PROJECT SUMMARY / ABSTRACT A common and deadly form of familial heart disease is dilated cardiomyopathy (DCM), which is typically characterized by adverse cellular and ventricular remodeling and systolic dysfunction. DCM is often associated with loss-of-function mutations in genes encoding sarcomeric or cytoskeletal proteins. Mechanotransmission and mechanosignaling in cardiomyocytes (CMs) rely on these protein networks, particularly in the costamere, which provides a direct mechanical link between the extracellular matrix (ECM) and the Z-disk of the sarcomere. The costamere may therefore regulate both ‘inside-out’ mechanotransmission (transmitting sarcomere-born forces out to the ECM) and ‘outside-in’ mechanosignaling (transmitting/transducing extracellular mechanical signals into the CM)—the dysfunction of either of which may be central to DCM progression. My overall hypothesis is that the costamere and cortical cytoskeleton of cardiomyocytes provide key mechanosensitive protein networks that regulate mechanical signalling pathways initiated by intracellular and extracellular forces, and that specific defects in these structures inhibits their ability to transmit and transduce mechanical forces, causing contractile dysfunction and pathological cell remodelling. Supporting this, the costamere protein Filamin C (FLNC) has recently been implicated in a variety of human cardiomyopathies, including DCM. During my F32 postdoctoral training, I used a new mouse model that exploits cardiac-specific and inducible homozygous FLNC deletion to trigger rapid DCM development. I found that a loss of FLNC causes significant reductions in the tissue- and cell- level contractility, as well as significant CM remodeling accompanied by a reduction in cortical cytoskeleton stiffness. However, whether FLNC mutations in humans with DCM cause similar defects in cortex structure and mechanics, systolic mechanotransmission, and mechanosensitive gene regulation requires further investigation. Thus, the goal of my proposed research is to integrate quantitative subcellular-level structural and biomechanical measurements with quantitative measurements of intracellular stress distributions and hypertrophic gene expression patterns in response to intra- and extra-cellular mechanical perturbations using human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) expressing a patient- specific FLNC-truncating mutation. To accomplish this, I will: (1) combine X-ray diffraction imaging, atomic force microscopy, and multiscale computational modeling to test the hypothesis that a loss of FLNC disrupts ‘inside-out’ mechanotransmission of sarcomeric forces by dysregulating myofilament lattice geometry via altered cortical cytoskeleton mechanics in murine CMs, (2) apply these biophysical methods and hypotheses to a new human DCM model made from gene-edited hiPSC-CMs expressing a patient-specific FLNC-truncating variant, and (3) combine FRET-based molecular tension sensor imag...