RNA Sequencing Research Articles

Effect of extracellular vesicles derived from inflammatory H9C2 cardiomyocytes on the polarization of macrophages and its mechanism

Abstract Objective The aim of this study was to investigate the effect of Extracellular vesicles (EVs) derived from inflammatory cardiomyocytes on the polarization of macrophages and its related mechanism. Methods EVs derived from the inflammatory and normal cardiomyocyte cell line H9C2 were extracted by differential centrifugation respectively. The extracted EVs were characterized by nanoparticle tracking analysis (NTA), transmission electron microscopy (TEM) and Western blot analysis (WB). The RAW264.7 macrophages in the inflammatory environment were treated with EVs, the proliferative activities were detected by CCK8, the expression of inflammatory factors were detected by qRT-PCR, and the differentiation of RAW264.7 macrophages into M1 and M2 was analyzed by flow cytometry. In vivo experiments, the animal model of viral myocarditis was established in Balb/c mice with intraperitoneal injection of CVB3. EVs derived from inflammatory H9C2 cardiomyocytes and medium were injected into mice by tail vein injection, respectively. The mice myocardium was stained by immunohistochemistry, and the total RNA of myocardial tissue was extracted for the detection of inflammatory factors by RT-PCR. In terms of mechanism research, mRNA expression profiles of RAW264.7 cells after EVs treatment were analyzed by RNA sequencing for functional annotation and mechanism study. At the same time, the extracted EVs derived from H9C2 normal group and the inflammatory group were subjected to 4D-Lable Free technology for quantitative proteomic analysis, and the results were analyzed bioinformatics, and then by WB analysis.Results We found that EVs derived from inflammatory H9C2 cardiomyocytes decreased the proliferative activity of RAW264.7 macrophages, the expression of pro-inflammatory cytokines IL-6, IL-1β, TNF-ɑ, and the proportion of M1 macrophages in the inflammatory state, while the proportion of M2 macrophages, which mainly inhibit inflammation, increased, and the expression levels of anti-inflammatory cytokines IL-10 and CD-206 increased. Results of immunohistochemical staining and RT-PCR showed that the EVs of inflammatory H9C2 regulated the heterogeneity of infiltrating macrophages and the expression of several inflammatory cytokines in mouse myocardium inflammatory tissue. Meanwhile, in vitro studies have found that the regulatory effect of EVs derived from inflammatory H9C2 cardiomyocytes on macrophage heterogeneity is mediated by MAPK signaling pathway. Conclusion EVs derived from inflammatory H9C2 cardiomyocytes can regulate the polarization of macrophages, change the expression of inflammatory factors, and regulate the myocardial inflammatory microenvironment, which may be achieved by mediating the p38 MAPK pathway through the delivery of PP2A.

Peripheral immune landscape of pediatric fulminant myocarditis revealed by single-cell sequencing

Abstract Fulminant myocarditis (FM) is one of the most feared diseases in children because of its fulminant nature and high mortality rate. However, the precise pathological immune subsets and molecular changes in FM are unknown. Here, we present the first comprehensive cellular and transcriptomic profiling of the peripheral blood mononuclear cells of children in FM acute and recovery phases using Single-cell RNA sequencing. 21 cell clusters are identified among 79542 cells. The proportion of T cells and NK cells increase, while myeloid cells and B cells reduce in acute phase, which were consistent with our flow cytometry data of 35 FM patients. Several unique cell types in peripheral blood are identified, including regulatory B cells, MAIT cells, adaptive NK cells and CD8+Tpex cells. The transcriptomic analysis exhibited elevated expression levels of chemokine receptor CXCR4 and S100A family genes almost in all cell types in FM acute phase, as well as MHC-II molecules in antigen-presenting cells. TCR and BCR exhibit remarkable amplification and obvious skewed usages of V genes in FM. Ligand receptor analysis show myeloid cells maintained active communication with other immune cells. Considering that CXCR4 may play an important role in myocarditis, we designed experiments to explore its function in mouse model. Vital myocarditis (VMC) mouse model was constructed using Coxsackie virus type3（CVB3）.We found that myocardial inflammation and myocardial injury of VMC mice were alleviated when treated with CXCR4 blocker. CXCR4 may be a potential therapeutic target for myocarditis. Our study will provide useful insights into key immune cell subsets and transcriptomic profiles implicated in pediatric fulminant myocarditis acute phase and recovery phase, thus providing several potential diagnostic and therapeutic targets for this disease.

Integrated plasma proteomics and myocardial tissue transcriptomics reveal elevated fibrosis markers in arrhythmogenic cardiomyopathy patients

Abstract Background Arrhythmogenic cardiomyopathy (ACM) is characterized by myocardial fibrofatty replacement and ventricular arrhythmias, with potential progression to heart failure. This study aims to identify ACM-specific biomarkers for improved prognosis and possible treatment strategies. Methods RNA sequencing and transcriptome analyses were performed in cardiomyocytes of 10 ACM patients, 10 dilated cardiomyopathy patients and 10 healthy controls. Analyses were performed using liquid chromatography and mass spectrometry. Additionally, plasma protein expression was assessed in 38 ACM patients and 24 healthy controls using the Proximity Extension Assay (Olink®). A standardized bicycle exercise test was performed in 11 ACM patients and controls and blood was obtained before and after exercise. Results Myocardial tissue transcriptomics identified several upregulated molecules associated with extracellular matrix formation and immune response in ACM. Plasma protein sequencing revealed upregulation of proteins involved in metabolic pathways, inflammation, and fibrosis. Integration of these analyses identified key soluble profibrotic markers such as Fibulin-3, Endostatin and C-X-C motif chemokine 10 (CXCL10) in plasma of ACM patients. After exercise, Fibulin-3 levels increased in ACM patients compared to controls. These findings were further validated using enzyme-linked immunosorbent assay (ELISA). Conclusion This comprehensive analysis revealed distinct proteomic and transcriptomic signatures in ACM patients' plasma and cardiomyocytes. The integrative analysis identified several upregulated profibrotic markers in ACM. Our Findings may advance diagnostic and prognostic strategies and offer potential therapeutic targets.

Signature of diabetic cardiomyopathy progression using high-fat diet and streptozotocin mouse model: from subclinical to clinical with three-hit mouse model

Abstract Background Diabetic cardiomyopathy (DbCM) is a pre-heart failure (pre-HF) condition that usually exhibits early diastolic dysfunction and evolving systolic dysfunction, along with left ventricular (LV) remodelling. Although DbCM shows distinct pathophysiology, the transition from the subclinical to the symptomatic stage is not straightforward and usually involves multifaceted processes which are not fully understood. Purpose To define the signature of DbCM progression in a novel mouse model using longitudinal echocardiography and multi-omics approaches. Methods Male mice (C57BL/6) were fed with high-fat diet (HFD) or control diet (CD) for 24 weeks, with single dose of streptozotocin (STZ) or vehicle injection at 8 weeks (n=10 each). Serial functional and metabolic parameters were measured prior to endpoint single nuclei RNA sequencing (snRNA-seq) and mass spectrometry-based proteomics analysis with comparison to cardiac remodelling to define detailed mechanisms underlying DbCM progression. Results HFD/STZ mice developed type 2 diabetes mellitus (T2DM) based on higher fasting blood glucose and HbA1C levels, insulin resistance and decreased beta-cell function (HOMA-IR and HOMA-beta, respectively) compared to controls. Progressive diastolic dysfunction was evident in HFD/STZ mice compared to CD mice based on enlarged LVPW;d from 12 weeks (P<0.05), reduced MV E/A ratio (P<0.01) and IVRT (P<0.05) from 16 weeks, and increased cardiac filling pressure (Left atrial (LA) area and LA volume) at 24 weeks, with preserved systolic function. This was paralleled by elevated collagen deposition (Picrosirius red), myocyte cross-sectional area and Col1a1 expression in HFD/STZ versus CD mice (all P<0.05). snRNA-seq analysis indicated preferential monocyte migration into diabetic hearts (P<0.01) whilst inflammatory pathways were activated across all cardiac cell types. Proteomics analysis revealed elevated expression of the established inflammation marker, plasma C-reactive protein (CRP), in HFD/STZ versus CD mice (P<0.05), which was correlated with worsening diastolic function (MV E/A ratio: r=-0.839, P<0.01) and LV hypertrophy (LVPW;d: r=0.861, P<0.01). Conclusion Our HFD/STZ mouse model offers a ‘three-hit’ approach which effectively captures key stages of clinical DbCM progression: 1) T2DM with insulin resistance, 2) progressive diastolic dysfunction with elevated chronic filling pressure and cardiac remodelling, and 3) systemic inflammation. This improved HFD/STZ mouse model is therefore appropriate to support a detailed study of mechanisms underlying DbCM progression and subsequent translation.

Loss of Y-chromosomal genes in macrophages induces paracrine phenotypic alterations in cardiac cells

Abstract Background At the intersection of gender medicine and ageing, mosaic loss of the Y chromosome (mLoY) in hematopoietic cells has emerged as a novel cardiovascular risk factor. As the most prevalent chromosomal aberration in men, mLoY is age-associated and characterised by interindividual varying frequencies of LoY cells in blood. It is associated with an increased risk for cardiovascular disease and mortality. A murine model of mLoY demonstrates myocardial fibrosis and reduced lifespan. However, the interactions between LoY hematopoietic and cardiac cells remain elusive. It is uncertain, whether the loss of single genes or combinations of multiple genes drive the observed effects, impeding the development of targeted therapies. Purpose We aimed to examine the impact of individual Y-chromosomal genes in macrophages concerning their paracrine effects on cardiac cells types. Methods and results To identify target genes, we analysed two single-nuclei RNA sequencing datasets of patient-derived PBMCs and THP-1 monocytic cells for their expression of Y-chromosomal genes. We identified nine genes that exhibited robust and concordant expression in both datasets. Seven were protein coding (RPS4Y, DDX3Y, EIF1AY, ZFY, UTY, USP9Y and KDM5D) and two were non-coding (TTTY14 and LINC00278). Using siRNA pools, we achieved >50% reductions in mRNA content in THP-1 M0 macrophages for all genes (p<0.05) and collected conditioned media (CM). As previous data suggested increased myocardial fibrosis in mLoY, we assessed the impact of CM on primary human cardiac fibroblasts. Notably, treatment with CM from ZFY, TTTY14 or LINC00278 silenced macrophages induced collagen 1A1 protein expression by 1.44-, 1.30- and 1.86-fold, respectively (all p<0.05), suggesting that loss of these genes might contribute to myocardial fibrosis. To explore potential effects on cardiomyocytes, neonatal rat cardiomyocytes were exposed to CM. We observed reduced beating frequencies upon treatment with CM from ZFY, TTTY14 or UTY silenced macrophages (relative fold-changes 0.45, 0.40, 0.53, respectively; all p<0.05). While suggestive of cardiomyocyte stress, we found no changes in the expression of the stress markers Nppa or Nppb, possibly indicating direct effects on ion transporters. Given the strong association of cardiovascular disease with endothelial cell dysfunction, we investigated the effects of CM on human umbilical vein endothelial cells (HUVEC). CM from ZFY and KDM5D silenced macrophages induced ICAM protein expression in HUVEC by 1.9- and 1.6-fold, respectively (both p<0.05). Conclusion Loss of individual Y-chromosomal genes in monocytic cells elicits distinct paracrine effects on various cardiac cell types. Particularly, silencing of ZFY in macrophages demonstrated profound effects on all investigated cell types, warranting further investigation. Additionally, two long non-coding RNAs, previously unexplored in cardiovascular disease, influenced paracrine activity of macrophages.

Effect and mechanism of Adipokine Omentin-1 on cardiac hypertrophy in heart failure with preserved ejection fraction

Abstract Background Omentin-1 secreted by epicardial adipose tissue can inhibit ventricular remodeling and improve early cardiac function after myocardial infarction. Our previous studies have identified the omentin-1 receptor as integrin receptors αvβ3 and αvβ5. However, the effect of omentin-1 on heart failure with preserved ejection fraction (HFpEF) remains poorly understood. Purpose The present study aims to study the role and mechanism of omentin-1 in cardiac function and myocardial cell structure in HFpEF. Methods and Results The influences of omentin-1 were investigated in HFpEF mice (HFD (60% calories from lard) and L-NAME (0.5 g/L in drinking water)), which were treated in vivo. Echocardiography showed better diastolic function in HFpEF mice treated with omentin-1 (5 ug/kg/h) subcutaneously for 4 weeks, and tissue staining showed alleviated myocardial fibrosis and myocardial hypertrophy. The RNA sequencing analysis revealed variations in the gene expression related to ketone body metabolism in HFpEF mice after omentin-1 treatment (600 ng/ml). The supernatant was obtained from mouse bone marrow macrophages treated with ultrapure endotoxin and ATP and was utilized as the macrophage-conditioned medium for culturing primary cardiomyocytes, which were stimulated with isoproterenol for 48 hours to construct the HFpEF cardiomyocyte model. In the heart tissue of HFpEF mice and HFpEF cardiomyocytes treated with omentin-1, there was a notable increase in the expression of a pivotal enzyme of ketone body metabolism- succinyl-CoA transferase (SCOT), and a decrease in the expression of hypertrophy signal. The myocardial hypertrophy model was established by stimulating the myocardial cell line H9C2 with isoproterenol. Following the inhibition of the integrin receptor and PI3K-AKT pathway, the impacts of omentin-1 on SCOT and myocardial hypertrophy signal expression in hypertrophic cardiomyocytes were eliminated. Conclusion This study provides evidence for the effects of omentin-1 on cardiac function and myocardial structure in HFpEF and proposes that omentin-1 may stimulate the downstream PI3K/AKT pathway via integrin receptors on cardiomyocytes, leading to increased expression of SCOT and improvement of cardiomyocyte hypertrophy.

Cells of the adult mouse cardiac conduction system in wild type and Nkx2-5 heterozygous loss-of-function mutation

Abstract Background Specialised cardiomyocytes in the cardiac conduction system (CCS) generate and spread the electrical impulse that initiates synchronised heartbeats. Disrupted signal transduction in the atrioventricular (AV) node, a key electrical connection between atria and ventricles, causes AV block. AV block potentially leads to electromechanical uncoupling and sudden cardiac death. Nkx2-5 encodes a cardiac transcription factor that is critical for embryonic CCS formation. Heterozygous loss-of-function mutation of Nkx2-5 (Nkx2-5+/–) results in a hypoplastic CCS in humans and mice and progressive AV block. Purpose We aimed to create a cell atlas of the adult mouse CCS and define the consequences of Nkx2-5 haploinsufficiency on the structural organisation and transcript expression of CCS cells. Methods We performed single-nucleus RNA sequencing on the sinoatrial nodes, AV nodes, His bundles, right bundle branches (RBB) and moderator bands of 17 wild type and 14 Nkx2-5+/– mice (aged 13 to 35 weeks, 5159 CCS cells). Employing single-molecule fluorescent RNA in situ hybridisation (smFISH), we validated selected transcripts and examined the spatial organisation of identified cell populations. Results In the AV node, we identified two subpopulations of pacemaker cells that were spatially separated in the proximal and distal pacemaker region (Fig1). Within these subpopulations, expressed transcripts indicated an atrial-to-ventricular gradient of electrical activity, corresponding to their respective topography. We determined two subpopulations in the His bundle and RBB that were enriched for either slow or fast conduction markers. Spatially, the slow-conducting populations seemed to surround the fast-conducting ones and might act as electrical isolation (Fig1). In Nkx2-5+/- cells of the pacemaker AV node, His bundle, RBB and Purkinje fibres, genes involved in Slit-Robo signalling were dysregulated. Slit-Robo signalling was previously reported in neuronal differentiation and embryonic heart lumen formation. Slit ligand 3 was downregulated in Nkx2-5+/- pacemaker AV node cells (Slit3, log2FC = -2.22, p = 7.0e-42), as was the roundabout guidance receptor 1 (Robo1, log2FC = -0.96, p = 1.6e-4). By contrast, there was upregulation of neural EGF-like 1 (Nell1, log2FC = 3.18, p = 5.3e-6), an alternative ligand of roundabout guidance receptors. Altered Nell1 and Slit3 expressions were confirmed by smFISH (Fig2). Conclusions Our cell atlas defines new CCS cell populations with informative transcriptomic profiles and spatial organisation, providing insights into subpopulations in the AV junction where electrical signals switch from slow to fast conduction. We identified disrupted Slit-Robo signalling in Nkx2-5+/-, possibly impairing differentiation and maturation of the CCS and altering electrophysiologic functions. Our normative anatomical and transcription data on the CCS can inform future studies of cardiac electrophysiology and arrhythmias.Fig1 - Subpopulations in AV junctionFig2 - Nell1 smFISH in pacemaker AV node

Role of inflammation and macrophages in the formation of the atrial cardiomyopathy associated with obesity

Abstract Introduction The atrial fibrillation (AF), the most frequent cardiac arrhythmia in clinical practice is often associated with an atrial cardiomyopathy (ACM) which is favoured by several clinical conditions including metabolic diseases such as obesity. Recently, the epicardial adipose tissue (EAT) has emerged as an important component of ACM notably in the context of metabolic diseases. Epicardial progenitor cells (EPC) are the main source of EAT. The replacement of EAT by fibrosis is arrhythmogenic and involved inflammation but not yet characterized. The hypothesis tested here is that macrophages are central players for both the recruitment of EPC and replacement of EAT by fibrosis. Purpose-Aims First to characterize subpopulation of macrophages involves in the progression of ACM. Second, to study how the inflammatory environment regulates EPC differentiation. Method A spatial gene expression was used to analyze human samples (n=4) (Agreement, CODECOH, Declaration or authorization of activities involving the preservation and preparation of human body parts for scientific purposes DC-2020-4183). A well-characterized mouse model of atrial cardiomyopathy associated with obesity induced by high fat diet (HFD) was used to characterize macrophages associated with progression of ACM by using immunolabeling together with single nuclei RNA sequencing approaches. ACM was characterized by using echocardiography and atrial burst pacing to evaluate determine AF vulnerability. Transgenic RGB-MAC mice with macrophages tagged with McApple-Csf1R and ECGFP-CX3CR1 or with a knock-out for CCR2 were used to follow infiltration of myeloid cells in the atrial at the different stages of the ACM. Bi-photon microscopy was used to visualize cell subpopulations in the distinct area of the atrial wall. In some experiments EPC and macrophages were cocultured with transwell. Results Macrophages were detected in subepicardium of human atrial samples in area of fibro-fatty infiltrates. After 2 months of HFD, monocytes were infiltrated atria and macrophages were overexpressed in atria from 1 to 4 months of HFD. Using Bi-photon microscopy it was possible to visualise resident ECGFP-CX3CR1+ macrophages at the contact of EAT whereas McApple-Csfr1r+ macrophages were at the junction with fibrosis (figure 1). Preliminary data generated by single nuclei RNA sequencing showed clusters of different subpopulations of macrophages at different time point of the ACM (figure 2). We will present results on the effects of suppression of the CCR2 subpopulation of macrophages on characteristics of the ACM. Conclusion Distinct populations of macrophages i.e resident and monocyte-derived macrophages, participate to the distinct stages of the ACM caused by obesity in mice.Myeloid cells infiltration in epicardiumCCR2+cells infiltration in EAT-fibrosis

Integrated analysis of bulk and single-cell RNA sequencing reveals the impact of nicotinamide and tryptophan metabolism on glioma prognosis and immunotherapy sensitivity

BackgroundNicotinamide and tryptophan metabolism play important roles in regulating tumor synthesis metabolism and signal transduction functions. However, their comprehensive impact on the prognosis and the tumor immune microenvironment of glioma is still unclear. The purpose of this study was to investigate the association of nicotinamide and tryptophan metabolism with prognosis and immune status of gliomas and to develop relevant models for predicting prognosis and sensitivity to immunotherapy in gliomas.MethodsBulk and single-cell transcriptome data from TCGA, CGGA and GSE159416 were obtained for this study. Gliomas were classified based on nicotinamide and tryptophan metabolism, and PPI network associated with differentially expressed genes was established. The core genes were identified and the risk model was established by machine learning techniques, including univariate Cox regression and LASSO regression. Then the risk model was validated with data from the CGGA. Finally, the effects of genes in the risk model on the biological behavior of gliomas were verified by in vitro experiments.ResultsThe high nicotinamide and tryptophan metabolism is associated with poor prognosis and high levels of immune cell infiltration in glioma. Seven of the core genes related to nicotinamide and tryptophan metabolism were used to construct a risk model, and the model has good predictive ability for prognosis, immune microenvironment, and response to immune checkpoint therapy of glioma. We also confirmed that high expression of TGFBI can lead to an increased level of migration, invasion, and EMT of glioma cells, and the aforementioned effect of TGFBI can be reduced by FAK inhibitor PF-573,228.ConclusionsOur study evaluated the effects of nicotinamide and tryptophan metabolism on the prognosis and tumor immune microenvironment of glioma, which can help predict the prognosis and sensitivity to immunotherapy of glioma.

Blockade of foam cell formation by an AP-1 inhibitor attenuates atherosclerosis in diabetes

Abstract Background Atherosclerosis is a major contributor to cardiovascular disease (CVD) related premature deaths in people with diabetes. Residual increased risk of CVD remains even after treatment of standard risk factors including hyperglycaemia. Therefore, there is an unmet clinical need of novel therapies that can directly target the intrinsic pathobiology of atherosclerotic plaque development. Macrophage associated pro-atherosclerotic processes including impaired cholesterol efflux resulting in foam cell formation are important therapeutic targets in atherosclerosis. Expression of genes linked to impaired cholesterol efflux can be regulated by multiple factors including transcription factors (TFs) such as activator protein 1 (AP-1) complex. Although, previous studies have implicated the role of foam cells in atherosclerosis, the role of AP-1 TF complex in foam cell formation in diabetes induced atherosclerosis is not known. Methods Diabetes was induced with streptozotocin (STZ) in atheroprone Apoe-/- mice and 10 weeks later aortae were subjected to single cell RNA sequencing (scRNA-seq) analysis using 10X Genomics platform. In vitro model mimicking diabetes-induced foam cell formation was established in human monocytes THP-1 derived macrophages using high glucose (25mM) and ox-LDL ± a small molecular inhibitor of AP-1, T-5224. AP-1 activity, gene expression and foam cell formation were assessed with activity assay, RT PCR and Oil Red O staining respectively in this setting. Complementary in vitro AP-1 knockdown experiments using shRNA were also performed. Furthermore, preclinical intervention studies were also performed where Apoe-/- mice made diabetic with STZ were treated 5-weeks post diabetes with T-5224 (30 mg/kg body weight daily for 5 weeks). Results scRNA-seq analysis identified a well-defined cluster of foam cells within macrophages with a diabetes specific proatherogenic transcriptomic profile. Differential gene expression analysis showed increased expression of AP-1 members cFos, Atf3, Atf4 and Junb exclusively in foam cell cluster in diabetes (cut off = FDR<0.05, Log2FC -2/2). Comparison with the ENCODE dataset for AP-1 target genes using Harmonizome version 3 showed that indeed multiple differentially expressed genes in diabetes were targets of AP-1. AP-1 members were also upregulated in the THP-1-based in vitro model of high glucose induced foam cell formation. THP-1 derived foam cells showed increased AP-1 activity and lipid uptake. Interestingly, AP-1 inhibition by T-5224 attenuated foam cell formation. AP-1 knockdown experiments validated experimental findings of AP-1 inhibition studies in THP-1 derived macrophages. Importantly, T-5224 treatment blocked foam cell formation resulting in reduced atherosclerosis in diabetic Apoe-/- mice in our intervention study design. Conclusion This study identified the AP-1 inhibition with T-5224 as a potential treatment for the diabetes induced foam cell formation and associated atherosclerosis.

Research trends and hotspots of the applications of single-cell RNA sequencing in cardiovascular diseases: a bibliometric and visualized study

Research trends and hotspots of the applications of single-cell RNA sequencing in cardiovascular diseases: a bibliometric and visualized study

Discovery of a cryptic founder variant in MYBPC3 associated with hypertrophic cardiomyopathy by promoting the most frequent natural missplicing events

Abstract Introduction MYBPC3 splicing errors are a common cause of hypertrophic cardiomyopathy (HCM). Most known actionable intronic variants are located near exons. However, genetic variants in deep intronic regions are also emerging as casuals factors of HCM. The previously undescribed variant MYBPC3 (NM_000256.3):c.2308+227G>A is not reported in gnomAD or dbSNP but is overrepresented in a cohort of probands with HCM from the same geographical area. This led us to suspect its clinical relevance despite the very low probability of being splice-altering as assigned by SpliceAI (Δ score = 0.03). Purpose To conduct a phenotypic and clinical description of the cohort, ascertain the pathogenicity of the variant, and investigate the existence of a common ancestor. Methods (1) Observational analysis of clinical data and familial evaluation results, (2) Functional study involving mRNA expression analysis in peripheral blood, along with in silico analysis of the splicing disruption mechanism, and (3) Haplotype analysis to evaluate the founder effect and assess the age of the mutation. Results MYBPC3:c.2308+227G>A was identified in 26 probands with HCM (69% male; mean age at diagnosis 53±10 years). Eighty relatives from 20 families were screened resulting in 17 cases diagnosed (mean age 49±19 years; 53% males), 19 positive genotype/negative phenotype and 38 non-carriers. The greatest cosegregation involved three cases carrying the variant, confirmed in four different families. Males were diagnosed at a younger age but without significant differences in clinical features or events during follow-up (table). RT-PCR expression analysis in patients' blood revealed that this variant disrupts pre-mRNA splicing by promoting the use of cryptic donor and acceptor splice sites, located downstream and upstream of the variant, respectively, which are already present in the intron 23 reference sequence. Strikingly, these cryptic splice sites represent one of the most frequent natural unannotated missplicing events in MYBPC3, as evidenced by SpliceVault analysis of high-throughput RNA sequencing databases. The direct primary event induced by the variant is expected to disrupt a regulatory RNA-protein binding site, thereby unblocking the use of preexisting cryptic splice sites and amplifying natural exonification missplicing events within intron 23 (figure). Finally, the haplotype analysis, based on the copy number of nine STRs covering a total of 29.2 Mb surrounding MYBPC3:c.2308+227G>A, revealed that 17 unrelated carriers shared a DNA segment of 10 Mb (10,067,150 bp). The origin of this mutation is estimated to be at least 82 generations ago and roughly 1649 years in the past (IC95%:1180-2300). Conclusions MYBPC3:c.2308+227G>A is an elusive novel deep intronic variant causing HCM by a molecular mechanism not previously described in this context. The founder effect of this variant is confirmed in our geographical area.Phenotypic and clinical characteristics

Local Controlled Delivery of IL-4 Decreases Inflammatory Bone Loss in a Murine Model of Periodontal Disease.

Chronic inflammatory diseases are a leading global health problem. In many of these diseases, the consistent presence of systemic low-grade inflammation induces tissue damage. This is true in conditions such as diabetes, arthritis, and autoimmune disorders, where an overactive and uncontrolled host immune response is a major driver of immunopathology. Central to this overactive and destructive host response are macrophages, the major phagocytic cells within the innate immune system. These cells exhibit a dual role in both host defense against invading pathogens and promotion of tissue repair during inflammation resolution. Those unique characteristics make macrophages an excellent target for therapeutic interventions in many chronic inflammatory conditions. Using periodontal disease as a model of chronic inflammation, we sought to assess the feasibility of using a controlled drug delivery strategy to target macrophages within the oral cavity. To that end, IL-4 was encapsulated within a biodegradable polymer carrier and locally delivered into the inflamed periodontal tissues. Our data indicate that local sustained delivery of IL-4 decreased inflammatory bone loss and promoted bone gain in the diseased mouse periodontium. Those effects correlated with a shift of local macrophage population toward a prorepair phenotype. Using single-cell RNA sequencing technology, we found that IL-4 delivery reversed several proinflammatory pathways associated with tissue destructive macrophages. Together, our data suggest that sustained delivery of IL-4 may be a viable therapeutic option for chronic diseases characterized by immune-mediated tissue damage.

Complement factor D deficiency ameliorates adverse cardiac remodelling by inhibiting C5a-C5aR mediated ISGs+ neutrophil activation post myocardial infarction

Abstract Background Exaggerated inflammatory response after myocardial infarction (MI) exacerbates myocardial injury and adverse cardiac remodeling, leading to heart failure. Current management of MI involves myocardial protection and antifibrotic therapy but do not target the inflammatory response per se. Complement factor D (CFD) is an upstream rate-limiting enzyme of complement system activation, which is the early event of inflammatory response post MI. We hypothesize that CFD can be an important modulator of the inflammation-remodeling cascade post MI, but the specific mechanism remains elusive. Purpose To investigate the role and mechanism of CFD in regulating inflammatory expansion and adverse remodeling post MI. Methods We generated Cfd-AKO (Cfdfl/fl Adipoq-cre) mice using the Cre-LoxP recombination system, as CFD is mainly secreted by adipocytes. Immune status and cardiac remodeling progress were assessed by cardiovascular magnetic resonance (CMR), histology, flow cytometry, immunofluorescence, and ELISA. Single cell RNA sequencing (scRNA-seq) analyzed non­cardiomyocytes isolated from Cfd-AKO and WT mice post MI or sham operation. Genetic ablation and co-culture experiments dissected the functional molecular properties and intercellular communication. An CFD inhibitor ACH-4471 was used to explore the role of CFD in ischemia-reperfusion (IR) injury. Results The survival of Cfd-AKO mice was strongly improved, while ventricular arrhythmia induction rates and number of episodes were significantly reduced compared with those of WT mice. CMR imaging showed that Cfd-AKO mice had higher ejection fraction and reduced left ventricular dilation than WTs. Masson trichrome and picrosirius red staining revealed that besides smaller infarct size, the extent of fibrosis in the border and distal remote area decreased in Cfd-AKO mice. Cfd loss reduced myocardial injury, inhibited the release of inflammatory factors, and limits the amplification of complement components C3a and C5a post MI. ScRNA-seq revealed that CFD modulated neutrophil chemotactic ability and pro-inflammatory function by inducing interferon-stimulated genes expressing (ISG+) neutrophil CXCL10 expression. Mechanistically, CFD activates the ISG transcriptional profile via the C5a/C5aR dependent STAT1/2-IRF9 pathway. Activated neutrophils promote myofibroblast transformation through IL1β/IL1R dependent intercellular communication, thus facilitating pathological fibrosis. Early pharmacological blocking of CFD in mice IR injury models inhibited inflammation, reduced infarct size and ameliorated cardiac dysfunction. Conclusions We demonstrate the crucial role of CFD in cardiac remodeling post MI. This process is critically regulated by C5a-C5aR, an important component of complement-inflammation cascade. This CFD-C5a mediated neutrophil pro-inflammatory and pro-fibrotic dual activity represents a novel pathway and an attractive therapeutic window for heart failure post MI.CFD in cardiac remodelling post MI

Caerin 1.1 and 1.9 peptides halt B16 melanoma metastatic tumours via expanding cDC1 and reprogramming tumour macrophages

BackgroundCancer immunotherapy, particularly immune checkpoint inhibitors (ICBs) such as anti-PD-1 antibodies, has revolutionised cancer treatment, although response rates vary among patients. Previous studies have demonstrated that caerin 1.1 and 1.9, host-defence peptides from the Australian tree frog, enhance the effectiveness of anti-PD-1 and therapeutic vaccines in a murine TC-1 model by activating tumour-associated macrophages intratumorally.MethodsWe employed a murine B16 melanoma model to investigate the therapeutic potential of caerin 1.1 and 1.9 in combination with anti-CD47 and a therapeutic vaccine (triple therapy, TT). Tumour growth of caerin-injected primary tumours and distant metastatic tumours was assessed, and survival analysis conducted. Single-cell RNA sequencing (scRNAseq) of CD45+ cells isolated from distant tumours was performed to elucidate changes in the tumour microenvironment induced by TT.ResultsThe TT treatment significantly reduced tumour volumes on the treated side compared to untreated and control groups, with notable effects observed by Day 21. Survival analysis indicated extended survival in mice receiving TT, both on the treated and distant sides. scRNAseq revealed a notable expansion of conventional type 1 dendritic cells (cDC1s) and CD4+CD8+ T cells in the TT group. Tumour-associated macrophages in the TT group shifted toward a more immune-responsive M1 phenotype, with enhanced communication observed between cDC1s and CD8+ and CD4+CD25+ T cells. Additionally, TT downregulated M2-like macrophage marker genes, particularly in MHCIIhi and tissue-resident macrophages, suppressing Cd68 and Arg1 expression across all macrophage types. Differential gene expression analysis highlighted pathway alterations, including upregulation of oxidative phosphorylation and MYC target V1 in Arg1hi macrophages, and activation of pro-inflammatory pathways in MHCIIhi and tissue-resident macrophages.ConclusionOur findings suggest that caerin 1.1 and 1.9, combined with immunotherapy, effectively modulate the tumour microenvironment in primary and secondary tumours, leading to reduced tumour growth and enhanced systemic immunity. Further investigation into these mechanisms could pave the way for improved combination therapies in advanced melanoma treatment.

Exploring tumor endothelial cells heterogeneity in hepatocellular carcinoma: insights from single-cell sequencing and pseudotime analysis

Objective This study aimed to explore the heterogeneity of tumor endothelial cells (TECs) in hepatocellular carcinoma (HCC) and their role in tumor progression, with the goal of identifying new therapeutic targets and strategies to improve patient prognosis. Methods Single-cell RNA sequencing data from nine primary liver cancer samples were analyzed, obtained from the Gene Expression Omnibus (GEO) database. Data preprocessing, normalization, dimensionality reduction, and batch effect correction were performed based on the Seurat package. HCC cell types were identified using uniform manifold approximation and projection (UMAP) and cluster analysis, and the different cell types were annotated using the CellMarker database. Pseudotime trajectory analysis was conducted with Monocle to explore the differentiation trajectory of TECs. MAPK signaling pathway activity and copy number variations (CNV) in TECs were analyzed in conjunction with data from The Cancer Genome Atlas (TCGA), the trans-well and wound healing assay was used for cell invasion and migration activity assessment. Results Two subgroups of TECs (TECs 1 and TECs 2) were identified, exhibiting distinct functional activities and signaling pathways. Specifically, TECs 1 may be involved in tumor cell proliferation and inflammatory responses, whereas TECs 2 is not only involved in cell proliferation pathways, but also enriched in pathways such as metabolic synthesis. Pseudotime analysis revealed dynamic changes in TECs subgroups during HCC progression, correlating specific gene expressions (such as PDGFRB, PGF, JUN, and NR4A1). Subsequently, the JUN gene was predicted by performing binding sites and was shown to act as a transcription factor that may regulate the expression of the PGF gene. CNV analysis highlighted key genes and pathways in TECs that might influence HCC progression, and the PGF as key regulatory factor mediated cell proliferation and migration. Conclusion The study revealed the heterogeneity of TECs in HCC and their potential roles in tumor progression, offering new perspectives and potential therapeutic targets for HCC molecular mechanisms. The findings emphasize the importance of further exploring TECs heterogeneity for understanding HCC pathogenesis and developing personalized treatment strategies.

Global deficiency in the inflammatory chemokine receptors 1,2,3 and 5 prevents vascular dysfunction in angiotensin II-induced hypertension and selectively modulates aortic myeloid cell phenotype

Abstract Background Leukocyte migration to the vasculature and the heart plays a critical role in hypertensive immune responses and is a carefully orchestrated process, regulated by the interactions of inflammatory chemokines with their receptors, notably CCR1, CCR2, CCR3, and CCR5 (collectively termed inflammatory chemokine receptors (iCCRs)). However, the inflammatory chemokine/receptor system is characterised by redundancy, ligand sharing and overlapping expression patterns. Consequently, our understanding of the specific and combinatory roles of chemokine receptors in cardiovascular inflammation remains incomplete, thus hindering the development of targeted therapies. To address this challenge, we have utilised two novel mouse models: iREP mice, carrying fluorescent reporters of inflammatory chemokine receptor expression, and TACKO mice, in which the entire iCcr locus containing Ccr1, Ccr2, Ccr3, and Ccr5 has been deleted. Purpose To evaluate the expression pattern of iCCRs in experimental hypertension and understand how the global deletion of iCCRs impacts disease progression. Methods We induced hypertension in mice by subcutaneously implanting osmotic minipumps administering angiotensin II (Ang II) for a duration of 14 days. Single-cell RNA sequencing was utilised for investigating iCCR expression in aortas during hypertension. Additionally, we employed flow cytometry and confocal microscopy to track iCCR expression and localization in aortas and hearts obtained from iREP mice. TACKO mice were utilised to assess the effect of global iCCr deficiency on hypertensive phenotypes. Blood pressure was measured via telemetry and vascular function was assessed using myography. Vascular and heart remodelling were evaluated through histology. Molecular mechanisms were analysed using Western Blotting and RT-qPCR, while immune cell changes in the aorta were evaluated through cytometry of time-of-flight (CyTOF). Results Ang II infusion induced higher iCCRs expression levels in both the aorta and the heart in comparison with sham treatment, particularly in fibrotic areas. After Ang II-treatment, global deletion of iCCRs in TACKO mice resulted in the prevention of endothelial dysfunction (n=5/group, P<0.05) and perivascular fibrosis (n=6-9, P<0.01), and a reduction in cardiomyocyte size (n=12-16, P<0.01) compared to wild type mice. However, no significant improvements were observed in other hypertensive phenotypes, including blood pressure, heart fibrosis, vascular NO synthase, and oxidative stress. CyTOF analysis revealed reductions in inflammatory monocytes and conventional type 2 dendritic cells in TACKO aorta, accompanied by a phenotypic switch in macrophages towards a less inflammatory phenotype. Conclusion Deficiency in iCCRs confers protective effects against vascular dysfunction in experimental hypertension, primarily by selectively reducing inflammatory myeloid cell homing to the aorta.

Macrophage-mediated interleukin-6 signaling drives ryanodine receptor-2 calcium leak in postoperative atrial fibrillation

Abstract Background Postoperative atrial fibrillation (poAF) is self-limited AF that occurs within days after cardiac surgery in one-third of patients. Clinical and preclinical studies have demonstrated that the magnitude of the perioperative increase in systemic inflammation, in particular mediated by interleukin (IL)-6, correlates with poAF risk. However, no studies to-date have mechanistically linked IL-6 signaling to fundamental arrhythmia mechanisms or identified the specific cell types driving postoperative atrial IL-6 signaling. Methods We performed single-cell RNA sequencing (scRNAseq), followed by pseudotime trajectory and cell-cell communication analyses on atrial non-myocytes comparing mice with and without poAF. We followed up our scRNAseq findings with pharmacologic and genetic approaches in mice to validate pathways related to macrophage-mediated IL-6 signaling. We used confocal imaging to assess calcium-signaling mechanisms at the single atrial cardiomyocyte (ACM) level and performed parallel patch-clamp studies in isolated human ACMs as well as biochemical analyses of human atrial tissue to assess the translational relevance of our findings. Results Our scRNAseq results demonstrated macrophages to be the most prominently altered cell type in the atria of mice with poAF. Pseudotime trajectory analyses revealed IL-6 to be one of the top inflammatory pathways implicated in macrophage transcriptional state. Indeed, both macrophage depletion and conditional knockout of IL-6 receptors in macrophages were sufficient to prevent poAF. We found that inhibition of STAT3, which is downstream of the IL-6Ra, with FDA-approved phospho-STAT3 inhibitor TTI-101 prevented poAF in mice. Lastly, we demonstrate that enhanced STAT3 activity in poAF promotes arrhythmogenic Ca2+ sparks and waves through ryanodine receptor-2 dysfunction, and that CaMKII inhibition is sufficient to ameliorate Ca2+ mishandling at the single atrial myocyte level. Conclusions Taken together, we use an integrated approach incorporating mouse and human biochemical, functional, and single-cell sequencing data to demonstrate that infiltrating atrial macrophages are fundamentally required for poAF. IL-6 was found to be a critical pathway upregulated in poAF leading to downstream ryanodine receptor-2 dysfunction and atrial arrhythmogenesis. Our findings portend significant therapeutic utility by providing robust mechanistic evidence that targeting the IL-6 axis may be used for poAF prevention and treatment in a clinically amenable timeframe.

Paired single-cell RNA sequencing and T cell receptor sequencing reveals both pro- and anti-inflammatory roles of T cells in patients with calcific aortic valve disease

Abstract Background Calcified aortic valve disease (CAVD) is a chronic unresolvable inflammatory disorder that affects 25% of adults over 65 years old, leading to aortic stenosis, heart failure, and death [1]. Since the molecular mechanisms of its pathogenesis are not well understood, therapies to treat root causes of the disease remain to be developed, with valve replacement as the major intervention strategy currently. Increasing evidence indicates that substantial T cells infiltrate in the aortic valve during calcification, and clonal expanded CD8+ T cells have been identified in the calcificed aortic valve by bulk T cell receptor (TCR) repertoire sequencing [2]. Purpose To decipher T cell heterogenity and understand the precise mechanisms of T cell subsets involving CAVD progression as a basis for novel therapeutic targets. Methods In Cohort 1, we collected aortic valves from CAVD (n=3) and healthy (n=2) patients for single-cell RNA sequencing (scRNA-seq). In Cohort 2, we performed paired scRNA-seq and TCR sequencing (scTCR-seq) on aortic valves obtained from CAVD (n=3) and healthy (n=3) patients to evaluate both gene expression and clonality. Pathway enrichment analysis and cell-cell interaction analysis elucidated the function of T cells in the calcificied valves. Immunohistochemistry and fluorescence-activated cell sorting were recruited to verify the key findings. Results scRNA-seq demonstrated a conversion from anti-inflammatory regulatory T (Treg) cells towards pro-inflammatory T helper 17 (Th17) cells in the calcificied aortic valve. Paired scRNA-seq and scTCR-seq revealed CAVD-related signaling pathways (osteoclast differentiation, NOD-like receptor signaling pathways, IL-17 signaling pathways) significantly upregulated in highly expanded CD8+ T cells in the calcified valve, with upregulation of pro-inflammatory mediators such as IFNG, CCL4 and CCL5, suggesting pro-inflammation functions in clonally expanded T cells. Moreover, we characterized tissue-resident memory T cells, which resembled similar transcriptome and TCR clonality as exhausted T cells in calcified valves. These cells up-regulated anti-inflammation-related transcriptomes but their cellular fraction decreased in calcified valves, implying anti-inflammatory functions of these tissue-resident memory and exhausted T cells. Conclusion This study elucidates transcriptomic and immune profiling of T cells in the calcified aortic valve, therefore shedding light on the pathophysiological mechanisms underlying aortic valve calcification from the novel perspective of tissue-specific T lymphocytes, providing promising targets for immune checkpoint-based therapeutic interventions.

Sinomenine hydrochloride alleviates autoimmune myocarditis via suppressing Th1 cell induced-M1 macrophage pyroptosis

Abstract Background Myocarditis is typical of the complex inflammatory response in the heart with high morbidity and mortality, accompanied by tissue damage and cardiac fibrosis. Previous studies have demonstrated that CD4+ T cells are crucial to the cardiac autoimmunity of myocarditis, but the role of different CD4+ T cell subsets remains unclear. Recently, sinomenine hydrochloride, a natural compound from the traditional Chinese herb Sinomenium acutum, is reported to inhibit T cell proliferation in some systemic autoimmune diseases such as rheumatoid arthritis. Given that autoimmune myocarditis is an organ-specific autoimmune disease, the effect of sinomenine hydrochloride on autoimmune myocarditis demands further study. Purpose This study aims to elucidate the role of CD4+ T-helper 1 (Th1) cells in regulating inflammatory cell death in autoimmune myocarditis and investigate the pharmacological effect of sinomenine hydrochloride on CD4+ Th1 cells and autoimmune myocarditis. Methods Male Balb/c mice were immunized with myosin heavy chain-α peptides to establish the experimental autoimmune myocarditis (EAM) model, followed by the treatments with sinomenine hydrochloride by gavage. Tohoku Hospital Pediatrics-1 (THP-1) cell line and murine T cells were used for in vitro experiments. Results In the acute phase of EAM, we observed plenty of CD4+ T cells infiltrating the heart tissue. RNA sequencing revealed that among the genes encoding typical transcription factors of CD4+ T cell subset, only Tbx21 and Stat4, both representing Th1 cells, were upregulated, which consisted with immunofluorescence staining. Further studies indicated that Th1 cells not only mediated classical activated macrophage (M1 macrophage) polarization by secreting interferon-γ (IFN-γ) but also associated with pro-inflammatory macrophage pyroptosis in the EAM, with the cleavage of gasdermin D (GSDMD), caspase-1 and IL-1β. Subsequent in vitro experiments confirmed that IFN-γ could dramatically promote macrophage pyroptosis combined with ATP, leading to the release of IL-1β and IL-18(Figure 1). To find an effective method for Th1 cell suppression and myocarditis remission, we identified that sinomenine hydrochloride impeded Th1 cell activation, differentiation and proliferation in vitro. Furthermore, sinomenine hydrochloride inhibited Th1 cell infiltration and M1 macrophage pyroptosis in the EAM heart, In the chronic phase, cardiac remodeling and fibrosis were suppressed, and cardiac systolic function was significantly improved in the treatment group (Figure 2). Conclusion Our study demonstrates that Th1 cells-induced M1 macrophage pyroptosis is an essential pathogenic mechanism of EAM. Sinomenine hydrochloride can alleviate the severity and progression of EAM, which may become a novel therapeutic strategy for treating myocarditis.Figure 1Figure 2