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
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
[Figure: see text].
Subject(s)
Blood Pressure/genetics , Kidney/metabolism , MicroRNAs/genetics , Transcriptome , Animals , Blood Pressure/physiology , Fibrosis/genetics , Gene Expression Profiling/methods , Gene Expression Regulation , Gene Ontology , Humans , Hypertension/genetics , Hypertension/physiopathology , Inflammation/genetics , Kidney/pathology , Kidney/physiopathology , Mice, Inbred C57BL , Mice, Knockout , MicroRNAs/metabolism , RNA-Seq/methods , Renin/genetics , Renin/metabolismABSTRACT
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
BACKGROUND: Cardiac fibrosis is a key antecedent to many types of cardiac dysfunction including heart failure. Physiological factors leading to cardiac fibrosis have been recognized for decades. However, the specific cellular and molecular mediators that drive cardiac fibrosis, and the relative effect of disparate cell populations on cardiac fibrosis, remain unclear. METHODS: We developed a novel cardiac single-cell transcriptomic strategy to characterize the cardiac cellulome, the network of cells that forms the heart. This method was used to profile the cardiac cellular ecosystem in response to 2 weeks of continuous administration of angiotensin II, a profibrotic stimulus that drives pathological cardiac remodeling. RESULTS: Our analysis provides a comprehensive map of the cardiac cellular landscape uncovering multiple cell populations that contribute to pathological remodeling of the extracellular matrix of the heart. Two phenotypically distinct fibroblast populations, Fibroblast-Cilp and Fibroblast-Thbs4, emerged after induction of tissue stress to promote fibrosis in the absence of smooth muscle actin-expressing myofibroblasts, a key profibrotic cell population. After angiotensin II treatment, Fibroblast-Cilp develops as the most abundant fibroblast subpopulation and the predominant fibrogenic cell type. Mapping intercellular communication networks within the heart, we identified key intercellular trophic relationships and shifts in cellular communication after angiotensin II treatment that promote the development of a profibrotic cellular microenvironment. Furthermore, the cellular responses to angiotensin II and the relative abundance of fibrogenic cells were sexually dimorphic. CONCLUSIONS: These results offer a valuable resource for exploring the cardiac cellular landscape in health and after chronic cardiovascular stress. These data provide insights into the cellular and molecular mechanisms that promote pathological remodeling of the mammalian heart, highlighting early transcriptional changes that precede chronic cardiac fibrosis.
