Project description:Single-cell RNA-seq (10X Genomics Chromium) to profile Adult cardiac interstitial cells sorted based on the presence of cKit, DDR2 and CD45 on cell membrane surface .
Project description:Two types of monocytes, inflammatory and patrolling, infiltrate the hearts in both human myocarditis and murine experimental autoimmune myocarditis (EAM) model. The fates and functions of these infiltrating monocytes governing the progression of heart failure remain unclear. Here, we created parabiotic EAM and naïve mice to show that cardiac inflammation facilitate monocyte-to-macrophage differentiation. Using a combination of flow cytometry, time lapsed imaging and transmission electron microscopy, we demonstrated in vitro that cardiac fibroblasts interact with monocytes and are instrumental in facilitating monocyte-to-macrophage differentiation. Moreover, IL-17A stimulated cardiac fibroblasts completely arrested Ly6Clo monocyte proliferation and inhibited both Ly6Chi and Ly6Clo monocyte-to-macrophage differentiation both in vitro and in vivo after intracardiac injections of monocytes into the hearts. Intriguingly, IL-17A signaling through cardiac fibroblasts also significantly downregulated Mer tyrosine kinase (MerTK) expressions on Ly6Chi monocyte-derived macrophages, thus jeopardizing their phagocytic abilities. Collectively, our results implicate divergent fates and functions of heart-infiltrating monocytes influenced by cardiac fibroblasts.
Project description:The overall data contains expression profile of rat genes of cardiac fibroblasts. G-protein signaling is regulated in cardiac fibroblasts. The expresssion of angiotensin II receptor or cytokine such as interleukin is regulated by PTX (which is suggested to suppress Gi function) treatment. Keywords: control or PTX treatment Rat cardiac fibroblasts were primarily isolated and plated onto 6 well plate. Cells were treated with water (Ctl 1-3) or PTX for 24 hr (PTX 1-3) and lysed by Buffer RLT. Total RNA was isolated using QIAGEN kit and gene expression was analysed using Affymetrix Gene Chip system and GCOS analysis.
Project description:Cardiac fibroblast (CF) population heterogeneity and plasticity present a challenge for categorization of biological and functional properties. Distinct molecular markers and associated signaling pathways provide valuable insight for CF biology and interventional strategies to influence injury response and aging-associated remodeling. Receptor tyrosine kinase c-Kit mediates cell survival, proliferation, migration, and is activated by pathological injury. However, the biological significance of c-Kit within CF population has not been addressed. An inducible reporter mouse detects c-Kit promoter activation with Enhanced Green Fluorescent Protein (EGFP) expression in cardiac cells. Coincidence of EGFP and c-Kit with the DDR2 fibroblast marker was confirmed using flow cytometry and immunohistochemistry. Subsequently, CFs expressing DDR2 with or without c-Kit was isolated and characterized. A subset of DDR2+ CFs also express c-Kit with coincidence in ~ 8% of total cardiac interstitial cells (CICs). Aging is associated with decreased number of c-Kit expressing DDR2+ CFs, whereas pathological injury induces c-Kit and DDR2 as well as the frequency of coincident expression in CICs. scRNA-Seq profiling reveals the transcriptome of c-Kit expressing CFs as cells with transitional phenotype. Cultured cardiac DDR2+ fibroblasts that are c-Kit+ exhibit morphological and functional characteristics consistent with youthful phenotypes compared to c-Kit- cells. Mechanistically, c-Kit expression correlates with signaling implicated in proliferation and cell migration, including phospho-ERK and pro-caspase 3. The phenotype of c-kit+ on DDR2+ CFs correlates with multiple characteristics of 'youthful' cells. To our knowledge, this represents the first evaluation of c-Kit biology within DDR2+ CF population and provides a fundamental basis for future studies to influence myocardial biology, response to pathological injury and physiological aging.
Project description:Duchenne muscular dystrophy (DMD) is a severe form of muscular dystrophy caused by mutations in the dystrophin gene. We characterized which isoforms of dystrophin were expressed by human induced pluripotent stem cell (hiPSC)-derived cardiac fibroblasts obtained from control and DMD patients. Distinct dystrophin isoforms were observed; however, the highest molecular weight isoform was absent in DMD patients carrying exon deletions or mutations in the dystrophin gene. The loss of the full-length dystrophin isoform in hiPSC-derived cardiac fibroblasts from DMD patients resulted in deficient formation of actin microfilaments and a metabolic switch from mitochondrial oxidation to glycolysis. The DMD hiPSC-derived cardiac fibroblasts exhibited a dysregulated mitochondria network and reduced mitochondrial respiration, with enhanced compensatory glycolysis to sustain cellular ATP production. This metabolic remodeling was associated with an exacerbated myofibroblast phenotype and increased fibroblast activation in response to pro fibrotic challenges. As cardiac fibrosis is a critical pathological feature of the DMD heart, the myofibroblast phenotype induced by the absence of dystophin may contribute to deterioration in cardiac function. Our study highlights therelationship between cytoskeletal dynamics, metabolism of the cell and myofibroblast differentiation and provides a new mechanism by which inactivation of dystrophin in non-cardiomyocyte cells may increase the severity of cardiopathy.
Project description:Reprogramming of mouse fibroblasts toward a myocardial cell fate by forced expression of cardiac transcription factors or microRNAs has recently been demonstrated. The potential clinical applicability of these findings is based on the minimal regenerative potential of the adult human heart and the limited availability of human heart tissue. An initial, but mandatory step toward clinical application of this approach is to establish conditions for conversion of adult human fibroblasts to a cardiac phenotype. Toward this goal, we sought to determine the optimal combination of factors necessary and sufficient for direct myocardial reprogramming of human fibroblasts. Here we show that four human cardiac transcription factors, including Gata4, Hand2, Tbx5, and myocardin, and two microRNAs, miR-1 and miR-133, activated cardiac marker expression in neonatal and adult human fibroblasts. After maintenance in culture for 4-11 weeks, human fibroblasts reprogrammed with these proteins and microRNAs displayed sarcomere-like structures and calcium transients, and a small subset of such cells exhibited spontaneous contractility. These phenotypic changes were accompanied by expression of a broad range of cardiac genes and suppression of non-myocyte genes. These findings indicate that human fibroblasts can be reprogrammed to cardiac-like myocytes by forced expression of cardiac transcription factors with muscle-specific microRNAs and represent a step toward possible therapeutic application of this reprogramming approach. Human foreskin fibroblasts were transduced with 5 transcription factors and total RNA was obtained 4 weeks later. total RNA was also obtained from human foreskin fibroblasts as a negative control and adult human heart tissue as a positive control. The expression level of genes in each sample was compared.
Project description:The developmental origin of the c-kit expressing progenitor cell pool in the adult heart has remained elusive. Recently, it has been discovered that the injured heart is enriched with c-kit+ cells, which also express the hematopoietic marker CD45. In this study, we characterize the phenotype and transcriptome of the c-kit+/CD45+ cell population, originating from the left atrial appendage. These cells are defined as cardiac macrophage progenitors. We also demonstrate that the c-kit+/CD45+ progenitor cell population activates heart development, neural crest and pluripotency associated pathways in vitro, in conjunction with CD45 down-regulation, and acquire a c-kit+/lin- phenotype. This spontaneous reprogramming progresses further to a highly proliferative, partially myogenic phenotype. Our data suggests that c-kit+/lin- cells and cardiac macrophages have a common lineage origin possibly resolving some current conundrums in the field of cardiac regeneration.
Project description:The developmental origin of the c-kit expressing progenitor cell pool in the adult heart has remained elusive. Recently, it has been discovered that the injured heart is enriched with c-kit+ cells, which also express the hematopoietic marker CD45. In this study, we characterize the phenotype and transcriptome of the c-kit+/CD45+ cell population, originating from the left atrial appendage. These cells are defined as cardiac macrophage progenitors. We also demonstrate that the c-kit+/CD45+ progenitor cell population activates heart development, neural crest and pluripotency associated pathways in vitro, in conjunction with CD45 down-regulation, and acquire a c-kit+/lin- phenotype. This spontaneous reprogramming progresses further to a highly proliferative, partially myogenic phenotype. Our data suggests that c-kit+/lin- cells and cardiac macrophages have a common lineage origin possibly resolving some current conundrums in the field of cardiac regeneration.