Cardiac tissue engineering has been limited by the inability to recreate native myocardial structural features. We hypothesized that heart cell elongation and alignment in 3D engineered cardiac constructs would be enhanced by using physiologic ratios of cardiomyocytes (CM) and cardiac fibroblasts (CF) via matrix metalloprotease (MMP)-dependent mechanisms. Co-cultured CM and CF constructs were compared to CM-enriched constructs using either basal media or media with a general MMP inhibitor for 8 days. Co-cultured constructs exhibited significantly increased cell alignment (p < 0.0002), which was eliminated by MMP inhibition. Co-cultured constructs expressed substantial active MMP-2 protein that was not present in CM-enriched constructs, increased pro-MMP-2 (p < 0.001), and reduced pro-MMP-9 (p < 0.001) expression. Apoptosis was decreased by co-culture (p < 0.05), independent of MMP inhibition. These results demonstrated that co-culture of CF in physiologic ratios within engineered cardiac constructs improved cell elongation and alignment via increased MMP-2 expression and activation, and also improved viability independent of MMP activity. (c) 2008 Elsevier Inc. All rights reserved.