Chamber‐Specific Decellularized Extracellular Matrices Differentially Modulate Cardiomyocyte Subtypes to Drive Engineered Heart Tissue Development and Function

Decellularized extracellular matrix (dECM) preserves native biochemical and biophysical cues and serves as a functional biomaterial for engineered tissue development. While tissue-specific extracellular matrix (ECM) has been widely studied, regional variation within the same organ remains poorly understood. Here, we investigate chamber-specific roles of ventricular (vtdECM) and atrial (atdECM) dECMs in engineered heart tissue (EHT) formation using induced pluripotent stem cell-derived cardiomyocyte (CM) subtypes. Proteomic analysis revealed distinct compositional profiles, with vtdECM enriched in ventricular development-related proteins and atdECM enriched in structural organization-related proteins. Ventricular CMs exhibited enhanced maturation and function in vtdECM, whereas atrial CMs showed limited responsiveness to ECM composition despite transcriptome-level differences. Encapsulation timing further modulated these effects, with early encapsulation promoting structural maturation and late encapsulation enhancing calcium handling. These findings demonstrate that chamber-specific ECM composition and developmental timing cooperatively regulate subtype-specific CM maturation, providing a framework for designing physiologically relevant EHTs.