Nanofiber Density Is a Higher-Order Architectural Regulator of Multidimensional Cardiomyocyte Maturation

Insufficient maturation of cardiomyocytes (CMs) remains a major challenge in cardiac tissue engineering. While electrospun nanofibers offer biomimetic topographical and mechanical cues, conventional high-density fiber architectures impose excessive mechanical constraints that restrict mechanotransduction and limit CM maturation. Here, we employed a suspended, aligned electrospun polycaprolactone (PCL) nanofiber platform to investigate how fiber density-categorized as low (LD; 15.89 ± 1.84 μg/cm2), medium (MD; 85.81 ± 12.74 μg/cm2), and high (HD; 404.90 ± 59.10 μg/cm2)-modulates the maturation of neonatal rat cardiomyocytes (NRCMs). The MD substrate was identified as the optimal configuration, effectively balancing topographical guidance with a permissive mechanical microenvironment for NRCM maturation. Compared with the HD substrate, NRCMs on MD substrates exhibited enhanced structural maturation, with a greater aspect ratio (5.65 ± 1.15) and sarcomere length (1.92 ± 0.17 μm). These structural improvements were accompanied by enhanced gap junction formation and improved electromechanical function, including a 4.03-fold higher contraction displacement and a 4.01-fold faster calcium decay rate. Furthermore, the MD substrate promoted metabolic maturation, evidenced by a 2.08-fold increase in mitochondrial membrane potential and a 2.42-fold higher ATP production. The LD substrate exhibited an intermediate maturation phenotype between MD and HD groups. Mechanistically, the maturation-promoting effects of the MD substrate were associated with increased YAP phosphorylation and cytoplasmic retention, together with enhanced PI3K-Akt signaling, all of which were attenuated by high-density fiber packing. This "Goldilocks effect" establishes fiber density as a critical architectural determinant of CM maturation and highlights optimization of this parameter to regulate mechanosignaling as an effective strategy for engineering functionally mature cardiomyocytes.