Background: Infiltration with inflammatory CD4+ T-cells and the accumulation of heterogeneous cardiac myofibroblasts are hallmarks of cardiac fibrosis and remodeling. The origin, identity, states, and functions of the resident cells involved in the transition from adaptive to maladaptive fibrotic remodeling, as well as the pathways of inflammatory regulation are unclear.
Methods: We performed mass cytometry profiling of resident human ventricular cardiac fibroblasts (hVCF) and determined the identity of cells contained in fibrotic right ventricle autopsy tissues from individuals diagnosed with pulmonary hypertension and tissue from SUGEN/hypoxia rats exhibiting cardiac fibrosis. We further characterized the resident cardiac fibroblast sub-population morphologically, structurally and functionally using transcriptome and secretome analysis of the secreted cytokines, chemokines, proteins, metabolites using milliplex panels, proteomics and metabolomics pipelines.
Results: Single-cell mass cytometry identified remarkable plasticity of resident human cardiac fibroblasts. We provide evidence of a sub-population of resident cardiac myofibroblasts expressing high levels of CD4+, a helper T-cell surface marker in addition to mesenchymal markers, αSMA and Vimentin in all the human donors. These cardiac cells co-expressing lymphoid CD4+and αSMA+ were localized to the fibrotic regions of the human right ventricular tissue and were a common feature in the interstitial and perivascular lesions of SUGEN/Hypoxia (SuHx) rats. CD3+CD4+ T-cell numbers were higher in the right ventricle compared with the left ventricle of SuHx, as determined by flow cytometry. In vitro, T-cell homing receptors CD44, Interleukin-1 receptor (IL-1R), and CCR2 were upregulated in cardiac fibroblasts in response to IL-1β. Exposure of cardiac fibroblasts to IL-1β led to upregulation of genes regulating extracellular matrix, collagen deposition and inflammation-related genes, and induced secretion of cytokines, chemokines, and metabolites involved in innate and adaptive humoral immune responses. Cell clustering, elevated phosphorylation of MAPK p38 and inflammatory NF-κB p65 and cell phenotype switching upon IL-1β stimulation reverted with the administration of an IL-1R antagonist.
Conclusions: Our data expand concepts of heterogeneity of resident cardiac fibroblasts and plasticity in response to pro-inflammatory cytokines by the demonstration of a unique subpopulation of cardiac fibroblasts exhibiting attributes of both mesenchymal and lymphoid cells. Exposure of cardiac fibroblasts to the pro-inflammatory cytokine, IL-1β, induces a robust phenotypic response linked to extracellular matrix deposition and up-regulates an immune-associated phenotype linked to expression of immune markers and secretion of immunomodulatory cytokines and chemokines. We also propose that resident cardiac fibroblast transdifferentiation and phenotype switching maybe the key process involved in adaptive to maladaptive remodeling leading to fibrosis and failure.
Non-standard abbreviations: CD4; Cluster of differentiation, αSMA; alpha smooth muscle actin, IL-1R; Interleukin-1-receptor, CCR2; C-X-C Motif Chemokine Receptor 2
Cardiac Fibroblasts and the Extracellular Matrix in Regenerative and Nonregenerative Hearts
