Patterning of cells is critical to the formation and function of the normal organ, and it appears to be dependent upon internal and external signals. Additionally, the formation of most tissues requires the interaction of several cell types. Indeed, both extracellular matrix (ECM) components and cellular components are necessary for three-dimensional (3-D) tissue formation in vitro. Using 3-D cultures we demonstrate that ECM arranged in an aligned fashion is necessary for the rod-shaped phenotype of the myocyte, and once this pattern is established, the myocytes were responsible for the alignment of any subsequent cell layers. This is analogous to the in vivo pattern that is observed, where there appears to be minimal ECM signaling, rather formation of multicellular patterns is dependent upon cell–cell interactions. Our 3-D culture of myocytes and fibroblasts is significant in that it models in vivo organization of cardiac tissue and can be used to investigate interactions between fibroblasts and myocytes. Furthermore, we used rotational cultures to examine cellular interactions. Using these systems, we demonstrate that specific connexins and cadherins are critical for cell–cell interactions. The data presented here document the feasibility of using these systems to investigate cellular interactions during normal growth and injury.

Cardiac fibroblasts are the most numerous cells in the heart and are critical in the formation and normal functioning of the organ. Cardiac fibroblasts are firmly attached to and surrounded by extracellular matrix (ECM). Mechanical forces transmitted through interaction with the ECM can result in changes of overall cellular shape, cytoskeletal organization, proliferation, and gene expression of cardiac fibroblasts. These responses may be different in the normally functioning heart, when compared with various pathological conditions, including inflammation or hypertrophy. It is apparent that cellular phenotype and physiology, in turn, are affected by multiple signal transduction pathways modulated directly by the state of polymerization of the actin cytoskeleton. Morphological changes in actin organization resulting from response to adverse conditions in fibroblasts and other cell types are basically descriptive. Some studies have approached quantifying changes in actin cytoskeletal morphology, but these have involved complex and difficult procedures. In this study, we apply image analysis and non-Euclidian geometrical fractal analysis to quantify and describe changes induced in the actin cytoskeleton of cardiac fibroblasts responding to mechanical stress. Characterization of these rapid responses of fibroblasts to mechanical stress may provide insight into the regulation of fibroblasts behavior and gene expression during heart development and disease.