Comparison of infarct-derived and control ovine cardiac myofibroblasts in culture: response to cytokines and natriuretic peptide receptor expression profiles

Abstract

This study investigated whether gene expression profiles of myofibroblasts derived from infarcted myocardium differ from normal cardiac fibroblasts. We compared the expression of cytoskeletal proteins in cultured ovine cardiac fibroblasts derived from infarcted (ID) and noninfarcted ovine myocardium (NID) and the levels of expression of the natriuretic peptide receptors (NPR)-A and NPR-B in response to treatment with transforming growth factor (TGF)-beta1 and/or platelet-derived growth factor (PDGF). Transformation of cultured cardiac fibroblasts to myofibroblasts, as indicated by alpha-smooth muscle actin and vimentin expression, was independent of the presence of TGF-beta1, PDGF, or cell origin. ID fibroblasts had higher basal levels than NID fibroblasts of NPR-A (ID: 58.0 +/- 32.2 arbitrary density units, NID: undetectable), NPR-B (ID: 780 +/- 155, NID: 330 +/- 38 arbitrary density units) and collagen I (ID: 17.2 +/- 0.5, NID: 10.5 +/- 1.7 pg mRNA/mug total RNA, P < 0.05) but lower levels of alpha-SMa expression (ID: 50.2 +/- 7.9, NID: 76.9 +/- 3.2 fluorescence units, P < 0.05). NPR-A mRNA in ID fibroblasts showed a rapid fourfold increase in response to TGF-beta1 and/or PDGF at 4 and 2 h, respectively, followed by a profound decline; in NID cells, NPR-A mRNA was undetectable. In ID fibroblasts, cytokines reduced NPR-B mRNA below control levels; in NID fibroblasts, TGF-beta1 and PDGF elicited prompt increments in expression: a fourfold increase with TGF-beta1 at 8 h and a twofold increase with PDGF at 4 h (P < 0.05). In summary, transformation of cardiac fibroblasts to myofibroblasts in culture is independent of cytokine treatment. Moreover, whether the cultured cardiac fibroblasts are from infarct tissue is a major determinant of NPR expression levels and cytokine responses, even after four to five passages.

Remodeling of cardiac fibroblasts following myocardial infarction results in increased gap junction intercellular communication

“…Fibroblasts are arguably the most important component of the “living scar” responsible for myocardial repair, infarct healing and scar formation [5–8]. Several studies have shown that specific phenotypic alterations in post-MI or failing fibroblasts are maintained in vitro through several passages [9–11]; thus, post-MI remodeled fibroblasts may be grown in culture to quantify alterations in intercellular communication and to study mechanisms underlying those alterations.…”

The Cardiac Fibroblast: Functional and Electrophysiological Considerations in Healthy and Diseased Hearts

“…The same study also showed an increase of platelet derived growth factor (PDGF) levels in response to tachypacing in fibroblasts isolated from atria but not ventricles. Similarly, another study by Jarvis et al20 using fibroblasts obtained from infarcted and non-infarcted ovine myocardium showed that the characteristics of cardiac fibroblasts in culture varied depending on the source. Their findings demonstrated lower levels of α-SMA, increased collagen I, higher expression levels of natriuretic peptide receptors (NPR-A and NPR-B) and differences in the response to TGF-β1 and PDGF treatment between cells obtained from infarcted and non-infarcted hearts.…”

“…Fibroblasts are phenotypically heterogeneous depending on developmental stage, organ and physiological conditions 14–20. In the heart, atrial and ventricular fibroblasts have different cell densities in intact tissue 17.…”

“…Recent studies have highlighted important differences between fibroblasts isolated from normal and fibrotic hearts maintained under similar culture conditions. Figure 1 summarizes the functional consequences of cardiac fibroblasts activation which include altered proliferation,17, 18, 35 migration,18, 19 release of signaling molecules,36 ECM deposition,18–20 adhesion,18, 19 and receptor expression 20. Importantly, fibroblast activation has recently been associated with significant electrophysiological changes 36, 37…”

“…Their findings demonstrated lower levels of α-SMA, increased collagen I, higher expression levels of natriuretic peptide receptors (NPR-A and NPR-B) and differences in the response to TGF-β1 and PDGF treatment between cells obtained from infarcted and non-infarcted hearts. In addition, several of these studies demonstrated that phenotypic differences between fibroblasts isolated from normal and diseased hearts were maintained in culture for several passages 18–20. These data suggest that the activation process due to culture conditions does not fully replicate the in vivo activation process.…”