Highlights d Deformation microscopy is developed by combining imaging and advanced mechanics d Modulation of nuclear LINC proteins or lamin A/C reveals altered intranuclear strain d Abnormal mechanical environments cause abnormal strain in high-density chromatin d Hyperosmotic conditions lead to nuclear strain asymmetry mediated by the cytoskeleton

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Biological tissues have a complex hierarchical architecture that spans organ to subcellular scales and comprises interconnected biophysical and biochemical machinery. Mechanotransduction, gene regulation, gene protection, and structure-function relationships in tissues depend on how force and strain are modulated from macro to micro scales, and vice versa. Traditionally, computational and experimental techniques have been used in common model systems (e.g., embryos) and simple strain measures were applied. But the hierarchical transfer of mechanical parameters like strain in mammalian systems is largely unexplored in vivo. Here, we experimentally probed complex strain transfer processes in mammalian skeletal muscle tissue over multiple biological scales using complementary in vivo ultrasound and optical imaging approaches. An iterative hyperelastic warping technique quantified the spatially-dependent strain distributions in tissue, matrix, and subcellular (nuclear) structures, and revealed a surprising increase in strain magnitude and heterogeneity in active muscle as the spatial scale also increased. The multiscale strain heterogeneity indicates tight regulation of mechanical signals to the nuclei of individual cells in active muscle, and an emergent behavior appearing at larger (e.g. tissue) scales characterized by dramatically increased strain complexity.

Understanding how cells remember previous mechanical environments to influence their fate, or mechanical memory, informs the design of biomaterials and therapies in medicine. Current regeneration therapies require two-dimensional (2D) cell expansion processes to achieve large cell populations critical for the repair of damaged (e.g. connective and musculoskeletal) tissues. However, the influence of mechanical memory on cell fate following expansion is unknown, and mechanisms defining how physical environments influence the therapeutic potential of cells remain poorly understood. Here, we show that the organization of histone H3 trimethylated at lysine 9 (H3K9me3) and expression of tissue-identifying genes in primary cartilage cells (chondrocytes) transferred to three-dimensional (3D) hydrogels depends on the number of previous population doublings on tissue culture plastic during 2D cell expansion. Decreased levels of H3K9me3 occupying promoters of dedifferentiation genes after the 2D culture were also retained in 3D culture. Suppression of H3K9me3 during expansion of cells isolated from a murine model similarly resulted in the loss of the chondrocyte phenotype and global remodeling of nuclear architecture. In contrast, increasing levels of H3K9me3 through inhibiting H3K9 demethylases partially rescued the chondrogenic nuclear architecture and gene expression, which has important implications for tissue repair therapies, where expansion of large numbers of phenotypically-suitable cells is required. Overall, our findings indicate mechanical memory in primary cells is encoded in the chromatin architecture, which impacts cell fate and the phenotype of expanded cells.SIGNIFICANCE STATEMENTTissue regeneration procedures, such as cartilage defect repair (e.g. Matrix-induced Autologous Chondrocyte Implantation) often require cell expansion processes to achieve sufficient cells to transplant into an in vivo environment. However, the chondrocyte cell expansion on 2D stiff substrates induces epigenetic changes that persist even when the chondrocytes are transferred to a different (e.g. 3D) or in vivo environment. Treatments to alter epigenetic gene regulation may be a viable strategy to improve existing cartilage defect repair procedures and other tissue engineering procedures that involve cell expansion.

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Mechanisms of cellular and nuclear mechanosensation are unclear, partially because of a lack of methods that can reveal dynamic processes. Here, we present a new concept for a low-cost, three-dimensionally printed device that enables high-magnification imaging of cells during stretch. We observed that nuclei of mouse embryonic skin fibroblasts underwent rapid (within minutes) and divergent responses, characterized by nuclear area expansion during 5% strain but nuclear area shrinkage during 20% strain. Only responses to low strain were dependent on calcium signaling, whereas actin inhibition abrogated all nuclear responses and increased nuclear strain transfer and DNA damage. Imaging of actin dynamics during stretch revealed similar divergent trends, with F-actin shifting away from (5% strain) or toward (20% strain) the nuclear periphery. Our findings emphasize the importance of simultaneous stimulation and data acquisition to capture mechanosensitive responses and suggest that mechanical confinement of nuclei through actin may be a protective mechanism during high mechanical stretch or loading.

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