Regulation of collagen deposition in the trout heart during thermal acclimation.

The passive mechanical properties of the vertebrate heart are controlled in part by the composition of the extracellular matrix (ECM). Changes in the ECM, caused by increased blood pressure, injury or disease can affect the capacity of the heart to fill with blood during diastole. In mammalian species, cardiac fibrosis caused by an increase in collagen in the ECM, leads to a loss of heart function and these changes in composition are considered to be permanent. Recent work has demonstrated that the cardiac ventricle of some fish species have the capacity to both increase and decrease collagen content in response to thermal acclimation. It is thought that these changes in collagen content help maintain ventricle function over seasonal changes in environmental temperatures. This current work reviews the cellular mechanisms responsible for regulating collagen deposition in the mammalian heart and proposes a cellular pathway by which a change in temperature can affect the collagen content of the fish ventricle through mechanotransduction. This work specifically focuses on the role of transforming growth factor ß1, MAPK signaling pathways, and biomechanical stretch in regulating collagen content in the fish ventricle. It is hoped that this work increases the appreciation of the use of comparative models to gain insight into phenomenon with biomedical relevance.

Short-term cyclical stretch phosphorylates p38 and ERK1/2 MAPKs in cultured fibroblasts from the hearts of rainbow trout, Oncorhynchus mykiss.

The form and function of the rainbow trout heart can remodel in response to various stressors including changes in environmental temperature and anemia. Previous studies have hypothesized that changes in biomechanical forces experienced by the trout myocardium as result of such physiological stressors could play a role in triggering the remodeling response. However, there has been no work examining the influence of biomechanical forces on the trout myocardium or of the cellular signals that would translate such a stimuli into a biological response. In this study, we test the hypothesis that the application of biomechanical forces to trout cardiac fibroblasts activate the cell signaling pathways associated with cardiac remodeling. This was done by cyclically stretching cardiac fibroblasts to 10% equibiaxial deformation at 0.33 Hz and quantifying the activation of the p38-JNK-ERK mitogen activated protein kinase (MAPK) pathway. After 20 min, p38 MAPK phosphorylation was elevated by 4.2-fold compared to control cells (P<0.05) and after 24 h of stretch, p38 MAPK phosphorylation remained elevated and extracellular-regulated kinase 1/2 was phosphorylated by 2.4-fold compared to control (P<0.05). Together, these results indicate that mechanotransductive pathways are active in cardiac fibroblasts, and lead to the activation of cell signaling pathways involved in cardiac remodeling.

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

Warm acclimation of rainbow trout can cause a decrease in the collagen content of the heart. This ability to remove cardiac collagen is particularly interesting considering that collagen deposition in the mammalian heart, following an injury, is permanent. We hypothesized that collagen removal can be facilitated by microRNA-29b (miR-29b), a highly conserved, small, non-coding RNA, as a reduction in this microRNA has been reported during the development of fibrosis in the mammalian heart. We also used a bioinformatics approach to investigate the binding potential of miR-29b to the seed sequences of vertebrate collagen isoforms. Cultured trout cardiac fibroblasts were transfected with zebrafish mature miR-29b mimic for 7 days with re-transfection occurring after 3 days. Transfection induced a 17.8-fold increase in miR-29b transcript abundance (P<0.05) as well as a 54% decrease in the transcript levels of the col1a3 collagen isoform, compared with non-transfected controls (P<0.05). Western blotting demonstrated that the level of collagen type I protein was 85% lower in cells transfected with miR-29b than in control cells (P<0.05). Finally, bioinformatic analysis suggested that the predicted 3'-UTR of rainbow trout col1a3 has a comparatively higher binding affinity for miR-29b than the 3'-UTR of col1a1 Together, these results suggest that miR-29b is a highly conserved regulator of collagen type I protein in vertebrates and that this microRNA decreases collagen in the trout heart by targeting col1a3.

Transforming growth factor-ß1 induces differentiation of rainbow trout (Oncorhynchus mykiss) cardiac fibroblasts into myofibroblasts.

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-ß1 (TGF-ß1). We have previously demonstrated that treatment of cultured rainbow trout cardiac fibroblasts with human TGF-ß1 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-ß1 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-ß1 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-ß1 treatment causes a 5.3-fold 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.

Transforming growth factor beta-1 (TGF-ß1) stimulates collagen synthesis in cultured rainbow trout cardiac fibroblasts.

Cold acclimation of rainbow trout, Oncorhynchus mykiss, causes collagen to increase within the extracellular matrix (ECM) of the myocardium, while warm acclimation has the opposite effect. The mechanism responsible for this remodelling response is not known. In mammals, transforming growth factor beta-1 (TGF-ß1) stimulates collagen deposition within the myocardial ECM. Therefore, we hypothesized that TGF-ß1 regulates trout myocardial ECM turnover and predicted that TGF-ß1 would induce collagen deposition in cultured rainbow trout cardiac fibroblasts. We found that treatment of trout cardiac fibroblasts with 15 ng ml-1 human recombinant TGF-ß1 caused an increase in total collagen at 48 and 72 h and an increase in collagen type I protein after 7 days. We also found that TGF-ß1 treatment caused an increase in the transcript abundance of tissue inhibitor of metalloproteinase 2 (timp-2) and matrix metalloproteinase 9 (mmp-9) at 24 h. Cells treated with TGF-ß1 also had lower levels of the gene transcript for mmp-2 after 48 h and higher levels of the gene transcript for collagen type I α1 (col1a1) after 72 h. These changes in gene expression suggest that the increase in collagen deposition is due to a decrease in the activity of matrix metalloproteinases and an increase in collagen synthesis. Together, these results indicate that TGF-ß1 is a regulator of ECM composition in cultured trout cardiac fibroblasts and suggest that this cytokine may play a role in regulating collagen content in the trout heart during thermal acclimation.

Cold acclimation alters the connective tissue content of the zebrafish (Danio rerio) heart.

Thermal acclimation can alter cardiac function and morphology in a number of fish species, but little is known about the regulation of these changes. The purpose of the present study was to determine how cold acclimation affects zebrafish (Danio rerio) cardiac morphology, collagen composition and connective tissue regulation. Heart volume, the thickness of the compact myocardium, collagen content and collagen fiber composition were compared between control (27°C) and cold-acclimated (20°C) zebrafish using serially sectioned hearts stained with Picrosirius Red. Collagen content and fiber composition of the pericardial membrane were also examined. Cold acclimation did not affect the volume of the contracted heart; however, there was a significant decrease in the thickness of the compact myocardium. There was also a decrease in the collagen content of the compact myocardium and in the amount of thick collagen fibers throughout the heart. Cold-acclimated zebrafish also increased expression of the gene transcript for matrix metalloproteinase 2, matrix metalloproteinase 9, tissue inhibitor of metalloproteinase 2 and collagen Type I α1. We propose that the reduction in the thickness of the compact myocardium as well as the change in collagen content may help to maintain the compliance of the ventricle as temperatures decrease. Together, these results clearly demonstrate that the zebrafish heart undergoes significant remodeling in response to cold acclimation.

Chronic hypoxia exposure of trout embryos alters swimming performance and cardiac gene expression in larvae.

Hypoxia exposure during embryonic development of rainbow trout causes developmental delay and bradycardia and alters the ontogeny of cardiac regulatory control mechanisms. The purpose of this study was to characterize how hypoxia exposure from the day of fertilization until stage 34 (57 d postfertilization) affects the aerobic fitness and growth of the hatched fish at multiple stages. In addition, we characterized the expression of gene transcripts for seven troponin I (TnI) isoforms to examine the effect of hypoxia treatment on cardiac muscle development. Results demonstrate that the critical swimming speed of the hypoxia-exposed fish was significantly less than that of the control group at stage 35 and the fry stage. Growth was reduced in the hypoxia-treated fish between stages 35 and 37, as was the relative lipid content at stage 37. Finally, six TnI isoforms were found in all hearts. One of these isoforms, RTcTnI, decreased in abundance between stage 35 and the fry stage, but hypoxia-exposed fish had higher levels than did controls at the fry stage. The abundance of AScTnI2 was significantly lower in hypoxia-exposed fry fish than in controls. These results indicate that chronic hypoxia exposure during embryonic development has long-term consequences on aerobic fitness, growth, and cardiac gene expression following hatch.