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<p>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.
Pre-engineering 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.</p><p>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. </p><p>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 N-cadherin 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. </p><p>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.</p><p>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.</p>
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