Influence of ECM proteins and their analogs on cells cultured on 2-D hydrogels for cardiac muscle tissue engineering

This study assessed the role of immobilized cell adhesion moieties on controlling the cellular attachment, adhesion and phenotype of cardiac muscle cells towards developing scaffolds for cardiac muscle tissue engineering. Collagen I, laminin and the cell-adhesive oligopeptide, arginine-glycine-aspartic acid (RGD) at concentrations of 0.5 and 5 mM were covalently bound to flexible two-dimensional hydrogels. A robust skeletal myoblast cell line demonstrated good bioactivity for the modified hydrogels, resulting in myoblast attachment and development of an intracellular contractile network after 1 day. Primary neonatal rat ventricular myocytes cultured for up to 7 days, however, were more sensitive to the different modified substrates. Although total cardiomyocyte DNA content did not vary significantly with surface modification, immunostaining for the contractile protein Troponin I and focal adhesion protein vinculin revealed marked improvements in spreading and intracellular contractile protein deposition for cells attached to protein-modified hydrogels over those modified with RGD, regardless of RGD concentration. On the RGD-modified surfaces, cardiomyocytes self-associated, forming aggregates that exhibited a disorganized cytoarchitecture. Cardiomyocyte maturation was assessed through the fetal gene program where expression for atrial natriuretic peptide decreased and sarco(endo)plasmic reticulum Ca²⁺ increased with culture time for the protein-modified surfaces, indicating a trend towards maturation, while the α/β-myosin heavy-chain ratio remained near fetal expression levels for all surfaces. Overall, our findings suggest that whole proteins, collagen and laminin, are effective in promoting cardiomyocyte interaction with hydrogels and cardiomyocyte maturation while RGD does not provide adequate extracellular matrix cues for cardiomyocytes.