Hierarchical architecture influences calcium dynamics in engineered cardiac muscle.

Changes in myocyte cell shape and tissue structure are concurrent with changes in electromechanical function in both the developing and diseased heart. While the anisotropic architecture of cardiac tissue is known to influence the propagation of the action potential, the influence of tissue architecture and its potential role in regulating excitation-contraction coupling (ECC) are less well defined. We hypothesized that changes in the shape and the orientation of cardiac myocytes induced by spatial arrangement of the extracellular matrix (ECM) affects ECC. To test this hypothesis, we isolated and cultured neonatal rat ventricular cardiac myocytes on various micropatterns of fibronectin where they self-organized into tissues with varying degrees of anisotropy. We then measured the morphological features of these engineered myocardial tissues across several hierarchical dimensions by measuring cellular aspect ratio, myocyte area, nuclear density and the degree of cytoskeletal F-actin alignment. We found that when compared with isotropic tissues, anisotropic tissues have increased cellular aspect ratios, increased nuclear densities, decreased myocyte cell areas and smaller variances in actin alignment. To understand how tissue architecture influences cardiac function, we studied the role of anisotropy on intracellular calcium ([Ca(2+)](i)) dynamics by characterizing the [Ca(2+)](i)-frequency relationship of electrically paced tissues. When compared with isotropic tissues, anisotropic tissues displayed significant differences in [Ca(2+)](i) transients, decreased diastolic baseline [Ca(2+)](i) levels and greater [Ca(2+)](i) influx per cardiac cycle. These results suggest that ECM cues influence tissue structure at cellular and subcellular levels and regulate ECC.