Simulating Cardiometabolic Disease in a 3D-Microenvironment: Human Cardiac Fibroblast Response to Nutritional Stress

Cell behavior is influenced by substrate stiffness and cell-cell and cell-environment interactions. The limitations of 2D culture, such as its inability to fully capture the complexity of cell interactions and tissue structure, underscore the necessity of 3D cell culture. While still in its infancy, 3D cell culture helps overcome these caveats of 2D culture systems and provides information of significant physiological relevance. This explicitly applies to ‘disease modeling in a dish’ platforms for pre-clinical and reverse-translation studies. 3D bioprinting has demonstrated significant potential in replicating the intricate physiological environments of human tissues associated with both healthy and pathological states. This allows for improved recapitulation of the complex biochemical interactions and physiological environment vital for understanding the pathophysiology and drug responses. Our study used the state-of-the-art 3D bioprinting technology to understand the response of 3D matrix-embedded human cardiac fibroblasts (HCFs) and 2D cultures to a ‘high-fat diet’ mimic. We hypothesized that 3D-embedded HCFs would closely mimic human myocardium more than the 2D-HCFs, both in uninjured state as well as in response to stressors such as high-caloric nutritional load. It is well established that overnutrition can contribute to cardiometabolic disease. To capture this in vitro, HCFs in 2D were exposed to fatty-acid (FA) rich medium for 48 hours and processed for assay for transpose accessible chromatin with sequencing (ATAC-seq) to identify chromatin accessibility and regulatory genes. These signatures were validated by gene and protein expression assays. HCFs in 3D matrix proliferated slower than in 2D cultures, with substrate stiffness ranging from low kilopascal (3D) to gigapascals (2D). The transcriptional profiles of both 2D and 3D cultured cells varied dramatically, including fibroblast activation markers and mechanosensing-responsive genes. To investigate this mechanical signaling further, we performed mitogen-activated protein kinase (MAPK) protein arrays. Extracellular matrix (ECM) homeostasis, critical for maintaining healthy tissue structure and function, is deregulated in response to excess FA in the cellular microenvironment. The pro-fibrotic agent transforming growth factor β (TGF-β) orchestrates the upregulation of ECM genes via canonical (Smad-dependent) and non-canonical (MAPK-dependent) pathways. Compared to 2D cultures, reduced activation of MAPK pathway members in 3D cultures closely mimicked the in vivo healthy microenvironment of the human heart and physiological response to FA overload. The significance of our findings is two-fold. Firstly, we establish that 3D matrix embedded HCFs have striking resemblance in form and function to the human heart tissue. Secondly, 3D-cultured HCFs accurately capture the response to acute stressors such as overnutrition, which may be insufficiently estimated by already activated 2D-HCFs. Our study, therefore, identifies key mechanisms of nutritional stress response in cardiac fibroblasts, and provides the foundation for a robust and physiologically relevant drug-screening platform with high translational capability for cardiometabolic diseases. This work is supported by UAMS/ABI Faculty Start-up funds and the American Heart Association Career Development Award to RAB. This abstract was presented at the American Physiology Summit 2025 and is only available in HTML format. There is no downloadable file or PDF version. The Physiology editorial board was not involved in the peer review process.