucial for diagnosing and developing effective treatments. NLR Family CARD Domain Containing 4 (NLRC4) encodes the key components of inflammasomes that function as pattern recognition receptors. The purpose of this study was to investigate the potential of NLRC4 methylation as a biomarker for KD. METHODS: In this study, pyrosequencing was utilized to analyze NLRC4 promoter methylation in blood samples from 44 children with initial complete KD and 51 matched healthy controls. Methylation at five CpG sites within the NLRC4 promoter region was evaluated. RESULTS: Compared to controls, NLRC4 methylation significantly decreased in KD patients (CpG1: p = 2.93E-06; CpG2: p = 2.35E-05; CpG3: p = 6.46E-06; CpG4: p = 2.47E-06; CpG5: p = 1.26E-05; average methylation: p = 5.42E-06). These changes were significantly reversed after intravenous immunoglobulin (IVIG) treatment. ROC curve analysis demonstrated remarkable diagnostic capability of mean NLRC4 gene methylation for KD (areas under ROC curve = 0.844, sensitivity = 0.75, p = 9.61E-06, 95% confidence intervals were 0.762-0.926 for mean NLRC4 methylation). In addition, NLRC4 promoter methylation was shown to be significantly negatively correlated with the levels of central granulocyte percentage, age, mean haemoglobin quantity and mean erythrocyte volume. Besides, NLRC4 promoter methylation was positively correlated with lymphocyte percentage, lymphocyte absolute value. CONCLUSIONS: Our work revealed the role of peripheral NLRC4 hypomethylation in KD pathogenesis and IVIG treatment response, could potentially serve as a treatment monitoring biomarker, although its precise functions remain to be elucidated.

Adaptive introgression of a visual preference gene.

Visual preferences are important drivers of mate choice and sexual selection, but little is known of how they evolve at the genetic level. In this study, we took advantage of the diversity of bright warning patterns displayed by Heliconius butterflies, which are also used during mate choice. Combining behavioral, population genomic, and expression analyses, we show that two Heliconius species have evolved the same preferences for red patterns by exchanging genetic material through hybridization. Neural expression of regucalcin1 correlates with visual preference across populations, and disruption of regucalcin1 with CRISPR-Cas9 impairs courtship toward conspecific females, providing a direct link between gene and behavior. Our results support a role for hybridization during behavioral evolution and show how visually guided behaviors contributing to adaptation and speciation are encoded within the genome.

Comparative transcriptomic analysis of circulating endothelial cells in sickle cell stroke.

Ischemic stroke (IS) is one of the most impairing complications of sickle cell anemia (SCA), responsible for 20% of mortality in patients. Rheological alterations, adhesive properties of sickle reticulocytes, leukocyte adhesion, inflammation and endothelial dysfunction are related to the vasculopathy observed prior to ischemic events. The role of the vascular endothelium in this complex cascade of mechanisms is emphasized, as well as in the process of ischemia-induced repair and neovascularization. The aim of the present study was to perform a comparative transcriptomic analysis of endothelial colony-forming cells (ECFCs) from SCA patients with and without IS. Next, to gain further insights of the biological relevance of differentially expressed genes (DEGs), functional enrichment analysis, protein-protein interaction network (PPI) construction and in silico prediction of regulatory factors were performed. Among the 2469 DEGs, genes related to cell proliferation (AKT1, E2F1, CDCA5, EGFL7), migration (AKT1, HRAS), angiogenesis (AKT1, EGFL7) and defense response pathways (HRAS, IRF3, TGFB1), important endothelial cell molecular mechanisms in post ischemia repair were identified. Despite the severity of IS in SCA, widely accepted molecular targets are still lacking, especially related to stroke outcome. The comparative analysis of the gene expression profile of ECFCs from IS patients versus controls seems to indicate that there is a persistent angiogenic process even after a long time this complication has occurred. Thus, this is an original study which may lead to new insights into the molecular basis of SCA stroke and contribute to a better understanding of the role of endothelial cells in stroke recovery.

Identifications of three novel alleles of Serrate in Drosophila.

The Notch signaling pathway, an evolutionarily highly conserved pathway, participates in various essential physiological processes in organisms. Activation of Notch signaling in the canonical manner requires the combination of ligand and receptor. There are two ligands of Notch in Drosophila: Delta (Dl) and Serrate (Ser). A mutation mf157 is identified for causing nicks of fly wings in genetic analysis from a mutant library (unpublished) that was established previously. Immunofluorescent staining illustrates that mf157 represses the expression of Cut and Wingless (Wg), the targets of Notch signaling. MARCM cloning analysis reveals that mf157 functions at the same level or the upstream of ligands of Notch in signaling sending cells. Sequencing demonstrates that mf157 is a novel allele of the Ser gene. Subsequently, mf553 and mf167 are also identified as new alleles of Ser from our library. Furthermore, the complementary assays and the examination of transcripts confirm the sequencing results. Besides, the repressed phenotypes of Notch signaling were reverted by transposon excision experiments of mf157. In conclusion, we identify three fresh alleles of Ser. Our works supply additional genetic resources for further study of functions of Ser and Notch signaling regulation.

Deletion of DWORF does not affect cardiac function in aging and in PLN-R14del cardiomyopathy.

The phospholamban (PLN) pathogenic gene variant p.Arg14del causes cardiomyopathy, which is characterized by perinuclear PLN protein clustering and can lead to severe heart failure (HF). Elevated expression of dwarf open reading frame (DWORF), a protein counteracting the function of PLN in the sarcoplasmic reticulum (SR), can delay disease progression in a PLN-R14del mouse model. Here, we evaluated whether deletion of DWORF (DWORF-/-) would have an opposite effect and accelerate age-dependent disease progression in wild-type (WT) mice and mice with a pathogenic PLN-R14del allele (R14Δ/+). We show that DWORF-/- mice maintained a normal left ventricular ejection fraction (LVEF) during aging and no difference with WT control mice could be observed up to 20 mo of age. R14Δ/+ mice maintained a normal cardiac function until 12 mo of age, but at 18 mo of age, LVEF was significantly reduced as compared with WT mice. Absence of DWORF did neither accelerate the R14Δ/+-induced reduction in LVEF nor enhance the increases in gene expression of markers related to cardiac remodeling and fibrosis and did not exacerbate cardiac fibrosis caused by the R14Δ/+ mutation. Together, these results demonstrate that absence of DWORF does not accelerate or exacerbate PLN-R14del cardiomyopathy in mice harboring the pathogenic R14del allele. In addition, our data indicate that DWORF appears to be dispensable for cardiac function during aging.NEW & NOTEWORTHY Although DWORF overexpression significantly delayed heart failure development and strongly prolonged life span in PLN-R14del mice, the current study shows that deletion of DWORF does not accelerate or exacerbate PLN-R14del cardiomyopathy in mice harboring the pathogenic R14del allele. In addition, DWORF appears to be dispensable for cardiac function during aging. Changes in DWORF gene expression are therefore unlikely to contribute to the clinical heterogeneity observed in patients with PLN-R14del cardiomyopathy.

Pericardial delta like non-canonical NOTCH ligand 1 (Dlk1) augments fibrosis in the heart through epithelial to mesenchymal transition.

BACKGROUND: Heart failure due to myocardial infarction (MI) involves fibrosis driven by epicardium-derived cells (EPDCs) and cardiac fibroblasts, but strategies to inhibit and provide cardio-protection remains poor. The imprinted gene, non-canonical NOTCH ligand 1 (Dlk1), has previously been shown to mediate fibrosis in the skin, lung and liver, but very little is known on its effect in the heart. METHODS: Herein, human pericardial fluid/plasma and tissue biopsies were assessed for DLK1, whereas the spatiotemporal expression of Dlk1 was determined in mouse hearts. The Dlk1 heart phenotype in normal and MI hearts was assessed in transgenic mice either lacking or overexpressing Dlk1. Finally, in/ex vivo cell studies provided knowledge on the molecular mechanism. RESULTS: Dlk1 was demonstrated in non-myocytes of the developing human myocardium but exhibited a restricted pericardial expression in adulthood. Soluble DLK1 was twofold higher in pericardial fluid (median 45.7 [34.7 (IQR)) µg/L] from cardiovascular patients (n = 127) than in plasma (median 26.1 µg/L [11.1 (IQR)]. The spatial and temporal expression pattern of Dlk1 was recapitulated in mouse and rat hearts. Similar to humans lacking Dlk1, adult Dlk1-/- mice exhibited a relatively mild developmental, although consistent cardiac phenotype with some abnormalities in heart size, shape, thorax orientation and non-myocyte number, but were functionally normal. However, after MI, scar size was substantially reduced in Dlk1-/- hearts as compared with Dlk1+/+ littermates. In line, high levels of Dlk1 in transgenic mice Dlk1fl/fl xWT1GFPCre and Dlk1fl/fl xαMHCCre/+Tam increased scar size following MI. Further mechanistic and cellular insight demonstrated that pericardial Dlk1 mediates cardiac fibrosis through epithelial to mesenchymal transition (EMT) of the EPDC lineage by maintaining Integrin ß8 (Itgb8), a major activator of transforming growth factor ß and EMT. CONCLUSIONS: Our results suggest that pericardial Dlk1 embraces a, so far, unnoticed role in the heart augmenting cardiac fibrosis through EMT. Monitoring DLK1 levels as well as targeting pericardial DLK1 may thus offer new venues for cardio-protection.

Satellite double-stranded RNA induces mesenchymal transition in pancreatic cancer by regulating alternative splicing.

Human satellite II (HSATII), composed of tandem repeats in pericentromeric regions, is aberrantly transcribed in epithelial cancers, particularly pancreatic cancer. Dysregulation of repetitive elements in cancer tissues can facilitate incidental dsRNA formation; however, it remains controversial whether dsRNAs play tumor-promoting or tumor-suppressing roles during cancer progression. Therefore, we focused on the double-stranded formation of HSATII RNA and explored its molecular function. The overexpression of double-stranded HSATII (dsHSATII) RNA promoted mesenchymal-like morphological changes and enhanced the invasiveness of pancreatic cancer cells. We identified an RNA-binding protein, spermatid perinuclear RNA-binding protein (STRBP), which preferentially binds to dsHSATII RNA rather than single-stranded HSATII RNA. The mesenchymal transition of dsHSATII-expressing cells was rescued by STRBP overexpression. Mechanistically, STRBP is involved in the alternative splicing of genes associated with epithelial-mesenchymal transition (EMT). We also confirmed that isoform switching of CLSTN1, driven by dsHSATII overexpression or STRBP depletion, induced EMT-like morphological changes. These findings reveal a novel tumor-promoting function of dsHSATII RNA, inducing EMT-like changes and cell invasiveness, thus enhancing our understanding of the biological significance of aberrant expression of satellite arrays in malignant tumors.

Missense variants in phospholamban and cardiac myosin binding protein identified in patients with a family history and clinical diagnosis of dilated cardiomyopathy.

As the genetic landscape of cardiomyopathies continues to expand, the identification of missense variants in disease-associated genes frequently leads to a classification of variant of uncertain significance (VUS). For the proper reclassification of such variants, functional characterization is an important contributor to the proper assessment of pathogenic potential. Several missense variants in the calcium transport regulatory protein phospholamban have been associated with dilated cardiomyopathy. However, >40 missense variants in this transmembrane peptide are currently known and most remain classified as VUS with little clinical information. Similarly, missense variants in cardiac myosin binding protein have been associated with hypertrophic cardiomyopathy. However, hundreds of variants are known and many have low penetrance and are often found in control populations. Herein, we focused on novel missense variants in phospholamban, an Ala15-Thr variant found in a 4-year-old female and a Pro21-Thr variant found in a 60-year-old female, both with a family history and clinical diagnosis of dilated cardiomyopathy. The patients also harbored a Val896-Met variant in cardiac myosin binding protein. The phospholamban variants caused defects in the function, phosphorylation, and dephosphorylation of this calcium transport regulatory peptide, and we classified these variants as potentially pathogenic. The variant in cardiac myosin binding protein alters the structure of the protein. While this variant has been classified as benign, it has the potential to be a low-risk susceptibility variant because of the structural change in cardiac myosin binding protein. Our studies provide new biochemical evidence for missense variants previously classified as benign or VUS.

DLK1/DIO3 locus upregulation by a ß-catenin-dependent enhancer drives cell proliferation and liver tumorigenesis.

The CTNNB1 gene, encoding ß-catenin, is frequently mutated in hepatocellular carcinoma (HCC, ∼30%) and in hepatoblastoma (HB, >80%), in which DLK1/DIO3 locus induction is correlated with CTNNB1 mutations. Here, we aim to decipher how sustained ß-catenin activation regulates DLK1/DIO3 locus expression and the role this locus plays in HB and HCC development in mouse models deleted for Apc (ApcΔhep) or Ctnnb1-exon 3 (ß-cateninΔExon3) and in human CTNNB1-mutated hepatic cancer cells. We identified an enhancer site bound by TCF-4/ß-catenin complexes in an open conformation upon sustained ß-catenin activation (DLK1-Wnt responsive element [WRE]) and increasing DLK1/DIO3 locus transcription in ß-catenin-mutated human HB and mouse models. DLK1-WRE editing by CRISPR-Cas9 approach impaired DLK1/DIO3 locus expression and slowed tumor growth in subcutaneous CTNNB1-mutated tumor cell grafts, ApcΔhep HB and ß-cateninΔExon3 HCC. Tumor growth inhibition resulted either from increased FADD expression and subsequent caspase-3 cleavage in the first case or from decreased expression of cell cycle actors regulated by FoxM1 in the others. Therefore, the DLK1/DIO3 locus is an essential determinant of FoxM1-dependent cell proliferation during ß-catenin-driven liver tumorigenesis. Targeting the DLK1-WRE enhancer to silence the DLK1/DIO3 locus might thus represent an interesting therapeutic strategy to restrict tumor growth in primary liver cancers with CTNNB1 mutations.

Hypoxic bone marrow mesenchymal stem cell exosomes promote angiogenesis and enhance endometrial injury repair through the miR-424-5p-mediated DLL4/Notch signaling pathway.

Background: Currently, bone marrow mesenchymal stem cells (BMSCs) have been reported to promote endometrial regeneration in rat models of mechanically injury-induced uterine adhesions (IUAs), but the therapeutic effects and mechanisms of hypoxic BMSC-derived exosomes on IUAs have not been elucidated. Objective: To investigate the potential mechanism by which the BMSCS-derived exosomal miR-424-5p regulates IUA angiogenesis through the DLL4/Notch signaling pathway under hypoxic conditions and promotes endometrial injury repair. Methods: The morphology of the exosomes was observed via transmission electron microscopy, and the expression of exosome markers (CD9, CD63, CD81, and HSP70) was detected via flow cytometry and Western blotting. The expression of angiogenesis-related genes (Ang1, Flk1, Vash1, and TSP1) was detected via RT‒qPCR, and the expression of DLL4/Notch signaling pathway-related proteins (DLL4, Notch1, and Notch2) was detected via Western blotting. Cell proliferation was detected by a CCK-8 assay, and angiogenesis was assessed via an angiogenesis assay. The expression of CD3 was detected by immunofluorescence. The endometrial lesions of IUA rats were observed via HE staining, and the expression of CD3 and VEGFA was detected via immunohistochemistry. Results: Compared with those in exosomes from normoxic conditions, miR-424-5p was more highly expressed in the exosomes from hypoxic BMSCs. Compared with those in normoxic BMSC-derived exosomes, the proliferation and angiogenesis of HUVECs were significantly enhanced after treatment with hypoxic BMSC-derived exosomes, and these effects were weakened after inhibition of miR-424-5p. miR-424-5p can target and negatively regulate the expression of DLL4, promote the expression of the proangiogenic genes Ang1 and Flk1, and inhibit the expression of the antiangiogenic genes Vash1 and TSP1. The effect of miR-424-5p can be reversed by overexpression of DLL4. In IUA rats, treatment with hypoxic BMSC exosomes and the miR-424-5p mimic promoted angiogenesis and improved endometrial damage. Conclusion: The hypoxic BMSC-derived exosomal miR-424-5p promoted angiogenesis and improved endometrial injury repair by regulating the DLL4/Notch signaling pathway, which provides a new idea for the treatment of IUAs.

Akt3 activation by R-Ras in an endothelial cell enforces quiescence and barrier stability of neighboring endothelial cells via Jagged1.

Communication between adjacent endothelial cells is important for the homeostasis of blood vessels. We show that quiescent endothelial cells use Jagged1 to instruct neighboring endothelial cells to assume a quiescent phenotype and secure the endothelial barrier. This phenotype enforcement by neighboring cells is operated by R-Ras through activation of Akt3, which results in upregulation of a Notch ligand Jagged1 and consequential upregulation of Notch target genes, such as UNC5B, and VE-cadherin accumulation in the neighboring cells. These signaling events lead to the stable interaction between neighboring endothelial cells to continue to fortify juxtacrine signaling via Jagged1-Notch. This mode of intercellular signaling provides a positive feedback regulation of endothelial cell-cell interactions and cellular quiescence required for the stabilization of the endothelium.

Early consequences of the phospholamban mutation PLN-R14del+/- in a transgenic mouse model.

AIMS: The heterozygous phospholamban (PLN) mutation R14del (PLN R14del+/- ) is associated with a severe arrhythmogenic cardiomyopathy (ACM) developing in the adult. "Superinhibition" of SERCA2a by PLN R14del is widely assumed to underlie the pathogenesis, but alternative mechanisms such abnormal energy metabolism have also been reported. This work aims to (1) to evaluate Ca2+ dynamics and energy metabolism in a transgenic (TG) mouse model of the mutation prior to cardiomyopathy development; (2) to test whether they are causally connected. METHODS: Ca2+ dynamics, energy metabolism parameters, reporters of mitochondrial integrity, energy, and redox homeostasis were measured in ventricular myocytes of 8-12 weeks-old, phenotypically silent, TG mice. Mutation effects were compared to pharmacological PLN antagonism and analyzed during modulation of sarcoplasmic reticulum (SR) and cytosolic Ca2+ compartments. Transcripts and proteins of relevant signaling pathways were evaluated. RESULTS: The mutation was characterized by hyperdynamic Ca2+ handling, compatible with a loss of SERCA2a inhibition by PLN. All components of energy metabolism were depressed; myocyte energy charge was preserved under quiescence but reduced during stimulation. Cytosolic Ca2+ buffering or SERCA2a blockade reduced O2 consumption with larger effect in the mutant. Signaling changes suggest cellular adaptation to perturbed Ca2+ dynamics and response to stress. CONCLUSIONS: (1) PLN R14del+/- loses its ability to inhibit SERCA2a, which argues against SERCA2a superinhibition as a pathogenetic mechanism; (2) depressed energy metabolism, its enhanced dependency on Ca2+ and activation of signaling responses point to an early involvement of metabolic stress in the pathogenesis of this ACM model.

ZBTB20 Regulates SERCA2a Activity and Myocardial Contractility Through Phospholamban.

BACKGROUND: Intracellular Ca2+ cycling determines myocardial contraction and relaxation in response to physiological demands. SERCA2a (sarcoplasmic/endoplasmic reticulum Ca2+-ATPase 2a) is responsible for the sequestration of cytosolic Ca2+ into intracellular stores during cardiac relaxation, and its activity is reversibly inhibited by PLN (phospholamban). However, the regulatory hierarchy of SERCA2a activity remains unclear. METHODS: Cardiomyocyte-specific ZBTB20 knockout mice were generated by crossing ZBTB20flox mice with Myh6-Cre mice. Echocardiography, blood pressure measurements, Langendorff perfusion, histological analysis and immunohistochemistry, quantitative reverse transcription-PCR, Western blot analysis, electrophysiological measurements, and chromatin immunoprecipitation assay were performed to clarify the phenotype and elucidate the molecular mechanisms. RESULTS: Specific ablation of ZBTB20 in cardiomyocyte led to a significant increase in basal myocardial contractile parameters both in vivo and in vitro, accompanied by an impairment in cardiac reserve and exercise capacity. Moreover, the cardiomyocytes lacking ZBTB20 showed an increase in sarcoplasmic reticular Ca2+ content and exhibited a remarkable enhancement in both SERCA2a activity and electrically stimulated contraction. Mechanistically, PLN expression was dramatically reduced in cardiomyocytes at the mRNA and protein levels by ZBTB20 deletion or silencing, and PLN overexpression could largely restore the basal contractility in ZBTB20-deficient cardiomyocytes. CONCLUSIONS: These data point to ZBTB20 as a fine-tuning modulator of PLN expression and SERCA2a activity, thereby offering new perspective on the regulation of basal contractility in the mammalian heart.