Search

Heart failure represents a major cause of morbidity and mortality worldwide. Single-cell transcriptomics have revolutionized our understanding of cell composition and associated gene expression. Through integrated analysis of single-cell and single-nucleus RNA-sequencing data generated from 27 healthy donors and 18 individuals with dilated cardiomyopathy, here we define the cell composition of the healthy and failing human heart. We identify cell-specific transcriptional signatures associated with age and heart failure and reveal the emergence of disease-associated cell states. Notably, cardiomyocytes converge toward common disease-associated cell states, whereas fibroblasts and myeloid cells undergo dramatic diversification. Endothelial cells and pericytes display global transcriptional shifts without changes in cell complexity. Collectively, our findings provide a comprehensive analysis of the cellular and transcriptomic landscape of human heart failure, identify cell type-specific transcriptional programs and disease-associated cell states and establish a valuable resource for the investigation of human heart failure.

BackgroundImmune checkpoint inhibitors (ICIs), antibodies targeting PD-1/PD-L1 or CTLA4 have revolutionized cancer management but are associated with devastating immune-related adverse events (irAEs) including myocarditis. The main risk factor for ICI myocarditis is the use of combination PD-1 and CTLA4 inhibition. ICI-myocarditis is often fulminant and is pathologically characterized by myocardial infiltration of T lymphocytes and macrophages. While much has been learned regarding the role of T-cells in ICI-myocarditis, little is understood regarding the identity, transcriptional diversity, and functions of infiltrating macrophages.MethodsWe employed an established murine ICI myocarditis model (Ctla4+/-Pdcd1-/-mice) to explore the cardiac immune landscape using single-cell RNA-sequencing, immunostaining, flow cytometry, in situ RNA hybridization and molecular imaging and antibody neutralization studies.ResultsWe observed marked increases in CCR2+monocyte-derived macrophages and CD8+T-cells in this model. The macrophage compartment was heterogeneous and displayed marked enrichment in an inflammatory CCR2+subpopulation highly expressingCxcl9,Cxcl10,Gbp2b, andFcgr4that originated from CCR2+monocytes. Importantly, a similar macrophage population expressingCXCL9,CXCL10, and CD16α (human homologue of mouse FcgR4) was found selectively expanded in patients with ICI myocarditis compared to other forms of heart failure and myocarditis.In silicoprediction of cell-cell communication suggested interactions between T-cells andCxcl9+Cxcl10+macrophages via IFN-γ and CXCR3 signaling pathways. Depleting CD8+T-cells, macrophages, and blockade of IFN-γ signaling blunted the expansion ofCxcl9+Cxcl10+macrophages in the heart and attenuated myocarditis suggesting that this interaction was necessary for disease pathogenesis.ConclusionThese data demonstrate that ICI-myocarditis is associated with the expansion of a specific population of IFN-γ induced inflammatory macrophages and suggest the possibility that IFN-γ blockade may be considered as a treatment option for this devastating condition.

Background: Recent studies have established that CCR2 (C-C chemokine receptor type 2) marks proinflammatory subsets of monocytes, macrophages, and dendritic cells that contribute to adverse left ventricle (LV) remodeling and heart failure progression. Elucidation of the effector mechanisms that mediate adverse effects of CCR2 + monocytes, macrophages, and dendritic cells will yield important insights into therapeutic strategies to suppress myocardial inflammation. Methods: We used mouse models of reperfused myocardial infarction, angiotensin II and phenylephrine infusion, and diphtheria toxin cardiomyocyte ablation to investigate CCL17 (C-C chemokine ligand 17). We used Ccl17 knockout mice, flow cytometry, RNA sequencing, biochemical assays, cell trafficking studies, and in vivo cell depletion to identify the cell types that generate CCL17, define signaling pathways that controlled its expression, delineate the functional importance of CCL17 in adverse LV remodeling and heart failure progression, and determine the mechanistic basis by which CCL17 exerts its effects. Results: We demonstrated that CCL17 is expressed in CCR2 + macrophages and cluster of differentiation 11b + conventional dendritic cells after myocardial infarction, angiotensin II and phenylephrine infusion, and diphtheria toxin cardiomyocyte ablation. We clarified the transcriptional signature of CCL17 + macrophages and dendritic cells and identified granulocyte-macrophage colony-stimulating factor (GM-CSF) signaling as a key regulator of CCL17 expression through cooperative activation of STAT5 (signal transducer and activator of transcription 5) and canonical NF-κB (nuclear factor κ-light-chain-enhancer of activated B cells) signaling. Ccl17 deletion resulted in reduced LV remodeling, decreased myocardial fibrosis and cardiomyocyte hypertrophy, and improved LV systolic function after myocardial infarction and angiotensin II and phenylephrine infusion. We observed increased abundance of regulatory T cells (Tregs) in the myocardium of injured Ccl17 knockout mice. CCL17 inhibited Treg recruitment through biased activation of CCR4. CCL17 activated Gq signaling and CCL22 (C-C chemokine ligand 22) activated both Gq and ARRB (β-arrestin) signaling downstream of CCR4. CCL17 competitively inhibited CCL22 stimulated ARRB signaling and Treg migration. We provide evidence that Tregs mediated the protective effects of Ccl17 deletion on myocardial inflammation and adverse LV remodeling. Conclusions: These findings identify CCL17 as a proinflammatory mediator of CCR2 + macrophages and dendritic cells and suggest that inhibition of CCL17 may serve as an effective strategy to promote Treg recruitment and suppress myocardial inflammation.

Inflammation and tissue fibrosis co-exist and are causally linked to organ dysfunction. However, the molecular mechanisms driving immune-fibroblast crosstalk in human cardiac disease remains unexplored and there are currently no therapeutics to target fibrosis. Here, we performed multi-omic single-cell gene expression, epitope mapping, and chromatin accessibility profiling in 38 donors, acutely infarcted, and chronically failing human hearts. We identified a disease-associated fibroblast trajectory marked by cell surface expression of fibroblast activator protein (FAP), which diverged into distinct myofibroblasts and pro-fibrotic fibroblast populations, the latter resembling matrifibrocytes. Pro-fibrotic fibroblasts were transcriptionally similar to cancer associated fibroblasts and expressed high levels of collagens and periostin (POSTN), thymocyte differentiation antigen 1 (THY-1), and endothelin receptor A (EDNRA) predicted to be driven by a RUNX1 gene regulatory network. We assessed the applicability of experimental systems to model tissue fibrosis and demonstrated that 3 different in vivo mouse models of cardiac injury were superior compared to cultured human heart and dermal fibroblasts in recapitulating the human disease phenotype. Ligand-receptor analysis and spatial transcriptomics predicted that interactions between C-C chemokine receptor type 2 (CCR2) macrophages and fibroblasts mediated by interleukin 1 beta (IL-1β) signaling drove the emergence of pro-fibrotic fibroblasts within spatially defined niches. This concept was validated through in silico transcription factor perturbation and in vivo inhibition of IL-1β signaling in fibroblasts where we observed reduced pro-fibrotic fibroblasts, preferential differentiation of fibroblasts towards myofibroblasts, and reduced cardiac fibrosis. Herein, we show a subset of macrophages signal to fibroblasts via IL-1β and rewire their gene regulatory network and differentiation trajectory towards a pro-fibrotic fibroblast phenotype. These findings highlight the broader therapeutic potential of targeting inflammation to treat tissue fibrosis and restore organ function.

Cardiac fibrosis is causally linked to heart failure pathogenesis and adverse clinical outcomes. However, the precise fibroblast populations that drive fibrosis in the human heart and the mechanisms that govern their emergence remain incompletely defined. Here, we performed Cellular Indexing of Transcriptomes and Epitomes by sequencing (CITE-seq) in 22 explanted human hearts from healthy donors, acute myocardial infarction (MI), and chronic ischemic and non-ischemic cardiomyopathy patients. We identified a fibroblast trajectory marked by fibroblast activator protein (FAP) and periostin (POSTN) expression that was independent of myofibroblasts, peaked early after MI, remained elevated in chronic heart failure, and displayed a transcriptional signature consistent with fibrotic activity. We assessed the applicability of cardiac fibrosis models and demonstrated that mouse MI, angiotensin II/phenylephrine infusion, and pressure overload models were superior compared to cultured human heart and dermal fibroblasts in recapitulating cardiac fibroblast diversity including pathogenic cell states. Ligand-receptor analysis and spatial transcriptomics predicted interactions between macrophages, T cells, and fibroblasts within spatially defined niches. CCR2+monocyte and macrophage states were the dominant source of ligands targeting fibroblasts. Inhibition of IL-1β signaling to cardiac fibroblasts was sufficient to suppress fibrosis, emergence, and maturation of FAP+POSTN+fibroblasts. Herein, we identify a human fibroblast trajectory marked by FAP and POSTN expression that is associated with cardiac fibrosis and identify macrophage-fibroblast crosstalk mediated by IL-1β signaling as a key regulator of pathologic fibroblast differentiation and fibrosis.

Recovery of cardiac function is the ultimate goal of heart failure therapy. Unfortunately, cardiac recovery remains a rare and poorly understood phemomenon. Herein, we performed single nucleus RNA-sequencing (snRNA-seq) from non-diseased donors and heart failure patients. By comparing patients who recovered LV systolic function following LV assist device implantation to those who did not recover and donors, we defined the cellular and transcriptional landscape and predictors of cardiac recovery. We sequenced 40 hearts and recovered 185,881 nuclei with 13 distinct cell types. Using pseudobulk differential expression analysis to explicate cell specific signatures of cardiac recovery, we observed that recovered cardiomyocytes do not revert to a normal state, and instead, retain transcriptional signatures observed in heart failure. Macrophages and fibroblasts displayed the strongest signatures of recovery. While some evidence of reversion to a normal state was observed, many heart failure associated genes remained elevated and recovery signatures were predominately indicative of a biological state that was unique from donor and heart failure conditions. Acquisition of recovery states was associated with improved LV systolic function. Pro-inflammatory macrophages and inflammatory signaling in fibroblasts were identified as negative predictors of recovery. We identified downregulation of RUNX1 transcriptional activity in macrophages and fibroblasts as a central event associated with and predictive of cardiac recovery. In silico perturbation of RUNX1 in macrophages and fibroblasts recapitulated the transcriptional state of cardiac recovery. This prediction was corroborated in a mouse model of cardiac recovery mediated by BRD4 inhibition where we observed a decrease in macrophage and fibroblast Runx1 expression, diminished chromatin accessibility within peaks linked to the Runx1 locus, and acquisition of recovery signatures. These findings suggest that cardiac recovery is a unique biological state and identify RUNX1 as a possible therapeutic target to facilitate cardiac recovery.

Immune checkpoint inhibitors (ICI) targeting cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) or programmed cell death protein 1(PD-1)/ programmed death-ligand 1(PD-L1) have revolutionized cancer management since the approval. However, they are associated with immune-related adverse events (irAEs), including myocarditis. ICI-associated myocarditis is often fulminant and fatal. The associated major risk factor is the combination of CTLA-4 and PD-1 inhibition. The mechanistic basis of ICI-associated myocarditis is unclear and mechanism-based treatment strategies are needed. Crosstalk between T-cells and macrophages may represent one pathogenic mechanism underlying ICI-associated myocarditis. We employed an established mouse model Ctla4 +/- Pdcd1 -/- to study the crosstalk between T-cells and macrophage in the heart and its impact on reshaping cardiac immune landscape using unbiased single cell-RNA-seq technology. We discovered a more inflammatory cardiac microenvironment in ICI-associated myocarditis mouse, characterized by CD68 + cells infiltration, robust CCR2 signal, enhanced T-cells activation with interferon gamma (IFN-γ) secretion and increased frequency of Cxcl9 + Cxcl10 + macrophages with an activated phenotype enriched in response to IFN-γ. Importantly, the existence of Cxcl9 + Cxcl10 + macrophages (characterized by CD16α and CXCL9/CXCL10 expression) was confirmed in human ICI-associated myocarditis specimens which establishes a connection between mouse model and human disease. Analysis of cell communication between myeloid cells and T-cells predicted that Cxcl9 + Cxcl10 + macrophages communicate with T-cells via IFN-γ-IFN-γR and Cxcl9/Cxcl10-CXCR3 signals. Pseudotime time analysis suggests Cxcl9 + Cxcl10 + macrophages were derived from monocytes. Blocking IFN-γ signal prolonged the survival time of ICI-associated myocarditis mouse and reduced emergence of Cxcl9 + Cxcl10 + macrophages in the heart suggesting specific blocking the mediator of crosstalk between T-cells and macrophages attenuated ICI-associated myocarditis. This study will provide support to the usage of IFN-γ as a potential target to prevent ICI associated myocarditis during or at the end of ICI therapy in cancer patients.

Previous human genetic studies have provided insight into the genes and pathways involved in atherosclerotic CAD, however, there remains an incomplete understanding of the precise cell types that drive disease pathogenesis. To generate a cellular atlas for CAD, we performed CITE-seq on left human coronary arteries with obstructive CAD (n=5), CAD with coronary stenting (n=4), or controls without CAD (n=5); single-nuclei RNA sequencing was performed on a subset (n=4) of these samples. Standard histopathology was used to assess the degree of atherosclerotic plaque burden and annotate disease status (Fig b). Sequencing yielded 65,437 cells with 23 distinct cell types (Fig a) and canonical marker genes (Fig c). Within the stroma we found prominent transcriptional changes in endothelial cells and fibroblasts along with the emergence of a modulated smooth muscle cell (SMC) state in CAD tissue. The modulated SMC cells were enriched with fibroblast activator protein surface expression. Additionally, we noted the emergence of a proliferative SMC population in stented arteries. Two distinct fibroblast populations were present: one was specific to coronary arteries in GTEx and this population was enriched for SVEP1 expression in the CAD samples. Furthermore, pseudotime analysis and integration of published single-cell SMC lineage tracing data in atherosclerotic mice show that this population is SMC-derived. Within the immune cells, we find expansion of B- and T-cell populations with transcriptional changes within myeloid subsets. We found 4 distinct macrophage populations: inflammatory, TREM2, HLA-II high, and resident-like macrophages. CAD macrophages had enriched inflammatory and foam-cell like signatures compared to controls. Furthermore, foam cell genes were localized to the inflammatory and TREM2 macrophages. Together, our results provide the first multi-omic single cell atlas of human coronary artery tissue with insights into disease pathogenesis.