Temporal Impact of Substrate Anisotropy on Differentiating Cardiomyocyte Alignment and Functionality.

Anisotropic biomaterials can affect cell function by driving cell alignment, which is critical for cardiac engineered tissues. Recent work, however, has shown that pluripotent stem cell-derived cardiomyocytes may self-align over long periods of time. To determine how the degree of biomaterial substrate anisotropy impacts differentiating cardiomyocyte structure and function, we differentiated mouse embryonic stem cells to cardiomyocytes on nonaligned, semialigned, and aligned fibrous substrates and evaluated cell alignment, contractile displacement, and calcium transient synchronicity over time. Although cardiomyocyte gene expression was not affected by fiber alignment, we observed gradient- and threshold-based differences in cardiomyocyte alignment and function. Cardiomyocyte alignment increased with the degree of fiber alignment in a gradient-based manner at early time points and in a threshold-based manner at later time points. Calcium transient synchronization tightly followed cardiomyocyte alignment behavior, allowing highly anisotropic biomaterials to drive calcium transient synchronization within 8 days, while such synchronized cardiomyocyte behavior required 20 days of culture on nonaligned biomaterials. In contrast, cardiomyocyte contractile displacement had no directional preference on day 8 yet became anisotropic in the direction of fiber alignment on aligned fibers by day 20. Biomaterial anisotropy impact on differentiating cardiomyocyte structure and function is temporally dependent. Impact Statement This work demonstrates that biomaterial anisotropy can quickly drive desired pluripotent stem cell-derived cardiomyocyte structure and function. Such an understanding of matrix anisotropy's time-dependent influence on stem cell-derived cardiomyocyte function will have future applications in the development of cardiac cell therapies and in vitro cardiac tissues for drug testing. Furthermore, our work has broader implications concerning biomaterial anisotropy effects on other cell types in which function relies on alignment, such as myocytes and neurons.