- Numerous diseases, including those of the heart, are characterized by increased stiffness due to excessive deposition of extracellular matrix proteins. Cardiomyocytes continuously adapt their morphology and function to the mechanical changes of their microenvironment. Because traditional cell culture is conducted on substrates that are many orders of magnitude stiffer than any environment encountered by a cardiomyocyte in health or disease, alternate culture systems are necessary to model these processes in vitro. Here, we employ photo-clickable thiol-ene poly(ethylene glycol) (PEG) hydrogels for three-dimensional cell culture of adult mouse cardiomyocytes. PEG hydrogels serve as versatile biocompatible scaffolds, whose stiffness can be precisely tuned to mimic physiological and pathological microenvironments. Compared to traditional culture, adult cardiomyocytes encapsulated in PEG hydrogels exhibited longer survival and preserved sarcomeric and T-tubular architecture. Culture in PEG hydrogels of varying stiffnesses regulated the subcellular localization of the mechanosensitive transcription factor, YAP, in adult cardiomyocytes, indicating PEG hydrogels offer a versatile platform to study the role of mechanical cues in cardiomyocyte biology.