PROJECT SUMMARY Despite the ubiquitous role of fibrosis in tissue dysfunction arising from aging and disease, no representative in vitro model of the fibrotic microenvironment exists. Fibrosis is characterized by excess extracellular matrix (ECM) deposition that stiffens the cellular microenvironment. Therefore, to model fibrosis in vitro, cell culture substrates that permit quantitative, dynamic tuning of matrix mechanics are necessary. However, existing dynamic hydrogel culture platforms generally rely on chemistries that may be toxic to cells or that simultaneously change multiple parameters, making it difficult to assign causal relationships between altered matrix properties and cell fate changes. Fibrotic stiffening occurs in a wide range of tissues, including the skeletal muscles, liver, lungs, and heart. Numerous genetic cardiomyopathies are characterized by progressive fibrotic stiffening that precedes heart failure. While fibrotic stiffening is known to impair the heart’s ability to pump blood, the impact of stiffening on the phenotype of individual cardiomyocytes remains poorly understood. The goal of this research proposal is to develop an in vitro model of tissue fibrosis based on dynamic hydrogel biomaterials that enables real time measurement of cellular dysfunction to determine how progressive fibrotic stiffening detrimentally impacts cell fate. As a model system, we will interrogate the effects of stiffening on human cardiomyocytes differentiated from induced pluripotent stem cells from Duchenne muscular dystrophy (DMD) patients. DMD is an ideal model system for studying outside-in mechanosignaling, as DMD arises from a lack of dystrophin, a structural protein linking the contractile cytoskeleton to the ECM. We will use the dynamic hydrogels developed during this research to assess contractile dysfunction, aberrant activation of mechanotransduction signaling, and novel molecular mechanisms of “mechanical memory” arising from fibrotic stiffening. In Aim 1, we will develop a synthetic hydrogel system that uses near-infrared light and bioorthogonal reactions to dynamically stiffen the gels, mimicking fibrosis. These hydrogels will be used to determine how contractile dysfunction arises from fibrotic stiffening. In Aim 2, we will determine how increased stiffness alters biochemical signaling in cardiomyocytes, focusing both on “canonical” mechanotransduction through Rho GTPases and YAP signaling and on a new mechanosensitive pathway in actively contracting cells that involves mechanical generation of reactive oxygen species (ROS), DNA damage, and impaired mitochondrial biogenesis. In Aim 3, we will investigate the first example of “mechanical memory” in cardiomyocytes. We will develop a hydrogel platform that is stiffened by one wavelength of light and subsequently softened by a second wavelength. This system will enable identification of molecular mechanisms by which exposure to a stiffened microenvironment causes persistent cellul...