Elucidating the Role of Microenvironment Mechanics in Regulating Cardiac Myofibroblast Plasticity

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PROJECT SUMMARY Fibrosis underlies a vast number of cardiac pathological conditions, ranging from genetic cardiomyopathies to ischemic heart failure. Although substantial progress has been made in identifying molecular signals that trigger the characteristic activation of quiescent cardiac fibroblasts (CFs) and their transdifferentiation into myofibroblasts (MyoFBs), far less is known about the mechanisms that govern their long-term fate and persistence, which presents major obstacles to the development of effective anti-fibrotic therapies. This K99/R00 application describes a five-year research training plan that proposes to leverage (i) human induced pluripotent stem cell-derived cardiac fibroblasts (iPSC-CFs), (ii) engineered biomaterials with tunable mechanical properties, and (ii) single-cell multiomics platforms to investigate molecular mechanisms that govern MyoFB fate and plasticity. Given the well-established sensitivity of CFs and MyoFBs to extracellular matrix (ECM) stiffness, the applicant Dr. Sangkyun Cho will test the hypothesis that modulation of ECM-mediated mechanical signaling potentiates the de-differentiation of MyoFBs, by synergizing with soluble factors known to regulate major pathways in fibrogenesis. In Aim 1 (K99), Dr. Cho will use reporter iPSC lines (with fluorescently tagged canonical MyoFB ‘marker’ genes, e.g., CFP-TAGLN) and a novel dynamically softening hydrogel system to characterize in real-time the effects of mechanical unloading on MyoFB fate. In Aim 2 (K99), Dr. Cho will investigate the synergy between ECM softening and the TGF-beta pathway in regulating MyoFB states, (i) by examining stiffness-dependent protein interactions among mechanosensitive transcription factors (e.g., yes- associated protein 1 (YAP)), and (ii) by identifying epigenetic regulators downstream of ECM stiffness with single- cell assay for transposase transposase-accessible chromatin (scATAC-seq). In Aim 3 (R00), Dr. Cho will identify potential druggable targets along the cell’s mechanosensory apparatus, and test candidate compounds in engineered heart tissues and a mouse model of pressure-overload induced hypertrophy and heart failure. The proposed studies build upon PI Dr. Sangkyun Cho’s well-suited prior training in biomaterials, proteomics, and ECM mechanobiology, while providing new training opportunities in (i) reporter iPSC-CFs, (ii) single-cell multiomics platforms, and (iii) animal models. Mentor Dr. Joseph Wu is a pioneer in iPSCs and cardiovascular biology, and co-mentor Dr. Sarah Heilshorn is a leading expert in biomaterials and regenerative medicine, whose mentorship complements that of Dr. Wu. Advisory Committee members Drs. Jeffery Molkentin (cardiac fibrosis), Joseph Hill (heart failure models), and Michal Snyder (single-cell genomics) provide additional expertise and guidance. In Summary, the well-tailored research training plan, exceptional mentoring team, and an outstanding Environment at Stanford University are anticipated to help propel Dr. Cho toward his long-term goal of establishing an independent research program at the intersection of bioengineering and cardiovascular stromal biology.