Regenerative cardiac tissue is a promising therapeutic option for myocardial repair after injury, however, poor electrical and contractile function has limited translational utility. Emerging research suggests that scaffolds that recapitulate the structure of the native myocardium improve physiological function. Engineered cardiac constructs with anisotropic extracellular architecture demonstrate improved tissue contractility, signaling synchronicity, and cellular organization when compared to constructs with reduced architectural order. The complexity of scaffold fabrication, however, limits isolated variation of individual structural and mechanical characteristics. Thus, the isolated impact of scaffold macroarchitecture on tissue function is poorly understood. Here, we produce isotropic and aligned collagen scaffolds seeded with embryonic stem cell derived cardiomyocytes (hESC-CM) while conserving all confounding physio-mechanical features to independently assess the effects of macroarchitecture on tissue function. We quantified spatiotemporal tissue function through calcium signaling and contractile strain. We further examined intercellular organization and intracellular development. Aligned tissue constructs facilitated improved signaling synchronicity and directional contractility as well as dictated uniform cellular alignment. Cells on aligned constructs also displayed phenotypic and genetic markers of increased maturity. Our results isolate the influence of scaffold macrostructure on tissue function and inform the design of optimized cardiac tissue for regenerative and model medical systems.

This zip folder contains bright-field (.tif files) for each construct morphology (aligned and isotropic). Bright-field videos were recorded on an Axiovert inverted microscope (Zeiss) using a Sony LEGRIA camera and the videos were converted into image stacks. Each image is of the circular surface of the construct. The zip folder also contains calcium cycling videos (.mp4 files) for each construct morphology. Videos were recorded on an Axiovert inverted microscope (Zeiss) using a Sony LEGRIA camera. Calcium cycling was observed using Fluo-4 AM. Each video is of the circular surface of the construct without stimulation. Microsoft Excel files of paced calcium dynamics are also included under the heading paced calcium cycling. Pacing occurred at frequencies of 1 and 1.5 Hz using c-PACE EM pace (IONOPTIX). Immunocytochemistry results are included as Microsoft Excel files for cell orientation (determined by phalloidin staining orientation), cell viability (determined by PrestoBlue Cell Viability Reagent), and sarcomere characteristic results. Micrographs were obtained using an SP-5 confocal microscope (LEICA) and analyzed using ImageJ as described in the methods section of the corresponding publication. Each of these files contains data for each construct morphology. The zip folder also contains gap junction results (.txt files) determined by connexin-43 staining for each construct morphology. Microsoft Excel files for qPCR results for phenotypic gene expression are also included for each construct morphology. X-ray micro-computed tomography (µCT) images (Skyscan 1172) were taken of each scaffold with a voltage of 25 kV, current of 138 mA, and a pixel size of 5.46 mm. Reconstructions of mCT images were performed with NRecon software by Skyscan and analysed in ImageJ. Results for scaffold alignment (.txt files) and scaffold pore size (.txt files) are also included in the zip file.