Background: Diabetes is associated with a significantly elevated risk of heart failure. However, the precise cellular and molecular protagonists underpinning the development of heart failure in diabetes remains unclear. Moreover, very little is known, of how disparate non-myocyte populations of the heart contribute to diabetic cardiomyopathy. Methodology: To address this gap in knowledge, we conducted single-cell transcriptomic analysis of non-myocytes from heart ventricles of spontaneous type-2 diabetic ( db/db ) male mice. Findings were corroborated by flow cytometry, histology and computational analysis of publically available bulk RNA sequencing datasets from alternative diabetes models. Results: Single-cell transcriptomic analysis of db/db mouse hearts revealed a concerted diabetes-induced cellular response driving cardiac pathological remodelling. We identified diabetes-induced up-regulation of pathways contributing to known features of diabetic cardiomyopathy such as dysregulation of vascular homeostasis and lipid metabolism, as well as augmented inflammation, in cell specific contexts. We also identified unexpected characteristics in the diabetic heart, including impaired protein folding and cellular trafficking within lymphatic vessels. Using flow cytometry and histology, increase in inflammatory cells, such as Ly6C hi monocytes, shifts in macrophage phenotype, and increased abundances of fibroblasts and endocardial cells were confirmed. Finally, integration of single-cell transcriptomic data with publically available bulk-RNA sequencing datasets from alternative diabetes models revealed shared hallmarks of diabetic heart and disease context-dependent features. Conclusions: Together, this work offers a new perspective for understanding the cellular and molecular mediators of diabetes-induced cardiac pathology. Targeting these mediators may offer new therapeutic avenues to address the cardiac complications associated with diabetes.
ABSTRACT Cardiac fibrosis is a major cause of cardiac dysfunction. Recently, single-cell genomic approaches have revealed in unprecedented resolution the orchestrated cellular responses driving cardiac fibrosis. Yet, the fibrosis-causing phenotypes that emerge in the heart following non-ischemic cardiac stress, and the transcriptional circuits that govern cell identity and drive fibrosis, are not well understood. Applying a paired multiomic approach, we reveal key transcriptional circuits, in mouse and human hearts, which are associated with fibrosis development following non-ischemic cardiac insults, independent of disease model, species or biological sex. Strikingly, we find the key regulatory events driving fibrosis are reversible at the single-cell transcriptional and epigenomic level, further pointing to key factors regulating fibrosis development and resolution. The transcriptional regulators identified in this study represent promising targets to ameliorate the development of fibrosis in the context of chronic stressors such as aging and hypertension.
Background: Yolk sac (YS) progenitors are a source of macrophages and endothelial cells in some tissues that are thought to be maintained postnatally by self-renewal in their differentiated states. How this is achieved remains poorly understood. Methods and Results: Single-cell digests from mouse aortas selectively formed macrophage colony-forming units (CFU-M) in methylcellulose, which self-renewed in secondary cultures from single cells. CFU-M comprised a homogeneous population of Lin-CD45+/LoCD11b-F4/80-Sca-1+c-Kit+ progenitor cells that co-expressed fractalkine receptor (CX3CR1) and colony stimulating factor-1 receptor (CSF1R). These progenitors displayed high proliferative activity from adult aorta even at steady state. Flt3Cre lineage mapping revealed their independence from Flt3+ bone marrow hematopoietic progenitor cells. They were especially abundant in neonatal aorta, with subsequent age-related decline, suggesting prenatal seeding, which was confirmed by finding their emergence in YS after embryonic day (E) 7.5 and from aorta-gonad-mesonephros by E10.5. Inducible fate-mapping using Csf1rMer-iCre-Mer and Cx3cr1YFP-creER mice established that these progenitors originate from an E8.5 CSF1R+ and E8.5-9.5 CX3CR1+ source, together with macrophages and endothelial cells in the aortic wall. Complementary differentiation studies revealed aortic progenitors to be vasculogenic and bipotent for macrophages and endothelium, contributing to adventitial neovascularization in aortic ring assays and forming perfused blood vessels and macrophages after transfer into ischemic hindlimb. Single-cell RNA sequencing showed their relatively homogeneous myelopoietic and angiogenic gene expression profile without expression of mature myeloid or endothelial genes. Finally, we found that aortic progenitors also express angiotensin converting enzyme (ACE) and angiotensin II receptor, AGTR2, and established regulatory roles for angiotensin II, which augmented their proliferative, self-renewal and differentiation properties in vitro and expansion in aorta in vivo. Conclusion: Our discovery of aortic endothelial-macrophage progenitors adds to the recognized fate of YS progenitors in postnatal tissues. These bipotent cells may help explain the local renewal of YS-derived tissue-resident macrophages and endothelial cells after birth.
Abstract Coronary microvascular dysfunction (CMD) is associated with cardiac dysfunction and predictive of cardiac mortality in obesity, especially in females. Emerging evidence suggests development of heart failure with preserved ejection fraction in females with CMD and that mineralocorticoid receptor (MR) antagonism may be more efficacious in obese female, versus male, HFpEF patients. Accordingly, we examined the hypothesis that smooth muscle cell (SMC)-specific MR deletion prevents obesity-associated coronary and cardiac diastolic dysfunction in females. Obesity was induced in female mice via western diet (WD) feeding alongside littermates fed standard diet. Initial studies revealed that global MR blockade with spironolactone prevented impaired coronary vasodilation and diastolic dysfunction in obese females. Importantly, specific deletion of SMC-MR similarly prevented obesity-associated coronary and cardiac dysfunction. Cardiac gene expression profiling suggested reduced cardiac inflammation in WD-fed mice with SMC-MR deletion independent of blood pressure, aortic stiffening, and cardiac hypertrophy. Further mechanistic studies utilizing single-cell RNA sequencing of non-cardiomyocyte cell populations revealed novel impacts of SMC-MR deletion on the cardiac cellulome in obese mice. Specifically, WD feeding induced inflammatory gene signatures in multiple non-myocyte populations (B/T cells, macrophages, and endothelium), independent of cardiac fibrosis, that was prevented by SMC-MR deletion. Further, SMC-MR deletion induced a basal reduction in cardiac mast cells and prevented WD-induced cardiac pro-inflammatory chemokine expression and leukocyte recruitment. These data reveal a central role for SMC-MR signaling in obesity-associated coronary and cardiac dysfunction thus supporting the emerging paradigm of a vascular origin of cardiac dysfunction in obesity.
Summary Excessive adipose tissue expansion is often linked with type-2 diabetes. Despite recent efforts mapping adipose tissue changes in obesity using single-cell omics, an understanding of cellular and gene expression changes in a model of type 2 diabetes, and the transcriptional circuitry controlling it, is still lacking. Here, we use single-nucleus RNA sequencing to analyze the remodeling of gonadal white and interscapular brown adipose tissue from female and male mice with or without diabetes. Analysis of 51,877 nuclei revealed altered phenotypes in every cell population in type 2 diabetes. This included an immunoregulatory response, and changes in extracellular matrix, energy generation, and hormone responses. Key transcription factors were inferred as cell-specific and non-specific nodes controlling diabetes-linked phenotypes. Finally, female-to-male population heterogeneity and gene expression differences were observed. Here we provide a resource detailing how adipose tissue remodeling, and the molecular mechanisms governing it, may contribute to cardiometabolic disease.
Coronary microvascular dysfunction (CMD) is associated with cardiac dysfunction and predictive of cardiac mortality in obesity, especially in females. Clinical data further support that CMD associates with development of heart failure with preserved ejection fraction and that mineralocorticoid receptor (MR) antagonism may be more efficacious in obese female, versus male, HFpEF patients. Accordingly, we examined the impact of smooth muscle cell (SMC)-specific MR deletion on obesity-associated coronary and cardiac diastolic dysfunction in female mice. Obesity was induced in female mice via western diet (WD) feeding alongside littermates fed standard diet. Global MR blockade with spironolactone prevented coronary and cardiac dysfunction in obese females and specific deletion of SMC-MR was sufficient to prevent obesity-associated coronary and cardiac diastolic dysfunction. Cardiac gene expression profiling suggested reduced cardiac inflammation in WD-fed mice with SMC-MR deletion independent of blood pressure, aortic stiffening, and cardiac hypertrophy. Further mechanistic studies utilizing single-cell RNA sequencing of non-cardiomyocyte cell populations revealed novel impacts of SMC-MR deletion on the cardiac cellulome in obese mice. Specifically, WD feeding induced inflammatory gene signatures in non-myocyte populations including B/T cells, macrophages, and endothelium as well as increased coronary VCAM-1 protein expression, independent of cardiac fibrosis, that was prevented by SMC-MR deletion. Further, SMC-MR deletion induced a basal reduction in cardiac mast cells and prevented WD-induced cardiac pro-inflammatory chemokine expression and leukocyte recruitment. These data reveal a central role for SMC-MR signaling in obesity-associated coronary and cardiac dysfunction, thus supporting the emerging paradigm of a vascular origin of cardiac dysfunction in obesity.
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