<p>Acoustic radiation force impulses (ARFI) have been used to generated transverse-traveling mechanical waves in various biological tissues. The velocity of these waves is related to a medium's stiffness and thus can offer useful diagnostic information. Consequently, shear wave velocimetry has the potential to investigate cardiac disease states that manifest themselves as changes in tissue stiffness (e.g., ischemia).</p><p> The work contained herein focuses on employing ARFI-based shear wave velocimetry techniques, similar to those previously utilized on other organs (e.g., breast, liver), for the investigation of cardiac tissue. To this end, ARFI excitations were used to generate slow-moving (under 3 m/s) mechanical waves in exposed myocardium (with access granted through a thoracotomy); these waves were then tracked with ultrasonic methods. Imaging techniques to increase frame-rate, decrease transducer/tissue heating, and reduce the effects of physiological motion were developed. These techniques, along with two shear wave velocimetry methods (i.e., the Lateral Time-to-Peak and Radon sum transformation algorithms), were utilized to successfully track shear wave propagation through the mid-myocardial layer <italic>in vitro</italic> and <italic>in vivo</italic>. <italic>In vitro</italic> experiments focused on the investigation of a shear wave anisotropy through the myocardium. This experimentation suggests a moderate shear wave velocity anisotropy through regions of the mid-myocardial layer. <italic>In vivo</italic> experiments focused on shear wave anisotropy (which tend to corroborate the aforementioned <italic>in vitro</italic> results), temporal/spatial stability of shear wave velocity estimates, and estimation of wave velocity through the cardiac cycle. Shear wave velocity was found to cyclically vary through the cardiac cycle, with the largest estimates occurring during systole and the smallest occurring during diastole. This result suggests a cyclic stiffness variation of the myocardium through the cardiac cycle. A novel, on-axis technique, the displacement ratio rate (DRR) method, was developed and compared to conventional shear wave velocitmetry and ARFI imaging results; all three techniques suggest a similar cyclic stiffness variation.</p><p> Shear wave velocimetry shows promise in future investigations of myocardial elasticity. The DRR method may offer a means for transthoracic characterization of myocardial stiffness. Additionally, the future use of transesophageal and catheter-based transducers presents a way of generating and tracking shear waves in a clinical setting (i.e., when epicardial imaging is not feasible). Lastly, it is hoped that continued investigations into the physical basis of these ARFI-generated mechanical waves may further clarify the relationship between their velocity in myocardium and material stiffness.</p> / Dissertation

Cardiovascular disease is the leading cause of death in the US, which can result in blockage of a coronary artery, triggering a myocardial infarction (MI). After a MI, hypoxic ventricular myocardial tissue dies, resulting in the deposition of non-contractile scar tissue and remodeling of the ventricle, leading to decreased cardiac output and ultimately heart failure. Currently, the gold-standard solution for total heart failure is a heart transplant. As donor hearts are in short supply, an alternative to total-organ transplantation is surgically remodeling the ventricle with the implantation of a cardiac patch. Acellular cardiac patches have previously been investigated using synthetic or decellularized native materials in effort to improve cardiac function. However, a limitation of this strategy is that acellular cardiac patches only reshape the ventricle and do not increase cardiac contractile function. By incorporating the use of a clinically relevant cell type and by matching native architecture, we propose the use of a highly aligned fibrin scaffold to support the maturation of human induced pluripotent stem cell cardiomyocytes (hiPS-CM) for use as a cell-populated cardiac patch. By micropatterning fibrin hydrogels, hiPS-CM seeded on the surface of this scaffold become highly aligned, which is crucial for increased contractile output. Our lab previously developed a composite fibrin hydrogel and microthread cardiac patch matching mechanical properties of native myocardium. By micropatterning fibrin hydrogel alone, we were able to match cellular alignment of hiPS-CM to that of native myocardium. hiPS-CMs seeded on this surface were found to express distinct sarcomere alignment and circumferential connexin-43 staining at 14 days of culture as well as cellular elongation, which are necessary for mature contractile properties. Constructs were also cultured under electrical stimulation to promote increased contractile properties. After 7 days of stimulation, contractile strains of micropatterned constructs were significantly higher than unpatterned controls. These results suggest that the use of topographical cues on fibrin scaffolds may be a promising strategy for creating engineered myocardial tissue to repair damaged myocardium.

Diseases of the heart have been the most common cause of death in the United States since the middle of the 20th century. The development of engineered cardiac tissue over the last three decades has yielded human induced pluripotent stem cell-derived (hiPSC) cardiomyocytes (CMs), microscale “heart-on-a-chip” platforms, optical interrogation techniques, and more. Having spawned its own scientific field, ongoing research promises lofty goals to address the heart disease burden around the world, such as patient-specific disease models, and clinical trials on chip-based platforms. The greatest academic pursuit for engineered cardiac tissues is to increase their maturity, thereby increasing relevance to native adult tissue. Investigation of cardiomyocyte maturity necessitates the development of 3D-tissue compatible techniques for measuring and perturbing cardiac biology with enhanced precision. This dissertation focuses on the development of biological microelectromechanical systems (BioMEMS) for precision measurement and perturbation of cardiac tissue. We discuss three unique approaches to interfacing MEMS-based tools with cardiac biology. The first is a high resolution magnetic sensor, which directly measures the spatial gradient of a magnetic field. This has an ideal application in magnetocardiography (MCG), as the flux of ions during cardiac contractions produces measurable magnetic signals around the tissue and can be leveraged for noncontact diagnosis. The second is a highly functionalized heart-on-a-chip platform, wherein the mechanical contractions of cardiac microtissues can be simultaneously recorded and actuated. Contractile dynamics are leading indicators of maturity in engineered cardiac tissue and mechanical conditioning has shown recent promise as a critical component of cardiac maturation. The third is the imaging of contractile nanostructures in engineered cardiomyocytes at depth in a 3D microtissue. We use small angle X-ray scattering (SAXS) to discern the periodic arrangement of myofilaments in their native 3D environment. We enable a significant structural analysis to provide insight for functional maturation. Enabling these three thrusts required developing two supporting technologies. The first is the engineered control of dynamic second order systems, a foundational element of all our MEMS and magnetic techniques. We demonstrate numerous algorithms to improve settling time or decrease dead-time such that samples with fast temporal effects can be measured. The second is a microscale gluing technique for integrating myriad of materials with MEMS devices, yielding unique sensors and actuators. / 2022-05-15T00:00:00Z

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第20972号 / 医博第4318号 / 新制||医||1026(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 長船 健二, 教授 渡邊 直樹, 教授 江藤 浩之 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

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This thesis is devoted to the study of the dynamics of spiral and scroll waves in a mathematical model for cardiac tissue. We study the effects of the presence of heterogeneities on electrical-wave dynamics. The heterogeneities in the medium occur because of the variation in the electrophysiological properties of the constituent myocytes in the tissue, or because of the presence of cells like fibroblasts and pathological myocytes that can trigger early afterdepolarizations (EADs). We study how these heterogeneities can lead to the formation of spiral and scroll waves and how they can affect the stability of the spiral and scroll waves in cardiac tissue. We also investigate the role of abnormal cells, which can trigger pathological excitations like EADs, on the formation of spiral and scroll waves, and how such cells can trigger premature electrical pulses like premature-ventricular-complexes (PVCs) in cardiac tissue. Earlier studies have examined the role of ionic heterogeneities on spiral-wave initiation and their effects on spiral-wave stability. However, none of these studies has calculated, in a controlled way, the effects of individual ion-channel conductances on spiral- and scroll-wave properties, such as the frequency of these waves, and the effects of the spatial gradients, in each ion-channel conductance, on their stability; we present these results in Chapter 2. Although many studies in the past have studied the effects of fibroblast coupling on wave-dynamics in cardiac tissue, a detailed study of spiral-wave dynamics in a medium with a well-defined, heterogeneous distribution of fibroblasts (e.g., with a gradient in the fibroblast density (GFD)) has not been performed; therefore, in Chapter 3 we present the effects of such GFD on spiral- and scroll-wave dynamics. Then, in Chapter 4, we present a systematic study of how a clump of fibroblasts can lead to spiral waves via high-frequency pacing. Some studies in the past have studied the role of early afterdepolarizations (EADs) in the formation of arrhythmias in cardiac tissue; we build on such studies and present a detailed study of the effects of EADs on the formation of spiral waves and their dynamics, in Chapter 5. Finally, in Chapter 6 we provide the results of our detailed investigation of the factors that assist the triggering of abnormal electrical pulses like premature ventricular complexes by a cluster of EAD-capable cells. A brief summary of the chapters is provided below: Chapter 2: In this chapter we investigate the effects of spatial gradients in the ion-channel conductances of various ionic currents on spiral-and scroll-wave dynamics. Ionic heterogeneities in cardiac tissue arise from spatial variations in the electrophysiological properties of cells in the tissue. Such variations, which are known to be arrhythmogenic, can be induced by diseases like ischemia. It is important, therefore, to understand the effects of such ionic heterogeneities on electrical-wave dynamics in cardiac tissue. To investigate such effects systematically, of changing the ion-channel properties by modifying the conductances of each ionic currents, on the action-potential duration (APD) of a myocyte cell. We then study how these changes in the APD affect the spiral-wave frequency ω in two-dimensional tissue. We also show that changing the ion-channel conductance not only changes ω but also the meandering pattern of the spiral wave. We then study how spatial gradients in the ion-channel conductances affect the spiral-wave stability. We find that the presence of this ionic gradient induces a spatial variation of the local ω, which leads to an anisotropic reduction of the spiral wavelength in the low-ω region and, thereby, leads to a breakup of the spiral wave. We find that the degree of the spiral-wave stability depends on the magnitude of the spatial variation in ω, induced by the gradient in the ion-channel conductances. We observe that ω varies most drastically with the ion-channel conductance of rapid delayed rectifier K+ current GKr, and, hence, a spiral wave is most unstable in the presence of a gradient in GKr (as compared to other ion-channel conductances). By contrast, we find that ω varies least prominently with the conductances of the transient outward K+ current Gto and the fast inward Na+ current (GNa); hence, gradients in these conduc-tances are least likely to lead to spiral-wave breaks. We also investigate scroll-wave instability in an anatomically-realistic human-ventricular heart model with an ionic gradient along the apico-basal direction. Finally, we show that gradients in the ion-channel densities can also lead to spontaneous initiation of spiral waves when we pace the medium at high frequency. Chapter 3: In this chapter we study the effects of gradients in the density of fibroblasts on wave-dynamics in cardiac tissue. The existence of fibroblast-myocyte coupling can modulate electrical-wave dynamics in cardiac tissue. In diseased hearts, the distribution of fibroblasts is heterogeneous, so there can be gradients in the fibroblast density (henceforth we call this GFD) especially from highly injured regions, like infarcted or ischemic zones, to less-wounded regions of the tissue. Fibrotic hearts are known to be prone to arrhythmias, so it is important to understand the effects of GFD in the formation and sustenance of arrhythmic re-entrant waves, like spiral or scroll waves. Therefore, we investigate the effects of GFD on the stability of spiral and scroll waves of electrical activation in a state-of-the-art mathematical model for cardiac tissue in which we also include fibroblasts. By introducing GFD in controlled ways, we show that spiral and scroll waves can be unstable in the presence of GFDs because of regions with varying spiral or scroll-wave frequency ω, induced by the GFD. We examine the effects of the resting membrane potential of the fibroblast and the number of fibroblasts attached to the myocytes on the stability of these waves. Finally, we show that the presence of GFDs can lead to the formation of spiral waves at high-frequency pacing. Chapter 4: In this chapter we study the arrhythmogenic effects of lo-calized fibrobblast clumps. Localized heterogeneities, caused by the regional proliferation of fibroblasts, occur in mammalian hearts because of diseases like myocardial infarction. Such fibroblast clumps can become sources of pathological reentrant activities, e.g., spiral or scroll waves of electrical activation in cardiac tissue. The occurrence of reentry in cardiac tissue with heterogeneities, such as fibroblast clumps, can depend on the frequency at which the medium is paced. Therefore, it is important to study the reentry-initiating potential of such fibroblast clumps at different frequencies of pacing. We investigate the arrhythmogenic effects of fibroblast clumps at high- and low-frequency pacing. We find that reentrant waves are induced in the medium more prominently at high-frequency pacing than with low-frequency pacing. We also study the other factors that affect the potential of fibroblast clumps to induce reentry in cardiac tissue. In particular, we show that the ability of a fibroblast clump to induce reentry depends on the size of the clump, the distribution and percentage of fibroblasts in the clump, and the excitability of the medium. We study the process of reentry in two-dimensional and a three-dimensional mathematical models for cardiac tissue. Chapter 5: In this chapter we investigate the role of early afterdepolarizations (EADs) on the formation of spiral and scroll waves. Early after depolarizations, which are abnormal oscillations of the membrane poten-tial at the plateau phase of an action potential, are implicated in the de-velopment of cardiac arrhythmias like Torsade de Pointes. We carry out extensive numerical simulations of the TP06 and ORd mathematical models for human ventricular cells with EADs. We investigate the different regimes in both these models, namely, the parameter regimes where they exhibit (1) a normal action potential (AP) with no EADs, (2) an AP with EADs, and (3) an AP with EADs that does not go back to the resting potential. We also study the dependence of EADs on the rate of at which we pace a cell, with the specific goal of elucidating EADs that are induced by slow or fast rate pacing. In our simulations in two- and three-dimensional domains, in the presence of EADs, we find the following wave types: (A) waves driven by the fast sodium current and the L-type calcium current (Na-Ca-mediated waves); (B) waves driven only by the L-type calcium current (Ca-mediated waves); (C) phase waves, which are pseudo-travelling waves. Furthermore, we compare the wave patterns of the various wave-types (Na-Ca-mediated, Ca-mediated, and phase waves) in both these models. We find that the two models produce qualitatively similar results in terms of exhibiting Na-Ca- mediated wave patterns that are more chaotic than those for the Ca-mediated and phase waves. However, there are quantitative differences in the wave patterns of each wave type. The Na-Ca-mediated waves in the ORd model show short-lived spirals but the TP06 model does not. The TP06 model supports more Ca-mediated spirals than those in the ORd model, and the TP06 model exhibits more phase-wave patterns than does the ORd model. Chapter 6: In this chapter we study the role of EAD-capable cells, and fibroblasts on the trigerring of abnormal electrical pulses called premature ventricular complexes (PVCs). Premature ventricular complexes, which are abnormal impulse propagations in cardiac tissue, can develop because of various reasons including early afterdepolarizations (EADs). We show how a cluster of EAD-generating cells (EAD clump) can lead to PVCs in a model of cardiac tissue, and also investigate the factors that assist such clumps in triggering PVCs. In particular, we study, through computer simulations, the effects of the following factors on the PVC-triggering ability of an EAD clump: (1) the repolarization reserve (RR) of the EAD cells; (2) the size of the EAD clump; (3) the coupling strength between the EAD cells in the clump; and (4) the presence of fibroblasts in the EAD clump. We find that, although a low value of RR is necessary to generate EADs and hence PVCs, a very low value of RR leads to low-amplitude EAD oscillations that decay with time and do not lead to PVCs. We demonstrate that a certain threshold size of the EAD clump, or a reduction in the coupling strength between the EAD cells, in the clump, is required to trigger PVCs. We illustrate how randomly distributed inexcitable obstacles, which we use to model collagen deposits, affect PVC-triggering by an EAD clump. We show that the gap-junctional coupling of fibroblasts with myocytes can either assist or impede the PVC-triggering ability of an EAD clump, depending on the resting membrane potential of the fibroblasts and the coupling strength between the myocyte and fibroblasts. We also find that the triggering of PVCs by an EAD clump depends sensitively on factors like the pacing cycle length and the distribution pattern of the fibroblasts.

The heart does not regenerate new functional tissue when myocardium dies following coronary artery occlusion, or is defective. Ventricular restoration involves excising the infarct and replacing it with a cardiac patch to restore the heart to a more efficient condition. The goal of this study was to design and develop a myocardial patch to replace myocardial infarctions. A basic design was developed that is composed of 3D microfibrous mats that house mesenchymal stem cells (MSCs) from umbilical cord matrix (Wharton&rsquo / s Jelly) aligned parallel to each other, and biodegradable macroporous tubings to supply growth media into the structure. Poly(glycerol sebacate) (PGS) prepolimer was synthesized and blended with P(L-D,L)LA and/or PHBV, to produce aligned microfiber (dia 1.16 - 1.37 &amp / #956 / m) mats and macroporous tubings. Hydrophilicity and softness of the polymer blends were found to be improved as a result of PGS introduction. The Wharton&rsquo / s Jelly (WJ) MSCs were characterized by determination of their cell surface antigens with flow cytometry and by differentiating them into cells of mesodermal lineage (osteoblasts, adipocytes, chondrocytes). Cardiomyogenic differentiation potential of WJ MSCs in presence of differentiation factors was studied with RT-PCR and immunocytochemistry. WJ MSCs expressed cardiomyogenic transcription factors even in their undifferentiated state. Expression of a ventricular sarcomeric protein was observed upon differentiation. The electrospun, aligned microfibrous mats of PHBV-P(L-D,L)LA-PGS blends allowed penetration of WJ MSCs and improved cell proliferation. To obtain the 3D myocardial graft, the WJ MSCs were seeded on the mats, which were then wrapped around macroporous tubings. The 3D construct (4 mm x 3.5 cm x 2 mm) was incubated in a bioreactor and maintained the uniform distribution of aligned cells for 2 weeks. The positive effect of nutrient flow within the 3D structure was significant. This study represents an important step towards obtaining a thick, autologous myocardial patch, with structure similar to native tissue and capability to grow, for ventricular restoration.

In this work, a new polyurethane (PU) chain extender was developed to incorporate a Glycine-Leucine (Gly-Leu) dipeptide, the cleavage site of several matrix metalloproteinases. PUs were synthesized with either the Gly-Leu-based chain extender (Gly-Leu PU) or a phenylalanine-based chain extender (Phe PU). Both PUs had high molecular weight averages (Mw > 125,000 g/mol) and were phase segregated, semi-crystalline polymers (Tm ~ 42°C) with a low soft segment glass transition temperature (Tg < -50°C). Uniaxial tensile testing of PU films revealed that the polymers could withstand high ultimate tensile strengths (~ 8-13 MPa) and were flexible with breaking strains of ~ 870-910% but the two PUs exhibited a significant difference in mechanical properties. The Phe and Gly-Leu PUs were electrospun into porous scaffolds for degradation and cell-based studies. Fibrous Phe and Gly-Leu PU scaffolds were formed with randomly organized fibers and an average fiber diameter of approximately 3.6 µm. In addition, the Phe PU was electrospun into scaffolds of varying architecture to investigate how fiber alignment affects the orientation response of cardiac cells. To achieve this, the Phe PU was electrospun into aligned and unaligned scaffolds and the physical, thermal, and mechanical properties of the scaffolds were investigated. The degradation of the Phe and Gly-Leu PU scaffolds was investigated in the presence of active MMP-1, active MMP-9, and a buffer solution over 28 days to test MMP-mediated and passive hydrolysis of the PUs. Mass loss and structural assessment suggested that neither PU experienced significant hydrolysis to observe degradation over the course of the experiment. In cell-based studies, Phe and Gly-Leu PU scaffolds successfully supported a high density of viable and adherent mouse embryonic fibroblasts (MEFs) out to at least 28 days. Culturing murine embryonic stem cell-derived cardiomyocytes (mESCDCs) alone and with MEFs on aligned and unaligned Phe PU scaffolds revealed both architectures supported adherent and functionally contractile cells. Importantly, fiber alignment and coculture with MEFs improved the organization and differentiation of mESCDCs suggesting these two parameters are important for developing engineered myocardial constructs using mESCDCs and PU scaffolds.

In this work, a new polyurethane (PU) chain extender was developed to incorporate a Glycine-Leucine (Gly-Leu) dipeptide, the cleavage site of several matrix metalloproteinases. PUs were synthesized with either the Gly-Leu-based chain extender (Gly-Leu PU) or a phenylalanine-based chain extender (Phe PU). Both PUs had high molecular weight averages (Mw > 125,000 g/mol) and were phase segregated, semi-crystalline polymers (Tm ~ 42°C) with a low soft segment glass transition temperature (Tg < -50°C). Uniaxial tensile testing of PU films revealed that the polymers could withstand high ultimate tensile strengths (~ 8-13 MPa) and were flexible with breaking strains of ~ 870-910% but the two PUs exhibited a significant difference in mechanical properties. The Phe and Gly-Leu PUs were electrospun into porous scaffolds for degradation and cell-based studies. Fibrous Phe and Gly-Leu PU scaffolds were formed with randomly organized fibers and an average fiber diameter of approximately 3.6 µm. In addition, the Phe PU was electrospun into scaffolds of varying architecture to investigate how fiber alignment affects the orientation response of cardiac cells. To achieve this, the Phe PU was electrospun into aligned and unaligned scaffolds and the physical, thermal, and mechanical properties of the scaffolds were investigated. The degradation of the Phe and Gly-Leu PU scaffolds was investigated in the presence of active MMP-1, active MMP-9, and a buffer solution over 28 days to test MMP-mediated and passive hydrolysis of the PUs. Mass loss and structural assessment suggested that neither PU experienced significant hydrolysis to observe degradation over the course of the experiment. In cell-based studies, Phe and Gly-Leu PU scaffolds successfully supported a high density of viable and adherent mouse embryonic fibroblasts (MEFs) out to at least 28 days. Culturing murine embryonic stem cell-derived cardiomyocytes (mESCDCs) alone and with MEFs on aligned and unaligned Phe PU scaffolds revealed both architectures supported adherent and functionally contractile cells. Importantly, fiber alignment and coculture with MEFs improved the organization and differentiation of mESCDCs suggesting these two parameters are important for developing engineered myocardial constructs using mESCDCs and PU scaffolds.

In this dissertation, I explored the effects of developmental hypoxia on heart contractility in three separate species of ectotherms: the common snapping turtle (Chelydra serpentina), the American alligator (Alligator mississippiensis), and the zebrafish (Danio rerio). I began with the common snapping turtle and tested whether the utilization of the sarcoplasmic reticulum was altered in response to developmental hypoxia. In the next two chapters, developmental hypoxia of the American alligator was explored studying how the cardiac tissue was affected, specifically in physiological stressors, sarcoplasmic reticulum utilization and sensitivity to pharmacological increases in contractility. The last chapter explored how zebrafish heart contractility was altered in response to chronic hypoxia from egg to adult. Findings from these chapters suggest that while developmental hypoxia did alter cardiac contractility, it did not alter the response of the heart to physiological stressors such as increased heart rate or under hypoxia. Overall, these findings contribute to increasing the current understanding of how developmental hypoxia alters the cardiovascular system but with an emphasis on the cardiac tissue level.