Robust T-tubulation and maturation of cardiomyocytes using tissue-engineered epicardial mimetics

Abstract

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.

Inspiration from heart development: Biomimetic development of functional human cardiac organoids

“…This suggested that the developing stage ratio provided a favorable microenvironment for hiPSC-CM development and supported the developmental basis for cardiac organoid fabrication. This finding is consistent with previous engineered findings showing that cardiac constructs containing a majority of cardiomyocytes of the construct supports increased hiPSC-CM functions (15, 17, 22, 48). The developing stage cell ratio for cardiac organoids was used for the remainder of the experiments.…”

The extracellular matrix protein agrin promotes heart regeneration in mice

“…2b–d). Agrin-cKO cardiomyocytes exhibited increased maturation of sarcomeric structures as determined by colocalization of sarcomeric α-actinin and Cav3, a marker of transverse tubules (T-tubules) 14 (Fig. 2e).…”

3D bioprinting for cardiovascular regeneration and pharmacology

“…This would allow for precise control over cell alignment, cell-cell interactions, ECM composition, and microenvironment geometry [2]. The relevant topographic tissue cues have been widely studied in 2D patterning culture previously [139,142,143]. Significant observations in calcium handling, action potentials, conductional velocities, cardiomyogenesis and myofibrillogenesis have been shown to better resemble the native myocardium in aligned CMs as compared to those grown in randomly oriented cultures [2].…”

“…Microfabrication-based patterning techniques (such as surface topography, micromolding/microchannels, microcontact printing, sacrificial template methods, high temperature molding, and laser patterned electrospinning), have been developed in some pioneering studies to confine colony geometry, regulate cell morphology and functions, and support high-throughput analysis [2]. However, these 2D culture systems lack the full architecture and functionality of 3D human tissues and organs [2,139,142,144]. Alternatively, 3D bioprinting not only can accomplish this anisotropy in 3D architecture, but also cells can be directly encapsulated into the constructs to form cellularized tissue.…”