Browsing by Author "Bursac, Nenad"

Tissue-engineered skeletal muscle presents promising opportunities for developing high-fidelity in vitro models for investigating human muscle biology in the areas of regeneration and disease. In muscle regeneration, satellite cells (SCs) are essential for new muscle fiber formation; however, they lose their native quiescent state upon isolation, making in vitro studies of human SC function challenging. To optimally promote SC quiescence and enable exploration of SC dynamics in vitro, engineered muscle needs to recapitulate the native muscle microenvironment, which is comprised of muscle fibers, extracellular matrix, and other biochemical and mechanical cues. In disease modeling, mechanistic studies and therapeutic development are still extensively evaluated in animal models, which have limited translational relevance to patients. Specifically, Pompe disease is caused by a variety of mutations in the lysosomal enzyme acid alpha-glucosidase (GAA) that varyingly affect residual GAA activity and cannot be captured in the current GAA-/- mouse model. Therefore, human in vitro models are needed to enhance our mechanistic understanding of diseases and stimulate the development of effective therapies. To overcome these limitations, we set the dissertation goals to: 1) generate a pool of quiescent SCs and explore the mechanisms governing their formation and activation using an engineered skeletal muscle microenvironment and 2) develop a high-fidelity tissue-engineered skeletal muscle model for Pompe disease to investigate pathological mechanisms and test candidate therapies. To achieve these goals, we first compared methods for primary human myoblast expansion and found that p38 inhibition significantly increases the formation of Pax7+ cells in engineered 3D skeletal muscle tissues (“myobundles”). Gene expression analysis suggested that within the myobundle environment the Pax7+ cells adopt a quiescent phenotype (3D SCs), characterized by increased Pax7 expression, cell cycle exit, and Notch signaling activation relative to the original 2D expanded myoblasts. We then compared 3D SCs to previously described satellite-like cells that form alongside myotubes in 2D culture, termed reserve cells (RCs). Compared to RCs, 3D SCs showed an advanced quiescent phenotype characterized by a higher Pax7, Spry1, and Notch3 expression, as well as increased functional myogenesis demonstrated by formation of myobundles with higher contractile strength. To examine 3D SC activation, we tested several myobundle injury methods and identified treatment with a bee toxin, melittin, to robustly induce myofiber fragmentation, functional decline, and 3D SC proliferation. To further investigate the transcriptional processes describing how 2D myoblasts acquire 3D SC phenotype (i.e. deactivate) and how 3D SCs respond to injury (i.e. reactivate), we applied single cell RNA-sequencing (scRNA-seq) from which we discovered the existence of two subpools of 3D SCs—“quiescent” (qSC) and “activated” (aSC). The qSC subpool possessed greater expression of quiescence genes Pax7, Spry1, and Hey1, whereas the aSC subpool exhibited increased expression of inflammatory and differentiation markers. Furthermore, we performed trajectory inference along the deactivation process from 2D myoblasts to qSCs and identified deactivation-associated genes, included downregulated genes for proliferation, cytoskeletal reorganization, and myogenic differentiation. In response to tissue injury, we observed a decrease in the proportion of qSCs and an increase in the proportion of aSCs and committed myogenic progenitor cells suggestive of myogenic differentiation. In addition, we observed transcriptional changes within the aSC population reflective of SC activation in vivo, namely increased TNF- signaling, proliferation, and glycolytic and oxidative metabolism. These results strongly suggested that 3D SC heterogeneity and function recapitulate several aspects of native human SCs and could be applied to study human muscle regeneration and disease-associated SC dysfunction. To evaluate the myobundle system in the context of disease modeling, we developed the first 3D tissue-engineered skeletal muscle model of infantile onset Pompe disease (IOPD), the most severe form of Pompe disease. Diseased myobundles demonstrated characteristic GAA enzyme deficiency, accumulation of the GAA target glycogen, and lysosome enlargement. Despite exhibiting these key biochemical and structural hallmarks of disease, IOPD myobundles did not show deficits in contractile force generation or autophagic buildup. We therefore identified metabolic stress conditions that acutely targeted disease-associated abnormalities in the lysosomes and glycogen metabolism, which revealed impairments in contractile function and glycogen mobilization. To further elucidate the biological mechanisms underlying the phenotype of IOPD myobundles, we applied RNA sequencing (RNA-seq) and observed enrichment for terms consistent with Pompe disease phenotype including downregulation of gene sets involved in muscle contraction, increased endoplasmic reticulum stress, and reduced utilization of specific metabolic pathways. We then compared the transcriptomic profiles of GAA-/-¬ and wild-type mice to identify a Pompe disease signature and confirmed the presence of this signature in IOPD myobundles. Finally, treating IOPD myobundles with clinically used recombinant protein (rhGAA) therapy resulted in increased GAA activity, glycogen clearance, and a partial reversal of the disease signature, further confirming the utility of the myobundle system for studies of Pompe disease and therapy. In summary, this dissertation describes novel strategies for the formation and characterization of quiescent human SCs using the myobundle system. We present first-time application of scRNA-seq to engineered skeletal muscle, and uncover transcriptional descriptors of human myoblast deactivation and SC heterogeneity and activation. When utilizing human myobundles as a novel model of Pompe disease, we identified disease hallmarks and responses to therapy consistent with observations in Pompe patients. We anticipate that the findings and methods developed in this work will serve as a useful framework for the future engineering of regenerative human muscle for therapeutic and disease modeling applications.

Cardiac fibroblasts represent an important connective tissue population in the heart with increasingly recognized roles in cardiac development, homeostasis, and disease. Heart failure, in particular, is debilitating cardiac disorder with hallmark features of extensive myocyte death, pathological remodeling, and fibrosis. In both pathological and age-related cardiac fibrotic states, resident cardiac fibroblasts could serve as a novel therapeutic target to reduce pathological remodeling or generate new functional cardiomyocytes. However, the detailed mechanistic understanding of how disease-driven phenotypic changes in cardiac fibroblasts influence their functional crosstalk with cardiomyocytes remains largely unknown. In addition, as an appealing therapeutic strategy, cardiac fibroblasts could be directly reprogrammed into functional cardiomyocytes. However, the direct reprogramming in two-dimensional (2D) culture remains ineffective and whether three-dimensional (3D) culture milieu could improve the reprogramming efficiency remains unexplored. Therefore, the primary goals of this dissertation have been to: 1) engineer a versatile 3D engineered co-culture system approximating environment of native cardiac tissue to systematically investigate age-dependent effects of cardiac fibroblasts on the function and molecular properties of surrounding cardiomyocytes, 2) identify subpopulations of pathologically activated cardiac fibroblasts in response to pressure overload and how they differentially affect cardiac function and fibrosis, and 3) determine whether the reprogramming efficiency of fibroblasts to cardiomyocytes can be enhanced in 3D engineered tissue environment.

To achieve these goals, we first developed a versatile and physiologically relevant hydrogel-based 3D cardiomimetic co-culture platform to systematically assess the direct cardiac fibroblast-induced effects on surrounding cardiomyocytes. By using this 3D co-culture system, we showed that the age of cardiac fibroblasts is a strong determinant of the structure, function, and molecular properties of co-cultured cardiomyocytes. In particular, adult, but not fetal, cardiac fibroblasts significantly deteriorated electrical and mechanical function of the co-cultured cardiomyocytes, as evidenced by slower action potential conduction, prolonged action potential duration, weaker contractions, higher tissue stiffness, and reduced calcium transient amplitude. This functional deficit was associated with structural and molecular signatures of pathological remodeling including fibroblast proliferation, interstitial collagen deposition, and upregulation of pro-fibrotic markers.

In response to cardiac insult, quiescent cardiac fibroblasts become pathologically activated myofibroblasts leading to dysregulated ECM deposition and eventually deterioration of cardiac function. Specifically targeting the pathologically activated myofibroblasts represent an appealing therapeutic goal to delay or reverse the progression of heart failure and pathological fibrosis. We thus set out to explore a possibility that there exist functional distinct subpopulations of cardiac fibroblasts in response to pressure overload. By utilizing a pressure-overload mouse model and flow cytometry-based cell sorting, we identified the previously uncharacterized Thy1neg (Thy1-/MEFSK4+/CD45-/CD31-) fibroblast population that displayed a more pathological activated phenotype and deteriorated cardiomyocyte funciotn in 3D co-culture system. Additionally, in response to pressure overload, mice with global knockout of Thy1 developed more severe cardiac dysfunction and fibrosis compared to wild-type counterparts, further suggesting a functional role of Thy1 in cardiac pathogenesis.

Finally, we explored whether a tissue-engineered 3D hydrogel microenvironment would enhance direct reprogramming efficiency of fibroblasts into induced cardiomyocytes beyond what has been achievable in traditional 2D culture. We demonstrated that culturing cardiac fibroblasts reprogrammed by a cocktail of microRNAs (miR combo) within a tissue-engineered 3D environment dramatically enhanced the efficiency of reprogramming, which was associated with significant increases in the expression of several matrix metalloproteinases (MMPs). Pharmacological inhibition of MMPs blocked the enhanced reprogramming effects of the 3D environment suggesting a potential mechanism for this observation.

In summary, this dissertation established and utilized a physiologically relevant 3D co-culture platform for optimizing engineered cardiac tissue function, studying fibrotic heart disease, and improving fibroblast-reprogramming based cardiac therapeutics. Our studies provide the first evidence of critical roles of the age of supporting cells in engineering functional cardiac tissues, reveal a pathogenic subpopulation of cardiac fibroblasts characterized by lack of Thy1 expression, and demonstrate that 3D tissue-engineered environment can enhance the direct reprogramming of fibroblasts to cardiomyocytes via a MMP-dependent mechanism. These findings provide the foundation for the further development of novel fibroblast-targeted therapeutic strategies for myocardial infarction and pressure-overload induced heart failure.

Excess cardiac myofibroblasts in fibrotic heart diseases as well as cell-based therapies involving implantation of stem cells or genetically engineered somatic cells in the heart may all lead to a situation where a cardiomyocyte becomes electrically coupled to an unexcitable cell. In these settings, electrotonic loading of cardiomyocytes by unexcitable cells can affect cardiac action potential generation, propagation, and repolarization depending on the properties of both cardiomyocytes and unexcitable cells. The objective of this dissertation was to advance our understanding of the electrical interactions between cardiomyocytes and unexcitable cells using a variety of electrophysiological, molecular, and cell culture techniques.

First, we utilized aligned cardiomyocyte monolayers covered with unexcitable cardiac fibroblasts or human embryonic kidney-293 (HEK) cells that expressed similar levels of the gap junction protein connexin-45. These cells weakly coupled to cardiomyocytes and marginally slowed cardiac conduction only at high coverage density, while producing no other measurable electrophysiological changes in cardiomyocytes. In contrast, unexcitable HEK cells genetically engineered to stably express the more conductive connexin-43 channels (Cx43 HEK) strongly coupled to cardiomyocytes, depolarized cardiac resting membrane potential, significantly slowed impulse propagation, decreased maximum capture rate, and increased action potential duration (APD) at high coverage density. None of the studied unexcitable cells significantly altered conduction velocity anisotropy ratio or the relatively low incidence of pacemaking activity of cardiac monolayers at any coverage density.

Next, we utilized individual micropatterned cell pairs consisting of a cardiomyocyte and an unexcitable Cx43 HEK cell with or without stably overexpressed inward rectifier potassium channels (Kir2.1+Cx43 HEK). By systematically varying the relative sizes of micropatterned cells, we showed that Cx43 HEK cells significantly depolarized cardiomyocytes, reduced maximum upstroke velocity and action potential amplitude, prolonged APD, and modulated beating rate as a function of HEK:CM area ratio. In contrast, in cell pairs formed between cardiomyocytes and Kir2.1+Cx43 HEK cells we observed significant reduction in cardiomyocyte action potential amplitude, duration, and maximum upstroke velocity, but no change in other measured parameters.

Finally, we utilized a hybrid dynamic clamp setting consisting of a live micropatterned cardiomyocyte coupled in real time to a virtual model of capacitive and/or ionic current components of Cx43 HEK or Kir2.1+Cx43 HEK cells. We found that coupling of cardiomyocytes to the ionic current components of Cx43 HEK or Kir2.1+Cx43 HEK cells was sufficient to reproduce the dependence of cardiomyocyte maximal diastolic potential and pacemaking behavior on HEK:CM area ratio observed in micropatterned cell pairs, but did not replicate the observed changes in action potential upstroke or duration. The pure capacitance model with no ionic current, on the other hand, significantly decreased cardiomyocyte maximum upstroke velocity and prolonged cardiomyocyte APD as function of HEK:CM area ratio without affecting maximal diastolic potential or pacemaking behavior. When the unexcitable cell model containing both capacitive and ionic currents was connected to cardiomyocytes, all changes in action potential shape observed in micropatterned cell pairs were accurately reproduced.

These studies describe how coupling of unexcitable cells to cardiomyocytes can alter cardiomyocyte electrophysiological properties dependent on the unexcitable cell connexin isoform expression, ion channel expression, and cell size. This knowledge is expected to aid in the design of safe and efficient cell and gene therapies for myocardial infarction, fibrotic heart disease, and cardiac arrhythmias.

Tissue engineering of biomimetic skeletal muscle may lead to development of new therapies for myogenic repair and generation of improved in vitro models for studies of muscle function, regeneration, and disease. For the optimal therapeutic and in vitro results, engineered muscle should recreate the force-generating and regenerative capacities of native muscle, enabled respectively by its two main cellular constituents, the mature myofibers and satellite cells (SCs). Still, after 20 years of research, engineered muscle tissues fall short of mimicking contractile function and self-repair capacity of native skeletal muscle. To overcome this limitation, we set the thesis goals to: 1) generate a highly functional, self-regenerative engineered skeletal muscle and 2) explore mechanisms governing its formation and regeneration in vitro and survival and vascularization in vivo.

By studying myogenic progenitors isolated from neonatal rats, we first discovered advantages of using an adherent cell fraction for engineering of skeletal muscles with robust structure and function and the formation of a SC pool. Specifically, when synergized with dynamic culture conditions, the use of adherent cells yielded muscle constructs capable of replicating the contractile output of native neonatal muscle, generating >40 mN/mm2 of specific force. Moreover, tissue structure and cellular heterogeneity of engineered muscle constructs closely resembled those of native muscle, consisting of aligned, striated myofibers embedded in a matrix of basal lamina proteins and SCs that resided in native-like niches. Importantly, we identified rapid formation of myofibers early during engineered muscle culture as a critical condition leading to SC homing and conversion to a quiescent, non-proliferative state. The SCs retained natural regenerative capacity and activated, proliferated, and differentiated to rebuild damaged myofibers and recover contractile function within 10 days after the muscle was injured by cardiotoxin (CTX). The resulting regenerative response was directly dependent on the abundance of SCs in the engineered muscle that we varied by expanding starting cell population under different levels of basic fibroblast growth factor (bFGF), an inhibitor of myogenic differentiation. Using a dorsal skinfold window chamber model in nude mice, we further demonstrated that within 2 weeks after implantation, initially avascular engineered muscle underwent robust vascularization and perfusion and exhibited improved structure and contractile function beyond what was achievable in vitro.

To enhance translational value of our approach, we transitioned to use of adult rat myogenic cells, but found that despite similar function to that of neonatal constructs, adult-derived muscle lacked regenerative capacity. Using a novel platform for live monitoring of calcium transients during construct culture, we rapidly screened for potential enhancers of regeneration to establish that many known pro-regenerative soluble factors were ineffective in stimulating in vitro engineered muscle recovery from CTX injury. This led us to introduce bone marrow-derived macrophages (BMDMs), an established non-myogenic contributor to muscle repair, to the adult-derived constructs and to demonstrate remarkable recovery of force generation (>80%) and muscle mass (>70%) following CTX injury. Mechanistically, while similar patterns of early SC activation and proliferation upon injury were observed in engineered muscles with and without BMDMs, a significant decrease in injury-induced apoptosis occurred only in the presence of BMDMs. The importance of preventing apoptosis was further demonstrated by showing that application of caspase inhibitor (Q-VD-OPh) yielded myofiber regrowth and functional recovery post-injury. Gene expression analysis suggested muscle-secreted tumor necrosis factor-α (TNFα) as a potential inducer of apoptosis as common for muscle degeneration in diseases and aging in vivo. Finally, we showed that BMDM incorporation in engineered muscle enhanced its growth, angiogenesis, and function following implantation in the dorsal window chambers in nude mice.

In summary, this thesis describes novel strategies to engineer highly contractile and regenerative skeletal muscle tissues starting from neonatal or adult rat myogenic cells. We find that age-dependent differences of myogenic cells distinctly affect the self-repair capacity but not contractile function of engineered muscle. Adult, but not neonatal, myogenic progenitors appear to require co-culture with other cells, such as bone marrow-derived macrophages, to allow robust muscle regeneration in vitro and rapid vascularization in vivo. Regarding the established roles of immune system cells in the repair of various muscle and non-muscle tissues, we expect that our work will stimulate the future applications of immune cells as pro-regenerative or anti-inflammatory constituents of engineered tissue grafts. Furthermore, we expect that rodent studies in this thesis will inspire successful engineering of biomimetic human muscle tissues for use in regenerative therapy and drug discovery applications.

Voltage-gated sodium channels (VGSCs) enable generation and spread of action potentials in electrically excitable cells and tissues of all metazoans, from jellyfish to humans. The functional, pore-forming α-subunit of eukaryotic VGSCs is formed from a large polypeptide chain of ~2000 amino acids (~260 kDa), comprising four homologous domains. In humans, VGSC loss-of-function mutations are associated with various neuronal, cardiac, and skeletal muscle disorders characterized by a decrease or complete loss of tissue excitability. Similarly, permanent excitability loss due to acute tissue injuries (e.g. stroke, spinal cord injury, heart attack) could lead to long-term disability and death. Whilst an increase in sodium current through stable gene transfer could improve such conditions, eukaryotic VGSC genes are too large (>6 kbp) to be efficiently delivered to cells by existing viral vectors. In contrast, prokaryotic voltage-gated sodium channels (BacNav) consist of four identical subunits, individually transcribed and translated from single genes of only ~800 bp in size. Therefore, it is plausible that small BacNav genes can be efficiently packaged into viral vectors, either alone or with other ion channel genes, and used to stably introduce or modify electrical excitability of primary human cells. The objective of this thesis is thus to develop the methodology to screen, optimize, and assess BacNav channels as potential substitutes for eukaryotic VGSCs. Specifically, we sought to utilize engineered BacNav to create de novo excitable human tissues and to rescue impaired action potential conduction in vitro.

First, by using a monoclonal HEK293 line stably expressing the potassium channel Kir2.1 and gap junction channel Cx43, we were able to select, among various BacNav orthologs and variants, the channel NavRosD G217A that yielded action potential propagation with highest maximum capture rate. Lentiviral transduction of each of the three channels (NavRosD G217A, Kir2.1, and Cx43) into human fibroblasts yielded robust expression and expected electrical properties as confirmed by patch clamp recordings. By co-expressing all three channels, we were able for the first time to stably convert human fibroblasts into electrically excitable and actively conducting cells. However, the conduction velocity of engineered fibroblast tissue was low, largely due to the slow activation kinetics of NavRosD channel.

In order to improve the conduction properties of engineered fibroblasts, we shifted our focus to NavSheP channel, currently the fastest known BacNav ortholog. Due to the overly hyperpolarized voltage dependency of the wild-type NavSheP channel, we generated a library of NavSheP mutants exhibiting a wide range of shifts in voltage-dependent activation and inactivation and, with the guidance from computational modeling, identified three mutants that yielded ~2.5-fold increases in conduction velocity compared to NavRosD G217A. Importantly, we demonstrated that engineered fibroblasts retained stable functional properties despite extensive expansion or differentiation into myofibroblasts and exhibited strong viability while supporting AP propagation in 3D settings. Furthermore, in an in vitro model of interstitial fibrosis, engineered excitable and actively-conducting fibroblasts rescued impaired cardiac conduction to healthy level. These results strongly suggested that engineered fibroblasts could be used as a robust source for potential cell-based therapies for cardiac diseases.

In addition to the generation of excitable fibroblasts, BacNav channels could also serve as potential substitutes for impaired VGSC in various excitable tissue disorders. The channel NavSheP D60A (ShePA) was chosen for direct expression in mammalian excitable tissues as it yielded fastest conduction in previous studies. By performing codon optimization and adding appropriate endoplasmic-reticulum export signal, we were able to significantly improve membrane expression of ShePA channels. Expression of ShePA in excitable HEK293 tissue (Ex293) rescued impaired conduction upon membrane depolarization and decoupling. Furthermore, cultures of neonatal rat ventricular myocytes (NRVMs) transduced with ShePA virus exhibited enhanced conduction properties and increased resistance to conduction failure in an in vitro model of regional ischemia. Lastly, ShePA expression in highly-arrhythmogenic cardiomyocyte-fibroblast co-cultures led to significant reduction in incidence of reentry. Taken together, these results demonstrated the potential applications of engineered BacNav channels for cardiac gene therapies.

In summary, this dissertation presents the first experimental evidences supporting the use of prokaryotic sodium channels for the induction, control, and rescue of mammalian tissue excitability. The encouraging in vitro results shown in these studies will stimulate the development of BacNav-based therapies for the treatment of cardiac diseases. Furthermore, the experimental methodology developed in this work will serve as a useful framework for the screening, optimization, and assessment of engineered BacNav for specific therapeutic applications.

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.

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 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.

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.

Disruption of coordinated impulse propagation in the heart as a result of fibrosis or myocardial infarction can create an asynchronous substrate with poor conduction and impaired contractility. This can ultimately lead to cardiac failure and make the heart more vulnerable to life-threatening arrhythmias and sudden cardiac death. The transplantation of exogenous cells into the diseased myocardium, "cardiac cell therapy," has been proposed as a treatment option to improve compromised cardiac function. Clinical trials of stem cell-based cardiac therapy have shown promising results, but also raised concerns about our inability to predict or control the fate of implanted cells and the electrical consequences of their interactions with host cardiomyocytes. Alternatively, genetically engineered somatic cells could be implanted to selectively and safely modify the cardiac electrical substrate, but their unexcitable nature makes them incapable of electrically repairing large conduction defects. The objective of this thesis was thus to develop a methodology to generate actively conducting excitable cells from an unexcitable somatic cell source and to demonstrate their utility for studies of basic electrophysiology and cardiac cell therapy.

First, based on the principles of cardiac action potential propagation, we applied genetic engineering techniques to convert human unexcitable cells (HEK-293) into an autonomous source of excitable and conducting cells by the stable forced expression of only three genes encoding an inward rectifier potassium (Kir2.1), a fast sodium (Na_v1.5), and a gap junction (Cx43) channel. Systematic pharmacological and electrical pacing studies in these cells revealed the individual contributions of each expressed channel to action potential shape and propagation speed. Conduction slowing and instability of induced arrhythmic activity was shown to be governed by specific mechanisms of I_Na inhibition by TTX, lidocaine, or flecainide. Furthermore, expression of the Na_v1.5 A1924T mutant sodium channel or Ca_v3.3 T-type calcium channel was utilized to study the specific roles of these channels in action potential conduction and demonstrate that genetic modifications of the engineered excitable cells in this platform allow quantitative correlations between single-cell patch clamp data and tissue-level function.

We further performed proof-of-concept experiments to show that networks of biosynthetic excitable cells can successfully repair large conduction defects within primary excitable tissue cultures. Specifically, genetically engineered excitable cells supported active action potential propagation between neonatal rat ventricular myocytes (NRVMs) separated by at least 2.5 cm in 2-dimensional and 1.3 cm in 3-dimensional cocultures. Using elastic films with micropatterned zig-zag NRVM networks that mimicked the tortuous conduction patterns observed in cardiac fibrosis, we showed that electrical resynchronization of cardiomyocyte activation by application of engineered excitable cells improved transverse conduction by 370% and increased cardiac twitch force amplitude by 64%. This demonstrated that despite being noncontractile, engineered excitable cells could potentially improve both the electrical and mechanical function of diseased myocardial tissue.

Lastly, we investigated how activation and repolarization gradients at the interface between cardiomyocytes and other excitable cells influence the vulnerability to conduction block. Microscopic optical mapping of action potential propagation was used to quantify dispersion of repolarization (DOR) in micropatterned heterocellular strands in which either well-coupled or poorly-coupled engineered excitable cells with a short action potential duration (APD), seamlessly interfaced with NRVMs that had a significantly longer APD. The resulting electrical gradients originating from the underlying heterogeneity in intercellular coupling and APD dispersion were further manipulated by the application of barium chloride (BaCl2) to selectively prolong APD in the engineered cells. We measured how the parameters of DOR affected the vulnerable time window (VW) of conduction block and found a strong linear correlation between the size of the repolarization gradient and VW. Reduction of DOR by BaCl2 significantly reduced VW and showed that VW correlated directly with dispersion height but not width. Conversely, at larger DOR, VW was inversely correlated with the dispersion width but independent of the dispersion height. In addition, despite their similar APDs, poorly-coupled excitable cells were found to significantly increase the maximum repolarization gradient and VW compared to well-coupled excitable cells, but only at larger DOR.

In summary, this thesis presents the novel concept of genetically engineering membrane excitability and impulse conduction in previously unexcitable somatic cells. This biosynthetic excitable cell platform is expected to enable studies of ion channel function in a reproducible tissue-level setting, promote the integration of theoretical and experimental studies of action potential propagation, and stimulate the development of novel gene and cell-based therapies for myocardial infarction and cardiac arrhythmias.

Over recent decades, the continued clinical burden of ischemic heart disease has created a need for alternative therapies to improve the function of diseased myocardium. While the existing clinical interventions are often aimed at restoring blood supply to infarcted myocardium, they have been unable to effectively replace damaged muscle with healthy new tissue. Transplantation of a pre-formed, tissue-engineered cardiac muscle has been proposed as a potential strategy to remuscularize the infarcted heart and prevent adverse remodeling leading to heart failure. Human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) are currently the only robust cell source for engineering of functional cardiac tissues. Despite more than 15 years of research with hPSC-CMs, human engineered cardiac tissues still fall short of replicating the mature electrical and contractile properties of adult human myocardium important for safety and efficacy of the future therapies. Furthermore, most of engineered cardiac tissues are currently engineered to be avascular and are too small for use in clinical applications. As such, the primary goals of this thesis were to: 1) generate mature engineered cardiac tissues from human pluripotent stem cells, 2) scale-up engineered tissue size to clinically relevant dimensions without loss of function, and 3) pre-vascularize engineered cardiac tissues in vitro and test their survival and ability to remain functional in vivo.

To address these goals, we first utilized our established hydrogel-based method to engineer 3D human cardiac tissues (“Cardiopatches”) and determine the effects of cardiomyocyte purity and culture conditions on the tissue structure and function. After 2wks of culture, hPSC-CMs in 3D cardiopatches exhibited faster conduction velocity (CV), longer sarcomeres, and increased expression of genes involved in cardiac contractile function compared to hPSC-CMs cultured in age- and purity-matched 2D monolayers. Furthermore, higher hPSC-CM fraction in cardiopatches yielded faster CV, while maximum forces of contraction were achieved for a particular range of hPSC-CM purities (60-80%). Furthermore, engineered cardiopatches demonstrated a positive inotropic response to β-adrenergic stimulation and generated contractile stresses in excess of 10mN/mm2. These functional results were reproducibly achieved using 4 independent hPSC lines.

Through further optimization, we identified low seeding density and transition from serum-free to serum-containing media as critical factors for accelerated maturation in vitro. These modifications yielded cardiopatches with improved electrical and mechanical function, with average contractile stresses (>20 mN/mm2) and CVs (>28 cm/s) approaching values in adult myocardium. Ultrastructural analysis of cardiopatches revealed highly organized sarcomeric structures, characterized by consistent H-zone and I-bands, frequent intercalated discs, abundant mitochondria, and occasional appearance of T-tubules and central M-bands. Continual increase in functional output during 3-week cardiopatch culture was associated with significant upregulation of molecular maturation markers that, in many instances, reached near-adult levels of expression. Under these optimized conditions, we successfully scaled up engineered cardiopatches to clinically relevant dimensions (4x4cm), while preserving high CVs and contractile strength (with absolute forces exceeding 20 mN) and lacking spontaneous or pacing-induced arrhythmias.

Finally, we explored the ability of cardiopatches to survive and undergo vascularization in vivo using a dorsal skinfold window chamber model in immunocompromised mice and following epicardial implantation onto healthy rat hearts. Within 2 weeks post-implantation into window chambers, initially avascular cardiopatches underwent robust vascularization in vivo and maintained ability to fire Ca2+ transients, albeit at an expense of declined electrical function. In an attempt to improve the vascularization and function in vivo, we developed methods to pre-vascularize cardiopatches with highly branched vascular networks made of hPSC-derived endothelial cells (hPSC-ECs). Upon implantation in window chambers, hPSC-ECs in cardiopatches co-localized with host vasculature and formed hybrid microvessels, indicative of host-donor vascular integration. To further establish their translational potential, we implanted cardiopatches into the mechanically active environment of healthy rat hearts, and demonstrated their survival, vascular integration, and higher levels of electrical function relative to those in dorsal window chambers. Lastly, as a proof-of-concept study, we implanted cardiopatches into a porcine model of myocardial infarction and demonstrated evidence of hiPSC-CM survival, thus providing a foundation for future large animal studies aimed at clinical translation.

In summary, this thesis describes novel methodologies to engineer human cardiac tissues with near-adult levels of electrical and mechanical function, capacity for pre-vascularization, scalability to clinical dimensions, and ability to survive, vascularize, and remain functional in vivo. To the best of our knowledge, the reported functional parameters of cardiopatches are the highest in the field, and our scale-up and pre-vascularization of cardiopatches without loss of function are the first reported in the field. As such, this thesis represents a significant advancement in human heart tissue engineering research that will enable development of next generation cell-based therapies for cardiac repair.

Despite continued progress, therapies to augment contractile function and prevent arrhythmias in patients with ischemic and non-ischemic heart disease remain limited. With growing understanding of the complex molecular mechanisms underlying cardiac function and dysfunction, gene therapies have emerged as a promising strategy to treat and potentially cure heart diseases. Particularly, therapies for cardiac arrhythmias and heart failure could greatly benefit from approaches that can augment peak cardiac Na+ or Ca2+ current in cardiomyocytes (CMs) by stably overexpressing mammalian voltage-gated Na+ or Ca2+ channels. However, these channels are encoded by large (>6kb) genes that cannot be packaged in adeno-associated viral vectors (AAV; limit 4.7kb), currently a standard delivery vehicle for stable expression of exogenous genes in the heart. Unlike their mammalian counterparts, prokaryotic voltage-gated sodium channels (BacNav) are homotetrameric proteins encoded by genes that are <1kb in size, suitable for packaging in any type of recombinant viral vector, including AAV. Thus, the objective of this thesis has been to optimize and evaluate BacNav gene therapy as a novel strategy for the treatment of heart failure and cardiac arrhythmias. Towards this goal, we first performed proof-of-concept experiments to show that lentiviral transduction of BacNav into cultured neonatal rat ventricular myocytes (NRVMs) can yield robust expression of functional channels and significantly augment cardiac action potential conduction via increase in peak Na+ current. Moreover, in vitro BacNav gene therapy in fibrotic NRVM cell cultures reduced occurrence of conduction block and reentrant arrhythmias. Furthermore, we showed that functional BacNav channels can be stably expressed in healthy mouse hearts six weeks following their delivery by intravenous injection of self-complementary AAV (scAAV) vectors coding BacNav gene, which in turn caused no adverse effects on cardiac electrophysiology. These results collectively demonstrated that BacNav channels can be directly, specifically, and stably expressed in CMs through viral gene delivery to augment myocardial excitability and conduction. In addition to enhanced peak Na+ current amplitude and CM excitability, based on the principles of cardiac excitation-contraction coupling, we assessed the ability of BacNav expression to also augment Ca2+ transient amplitude and contractility of CMs, as a potential two-pronged gene therapy strategy for treatment of heart failure. In multi-species studies in vitro and ex vivo, we showed that expression of BacNav enhanced Ca2+ transient amplitude and contractility of neonatal rat, mouse, and human CMs in a dose-depend manner by modulating the activity of the Na+/Ca2+ exchanger and increasing sarcoplasmic reticulum Ca2+ stores. This mechanism of BacNav action was further corroborated in silico, using a computational model of rabbit cardiac action potential. Importantly, to further support translational potential of in vivo BacNav therapy, we showed that AAV9-mediated BacNav expression rescued contractile deficit and prevented arrhythmogenicity in the settings of chronic cardiac pressure-overload in mice and acute myocardial infarction in non-human primates (NHPs). Furthermore, we established the safety of systemic and intramyocardial delivery of AAV9-BacNav in mice and NHPs, demonstrating the promise of BacNav gene delivery as a novel therapy for heart failure. Finally, we functionally screened previously uncharacterized BacNav variants, aiming to expand the therapeutic BacNav pool with novel channel candidates exhibiting faster gating kinetics that better resembles kinetics of mammalian cardiac Nav channels. One of the identified BacNav candidates from this screen, NavRhi, demonstrated improved gating kinetics and gain-of-excitability properties compared to our previously characterized NavSheP channel. We further optimized the membrane expression of NavRhi by comparing different human codon-optimized sequences, with additional enhancements achieved by creating a multicistronic vector incorporating two copies of NavRhi. By incorporating an Ankyrin G binding motif at the channel C-terminus, we also improved trafficking of NavRhi to the lateral membrane and intercalated disk, with the potential to further enhance cardiac conduction in vivo. In summary, this dissertation presents a novel therapeutic strategy to genetically augment cardiac conduction and contraction by revealing a unique dual-action mechanism of BacNav expression in cardiomyocytes. The encouraging in vivo results using diverse heart failure etiologies (pressure overload, myocardial infarction), animal models (mouse, NHP) and gene delivery routes (intravenous, intramyocardial) pave the way for future clinical translation of BacNav-based therapies for the treatment of heart disease. Moreover, this work establishes a comprehensive multi-species preclinical platform which will be instrumental in designing and validating innovative therapeutic strategies for heart failure and conduction disorders, offering a translational pipeline towards future clinical applications.