Hypertrophy, gene expression, and beating of neonatal cardiac myocytes are affected by microdomain heterogeneity in 3D

Cardiac myocytes are known to be influenced by the rigidity and topography of their physical microenvironment. It was hypothesized that 3D heterogeneity introduced by purely physical microdomains regulates cardiac myocyte size and contraction. This was tested in vitro using polymeric microstructures (G′ = 1.66 GPa) suspended with random orientation in 3D by a soft Matrigel matrix (G′ = 22.9 Pa). After 10 days of culture, the presence of 100 μm-long microstructures in 3D gels induced fold increases in neonatal rat ventricular myocyte size (1.61 ± 0.06, p < 0.01) and total protein/cell ratios (1.43 ± 0.08, p < 0.05) that were comparable to those induced chemically by 50 μM phenylephrine treatment. Upon attachment to microstructures, individual myocytes also had larger cross-sectional areas (1.57 ± 0.05, p < 0.01) and higher average rates of spontaneous contraction (2.01 ± 0.08, p < 0.01) than unattached myocytes. Furthermore, the inclusion of microstructures in myocyte-seeded gels caused significant increases in the expression of beta-1 adrenergic receptor (β1-AR, 1.19 ± 0.01), cardiac ankyrin repeat protein (CARP, 1.26 ± 0.02), and sarcoplasmic reticulum calcium-ATPase (SERCA2, 1.59 ± 0.12, p < 0.05), genes implicated in hypertrophy and contractile activity. Together, the results demonstrate that cardiac myocyte behavior can be controlled through local 3D microdomains alone. This approach of defining physical cues as independent features may help to advance the elemental design considerations for scaffolds in cardiac tissue engineering and therapeutic microdevices.