3-D biomechanics and epigenomics reveal atypical fibroblast responses in cardiometabolic disease.

Cell behavior is influenced by substrate stiffness and cell-cell and cell-environment interactions. The limitations of two-dimensional (2-D) culture, such as its inability to fully capture the complexity of cell interactions and tissue structure, highlight the necessity of three-dimensional (3-D) cell culture. This explicitly applies to "disease modeling in a dish" platforms for translational studies. 3-D bioprinting demonstrates significant potential in recapitulating the intricate physiological environments of human tissues in both healthy and pathological states. With the alarming rise in obesity, addressing systemic pathophysiological dysfunction beyond adipose tissue itself, such as the heart, is inevitable. To capture cellular and tissue-level responses to overnutrition, we used state-of-the-art 3-D bioprinting technology to understand the acute response of 3-D matrix-embedded human cardiac fibroblasts to a "high-fat diet" mimic. Chromatin accessibility profiling revealed that excess fatty acid (FA) exposure in 2-D induces a noncanonical extracellular matrix gene program that is minimally expressed in healthy adult myocardium. In contrast, 3-D cultures exhibited reduced fibroblast proliferation and blunted transcriptional responses to the impact of biomechanical cues under metabolic stress, reflecting a more quiescent and physiologically relevant phenotype. Furthermore, we incorporated human induced pluripotent stem cell-derived cardiac fibroblasts (iPSC-CFs), which mirrored key transcriptional changes, including sex-dependent gene regulation. Notably, male iPSC-CFs showed stronger fibrotic gene induction than females, reinforcing the need to account for biological sex in disease modeling. Together, our results highlight the limitations of 2-D systems and demonstrate that 3-D-bioprinted platforms provide a scalable, physiologically relevant tool for investigating cardiometabolic diseases and therapeutic targets.NEW & NOTEWORTHY Biomechanical cues in conventional 2-D systems can artificially prime fibroblasts toward activation, whereas 3-D-bioprinted hydrogels better preserve physiological phenotypes. The use of Assay for Transposase-Accessible Chromatin with high-throughput sequencing (ATAC-seq) to profile chromatin accessibility uncovered noncanonical epigenomic remodeling in response to FA overload, highlighting novel fibrotic gene programs not captured by traditional assays. The identification of sex-specific transcriptional responses in iPSC-derived fibroblasts highlights the importance of incorporating biological variables for personalized cardiometabolic disease modeling and accelerating therapeutic development.