Cellular mechanotransduction plays a central role in fibroblast activation during fibrotic disease progression, leading to increased tissue stiffness and reduced organ function. While the role of epigenetics in disease mechanotransduction has begun to be appreciated, little is known about how substrate mechanics, particularly the timing of mechanical inputs, regulate epigenetic changes such as DNA methylation and chromatin reorganization during fibroblast activation. In this work, we engineered a hyaluronic acid hydrogel platform with independently tunable stiffness and viscoelasticity to model normal (storage modulus, G' ∼ 0.5 kPa, loss modulus, G'' ∼ 0.05 kPa) to increasingly fibrotic (G' ∼ 2.5 and 8 kPa, G'' ∼ 0.05 kPa) lung mechanics. Human lung fibroblasts exhibited increased spreading and nuclear localization of myocardin-related transcription factor-A (MRTF-A) with increasing substrate stiffness within 1 day, with these trends holding steady for longer cultures. However, fibroblasts displayed time-dependent changes in global DNA methylation and chromatin organization. Fibroblasts initially displayed increased DNA methylation and chromatin decondensation on stiffer hydrogels, but both of these measures decreased with longer culture times. To investigate how culture time affected the responsiveness of fibroblast nuclear remodeling to mechanical signals, we engineered hydrogels amenable to in situ secondary crosslinking, enabling a transition from a compliant substrate mimicking normal tissue to a stiffer substrate resembling fibrotic tissue. When stiffening was initiated after only 1 day of culture, fibroblasts rapidly responded and displayed increased DNA methylation and chromatin decondensation, similar to fibroblasts on static stiffer hydrogels. Conversely, when fibroblasts experienced later stiffening at day 7, they showed no changes in DNA methylation and chromatin condensation, suggesting the induction of a persistent fibroblast phenotype. These results highlight the time-dependent nuclear changes associated with fibroblast activation in response to dynamic mechanical perturbations and may provide mechanisms to target for controlling fibroblast activation.

Peyton C. Johnston

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