Cardiac fibroblasts influence cardiomyocyte phenotype in vitro

Abstract

Cardiac fibroblasts impact myocardial development and remodeling through intercellular contact with cardiomyocytes, but less is known about noncontact, profibrotic signals whereby fibroblasts alter cardiomyocyte behavior. Fibroblasts and cardiomyocytes were harvested from newborn rat ventricles and separated by serial digestion and gradient centrifugation. Cardiomyocytes were cultured in 1) standard medium, 2) standard medium diluted 1:1 with PBS, or 3) standard medium diluted 1:1 with medium conditioned ≥72 h by cardiac fibroblasts. Serum concentrations were held constant under all media conditions, and complete medium exchanges were performed daily. Cardiomyocytes began contracting within 24 h at clonal or mass densities with <5% of cells expressing vimentin. Immunocytochemical analysis revealed progressive expression of α-smooth muscle actin in cardiomyocytes after 24 h in all conditions. Only cardiomyocytes in fibroblast-conditioned medium stopped contracting by 72 h. There was a significant, sustained increase in vimentin expression specific to these cultures (means ± SD: conditioned 46.3 ± 6.0 vs. control 5.3 ± 2.9%, P < 0.00025) typically with cardiac myosin heavy chain coexpression. Proteomics assays revealed 10 cytokines (VEGF, GRO/KC, monocyte chemoattractant protein-1, leptin, macrophage inflammatory protein-1α, IL-6, IL-10, IL-12p70, IL-17, and tumor necrosis factor-α) at or below detection levels in unconditioned medium that were significantly elevated in fibroblast-conditioned medium. Latent transforming growth factor-β and RANTES were present in unconditioned medium but rose to higher levels in conditioned medium. Only granulocyte-macrophage colony-stimulating factor was present above threshold levels in standard medium but decreased with fibroblast conditioning. These data indicated that under the influence of fibroblast-conditioned medium, cardiomyocytes exhibited marked hypertrophy, diminished contractile capacity, and phenotype plasticity distinct from the dedifferentiation program present under standard culture conditions.

Neonatal rat cardiac fibroblasts express three types of voltage-gated K+ channels: regulation of a transient outward current by protein kinase C

“…For example, coculture of fibroblasts with myocytes dramatically enhances the expression of cytokines such as IL-6, IL-10, and tumor necrosis factor-␣, compared with cells cultured separately (2,15). These factors in turn alter the structural and contractile properties of the myocytes (2,15). Establishment of electrical contact between adjacent fibroblasts and myocytes may be of critical importance in the release of these cytokines and growth factors.…”

“…Although cardiac myocytes make up the bulk of the myocardial volume, the cardiac fibroblast is the most numerous cell type found in the heart and functions in the deposition of the extracellular matrix (2, 3). In addition, fibroblasts can serve as "sentinel cells" that coordinate the myocardial response to chemical and mechanical stimulation through the release of cytokines and growth factors (2,3,15). Release of these signals during myocardial injury may regulate the extent of cardiac hypertrophy and fibrosis during the healing process (5, 15).…”

The use of a cytokine panel to define the long-term risk stratification of heart failure/death in patients presenting with chest pain to the emergency department

“…Both IL-10 and VEGF have been reported to have a role in the development of ACS and in subsequent health outcomes [4,5]. In vitro data support the role of these proteins in influencing cardio-myocyte phenotype [6]. Furthermore, our previous data have demonstrated that both IL-6 and MCP-1 are useful for risk stratification for HF/death [2].…”

“…However, more specific and earlier mediators of the inflammatory process (e.g., IL-6, MCP-1) may provide better or additional independent information [2]. In addition to IL-6 and MCP-1 there are other cytokines that may be measured that reflect or are indices of inflammation, cardiac fibrosis and cardiomyocyte function that have not been evaluated in this context (e.g., IL-10, VEGF, EGF) [3][4][5][6]. For the present study the objective was to test the hypothesis that a combination of these biomarkers (i.e., IL-6, MCP-1, IL-10, VEGF, and EGF as a panel test) could be used to identify those at high risk for future HF/death.…”

Effects of interleukin-1 on cardiac fibroblast function: Relevance to post-myocardial infarction remodelling

“…CCL2/MCP-1) and CXC chemokines (e.g. CXCL1/GRO/KC and CXCL8/IL-8,) in a feed-forward loop driven by the inflammatory milieu, thereby exacerbating the initial inflammatory response (Turner et al, 2007;Turner et al, 2011;Demyanets et al, 2013;LaFramboise et al, 2007). Although further studies are required to determine the precise relative contribution of CF in this early inflammatory stage of remodelling compared with other cell types (mast cells, neutrophils, monocyte/macrophages etc), the available evidence to date suggests that CF play an important role .…”