Stretch-activated nuclear YAP localization in cardiomyocytes.

Abstract

Yes Associated Protein (YAP) is a mechanosensitive transcriptional activator regulating a variety of cell fates, including proliferation and cell growth. YAP is activated (localized to the nucleus) in both physiological development (e.g., organ growth mediated by cell proliferation) and pathological heart diseases (e.g., organ growth mediated by cell enlargement). Cardiomyocytes (CMs) generate the mechanical work that powers each heartbeat, and each contraction shortens the CM and strains the nuclei. Mechanical forces and nuclear deformation are considered putative drivers of YAP activation. However, the mechanoregulation of YAP's dual roles in organ growth - through proliferation during development and via hypertrophic enlargement in adulthood - remain unknown. Both contractile output and strain increase as the heart works harder under increased load (e.g., with exercise, disease, aging) and over time can lead to remodeling characteristic of hypertrophic cardiomyopathy. To mimic this additional loading, we first patterned extracellular matrix (Matrigel) single-cell rectangles of 7:1 aspect ratio on silicon substrate of a previously published, microfabricated, silicone pneumatic cell-stretching device (Hart, CAMB, 2021). Then, we then seeded human induced pluripotent stem cell-derived CMs (hiPSC-CM) on these patterns to elicit an elongated, anisotropic subcellular structure. We applied 15% stretch strain in static and cyclic, at 1 Hz frequency, for 10 mins to hiPSC-CM aligned in perpendicular or in parallel to the direction of stretch. Finally, we fixed and immunostained with YAP antibodies to quantify YAP localization. We quantified the ratio of nuclear/non-nuclear YAP in hiPSC-CM and found an upregulation of nuclear YAP in parallel, 97±10% in static and 42±5% in cyclic, and no significant change in regulation in perpendicular. Ultimately, these experiments will help us understand how mechanical cues and directionality of forces drive nuclear YAP, and the role of YAP signaling in driving hypertrophic hiPSC-CM remodeling.