京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第19595号 / 医博第4102号 / 新制||医||1014(附属図書館) / 32631 / 京都大学大学院医学研究科医学専攻 / (主査)教授 山下 潤, 教授 瀬原 淳子, 教授 前川 平 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

There is scarce information on the fate of cardiac progenitor cells (CPC) in the embryonic heart after chamber specification. Furthermore, the role of acetylcholinesterase (AChE) during heart development is unknown, despite record of its presence in the myocardium. Although three molecular variants of AChE (R, H and T) exist due to alternate splicing, temporal and spatial distribution of these splice variants during cardiac ontogeny is not well characterized. We hypothesized that the AChE “R” splice variant (AChE-R) is involved in directing lineage commitment of mouse ventricular CPCs to the conduction cell phenotype. It is possible that AChE may promote the breakdown of ACh and block the effects of ligand-binding via M2 receptors present on the surface of CPCs. Our study has also provided a platform to suggest that AChE may play a role in the molecular mechanisms underlying functional diversification of myocardial cells into conduction system cells during ontogenesis.

α-Crystallin B (cryAB) is the most abundant small heat shock protein in cardiomyocytes (CMs), where it has been shown to have potent anti-apoptotic properties. The mechanism by which cryAB prevents apoptosis has not been fully characterized. Therefore, I was interested in elucidating its protective mechanism in CMs. I identified its sub-cellular localization and its binding interactors following H2O2 exposure. I found that cryAB is found in the cytosol under control conditions and that following H2O2 exposure it becomes phosphorylated and translocates to the mitochondria. CryAB silencing resulted in increased apoptosis levels in CMs. Co-immunoprecipitation revealed an apparent increased interaction of cryAB and PcryAB with mitochondrial VDAC, caspase 12 and uncleaved caspase 3 in stressed hearts relative to controls. These results suggest that the cardio-protective effects of cryAB are mediated by its translocation to the mitochondria and its interaction with VDAC, caspase 12 and caspase 3 following exposure to H2O2.

α-Crystallin B (cryAB) is the most abundant small heat shock protein in cardiomyocytes (CMs), where it has been shown to have potent anti-apoptotic properties. The mechanism by which cryAB prevents apoptosis has not been fully characterized. Therefore, I was interested in elucidating its protective mechanism in CMs. I identified its sub-cellular localization and its binding interactors following H2O2 exposure. I found that cryAB is found in the cytosol under control conditions and that following H2O2 exposure it becomes phosphorylated and translocates to the mitochondria. CryAB silencing resulted in increased apoptosis levels in CMs. Co-immunoprecipitation revealed an apparent increased interaction of cryAB and PcryAB with mitochondrial VDAC, caspase 12 and uncleaved caspase 3 in stressed hearts relative to controls. These results suggest that the cardio-protective effects of cryAB are mediated by its translocation to the mitochondria and its interaction with VDAC, caspase 12 and caspase 3 following exposure to H2O2.

Mathematical modeling has been instrumental to the development of natural sciences over the last half-century. Through iterated interactions between modeling and real-world exper- imentation, these models have furthered our understanding of the processes in biology and chemistry that they seek to represent. In certain application domains, such as the field of car- diac biology, communities of modelers with common interests have emerged, leading to the development of many models that attempt to explain the same or similar phenomena. As these communities have developed, however, reporting standards for modeling studies have been in- consistent, often focusing on the final parameterized result, and obscuring the assumptions and data used during their creation. These practices make it difficult for researchers to adapt exist- ing models to new systems or newly available data, and also to assess the identifiability of said models - the degree to which their optimal parameters are constrained by data - which is a key step in building trust that their formulation captures truth about the system of study. In this thesis, we develop tools that allow modelers working with biological or chemical time series data to assess identifiability in an automated fashion, and embed these tools within a novel online community resource that enforces reproducible standards of reporting and facilitates exchange of models and data. We begin by exploring the application of Bayesian and approximate Bayesian inference methods, which parameterize models while simultaneously assessing uncertainty about these estimates, to assess the identifiability of models of the cardiac action potential. We then demon- strate how the side-by-side application of these Bayesian and approximate Bayesian methods can be used to assess the information content of experiments where system observability is limited to "summary statistics" - low-dimensional representations of full time-series data. We next investigate how a posteriori methods of identifiability assessment, such as the above inference techniques, compare against a priori methods based on model structure. We compare these two approaches over a range of biologically relevant experimental conditions, and high- light the cases under which each strategy is preferable. We also explore the concept of optimal experimental design, in which measurements are chosen in order to maximize model identifia- bility, and compare the feasibility of established a priori approaches against a novel a posteriori approach. Finally, we propose a framework for representing and executing modeling experiments in a reproducible manner, and use this as the foundation for a prototype "Modeling Web Lab" where researchers may upload specifications for and share the results of the types of inference explored in this thesis. We demonstrate the Modeling Web Lab's utility across multiple mod- eling domains by re-creating the results of a contemporary modeling study of the hERG ion channel model, as well as the results of an original study of electrochemical redox reactions. We hope that this works serves to highlight the importance of both reproducible standards of model reporting, as well as identifiability assessment, which are inherently linked by the desire to foster trust in community-developed models in disciplines across the natural sciences.

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第21455号 / 医博第4422号 / 新制||医||1033(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 山下 潤, 教授 湊谷 謙司, 教授 浅野 雅秀 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

Advancements in regenerative medicine have opened new possibilities for treating cardiovascular diseases. Using stem cell-derived cardiac cells has shown great promise in regenerating damaged heart tissue. However, the efficacy of this approach is limited by the inability to culture, differentiate, and mature these cells in a controlled and efficient manner. This work addresses some of these challenges by developing new tools and techniques for the culture and differentiation of human stem cell-derived cardiomyocytes. To address the above issues, we developed a novel bioreactor to deliver electrical stimulation and fluid mixing for enhanced nutrient transfer to improve the differentiation and maturation of stem cell-derived cardiomyocytes. This bioreactor was designed using computation modeling to optimize the applied electrical stimulation and fluid flow and constructed using low-cost, 3D-printed materials. Electrical stimulation in the bioreactor improves the differentiation and maturation of cardiomyocytes. Specifically, we tested how electrical stimulation can influence the subtype determination of stem cell-derived cardiomyocytes in vitro. In addition, we have developed conductive biomaterials in the form of transparent conductive films and conductive nanofibers to further aid in the maturation of cardiomyocytes. Overall, this study represents a significant step forward in developing new tools and techniques for the culture and differentiation of stem cell-derived cardiac cells. The bioreactor and conductive biomaterials developed in this study have the potential to improve the efficiency and effectiveness of stem cell-based therapies for the treatment of cardiovascular diseases, and the results of electrical stimulation experiments provide essential insights into the optimal stimulation parameters for the differentiation and maturation of stem cell-derived cardiac cells. Further research is needed to optimize these techniques and translate them into clinical practice, but this study provides an important foundation for future work in this area. / Bioengineering / Accompanied by one .zip file : 1) Licata_temple_0225E_171Supplemental_Videos.zip

Introdução:O papel das biopsias do miocárdio, tem tido relativa importância em cardiologia, sendo método diagnóstico fundamental em um pequeno número de patologias do coração como na doença por depósito de glicogênio, amiloidose, hemocromatose e nas miocardites. Ao longo dos anos, o desenvolvimento de diferentes procedimentos que possibilitam a obtenção de fragmentos do tecido cardíaco passou por diversos estágios e evoluída miocardiectomia a céu-aberto aos cateteres endovasculares, passando por procedimentos com agulhas de punção. Estes apesar de estar em desuso atualmente, têm particular importância por proporcional acesso ao miocárdio e a cavidade ventricular esquerda. Paralelamente, a terapia celular tem sido utilizada na recuperação e preservação da função cardíaca em coronariopatia crônica e na doença de chagas. As punções biópsias do coração ressurgem como possível método alternativo de acesso ao miocárdio e implante de material biológico para terapia celular. Objetivos:apresentar instrumento de punção, biopsia e injeção intramiocárdica de material biológico, padronizar a técnica e atestar a segurança do método.A adaptação consiste em um sistema de escarificação do miocárdio para permitir melhor fixação de células-tronco. O objetivo do presente trabalho visa apenas o desenvolvimento da agulha e testar histologicamente a qualidade das biópsias. Método: O instrumento para punção e injeção de material biológico é composto por uma agulha exterior (1), chamado de acoplamento de infusão, o qual contém na sua extremidade uma ponta romba (2) e vários orifícios de 0,5 mm de diâmetro (3). Internamente, está equipado com uma agulha com um mandril fechado, que quando introduzidas no exterior, pode ser mobilizada para dentro para encher os orifícios laterais ocluindo ou soltá-los. O procedimento para a produção de microlesões micro é feito através da troca do mandril de ponta romba (durante o procedimento) por um mandril escova (4), provido de microcerdas que são estruturalmente concebidas para preencher os orifícios com a exteriorização das pequenas cerdas (5). O instrumento está equipado com um mecanismo de bloqueio, que permite a sua mobilização perfeita como uma única unidade de microlesões ou, ainda pode ser utilizado somente como uma agulha externa, de modo que pode tornar-se um instrumento de injeção biológica. Resultado: A técnica já foi testada em modelo suíno vivo mostrando sua viabilidade e segurança. Como resultados são apresentados aspectos macroscópicos e microscópicos do coração (Corantes Hematoxilina eosina, Tricrômico Masson e Azul de Evans).Conclusão: No tocante a sua função o novo Instrumento de punção/infusão tem por característica principal o fato de ser multifuncional. Permite ao operador acessar a cavidade ventricular esquerda por via transtorácica sem risco de lesão (perfuração), das artérias coronárias. Permite penetrar o miocárdio sem laceração das fibras musculares pela divulsionamento das mesmas e escarificar o miocárdio. gerando micro-lesões musculares por intermédio de seu mandril com cerdas, promovendo a \"inflamação benéfica ao processo de transplante celular. / Introduction: The role of myocardial biopsy has had relative importance in cardiology, being fundamental diagnostic method in a small number of diseases of the heart as in glycogen deposit disease, amyloidosis, hemochromatosis and in miocardites. Over the years, the development of different procedures that allow obtaining cardiac tissue fragments went through several stages and evolved from open miocardiectomy to endovascular catheters, going through procedures with puncture needles. These despite being in disuse today, have particular importance for offering access to myocardium and left ventricular cavity. At the same time, the Cellular therapy has been used in the recovery and preservation of cardiac function in chronic coronary artery disease and Chagas disease. The puncture-heart biopsies to re-emerge as a possible alternative method of access to the myocardium and implantable biomaterial for cell therapy. Objectives: Objectives: to present puncture tool, biopsy and intramyocardial injection of biological material, standardizing the technical and certify the safety of the method. The adaptation allows in a myocardial scarification system for making possible a better stem cells fixation. The objective of this study covers only the development of needle and test, macroscopically and histologically the quality of biopsies. Methods: The instrument for puncturing and injection of biological material is composed of an external needle (1), called coupling infusion, which contains at its end a blunt tip (2) and multiple 0.5 mm diameter holes (3). Internally it is fitted with a blunt mandrill needle, which when introduced into the external, can be mobilized inside to fill the lateral holes occluding or releasing them. The procedure for producing micro lesions is done by exchanging the blunt mandrill (during the procedure) for a brush-mandrill (4), provided with micro bristles that are structurally designed to fill the holes with small exteriorization of bristles (5). The instrument is equipped with a locking mechanism, which allows its perfect mobilization as one single unit for micro lesions or it can be used only as an external needle so it can become a biological injection instrument.Result: The technique has been tested in vivo pig model showing its feasibility and safety. The results are presented through macroscopic and microscopic aspects of the heart (dyes hematoxylin eosin, Masson Masson and Evans blue).Conclusion: Regarding its function the new instrument is to be multifunctional main feature. It allows the operator to access the left ventricular cavity through transthoracic without risk of injury (perforation) of the coronary arteries. It allows penetrate the myocardial laceration of the muscle fibers by divulsionamento of them and rip the myocardium. generating muscle micro-injuries through its arbor with bristles, promoting \"inflammation beneficial to the cell transplant process.Key words: heart biopsy puncture, infusion of stem cells in the heart.

<p>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.</p><p>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.</p><p>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. </p><p>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.</p><p>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.</p> / Dissertation

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