ABSTRACT
Diabetes is associated with a significantly elevated risk of heart failure. However, despite extensive efforts to characterize the phenotype of the diabetic heart, the molecular and cellular protagonists that underpin cardiac pathological remodeling in diabetes remain unclear, with a notable paucity of data regarding the impact of diabetes on non-myocytes within the heart. Here we aimed to define key differences in cardiac non-myocytes between spontaneously type-2 diabetic (db/db) and healthy control (db/h) mouse hearts. Single-cell transcriptomic analysis revealed a concerted diabetes-induced cellular response contributing to cardiac remodeling. These included cell-specific activation of gene programs relating to fibroblast hyperplasia and cell migration, and dysregulation of pathways involving vascular homeostasis and protein folding. This work offers a new perspective for understanding the cellular mediators of diabetes-induced cardiac pathology, and pathways that may be targeted to address the cardiac complications associated with diabetes.
ABSTRACT
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.
Subject(s)
Cardiomyopathies , Heart Failure , Male , Female , Mice , Animals , Mice, Obese , Heart Failure/complications , Multiomics , Receptors, Mineralocorticoid/genetics , Receptors, Mineralocorticoid/metabolism , Stroke Volume , Mineralocorticoid Receptor Antagonists/pharmacology , Obesity/metabolismABSTRACT
This protocol features parallel isolation of myocytes and non-myocytes from murine hearts. It was designed with considerations for (1) time required to extract cardiac cells, (2) cell viability, and (3) protocol scalability. Here, a peristaltic pump and 3D-printed elements are combined to perfuse the heart with enzymes to dissociate cells. Myocytes and non-myocytes extracted using this protocol are separated by centrifugation and/or fluorescence-activated cell sorting for use in downstream applications including single-cell omics or other bio-molecular analyses. For complete details on the use and execution of this protocol, please refer to McLellan et al. (2020).
Subject(s)
Cell Separation/methods , Myocardium/cytology , Myocytes, Cardiac , Single-Cell Analysis/methods , Animals , Cell Culture Techniques , Cells, Cultured , Genomics , Male , Mice , Mice, Inbred C57BL , Myocytes, Cardiac/classification , Myocytes, Cardiac/cytologyABSTRACT
BACKGROUND: Diabetes is associated with a significantly elevated risk of cardiovascular disease and its specific pathophysiology remains unclear. Recent studies have changed our understanding of cardiac cellularity, with cellular changes accompanying diabetes yet to be examined in detail. This study aims to characterise the changes in the cardiac cellular landscape in murine diabetes to identify potential cellular protagonists in the diabetic heart. METHODS: Diabetes was induced in male FVB/N mice by low-dose streptozotocin and a high-fat diet for 26-weeks. Cardiac function was measured by echocardiography at endpoint. Flow cytometry was performed on cardiac ventricles as well as blood, spleen, and bone-marrow at endpoint from non-diabetic and diabetic mice. To validate flow cytometry results, immunofluorescence staining was conducted on left-ventricles of age-matched mice. RESULTS: Mice with diabetes exhibited hyperglycaemia and impaired glucose tolerance at endpoint. Echocardiography revealed reduced E:A and e':a' ratios in diabetic mice indicating diastolic dysfunction. Systolic function was not different between the experimental groups. Detailed examination of cardiac cellularity found resident mesenchymal cells (RMCs) were elevated as a result of diabetes, due to a marked increase in cardiac fibroblasts, while smooth muscle cells were reduced in proportion. Moreover, we found increased levels of Ly6Chi monocytes in both the heart and in the blood. Consistent with this, the proportion of bone-marrow haematopoietic stem cells were increased in diabetic mice. CONCLUSIONS: Murine diabetes results in distinct changes in cardiac cellularity. These changes-in particular increased levels of fibroblasts-offer a framework for understanding how cardiac cellularity changes in diabetes. The results also point to new cellular mechanisms in this context, which may further aid in development of pharmacotherapies to allay the progression of cardiomyopathy associated with diabetes.
Subject(s)
Diabetes Mellitus, Experimental/complications , Diabetic Cardiomyopathies/etiology , Fibroblasts/pathology , Myocardium/pathology , Ventricular Dysfunction, Left/etiology , Ventricular Function, Left , Animals , Blood Glucose/metabolism , Diabetes Mellitus, Experimental/metabolism , Diabetic Cardiomyopathies/metabolism , Diabetic Cardiomyopathies/pathology , Diabetic Cardiomyopathies/physiopathology , Diastole , Diet, High-Fat , Fibroblasts/metabolism , Hematopoietic Stem Cells/metabolism , Hematopoietic Stem Cells/pathology , Male , Mice , Monocytes/metabolism , Monocytes/pathology , Myocardium/metabolism , Myocytes, Smooth Muscle/metabolism , Myocytes, Smooth Muscle/pathology , Streptozocin , Ventricular Dysfunction, Left/metabolism , Ventricular Dysfunction, Left/pathology , Ventricular Dysfunction, Left/physiopathologyABSTRACT
Single-cell transcriptomics enables inference of context-dependent phenotypes of individual cells and determination of cellular diversity of complex tissues. Cardiac fibrosis is a leading factor in the development of heart failure and a major cause of morbidity and mortality worldwide with no effective treatment. Single-cell RNA-sequencing (scRNA-seq) offers a promising new platform to identify new cellular and molecular protagonists that may drive cardiac fibrosis and development of heart failure. This review will summarize the application scRNA-seq for understanding cardiac fibrosis and development of heart failure. We will also discuss some key considerations in interpreting scRNA-seq data and some of its limitations.
