Electrical Propagation of Three-Dimensional Engineered Hearts using Decellularized Extracellular Matrix

Abstract

[Background] Construction of three-dimensional cardiac tissues using decellularized extracellular matrix could be a new technique to create an “organ-like” structure of the heart. To engineer functional artificial hearts comparable for orthotopic hearts, however, much remain to be solved including electrical stability for efficient contraction. To elucidate the points, we examined electrophysiological properties of recellularized heart tissues. [Methods] Entire hearts of adult Wistar rats were decellularized using 0.5% SDS and 1% triton-X, and then recellularized with enzymatically-dispersed neonatal rat cardiac cells (1x10⊥8 cells) through antegrade coronary circulation. Three and seven days later after cell seeding, we observed excitation of spontaneous and pacing-induced beatings of recellularized heart tissues expressing Ca2+-indicating protein (GCaMP2) using high resolution cameras. We also conducted immunofluorescence staining to examine morphological aspects of engineered tissues. [Results] Live tissue fluorescence imaging revealed that GFP-labeled-isolated cardiac cells were dispersed into interstitial spaces through extravasation from coronary arteries. Engineered hearts seeded with GCaMP2-expressing cardiac cells started showing spontaneous beating, and were subjected to further electrophysiological experiments using optical imaging system. Both in spontaneous and pacing-induced beating hearts, we observed well-organized conduction of stable excitation in substantial areas of the engineered heart tissues, whereas we also recorded disorganized propagation of asynchronous excitation with multiple origins. Conduction velocity was markedly decreased (∼5 cm/sec) during early stages (up to nine days) after recellularization. Immunofluorescence study revealed randomly-mixed alignment of cardiomyocytes, endothelial cells and smooth muscle cells, stained with alpha-actinin, CD31 and sm-actin, respectively. Recellularized heart tissues also showed disarray of cardiomyocytes and markedly decreased expression of connexin43. [Conclusion] Three-dimensional engineered hearts showed electrical heterogeneity and proarrhythmic propensity during early stages of culture. These findings may be due to, at least in part, disorganized alignment of microstructure and immature formation of gap junction.