Age-Dependent RGS5 Loss in Pericytes Induces Cardiac Dysfunction and Fibrosis.

BACKGROUND: Pericytes are capillary-associated mural cells involved in the maintenance and stability of the vascular network. Although aging is one of the main risk factors for cardiovascular disease, the consequences of aging on cardiac pericytes are unknown. METHODS: In this study, we have combined single-nucleus RNA sequencing and histological analysis to determine the effects of aging on cardiac pericytes. Furthermore, we have conducted in vivo and in vitro analysis of RGS5 (regulator of G-protein signaling 5) loss of function and finally have performed pericytes-fibroblasts coculture studies to understand the effect of RGS5 deletion in pericytes on the neighboring fibroblasts. RESULTS: Aging reduced the pericyte area and capillary coverage in the murine heart. Single-nucleus RNA sequencing analysis further revealed that the expression of Rgs5 was reduced in cardiac pericytes from aged mice. In vivo and in vitro studies showed that the deletion of RGS5 impaired cardiac function, induced fibrosis, and morphological changes in pericytes characterized by a profibrotic gene expression signature and the expression of different ECM (extracellular matrix) components and growth factors, for example, TGFB2 and PDGFB. Indeed, culturing fibroblasts with the supernatant of RGS5-deficient pericytes induced their activation as evidenced by the increased expression of αSMA (alpha smooth muscle actin) in a TGFß (transforming growth factor beta)2-dependent mechanism. CONCLUSIONS: Our results have identified RGS5 as a crucial regulator of pericyte function during cardiac aging. The deletion of RGS5 causes cardiac dysfunction and induces myocardial fibrosis, one of the hallmarks of cardiac aging.

Improved integration of single-cell transcriptome data demonstrates common and unique signatures of heart failure in mice and humans.

BACKGROUND: Cardiovascular research heavily relies on mouse (Mus musculus) models to study disease mechanisms and to test novel biomarkers and medications. Yet, applying these results to patients remains a major challenge and often results in noneffective drugs. Therefore, it is an open challenge of translational science to develop models with high similarities and predictive value. This requires a comparison of disease models in mice with diseased tissue derived from humans. RESULTS: To compare the transcriptional signatures at single-cell resolution, we implemented an integration pipeline called OrthoIntegrate, which uniquely assigns orthologs and therewith merges single-cell RNA sequencing (scRNA-seq) RNA of different species. The pipeline has been designed to be as easy to use and is fully integrable in the standard Seurat workflow.We applied OrthoIntegrate on scRNA-seq from cardiac tissue of heart failure patients with reduced ejection fraction (HFrEF) and scRNA-seq from the mice after chronic infarction, which is a commonly used mouse model to mimic HFrEF. We discovered shared and distinct regulatory pathways between human HFrEF patients and the corresponding mouse model. Overall, 54% of genes were commonly regulated, including major changes in cardiomyocyte energy metabolism. However, several regulatory pathways (e.g., angiogenesis) were specifically regulated in humans. CONCLUSIONS: The demonstration of unique pathways occurring in humans indicates limitations on the comparability between mice models and human HFrEF and shows that results from the mice model should be validated carefully. OrthoIntegrate is publicly accessible (https://github.com/MarianoRuzJurado/OrthoIntegrate) and can be used to integrate other large datasets to provide a general comparison of models with patient data.

Aging impairs the neurovascular interface in the heart.

Aging is a major risk factor for impaired cardiovascular health. Because the aging myocardium is characterized by microcirculatory dysfunction, and because nerves align with vessels, we assessed the impact of aging on the cardiac neurovascular interface. We report that aging reduces nerve density in the ventricle and dysregulates vascular-derived neuroregulatory genes. Aging down-regulates microRNA 145 (miR-145) and derepresses the neurorepulsive factor semaphorin-3A. miR-145 deletion, which increased Sema3a expression or endothelial Sema3a overexpression, reduced axon density, mimicking the aged-heart phenotype. Removal of senescent cells, which accumulated with chronological age in parallel to the decline in nerve density, rescued age-induced denervation, reversed Sema3a expression, preserved heart rate patterns, and reduced electrical instability. These data suggest that senescence-mediated regulation of nerve density contributes to age-associated cardiac dysfunction.

Single-nuclear transcriptome profiling identifies persistent fibroblast activation in hypertrophic and failing human hearts of patients with longstanding disease.

AIMS: Cardiac fibrosis drives the progression of heart failure in ischaemic and hypertrophic cardiomyopathy. Therefore, the development of specific anti-fibrotic treatment regimens to counteract cardiac fibrosis is of high clinical relevance. Hence, this study examined the presence of persistent fibroblast activation during longstanding human heart disease at a single-cell resolution to identify putative therapeutic targets to counteract pathological cardiac fibrosis in patients. METHODS AND RESULTS: We used single-nuclei RNA sequencing with human tissues from two samples of one healthy donor, and five hypertrophic and two failing hearts. Unsupervised sub-clustering of 7110 nuclei led to the identification of 7 distinct fibroblast clusters. De-convolution of cardiac fibroblast heterogeneity revealed a distinct population of human cardiac fibroblasts with a molecular signature of persistent fibroblast activation and a transcriptional switch towards a pro-fibrotic extra-cellular matrix composition in patients with established cardiac hypertrophy and heart failure. This sub-cluster was characterized by high expression of POSTN, RUNX1, CILP, and a target gene adipocyte enhancer-binding protein 1 (AEBP1) (all P < 0.001). Strikingly, elevated circulating AEBP1 blood level were also detected in a validation cohort of patients with confirmed cardiac fibrosis and hypertrophic cardiomyopathy by cardiac magnetic resonance imaging (P < 0.01). Since endogenous AEBP1 expression was increased in patients with established cardiac hypertrophy and heart failure, we assessed the functional consequence of siRNA-mediated AEBP1 silencing in human cardiac fibroblasts. Indeed, AEBP1 silencing reduced proliferation, migration, and fibroblast contractile capacity and α-SMA gene expression, which is a hallmark of fibroblast activation (all P < 0.05). Mechanistically, the anti-fibrotic effects of AEBP1 silencing were linked to transforming growth factor-beta pathway modulation. CONCLUSION: Together, this study identifies persistent fibroblast activation in patients with longstanding heart disease, which might be detected by circulating AEBP1 and therapeutically modulated by its targeted silencing in human cardiac fibroblasts.

Cardiomyocyte hyperplasia and immaturity but not hypertrophy are characteristic features of patients with RASopathies.

AIMS: RASopathies are caused by mutations in genes that alter the MAP kinase pathway and are marked by several malformations with cardiovascular disorders as the predominant cause of mortality. Mechanistic insights in the underlying pathogenesis in affected cardiac tissue are rare. The aim of the study was to assess the impact of RASopathy causing mutations on the human heart. METHODS AND RESULTS: Using single cell approaches and histopathology we analyzed cardiac tissue from children with different RASopathy-associated mutations compared to age-matched dilated cardiomyopathy (DCM) and control hearts. The volume of cardiomyocytes was reduced in RASopathy conditions compared to controls and DCM patients, and the estimated number of cardiomyocytes per heart was ∼4-10 times higher. Single nuclei RNA sequencing of a 13-year-old RASopathy patient (carrying a PTPN11 c.1528C > G mutation) revealed that myocardial cell composition and transcriptional patterns were similar to <1 year old DCM hearts. Additionally, immaturity of cardiomyocytes is shown by an increased MYH6/MYH7 expression ratio and reduced expression of genes associated with fatty acid metabolism. In the patient with the PTPN11 mutation activation of the MAP kinase pathway was not evident in cardiomyocytes, whereas increased phosphorylation of PDK1 and its downstream kinase Akt was detected. CONCLUSION: In conclusion, an immature cardiomyocyte differentiation status appears to be preserved in juvenile RASopathy patients. The increased mass of the heart in such patients is due to an increase in cardiomyocyte number (hyperplasia) but not an enlargement of individual cardiomyocytes (hypertrophy).

Acute injury to the mouse carotid artery provokes a distinct healing response.

Treatment of vascular stenosis with angioplasty results in acute vascular damage, which may lead to restenosis. Owing to the highly complex cellularity of blood vessels, the healing response following this damage is incompletely understood. To gain further insight into this process, scRNA-seq of mouse carotid tissue after wire injury was performed. Stages of acute inflammation, resolution and remodeling were recapitulated in these data. To identify cell types which give rise to neointima, analyses focused on smooth muscle cell and fibroblast populations, and included data integration with scRNA-seq data from myocardial infarction and atherosclerosis datasets. Following carotid injury, a subpopulation of smooth muscle cells which also arises during atherosclerosis and myocardial infarction was identified. So-called stem cell/endothelial cell/monocyte (SEM) cells are candidates for repopulating injured vessels, and were amongst the most proliferative cell clusters following wire-injury of the carotid artery. Importantly, SEM cells exhibit specific transcriptional profiles which could be therapeutically targeted. SEM cell gene expression patterns could also be detected in bulk RNA-sequencing of neointimal tissue isolated from injured carotid vessels by laser capture microdissection. These data indicate that phenotypic plasticity of smooth muscle cells is highly important to the progression of lumen loss following acute carotid injury. Interference with SEM cell formation could be an innovative approach to combat development of restenosis.

Why is endothelial resilience key to maintain cardiac health?

Myocardial injury as induced by myocardial infarction results in tissue ischemia, which critically incepts cardiomyocyte death. Endothelial cells play a crucial role in restoring oxygen and nutrient supply to the heart. Latest advances in single-cell multi-omics, together with genetic lineage tracing, reveal a transcriptional and phenotypical adaptation to the injured microenvironment, which includes alterations in metabolic, mesenchymal, hematopoietic and pro-inflammatory signatures. The extent of transition in mesenchymal or hematopoietic cell lineages is still debated, but it is clear that several of the adaptive phenotypical changes are transient and endothelial cells revert back to a naïve cell state after resolution of injury responses. This resilience of endothelial cells to acute stress responses is important for preventing chronic dysfunction. Here, we summarize how endothelial cells adjust to injury and how this dynamic response contributes to repair and regeneration. We will highlight intrinsic and microenvironmental factors that contribute to endothelial cell resilience and may be targetable to maintain a functionally active, healthy microcirculation.

Comparative analysis of common alignment tools for single-cell RNA sequencing.

BACKGROUND: With the rise of single-cell RNA sequencing new bioinformatic tools have been developed to handle specific demands, such as quantifying unique molecular identifiers and correcting cell barcodes. Here, we benchmarked several datasets with the most common alignment tools for single-cell RNA sequencing data. We evaluated differences in the whitelisting, gene quantification, overall performance, and potential variations in clustering or detection of differentially expressed genes. We compared the tools Cell Ranger version 6, STARsolo, Kallisto, Alevin, and Alevin-fry on 3 published datasets for human and mouse, sequenced with different versions of the 10X sequencing protocol. RESULTS: Striking differences were observed in the overall runtime of the mappers. Besides that, Kallisto and Alevin showed variances in the number of valid cells and detected genes per cell. Kallisto reported the highest number of cells; however, we observed an overrepresentation of cells with low gene content and unknown cell type. Conversely, Alevin rarely reported such low-content cells. Further variations were detected in the set of expressed genes. While STARsolo, Cell Ranger 6, Alevin-fry, and Alevin produced similar gene sets, Kallisto detected additional genes from the Vmn and Olfr gene family, which are likely mapping artefacts. We also observed differences in the mitochondrial content of the resulting cells when comparing a prefiltered annotation set to the full annotation set that includes pseudogenes and other biotypes. CONCLUSION: Overall, this study provides a detailed comparison of common single-cell RNA sequencing mappers and shows their specific properties on 10X Genomics data.

The hydrogen-peroxide producing NADPH oxidase 4 does not limit neointima development after vascular injury in mice.

OBJECTIVE: The NADPH oxidase Nox4 is an important source of H2O2. Nox4-derived H2O2 limits vascular inflammation and promotes smooth muscle differentiation. On this basis, the role of Nox4 for restenosis development was determined in the mouse carotid artery injury model. METHODS AND RESULTS: Genetic deletion of Nox4 by a tamoxifen-activated Cre-Lox-system did not impact on neointima formation in the carotid artery wire injury model. To understand this unexpected finding, time-resolved single-cell RNA-sequencing (scRNAseq) from injured carotid arteries of control mice and massive-analysis-of-cDNA-ends (MACE)-RNAseq from the neointima harvested by laser capture microdissection of control and Nox4 knockout mice was performed. This revealed that resting smooth muscle cells (SMCs) and fibroblasts exhibit high Nox4 expression, but that the proliferating de-differentiated SMCs, which give rise to the neointima, have low Nox4 expression. In line with this, the first weeks after injury, gene expression was unchanged between the carotid artery neointimas of control and Nox4 knockout mice. CONCLUSION: Upon vascular injury, Nox4 expression is transiently lost in the cells which comprise the neointima. NADPH oxidase 4 therefore does not interfere with restenosis development after wire-induced vascular injury.

Single Nuclei Sequencing Reveals Novel Insights Into the Regulation of Cellular Signatures in Children With Dilated Cardiomyopathy.

BACKGROUND: Dilated cardiomyopathy (DCM) is a leading cause of death in children with heart failure. The outcome of pediatric heart failure treatment is inconsistent, and large cohort studies are lacking. Progress may be achieved through personalized therapy that takes age- and disease-related pathophysiology, pathology, and molecular fingerprints into account. We present single nuclei RNA sequencing from pediatric patients with DCM as the next step in identifying cellular signatures. METHODS: We performed single nuclei RNA sequencing with heart tissues from 6 children with DCM with an age of 0.5, 0.75, 5, 6, 12, and 13 years. Unsupervised clustering of 18 211 nuclei led to the identification of 14 distinct clusters with 6 major cell types. RESULTS: The number of nuclei in fibroblast clusters increased with age in patients with DCM, a finding that was confirmed by histological analysis and was consistent with an age-related increase in cardiac fibrosis quantified by cardiac magnetic resonance imaging. Fibroblasts of patients with DCM >6 years of age showed a profoundly altered gene expression pattern with enrichment of genes encoding fibrillary collagens, modulation of proteoglycans, switch in thrombospondin isoforms, and signatures of fibroblast activation. In addition, a population of cardiomyocytes with a high proregenerative profile was identified in infant patients with DCM but was absent in children >6 years of age. This cluster showed high expression of cell cycle activators such as cyclin D family members, increased glycolytic metabolism and antioxidative genes, and alterations in ß-adrenergic signaling genes. CONCLUSIONS: Novel insights into the cellular transcriptomes of hearts from pediatric patients with DCM provide remarkable age-dependent changes in the expression patterns of fibroblast and cardiomyocyte genes with less fibrotic but enriched proregenerative signatures in infants.

Single cell sequencing reveals endothelial plasticity with transient mesenchymal activation after myocardial infarction.

Endothelial cells play a critical role in the adaptation of tissues to injury. Tissue ischemia induced by infarction leads to profound changes in endothelial cell functions and can induce transition to a mesenchymal state. Here we explore the kinetics and individual cellular responses of endothelial cells after myocardial infarction by using single cell RNA sequencing. This study demonstrates a time dependent switch in endothelial cell proliferation and inflammation associated with transient changes in metabolic gene signatures. Trajectory analysis reveals that the majority of endothelial cells 3 to 7 days after myocardial infarction acquire a transient state, characterized by mesenchymal gene expression, which returns to baseline 14 days after injury. Lineage tracing, using the Cdh5-CreERT2;mT/mG mice followed by single cell RNA sequencing, confirms the transient mesenchymal transition and reveals additional hypoxic and inflammatory signatures of endothelial cells during early and late states after injury. These data suggest that endothelial cells undergo a transient mes-enchymal activation concomitant with a metabolic adaptation within the first days after myocardial infarction but do not acquire a long-term mesenchymal fate. This mesenchymal activation may facilitate endothelial cell migration and clonal expansion to regenerate the vascular network.

Mapping the Endothelial Cell S-Sulfhydrome Highlights the Crucial Role of Integrin Sulfhydration in Vascular Function.

BACKGROUND: In vascular endothelial cells, cysteine metabolism by the cystathionine γ lyase (CSE), generates hydrogen sulfide-related sulfane sulfur compounds (H2Sn), that exert their biological actions via cysteine S-sulfhydration of target proteins. This study set out to map the "S-sulfhydrome" (ie, the spectrum of proteins targeted by H2Sn) in human endothelial cells. METHODS: Liquid chromatography with tandem mass spectrometry was used to identify S-sulfhydrated cysteines in endothelial cell proteins and ß3 integrin intraprotein disulfide bond rearrangement. Functional studies included endothelial cell adhesion, shear stress-induced cell alignment, blood pressure measurements, and flow-induced vasodilatation in endothelial cell-specific CSE knockout mice and in a small collective of patients with endothelial dysfunction. RESULTS: Three paired sample sets were compared: (1) native human endothelial cells isolated from plaque-free mesenteric arteries (CSE activity high) and plaque-containing carotid arteries (CSE activity low); (2) cultured human endothelial cells kept under static conditions or exposed to fluid shear stress to decrease CSE expression; and (3) cultured endothelial cells exposed to shear stress to decrease CSE expression and treated with solvent or the slow-releasing H2Sn donor, SG1002. The endothelial cell "S-sulfhydrome" consisted of 3446 individual cysteine residues in 1591 proteins. The most altered family of proteins were the integrins and focusing on ß3 integrin in detail we found that S-sulfhydration affected intraprotein disulfide bond formation and was required for the maintenance of an extended-open conformation of the ß leg. ß3 integrin S-sulfhydration was required for endothelial cell mechanotransduction in vitro as well as flow-induced dilatation in murine mesenteric arteries. In cultured cells, the loss of S-sulfhydration impaired interactions between ß3 integrin and Gα13 (guanine nucleotide-binding protein subunit α 13), resulting in the constitutive activation of RhoA (ras homolog family member A) and impaired flow-induced endothelial cell realignment. In humans with atherosclerosis, endothelial function correlated with low H2Sn generation, impaired flow-induced dilatation, and failure to detect ß3 integrin S-sulfhydration, all of which were rescued after the administration of an H2Sn supplement. CONCLUSIONS: Vascular disease is associated with marked changes in the S-sulfhydration of endothelial cell proteins involved in mediating responses to flow. Short-term H2Sn supplementation improved vascular reactivity in humans highlighting the potential of interfering with this pathway to treat vascular disease.

The histone demethylase JMJD2B regulates endothelial-to-mesenchymal transition.

Endothelial cells play an important role in maintenance of the vascular system and the repair after injury. Under proinflammatory conditions, endothelial cells can acquire a mesenchymal phenotype by a process named endothelial-to-mesenchymal transition (EndMT), which affects the functional properties of endothelial cells. Here, we investigated the epigenetic control of EndMT. We show that the histone demethylase JMJD2B is induced by EndMT-promoting, proinflammatory, and hypoxic conditions. Silencing of JMJD2B reduced TGF-ß2-induced expression of mesenchymal genes, prevented the alterations in endothelial morphology and impaired endothelial barrier function. Endothelial-specific deletion of JMJD2B in vivo confirmed a reduction of EndMT after myocardial infarction. EndMT did not affect global H3K9me3 levels but induced a site-specific reduction of repressive H3K9me3 marks at promoters of mesenchymal genes, such as Calponin (CNN1), and genes involved in TGF-ß signaling, such as AKT Serine/Threonine Kinase 3 (AKT3) and Sulfatase 1 (SULF1). Silencing of JMJD2B prevented the EndMT-induced reduction of H3K9me3 marks at these promotors and further repressed these EndMT-related genes. Our study reveals that endothelial identity and function is critically controlled by the histone demethylase JMJD2B, which is induced by EndMT-promoting, proinflammatory, and hypoxic conditions, and supports the acquirement of a mesenchymal phenotype.

Dissection of heterocellular cross-talk in vascularized cardiac tissue mimetics.

Cellular specialization and interaction with other cell types in cardiac tissue is essential for the coordinated function of cell populations in the heart. The complex interplay between cardiomyocytes, endothelial cells and fibroblasts is necessary for adaptation but can also lead to pathophysiological remodeling. To understand this complex interplay, we developed 3D vascularized cardiac tissue mimetics (CTM) to study heterocellular cross-talk in hypertrophic, hypoxic and fibrogenic environments. This 3D platform responds to physiologic and pathologic stressors and mimics the microenvironment of diseased tissue. In combination with endothelial cell fluorescence reporters, these cardiac tissue mimetics can be used to precisely visualize and quantify cellular and functional responses upon stress stimulation. Utilizing this platform, we demonstrate that stimulation of α/ß-adrenergic receptors with phenylephrine (PE) promotes cardiomyocyte hypertrophy, metabolic maturation and vascularization of CTMs. Increased vascularization was promoted by conditioned medium of PE-stimulated cardiomyocytes and blocked by inhibiting VEGF or upon ß-adrenergic receptor antagonist treatment, demonstrating cardiomyocyte-endothelial cross-talk. Pathophysiological stressors such as severe hypoxia reduced angiogenic sprouting and increased cell death, while TGF ß2 stimulation increased collagen deposition concomitant to endothelial-to-mesenchymal transition. In sum, we have developed a cardiac 3D culture system that reflects native cardiac tissue function, metabolism and morphology - and for the first time enables the tracking and analysis of cardiac vascularization dynamics in physiology and pathology.

Transcriptional heterogeneity of fibroblasts is a hallmark of the aging heart.

Aging is a major risk factor for cardiovascular disease. Although the impact of aging has been extensively studied, little is known regarding the aging processes in cells of the heart. Here we analyzed the transcriptomes of hearts of 12-week-old and 18-month-old mice by single-nucleus RNA-sequencing. Among all cell types, aged fibroblasts showed most significant differential gene expression, increased RNA dynamics, and network entropy. Aged fibroblasts exhibited significantly changed expression patterns of inflammatory, extracellular matrix organization angiogenesis, and osteogenic genes. Functional analyses indicated deterioration of paracrine signatures between fibroblasts and endothelial cells in old hearts. Aged heart-derived fibroblasts had impaired endothelial cell angiogenesis and autophagy and augmented proinflammatory response. In particular, expression of Serpine1 and Serpine2 were significantly increased and secreted by old fibroblasts to exert antiangiogenic effects on endothelial cells, an effect that could be significantly prevented by using neutralizing antibodies. Moreover, we found an enlarged subpopulation of aged fibroblasts expressing osteoblast genes in the epicardial layer associated with increased calcification. Taken together this study provides system-wide insights and identifies molecular changes of aging cardiac fibroblasts, which may contribute to declined heart function.