Abstract
Mechanical cues activate cardiac fibroblasts and induce differentiation into myofibroblasts, which are key steps for development of cardiac fibrosis. In vitro, the high stiffness of plastic culturing conditions will also induce these changes. It is therefore challenging to study resting cardiac fibroblasts and their activation in vitro. Here we investigate the extent to which disrupting mechanotransduction by culturing cardiac fibroblasts on soft hydrogels or in the presence of biochemical inhibitors can be used to maintain resting cardiac fibroblasts in vitro. Primary cardiac fibroblasts were isolated from adult mice and cultured on plastic or soft (4.5 kPa) polyacrylamide hydrogels. Myofibroblast marker gene expression and smooth muscle α-actin (SMA) fibers were quantified by real-time PCR and immunostaining, respectively. Myofibroblast differentiation was prevented on soft hydrogels for 9 days, but had occurred after 15 days on hydrogels. Transferring myofibroblasts to soft hydrogels reduced expression of myofibroblast-associated genes, albeit SMA fibers remained present. Inhibitors of transforming growth factor β receptor I (TGFβRI) and Rho-associated protein kinase (ROCK) were effective in preventing and reversing myofibroblast gene expression. SMA fibers were also reduced by blocker treatment although cell morphology did not change. Reversed cardiac fibroblasts maintained the ability to re-differentiate after the removal of blockers, suggesting that these are functionally similar to resting cardiac fibroblasts. However, actin alpha 2 smooth muscle (Acta2), lysyl oxidase (Lox) and periostin (Postn) were no longer sensitive to substrate stiffness, suggesting that transient treatment with mechanotransduction inhibitors changes the mechanosensitivity of some fibrosis-related genes. In summary, our results bring novel insight regarding the relative importance of specific mechanical signaling pathways in regulating different myofibroblast-associated genes. Furthermore, combining blocker treatment with the use of soft hydrogels has not been tested previously and revealed that only some genes remain mechano-sensitive after phenotypic reversion. This is important information for researchers using inhibitors to maintain a "resting" cardiac fibroblast phenotype in vitro as well as for our current understanding of mechanosensitive gene regulation.
Highlights
Activation of cardiac fibroblasts is a key step in development of cardiac fibrosis
We observed a rapid increase in the classical myofibroblast marker actin alpha 2 smooth muscle (Acta2) mRNA (Fig 1A) and protein (Fig 1B) in response to plastic culture conditions, resulting in the formation of clear smooth muscle α-actin (SMA) fibers in the cytoplasm (Fig 1C)
We previously showed that culturing cardiac fibroblasts on soft hydrogels, mimicking the stiffness of the healthy heart, prevented myofibroblast differentiation over the course of several days (5)
Summary
Activation of cardiac fibroblasts is a key step in development of cardiac fibrosis. In response to increased mechanical stress caused by increased left ventricular pressure or myocardial stiffening, cardiac fibroblasts differentiate into myofibroblasts characterized by de novo gene expression of actin alpha 2 smooth muscle (Acta2) and assembly of contractile smooth muscle α-actin (SMA) fibers, as well as enhanced production of typical cardiac extracellular matrix (ECM) genes including collagen (Col) 1a1, 1a2, 3a1 and the collagen cross-linking enzymes lysyl oxidase (Lox) [1] and Lox-like 2 [2]. The increases in myofibroblast markers are accompanied by decreased expression of transcription factor 21 (Tcf21), a marker of resting cardiac fibroblasts [4]. These changes in cardiac fibroblast phenotype alter ECM composition and structure leading to increased stiffness of the heart, which can compromise diastolic function. Studying cardiac fibroblast activation in vitro is hampered by rapid myofibroblast differentiation in response to the high stiffness of plastic culturing conditions [1]. We here investigate to what extent disrupting mechanotransduction can be used to maintain resting cardiac fibroblasts in vitro, thereby enabling the study of biomechanical activation of resting cardiac fibroblasts
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