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Mechanical regulation of cardiac fibroblast profibrotic phenotypes.

Molecular biology of the cell (2017-05-05)
Kate M Herum, Jonas Choppe, Aditya Kumar, Adam J Engler, Andrew D McCulloch
ABSTRACT

Cardiac fibrosis is a serious condition currently lacking effective treatments. It occurs as a result of cardiac fibroblast (CFB) activation and differentiation into myofibroblasts, characterized by proliferation, extracellular matrix (ECM) production and stiffening, and contraction due to the expression of smooth muscle α-actin. The mechanical properties of myocardium change regionally and over time after myocardial infarction (MI). Although mechanical cues are known to activate CFBs, it is unclear which specific mechanical stimuli regulate which specific phenotypic trait; thus we investigated these relationships using three in vitro models of CFB mechanical activation and found that 1) paracrine signaling from stretched cardiomyocytes induces CFB proliferation under mechanical conditions similar to those of the infarct border region; 2) direct stretch of CFBs mimicking the mechanical environment of the infarct region induces a synthetic phenotype with elevated ECM production; and 3) progressive matrix stiffening, modeling the mechanical effects of infarct scar maturation, causes smooth muscle α-actin fiber formation, up-regulation of collagen I, and down-regulation of collagen III. These findings suggest that myocyte stretch, fibroblast stretch, and matrix stiffening following MI may separately regulate different profibrotic traits of activated CFBs.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
Monoclonal Anti-Vinculin antibody produced in mouse, clone hVIN-1, ascites fluid
Sigma-Aldrich
GW2580, ≥98% (HPLC)
Sigma-Aldrich
Goat Serum Donor Herd, USA origin, sterile-filtered, suitable for cell culture
Sigma-Aldrich
Laminin from Engelbreth-Holm-Swarm murine sarcoma basement membrane, 1-2 mg/mL in Tris-buffered saline, 0.2 μm filtered, BioReagent, suitable for cell culture
Sigma-Aldrich
N-(3-Dimethylaminopropyl)-N′-ethylcarbodiimide, ≥97.0% (T)
Sigma-Aldrich
Anti-Actin, α-Smooth Muscle antibody, Mouse monoclonal, clone 1A4, purified from hybridoma cell culture