Complex three-dimensional (3-D) heart structure is an important determinant of cardiac electrical and mechanical function. In this study, we set to develop a versatile tissue-engineered system that can promote important aspects of cardiac functional maturation and reproduce variations in myofiber directions present in native ventricular epicardium. We cultured neonatal rat cardiomyocytes within a 3-D hydrogel environment using microfabricated elastomeric molds with hexagonal posts. By varying individual post orientations along the directions derived from diffusion tensor magnetic resonance imaging (DTMRI) maps of human ventricle, we created large (2.5 × 2.5 cm2) 3-D cardiac tissue patches with cardiomyocyte alignment that replicated human epicardial fiber orientations. After 3 weeks of culture, the advanced structural and functional maturation of the engineered 3-D cardiac tissues compared to age-matched 2-D monolayers was evident from: 1) the presence of dense, aligned and electromechanically-coupled cardiomyocytes, quiescent fibroblasts, and interspersed capillary-like structures, 2) action potential propagation with near-adult conduction velocity and directional dependence on local cardiomyocyte orientation, and 3) robust formation of T-tubules aligned with Z-disks, co-localization of L-type Ca2+ channels and ryanodine receptors, and accelerated Ca2+ transient kinetics. This biomimetic tissue-engineered platform can enable systematic in vitro studies of cardiac structure-function relationships and promote the development of advanced tissue engineering strategies for cardiac repair and regeneration.

The present study strongly suggests that in the LQTS, focal activity generated in subendocardial Purkinje tissue is the primary, if not the only, trigger for TdP VAs by acting on a substrate of three-dimensional dispersion of myocardial repolarization to induce re-entrant excitation.

Simultaneous mapping of transmembrane voltage (Vm) and intracellular Ca2+ concentration (Cai) has been used for studies of normal and abnormal impulse propagation in cardiac tissues. Existing dual mapping systems typically utilize one excitation and two emission bandwidths, requiring two photodetectors with precise pixel registration. In this study we describe a novel, single-detector mapping system that utilizes two excitation and one emission bandwidth for the simultaneous recording of action potentials and calcium transients in monolayers of neonatal rat cardiomyocytes. Cells stained with the Ca2+-sensitive dye X-Rhod-1 and the voltage-sensitive dye Di-4-ANEPPS were illuminated by a programmable, multicolor LED matrix. Blue and green LED pulses were flashed in opposite phase at a rate of 488.3Hz using a custom-built dual bandpass excitation filter that transmitted blue (482±6nm) and green (577±31nm) light. A long-pass emission filter (>605 nm) and a 504-channel photodiode array were used to record combined signals from cardiomyocytes. Green excitation yielded Cai transients without significant crosstalk from Vm. Crosstalk present in Vm signals obtained with blue excitation was removed by subtracting an appropriately scaled version of the Cai transient. This method was applied to study delay between onsets of action potentials and Cai transients in anisotropic cardiac monolayers.