Transforming growth factor-beta 1 (TGF-β1) induces the differentiation of rainbow trout (Oncorhynchus mykiss) cardiac fibroblasts into myofibroblasts

Abstract

The collagen content of the rainbow trout heart increases in response to cold acclimation and decreases with acclimation to warm temperatures. This ability to remodel the myocardial extracellular matrix (ECM) makes these fish useful models to study the cellular pathways involved in collagen regulation in the vertebrate heart. Remodelling of the ECM in the mammalian heart is regulated, in part, by myofibroblasts which arise from pre-existing fibroblasts in response to transforming growth factor-β1 (TGF-β1). We have previously demonstrated that treatment of cultured rainbow trout cardiac fibroblasts with human TGF-β1 causes an increase in collagen production. Here, we showed that repetitive treatment of rainbow trout cardiac fibroblasts with a physiologically relevant concentration of human recombinant TGF-β1 results in a ∼29-fold increase in phosphorylated small mothers against decapentaplegic 2 ( pSmad2); a 2.9-fold increase in vinculin protein, a 1.2-fold increase in cellular size and a 3-fold increase in filamentous actin (F-actin). These are common markers of the transition of fibroblasts to myofibroblasts. Cells treated with TGF-β1 also had highly organized cytoskeletal α-smooth muscle actin, as well as increased transcript abundances of mmp-9, timp-2 and col1a1. Furthermore, using gelatin zymography, we demonstrated that TGF-β1 treatment causes a 5.3fold increase in gelatinase activity. Together, these results suggest that trout cardiac fibroblasts have the capacity to differentiate into myofibroblasts and that this cell type can increase extracellular collagen turnover via gelatinase activity. Cardiac myofibroblasts are, therefore, likely involved in the remodelling of the cardiac ECM in the trout heart during thermal acclimation.

Proteasome inhibitor MG132 inhibits the process of renal interstitial fibrosis

“…Col III expression was significantly increased 24 h after TGF-β1 treatment and significantly decreased 6, 12 and 24 h after MG132 and TGF-β1 treatment compared with the control group (all P<0.05). The results indicated that 2.5 µM MG132 decreased the mRNA levels of fibrosis-associated factors following stimulation with 5 ng/ml TGF-β1, which causes the fibroblasts to differentiate into myofibroblasts associated with fibrotic processes ( 21 ).…”

Mitogen-activated protein kinases contribute to temperature induced cardiac remodelling in rainbow trout (Oncorhynchus mykiss) heart

“…The transcript levels of the target genes, col1a3, mmp-2, mmp-9, mmp-13 , were measured at multiple time points to establish a timeline of the cellular response during remodelling with respect to the signalling protein kinases and acclimation time. The primers used were those previously described by Johnston and Gillis (2018). ef1α and β-actin were determined to be the most suitable reference genes using the program GeNorm (Vandesompele et al, 2002).…”

“…The purpose of the present study was to determine if thermal acclimation of rainbow trout activates the MAPK signalling pathway, which is hypothesized to be responsible for initiating temperature induced cardiac remodelling. Building from our in vitro studies (Johnston and Gillis, 2020; Johnston et al 2019; Johnston and Gillis 2018; Johnston and Gillis, 2017), we specifically hypothesized that cold acclimation (4 °C) would cause an increase in the phosphorylation status of p38 MAPK and ERK, and induce changes in the expression of gene transcripts that would support an increase in collagen deposition. It was also hypothesized that warm acclimation (18 °C) would have the opposite effect.…”

microRNA-29b knocks down collagen type I production in cultured rainbow trout (Oncorhynchus mykiss) cardiac fibroblasts

“…Average normalized collagen protein levels between control, nonsense siRNA-and miR-29b-transfected groups were analysed with a one-way ANOVA at a confidence interval of 95%. For each treatment, n=3, where n represents a cell culture established from the same ventricle of a male rainbow trout, as previously described (Johnston and Gillis, 2018).…”