Thesis (M.S.)--West Virginia University, 2007. / Title from document title page. Document formatted into pages; contains x, 67 p. : ill. (some col.). Includes abstract. Includes bibliographical references (p. 37-38).

Thesis (Ph. D.)--University of Hong Kong, 1998. / Includes bibliographical references (leaves 128-149).

Thesis (M.S.)--West Virginia University, 1999. / Title from document title page. Document formatted into pages; contains viii, 91 p. : ill. (some col.) Includes abstract. Includes bibliographical references (p. 67-69).

Thesis (Ph. D.)--Ohio State University, 2005. / Available online via OhioLINK's ETD Center; full text release delayed at author's request until 2006 September 15.

Around 170000 patients worldwide receive cardiac valve substitutes each year. Valve replacement with mechanical or bioprosthetic devices enhances patient survival and quality of life. Bioprosthetic valves have a significant advantage over mechanical valves: they do not necessarily require long-term anticoagulant therapy however, dystrophic calcification can lead to early failure. The actual mechanism of calcification is still poorly understood despite several established possible factors associated with it. Amongst these is the glutaraldehyde pre-treatment of the valves during their manufacture. Glutaraldehyde has been used for the treatment of bioprosthetic valves for the last thirty years, as a cross-linking agent and a sterilant. Whilst it is assumed to introduce stable inter- and intra-fibrillar collagen cross-links, which contribute to the durability of these valves, the specific chemistry of the fixation process is not fully understood. Additionally, glutaraldehyde is thought to be involved somehow in the process of dystrophic calcification of these same bioprosthetic valves. The primary aim of this study was to gain a greater understanding of the chemistry involved in the treatment of collagenous valve tissue with glutaraldehyde. Amino acids, peptides and proteins were thus used to mimic the effect of the glutaraldehyde treatment and to model potential reactions involved in such treatment. Techniques such as MALDI-TOF MS, ESI MS, NMR, FTIR-ATR and Raman spectroscopy were utilised to study the products of the glutaraldehyde reaction and their relationship with the calcification process. Data obtained from the products of the reactions between glutaraldehyde and model compounds showed the presence of: aldol and aldol/Michael condensation products of glutaraldehyde, Schiff base moieties (including cross-links) and various cyclisation products incorporating pyridinium and dihydropyridine ring structures. Some of these structures are in agreement with the literature, whilst others are essentially new structures that have never been proposed. Glutaric acid, used to mimic the oxidation of glutaraldehyde that can occur in-vivo, was shown to have the ability to form complexes with cations such as calcium in-vitro. A similar result was found with aqueous dilute solutions of glutaraldehyde (similar concentrations to the ones used in valve manufacture), thus leading to the hypothesis of its strong role in the initiation of calcification in-vivo. However, an extrapolation of these results to the role of the nucleophilic groups of amino acids or peptides, that could behave as the collagen macromolecule, was difficult to assess using FTIR because of the complex infrared spectra. However some findings corroborated the hypothesis that amino acids of the collagen tissue may also play a role in the initiation of calcification. Secondly, methodology was developed to allow successful analysis of tissue calcification using environmental scanning electron microscopy (ESEM). This is thought to be an important step in the analysis of tissues in their native state. Investigation of the calcification process with samples from clinical investigations (explanted human calcified valves), in-vivo screening (rat subcutaneous implantation model) and in-vitro screening (pericardial tissue incubated in metastable calcification solution) was thus undertaken using ESEM, along with other techniques such as FTIR-ATR, Raman, XRD spectroscopy and ICP-OES. The data revealed both similarities and differences between in-vivo and in-vitro calcification, although the process is unequivocally different. Late calcific deposits were assigned to poorly crystalline hydroxyapatite with high Ca/P ratios due to the probable presence of carbonate and possibly cations such as silicon and magnesium. A picture of the onset of mineralisation was hypothesised involving precursors, containing various amounts of calcium and phosphate, along with the incorporation of magnesium and silicon. These precursors phases evolved with time of implantation to the poorly crystalline form of hydroxyapatite found in the late stage of calcification. This work has provided an insight into how glutaraldehyde reacts with valve tissue and a possible explanation as to why valves fail by non-calcific or calcific mechanisms. A new approach to the study of calcified valve tissue has also been developed using ESEM methodology.

The heart has the remarkable ability to adjust in response to varying stress stimuli and myocardium enlargement, referred to as cardiac hypertrophy, is a common form of stress adaptation. Divergent forms of hypertrophy can occur depending on the type and duration of the insult. The beneficial physiological form of hypertrophy is reversible and leads to improved cardiac function, while the pathological form is a maladaptive process that often transitions to heart failure. As a result of the prominence of cardiac disease, investigations into methods of reducing this detrimental form of cardiac remodeling are sought. Interestingly, pathological cardiac hypertrophy shares common features with the regulated form of cell death referred to as apoptosis. Here, we describe an essential role for apoptotic caspase-dependent signaling in the induction of pathological cardiac hypertrophy. Initially, we discovered that primary cardiomyocytes treated with hypertrophy agonists display transient activation of intrinsic-mediated apoptotic-signaling, including caspase 9 and caspase 3 activity. The necessity of functional caspase activation in hypertrophic signaling was shown by both in vitro and in vivo methods. We further investigated caspase cleavage targets histone deacetylase 3 (HDAC3) and gelsolin (GSN). HDAC3 cleavage was observed during early stages of hypertrophy and reduced in the presence of a caspase inhibitor. Caspase-mediated GSN cleavage occurred at latter stages, coincident with the cytoskeletal alterations that occur during this process. We demonstrated the requirement of GSN and its caspase-mediated processing by use of GSN expressing adenoviruses (AdVs). Use of a non-cleavable GSN-AdV provided evidence for not only the requirement of GSN in the hypertrophic response, but also for caspase mediated GSN cleavage. This body of work implicates caspase pathways and their targets as inductive signaling cues for pathological cardiac hypertrophy. These observations suggest that inhibitors that mute or suppress caspase activity and/or activity of its cognate substrates may offer novel therapeutic targets to limit the development of pathological hypertrophy.

Thesis (M.A.)--Boston University

INTRODUCTION: The Modified Blalock Taussig (BT) shunt is the most common palliative procedure in neonatal cardiac repair to secure pulmonary blood flow (3-6) despite high mortality and morbidity rates of 4-14% (3,5) and 9-18% (4,5) respectively. The high risk of thrombosis and stenosis associated with the polytetrafluoroethylene (PTFE) material that is currently used for BT shunts significantly contributes to these high mortality and morbidity rates. Thrombosis and stenosis occur in 3-12% of patients (1,5), primarily due to the lack of endothelium of the synthetic PTFE graft. This study hypothesizes that a preserved autologous umbilical vein could be a feasible replacement for the PTFE graft. Experiments were performed to examine and optimize preservation methods of umbilical veins for use as BT shunts. METHODS: Umbilical cords (n=45) were collected from healthy neonates and umbilical vein segments were preserved in either static (n=145) or flow conditions (n=84) for 7 days or 14 days in varying media. Samples of each vein were collected at time of harvest (Day 0) and at the end of culture and compared by burst pressure, histopathology, platelet adhesion and scanning electron microscopy (SEM). RESULTS: Burst pressure strength of veins from Day 0 to up to two weeks of preservation did not significantly differ (431 ± 229 mmHg vs 438 ± 244 mmHg). Overall histology demonstrated an improved morphology in endothelial and medial layers of the segments preserved in flow culture with UW + 5% HPL for 7 days. Platelet adhesion testing demonstrated significantly less platelet attachment in flow samples compared to static, indicating less endothelial injury. SEM showed greater cellular flow-alignment and consistency of the endothelium in flow samples. CONCLUSION: Flow culture utilizing UW plus 5% HPL adequately preserves morphology and function of the endothelium. Preserved autologous umbilical veins stand as a viable option to replace the current PTFE graft used for BT shunts due to the presence of an endothelium.

Mechanosensitivity describes the ability to respond to a mechanicalstimulus. The heart can respond to a mechanical stimulus through the action ofmechanosensitive ion channels (MSCs). MSCs provide a direct link betweenstretch and electrical activity. However, the heart does not react to stretch solelythrough the action of MSCs. The mammalian myocardium possesses a biphasicresponse to stretch: first, there is an immediately increase in the force of cardiaccontraction, known as the Frank-Starling response; secondly, there is a slowpositive inotropic response, known as the slow force response (SFR) that occursover the minutes following the initial stretch. Fish are unique amongst vertebrates as, with a few exceptions, they relymore heavily on changes in stroke volume than heart rate when regulatingcardiac output. Rainbow trout (Oncorhynchus mykiss) are particularly sensitiveto the Frank-Starling response and small increases in filling pressure lead to largeincreases in stroke volume (300 %) during strenuous exercise. The ability of fishhearts to undertake these large dilations makes them an ideal model whenlooking at the effect of stretch on cardiac muscle as they may exhibit morepronounced responses to mechanical stimuli. Despite this, the role of mechanicalregulation in the fish heart has undergone sparse investigation. The aim of this thesis was to investigate the mechanosensitivity of thefish heart at a number of resolutions. Chapter three looks at the effect of stretchon the isolated whole rainbow trout heart. I found that MSCs are activated atphysiological extremes of input and output pressures. The trout ortholog of acandidate MSC, TRPC1, was cloned and its presence in the heart was verified. Both MSCs and exaggerated cardiac transmural electrical heterogeneity cancause re-entrant arrhythmias in the mammalian heart. As the piscine heart hasshown resistance to these arrhythmias I examined the transmural electricalheterogeneity of the tuna heart in Chapter four. I found no evidence oftransmural electrical heterogeneity in the tuna heart which may explain thereduced susceptibility of the fish heart to re-enterant arrhythmias. In Chapter fiveI investigated the effect of stretch on ventricular trabecular bundle preparationsand isolated ventricular myocytes of the rainbow trout. This study was the first tofind a lack of a SFR in a vertebrate heart and provides evidence for theimportance of the Na+ /H+ -exchanger in the SFR. Finally the study in Chaptersix examined the length-dependent Ca2+ sensitivity of skinned ventricular rat andtrout myocytes. I show that the increased length-dependent Ca2+ sensitivity of thetrout myocytes may account for the extended functional limb of the piscinelength-tension relationship. Skinned trout myocytes were shown to develop ahigh passive tension that could not be explained by the trout titin isoform ratio,but may be explained by increased phosphorylation of titin in vivo. My PhD research has produced clear and novel evidence for theimportance of mechanosensitivity in the fish heart. From the level of the wholeheart to the level of the individual sarcomere, stretch induces physiologicalchanges in this vital organ. A greater understanding of piscine cardiacmechanosensitivity will greatly improve general knowledge ofmechanosensitivity in general and will provide an evolutionary point ofcomparison for the studies of mechanosensitivity in other organisms.

When modelling tissue-level cardiac electrophysiology, a continuum approximation to the discrete cell-level equations, known as the bidomain equations, is often used to maintain computational tractability. The bidomain equations are derived from the discrete equations using a mathematical technique known as homogenisation. As part of this derivation conductivity tensors are specified for use in the continuum model. Analysing the derivation of the bidomain equations allows us to investigate how microstructure, in particular gap junctions that electrically connect cells, affect tissue-level conductivity properties and model solutions. We perform two distinct but related strands of investigation in this thesis. In the first, we consider the effect of including gap junctions on the results of both discrete and continuum simulations, and identify when the continuum model fails to be a good approximation to the discrete model. Secondly, we perform a comprehensive study into how cell-level microstructure properties, such as cell shape, impact the homogenised conductivities to be used in a tissue-level continuum model. This will allow us to predict how the onset of a disease or a change in cellular microstructure will affect the propagation of action potentials. To do this, we first derive a modified version of the bidomain equations that explicitly takes gap junctions into account. We then derive analytic solutions for the homogenised conductivity tensors on a simplified two-dimensional geometry and find that diseased gap junctions have a large impact on the results of homogenisation. On this same geometry we compare the results of discrete and continuum simulations and find a significant discrepancy between model conduction velocities when we introduce gap junctions with lower coupling strength, or when we consider elongated cells. From this, we conclude that the bidomain equations are less likely to give an accurate representation of the underlying discrete system when modelling diseased states whose symptoms include reduced gap junction coupling or an increase in myocyte length. We then use a more realistic two-dimensional geometry and numerically approximate the homogenised conductivity tensors on this geometry. We discover that the packing of cells has a substantial effect on conduction, with a brick-wall geometry particularly beneficial for fast propagation, and that gap junctions also have a large effect on conduction. Finally, we consider a three-dimensional cellular geometry and show that the effect of changing gap junction properties is different when compared to two dimensions, and discover that the anisotropy ratios of the tissue are highly sensitive to changes in gap junction parameters. Overall, we conclude that gap junctions and cell structure have a large effect on discrete and continuum model results, and on homogenised conductivity calculations in tissue-level cardiac electrophysiology.