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Abstract We examined the mechanical deformation of valve interstitial cells (VICs) in the anterior leaflet of the tricuspid valve and explored the relationship between the extracellular matrix (ECM) structure and cellular mechanics. Fresh porcine hearts were used to prepare specimens, subjected to biaxial tensile testing, and imaged using confocal microscopy with VIC nuclei staining. A multi-scale computational framework was developed to analyze cellular deformation and orientation within the ECM, using nuclear aspect ratio (NAR) as a metric. Experimental results showed that NAR values increased with mechanical loading, from 2.57±0.72 in a traction-free state to 3.4±1.29 at 130 kPa. Model predictions aligned with experimental findings. They also highlighted the significant impact of ECM fiber orientation on VIC nuclei deformation. These results indicate that mechanical forces profoundly influence cellular morphology and, potentially, their function. Further development of models is necessary to understand the complex interplay between the mechanical environment and cellular responses, crucial for identifying how mechanical forces affect tricuspid valve function and malfunction.