陈晓
中国医学科学院阜外医院 心血管疾病国家重点实验室
Cardiac hypertrophy can develop to end-stage heart failure (HF), which inevitably leading to heart transplantation or death. Preserving cardiac function in cardiomyocytes (CMs) is essential for improving prognosis in hypertrophic cardiomyopathy (HCM) patients. Therefore, understanding transcriptomic heterogeneity of CMs in HCM would be indispensable to aid potential therapeutic targets investigation. We isolated primary CM from HCM patients who had extended septal myectomy, and obtained transcriptomes in 338 human primary CM with single-cell tagged reverse transcription (STRT-seq) approach. Our results revealed that CMs could be categorized into three subsets in nonfailing HCM heart: high energy synthesis cluster, high cellular metabolism cluster and intermediate cluster. The expression of electron transport chain (ETC) was up-regulated in larger-sized CMs from high energy synthesis cluster. Of note, we found the expression of Cytochrome c oxidase subunit 7B (COX7B), a subunit of Complex IV in ETC had trends of positively correlation with CMs size. Further, by assessing COX7B expression in HCM patients, we speculated that COX7B was compensatory up-regulated at early-stage but down-regulated in failing HCM heart. To test the hypothesis that COX7B might participate both in hypertrophy and HF progression, we used adeno associated virus 9 (AAV9) to mediate the expression of Cox7b in pressure overload-induced mice. Mice in vivo data supported that knockdown of Cox7b would accelerate HF and Cox7b overexpression could restore partial cardiac function in hypertrophy. Our result highlights targeting COX7B and preserving energy synthesis in hypertrophic CMs could be a promising translational direction for HF therapeutic strategy.
Journal of molecular and cellular cardiology 2024
BACKGROUND:Dilation may be the first right ventricular change and accelerates the progression of threatening ventricular tachyarrhythmias and heart failure for patients with arrhythmogenic right ventricular cardiomyopathy (ARVC), but the treatment for right ventricular dilation remains limited.METHODS:Single-cell RNA sequencing (scRNA-seq) of blood and biventricular myocardium from 8 study participants was performed, including 6 end-stage heart failure patients with ARVC and 2 normal controls. ScRNA-seq data was then deeply analyzed, including cluster annotation, cellular proportion calculation, and characterization of cellular developmental trajectories and interactions. An integrative analysis of our single-cell data and published genome-wide association study-based data provided insights into the cell-specific contributions to the cardiac arrhythmia phenotype of ARVC. Desmoglein 2 (Dsg2)mut/mut mice were used as the ARVC model to verify the therapeutic effects of pharmacological intervention on identified cellular cluster.RESULTS:Right ventricle of ARVC was enriched of CCL3+ proinflammatory macrophages and TNMD+ fibroblasts. Fibroblasts were preferentially affected in ARVC and perturbations associated with ARVC overlap with those reside in genetic variants associated with cardiac arrhythmia. Proinflammatory macrophages strongly interact with fibroblast. Pharmacological inhibition of Nod-like receptor protein 3 (NLRP3), a transcriptional factor predominantly expressed by the CCL3+ proinflammatory macrophages and several other myeloid subclusters, could significantly alleviate right ventricular dilation and dysfunction in Dsg2mut/mut mice (an ARVC mouse model).CONCLUSIONS:This study provided a comprehensive analysis of the lineage-specific changes in the blood and myocardium from ARVC patients at a single-cell resolution. Pharmacological inhibition of NLRP3 could prevent right ventricular dilation and dysfunction of mice with ARVC.
BMC medicine 2024
Macrophages play an essential role in the pathogenesis of autoimmune myocarditis, but the molecular mechanism remains largely unknown. Here, the role of Stimulator of interferon gene (Sting) in autoimmune myocarditis was investigated. Six-week-old male BALB/c mice received two subcutaneous injections of 250 μg α-MyHC peptide to establish experimental autoimmune myocarditis (EAM). With single-cell RNA sequencing analysis of cardiac immune (Cd45+) cells, Sting was found to initiate proinflammatory macrophage differentiation related to the acute EAM phase. Furthermore, proinflammatory macrophages contribute to the pathogenesis of EAM via hypoxia-inducible factor-1α (Hif1α). A higher expression level of Sting was detected in macrophages from myocarditis, which was positively correlated with Hif1α expression. Single-stranded DNA (ssDNA) accumulation in macrophages in myocarditis was observed in the hearts of EAM mice. Pharmacological blockade of STING by C-176 (a specific inhibitor) ameliorated the inflammatory response of EAM and reduced proinflammatory molecule (Ifn-β, Tnf-α, Ccl2, and F4/80) expression and Hif1α expression. In vitro studies revealed that ssDNA activated the expression of Sting; in turn, Sting accelerated proinflammatory molecule expression in mouse macrophages. Inhibition of Hif1α expression could reduce Sting-associated cardiac inflammation and proinflammatory molecule expression. In addition, the expression of STING and ssDNA accumulation in macrophages were observed in human autoimmune myocarditis heart samples. STING activated proinflammatory macrophage via HIF1A, promoting the development of autoimmune myocarditis. The STING signaling pathway might provide a novel mechanism of autoimmune myocarditis and serve as a potential therapeutic target for autoimmune myocarditis patients.
International immunopharmacology 2023
BACKGROUND:Thoracic aortic dissection (TAD) is a life-threatening disease caused by an intimal tear in the aorta. The histological characteristics differ significantly between the tear area (TA) and the distant area. Previous studies have emphasized that certain specific genes tend to cluster at the TA. Obtaining a thorough understanding of the precise molecular signatures near the TA will assist in discovering therapeutic strategies for TAD.METHODS:We performed a paired comparison of the pathological patterns in the TA with that in the remote area (RA). We used Tomo-seq, genome-wide transcriptional profiling with spatial resolution, to obtain gene expression signatures spanning from the TA to the RA. Samples from multiple sporadic TAD patients and animal models were used to validate our findings.RESULTS:Pathological examination revealed that the TA of TAD exhibited more pronounced intimal hyperplasia, media degeneration, and inflammatory infiltration compared to the RA. The TA also had more apoptotic cells and CD31+α-SMA+ cells. Tomo-seq revealed four distinct gene expression patterns from the TA to the RA, which were inflammation, collagen catabolism, extracellular matrix remodeling, and cell stress, respectively. The spatial distribution of genes allowed us to identify genes that were potentially relevant with TAD. NINJ1 encoded the protein-mediated cytoplasmic membrane rupture, regulated tissue remodeling, showed high expression levels in the tear area, and co-expressed within the inflammatory pattern. The use of short hairpin RNA to reduce NINJ1 expression in the beta-aminopropionitrile-induced TAD model led to a significant decrease in TAD formation. Additionally, it resulted in reduced infiltration of inflammatory cells and a decrease in the number of CD31+α-SMA+ cells. The NINJ1-neutralizing antibody also demonstrated comparable therapeutic effects and can effectively impede the formation of TAD.CONCLUSIONS:Our study showed that Tomo-seq had the advantage of obtaining spatial expression information of TAD across the TA and the RA. We pointed out that NINJ1 may be involved in inflammation and tissue remodeling, which played an important role in the formation of TAD. NINJ1 may serve as a potential therapeutic target for TAD.
BMC medicine 2023
AIMS:Our previous studies suggested that the complement system was critical in the prognosis of arrhythmogenic right ventricular cardiomyopathy (ARVC). The acylation-stimulating protein (ASP), generated through the alternate complement pathway, was reported to regulate lipogenesis and triglyceride storage. This study aimed to investigate the role of ASP in predicting adverse cardiac events in an ARVC cohort.METHODS AND RESULTS:We enrolled 111 ARVC patients and 106 healthy volunteers, and measured their plasma ASP levels using enzyme-linked immunosorbent assays. Plasma ASP levels were significantly higher in the ARVC patients than in the healthy controls (2325.22 ± 20.08 vs. 2189.75 ± 15.55, P < 0.001), with a similar trend observed in the myocardial explant assay. Spearman correlation analysis indicated plasma ASP level associated with cardiac structural (right ventricular internal dimension, P = 0.006) and functional remodelling (left ventricular ejection fraction, P = 0.002) in ARVC patients. The ARVC patients were followed up for an average of 17.79 ± 1.09 months. Heart failure-associated events (HFAEs) were defined as heart transplantation, on a cardiac transplant list, or death due to end-stage heart failure. Plasma ASP levels in patients with HFAEs were significantly higher than in those without clinical events (2486.03 ± 26.70 vs. 2268.83 ± 23.51, P < 0.001) or those with malignant arrhythmic events (2486.03 ± 26.70 vs. 2297.80 ± 60.46, P = 0.008). LASSO (least absolute shrinkage and selection operator) and multivariable Cox regression analyses showed the ASP level (HR = 1.004, 95% CI [1.002,1.006], P = 0.002) was an independent predictor for adverse HFAEs in ARVC patients. The spline-fitting procedure was applied to illustrate the HFAE-free probabilities at different time points.CONCLUSIONS:Our results suggest that plasma ASP may be a useful biomarker in prediction of adverse HF-associated events in ARVC patients.
ESC heart failure 2023
BACKGROUND:Perivascular adipose tissue (PVAT) is vital for vascular homeostasis, and PVAT dysfunction is associated with increased atherosclerotic plaque burden. But the mechanisms underlining coronary PVAT dysfunction in coronary atherosclerosis remain elusive.METHODS:We performed single-cell RNA sequencing of the stromal vascular fraction of coronary PVAT from 3 groups of heart transplant recipients with end-stage heart failure, including 3 patients with nonobstructive coronary atherosclerosis, 3 patients with obstructive coronary artery atherosclerosis, and 4 nonatherosclerosis control subjects. Bioinformatics was used to annotate the cellular populations, depict the cellular developmental trajectories and interactions, and explore the differences among 3 groups of coronary PVAT at the cellular and molecular levels. Pathological staining, quantitative real-time polymerase chain reaction, and in vitro studies were performed to validate the key findings.RESULTS:Ten cell types were identified among 67 936 cells from human coronary PVAT. Several cellular subpopulations, including SPP1+ (secreted phosphoprotein 1) macrophages and profibrotic fibroadipogenic progenitor cells, were accumulated in PVAT surrounding atherosclerotic coronary arteries compared with nonatherosclerosis coronary arteries. The fibrosis percentage was increased in PVAT surrounding atherosclerotic coronary arteries, and it was positively associated with the grade of coronary artery stenosis. Cellular interaction analysis suggested OPN (osteopontin) secreted by SPP1+ macrophages interacted with CD44 (cluster of differentiation 44)/integrin on fibroadipogenic progenitor cells. Strikingly, correlation analyses uncovered that higher level of SPP1 in PVAT correlates with a more severe fibrosis degree and a higher coronary stenosis grade. In vitro studies showed that conditioned medium from atherosclerotic coronary PVAT promoted the migration and proliferation of fibroadipogenic progenitor cells, while such effect was prevented by blocking CD44 or integrin.CONCLUSIONS:SPP1+ macrophages accumulated in the PVAT surrounding atherosclerotic coronary arteries, and they promoted the migration and proliferation of fibroadipogenic progenitor cells via OPN-CD44/integrin interaction and thus aggravated the fibrosis of coronary PVAT, which was positively correlated to the coronary stenosis burden. Therefore, SPP1+ macrophages in coronary PVAT may participate in the progression of coronary atherosclerosis.
Arteriosclerosis, thrombosis, and vascular biology 2023
Giant cell myocarditis (GCM) is a rare, usually rapidly progressive, and potentially fatal disease. Detailed inflammatory responses remain unknown, in particular the formation of multinucleate giant cells. We performed single-cell RNA sequencing analysis on 15,714 Cd45+ cells extracted from the hearts of GCM rats and normal rats. NETosis has been found to contribute to the GCM process. An inhibitor of NETosis, GSK484, alleviated GCM inflammation in vivo. MPO (a marker of neutrophils) and H3cit (a marker of NETosis) were expressed at higher levels in patients with GCM than in patients with DCM and healthy controls. Imaging mass cytometry analysis revealed that immune cell types within multinucleate giant cells included CD4+ T cells, CD8+ T cells, neutrophils, and macrophages but not B cells. We elucidated the role of NETosis in GCM pathogenesis, which may serve as a potential therapeutic target in the clinic.
iScience 2023
INTRODUCTION:Hypertensive nephropathy is characterized by glomerular and tubulointerstitial damage, but we know little about changes in cell-specific gene expression in the early stages of hypertensive kidney injury, which usually has no obvious pathological changes.METHODS:We performed unbiased single-cell RNA sequencing of rat kidney samples from hypertensive kidney injury to generate 10,602 single-cell transcriptomes from 2 control and 2 early stage hypertensive kidney injury samples.RESULTS:All major cell types of the kidney were represented in the final dataset. Side-by-side comparisons showed that cell type-specific changes in gene expression are critical for functional impairment of glomeruli and tubules and activation of immune cells. In particular, we found a significantly reduced gene expression profile of maintaining vascular integrity in glomerular cells of hypertensive kidney injury. Meanwhile, the expression of genes associated with oxidative stress injury and fibrosis in the renal tubules and collecting ducts was elevated, but the degree of tubular cells response to injury differed between parts. We also found a signature of immune cell infiltration in hypertensive kidney injury.CONCLUSION:Exploring the changes of gene expression in hypertension-injured kidneys may be helpful to identify the early biomarkers and signal pathways of this disease. Our data provide rich resources for understanding the pathogenesis of hypertensive renal injury and formulating effective treatment strategies.
Kidney & blood pressure research 2023
BACKGROUND:Tetralogy of Fallot (TOF) is a common congenital heart disease with high mortality. However, the medical imageology and liquidbiopsy techniques present certain limitations. Thus, this study investigated the plasma metabolic profiles to distinguish key metabolites for early diagnosis of TOF.METHODS:In total, 69 patients with TOF and 43 normal controls were enrolled for targeted metabolomics based on liquid chromatography-tandem mass spectroscopy (LC-MS/MS). Absolute quantification of metabolites was performed using our standard database. The differentially expressed metabolites (DEMs) were screened by fold change (FC), VIP value and pearson correlation coefficient of OPLS-DA model. Receiver operating characteristic curve (ROC) was used to evaluate predictive ability of DEMs.RESULTS:Different metabolic profiles were presented between TOF and Normal.The pathway analysis showed that significantly changed metabolites were enriched in nicotinamide and purine metabolism. Many intermediatesproductof purine and amido acid were higher in TOF than in Normal group, while energy substrates and electron carriers were lower in TOF than in Normal group. ROC analysis revealed a high diagnostic value of plasma FAD for differentiating TOF from Normal (AUC = 1).CONCLUSION:Our study quantitatively characterized plasma metabolites in patients with TOF and may help to develop reliable biomarkers that contribute to the early TOF screening.
Clinica chimica acta; international journal of clinical chemistry 2023
The benefits of IL2RA antagonists in heart transplant patients are controversial. We aimed to elucidate the effects of IL2RA antagonists and identify targets that could be better than IL2RA antagonists. By using single-cell RNA sequencing of immune cells at different time points in patients receiving IL2RA antagonists, we identified nineteen types of cells. We revealed higher IL2RA expression in regulatory T cells (Tregs), suggesting that IL2RA antagonists attenuated IL-2-induced Treg activation. CD4_C04_IFNGR1 and CD8_C05_IFITM2 which had more cytotoxic effects, remained elevated at later time points. IFNGR1 was upregulated in these two subtypes, but was not expressed in Treg. Ruxolitinib targeted the pathways of IFNGR1 (JAK1/2) while not affecting the pathway of IL-2-induced Tregs activation (JAK3). Ruxolitinib showed prolonged survival compared to IL2RA mAb-treated mice. Our study provided dynamic changes of immune cells after IL2RA antagonists treatment at single-cell resolution. Ruxolitinib has potential as a new immunoinduction therapy without affecting Treg.
Clinical immunology (Orlando, Fla.) 2023
