Ischemic heart disease is the leading cause of death worldwide, in part due to the heart’s limited capacity to regenerate. Transplantation of exogenous cells into the heart is a promising approach to restore cardiac function in ischemic disease. Preengineering of cells into a functional cardiac tissue patch prior to implantation is expected to maximize therapeutic benefits, however, the electrical and mechanical properties of engineered cardiac tissues are currently far inferior to those of native myocardium. Furthermore, the levels of functionality of engineered tissues following implantation on the heart have not been studied. To further the state-of-the-art in the field, the primary goals of this dissertation have been to engineer cardiac tissue with functional properties comparable to those of adult myocardium and to quantify electrical function of such engineered tissues following epicardial implantation.

To achieve these goals, we first developed dynamic, free-floating culture conditions for engineering "cardiobundles", 3-dimensional cylindrical tissues made from neonatal rat cardiomyocytes embedded in fibrin-based hydrogel. Compared to static conditions, 2-week dynamic culture of neonatal rat cardiobundles significantly increased expression of sarcomeric proteins, cardiomyocyte size (∼2.1-fold), contractile force (∼3.5-fold), and conduction velocity of action potentials (∼1.4-fold). The average contractile force per cross-sectional area (59.7 mN/mm²) and conduction velocity (CV=52.5 cm/s) matched or approached those of adult rat myocardium, respectively. The inferior function of statically cultured cardiobundles was rescued by transfer to dynamic conditions. This functional rescue, which could be blocked by rapamycin, was accompanied by an increase in mTORC1 activity and decline in AMPK phosphorylation. Furthermore, dynamic culture effects did not stimulate ERK1/2 pathway and were insensitive to blockers of mechanosensitive channels, suggesting increased nutrient availability rather than mechanical stimulation as the upstream activator of mTORC1. Direct comparison with phenylephrine treatment confirmed that dynamic culture promoted physiological cardiomyocyte growth rather than pathological hypertrophy.

We then combined 0.2 Hz electrical stimulation with application of thyroid hormone (5 nM triiodothyronine) to further mature dynamically cultured cardiobundles during 5-week culture. These conditions further increased myocardial volume and contractile force by ~40%, shortened action potential and twitch durations and increased maximum capture rate. Additional evidence of maturation included polarization of Ncadherin junctions, a switch to troponin isoforms expressed in the adult heart, and development of sarcolemmal T-tubular structures. Since cardiomyocytes in this system exited cell cycle by two weeks of culture (<1% of cycling cells per day), we utilized cardiobundles to screen factors that reactivate cardiomyocyte proliferation following injury by hydrogen peroxide (H2O2). Specifically, we expressed a pro-proliferative transcription factor, constitutively active Yes-associated protein 1 (caYAP), under the control of an enhancer element selectively activated during injury in zebrafish hearts. Application of H2O2 resulted in a transient activation of the injury-responsive enhancer in a subset of cardiomyocytes 1-2 days post-injury, but the resulting caYAP expression was insufficient to induce a significant mitogenic effects. Nonetheless, in vitro matured cardiobundles hold promise for use as a relatively high-throughput system for discovery of novel pro-regenerative factors in various cardiac injury settings.

Finally, we analyzed electrical function and integration of engineered cardiac tissues following epicardial implantation. Cardiac patches were generated from neonatal rat cardiomyocytes expressing a genetically-encoded calcium indicator (GCaMP6) and implanted in adult rats with normal heart function for up to 6 weeks. After 2 weeks of in vitro culture, engineered cardiac patches contained robustly coupled cardiomyocytes, generated maximum active forces of 18.0 ± 1.4 mN, and propagated action potentials with a conduction velocity of 32.3 ± 1.8 cm/s. From dual optical mapping of GCaMP6-labelled patch and RH237-stained heart, 85% patches survived implantation and conducted action potential with velocities not different from those pre-implantation. Asynchronous activation of the patch and the heart indicated a lack of graft-host electrical coupling consistent with the formation of non-cardiomyocyte scar tissue between the patch and heart. In a subcutaneous implantation model, scar tissue formation between the patch and native muscle could not be reduced by enhancement of patch-muscle contact area with a surgical mesh or co-implantation of bone marrow-derived macrophages within the patch.

In summary, using neonatal rat cardiomyocytes, we developed a novel methodology for engineering cylindrical cardiac tissues (cardiobundles) with a near-adult functional output. mTOR signaling was identified as an important mechanism for advancing cardiobundle maturation and function in vitro, along with the application of electrical stimulation and thyroid hormone supplementation. Cardiobundle injury model was established to allow screening of pro-regenerative factors and approaches in vitro. Epicardial implantation of engineered cardiac tissue patches served to develop an enhanced analysis method for graft-host integration in animal models of cell-based cardiac repair. Collectively, these methods and results are expected to aid advances in the field of cell-based cardiac therapy towards eventual clinical applications.