Myocardial reperfusion injury is thought to be caused by reperfusion induced i) cytosolic Ca²⁺ overload and/or, ii) the formation of oxygen derived freeradicals. At the start of this study, data implicating cytosolic Ca²⁺ overload in the genesis of reversible reperfusion injury were inconclusive. Although several workers have approached this problem by measurements of cytosolic calcium ions, it was my aim to examine the potential sources of such calcium overload. The experiments reported in this thesis were therefore designed to examine the role of altered intracellular and transsarcolemmal Ca²⁺ fluxes in the genesis of reperfusion stunning and arrhythmias. The study was also aimed at elucidating the possible sources and entry pathways contributing to this proposed cytosolic Ca²⁺ overload. In order to investigate the possible role of altered reperfusion Ca²⁺ fluxes in reperfusion injury, we exposed the isolated working, and Langendorff perfused rat heart model to ischaemia and reperfusion to induce reperfusion stunning and arrhythmias. Hearts were pre-treated (before ischaemia) or reperfused with pharmacological compounds, or by interventions known to enhance or inhibit intracellular or transsarcolemmal Ca²⁺ fluxes. The severity of reperfusion stunning (mechanical dysfunction) was measured by reperfusion aortic output, coronary flow and left ventricular pressure. The incidence of reperfusion ventricular arrhythmias was measured by the incidence of ventricular tachycardia and/ or fibrillation. In selected studies, the metabolic status of hearts was evaluated using biochemical assays performed on myocardial tissue samples. Data obtained in these studies indicate that increased Ca²⁺ fluxes through sarcolemmal L-type Ca²⁺ channels during early reperfusion exacerbate stunning, while inhibition of these fluxes with the Ca²⁺ antagonist drug nisoldipine or by Mg²⁺ or Mn²⁺ improve reperfusion function. These data also suggest that although interventions increasing Ca²⁺ fluxes early in reperfusion exacerbate reperfusion stunning, these same interventions improve reperfusion function when performed later. The data also indicate that Ca²⁺ may enter the myocyte indirectly via activation of the Na⁺/H⁺ and Na⁺/Ca²⁺ exchanger during reperfusion. Inhibition of Na⁺/H⁺ exchange activity by HOE 694 during reperfusion attenuated reperfusion stunning and arrhythmias. Both activation of the Na⁺/H⁺ (and Na⁺/Ca²⁺) exchanger and Ca²⁺ influx via the Ca²⁺ channel could contribute to reperfusion induced Ca²⁺ overload and subsequent injury. The study also showed that altered intracellular Ca²⁺ oscillations play a role in reperfusion stunning and arrhythmias as shown by the use of the SR Ca²⁺ release channel blocker, ryanodine. Inhibition of the sarcoplasmic reticulum Ca²⁺ A TP-ase pump by two novel inhibitors, thapsigargin and cyclopiazonic acid, during ischaemia and early reperfusion improved reperfusion function and reduced the incidence of ventricular arrhythmias. function when unphysiologically high concentrations of the peptide were infused into the heart during reperfusion. Taken together, these data suggest that: 1) Ca²⁺ fluxes during early reperfusion (intracellular and transsarcolemmal) play a role in reperfusion injury, 2) that both the Ca²⁺ channel and Na⁺/H⁺ exchange activity contribute to reperfusion injury by possibly contributing to cytosolic Ca²⁺ overload and that, 3) altered intracellular Ca²⁺ oscillations through the SR play a role in both stunning and arrhythmias. Thus the proposal is that modulation of Ca²⁺ fluxes through either the sarcolemma or the sarcoplasmic reticulum, lessen reperfusion injury (stunning and arrhythmias). Although these data do not provide direct evidence of reperfusion Ca²⁺ overload, they support the concept that calcium ions play a role in the genesis of reversible reperfusion injury.

The sarcoplasmic reticulum (SR) is an intracellular membrane system dedicated to the active regulation of cytosolic calcium in muscle. The opening of Ca²⁺ channels in the SR results in a rapid increase in the myoplasmic Ca²⁺ concentration and the initiation of contraction. Closure of these channels allows the SR to re-accumulate the released Ca²⁺ which results in muscle relaxation. While it is known that a muscle fiber is stimulated to contract by the depolarization of the sarcolemma, it is not understood how this signal is communicated to the SR. The focus of this dissertation is twofold. The first objective is to gain an understanding of the mechanism of Ca²⁺ release from the SR. To this end, three studies have been performed which indicate that Ca²⁺ release is mediated by an oxidation reaction. The second goal is to gain insight into the function of the Ca²⁺ release channel. This is addressed by a fourth study which characterizes the effect of the plant alkaloid, ryanodine on channel operation. The anthraquinones mitoxantrone , doxorubicin, daunorubicin, and rubidazone are shown to be potent stimulators of Ca²⁺ release from SR vesicles. Anthraquinoneinduced Ca²⁺ release is shown to be via a specific interaction with the Ca²⁺ release system of the SR. In addition, a strong interaction between anthraquinone and caffeine binding sites on the Ca²⁺ release channel is observed when monitoring Ca²⁺ fluxes across the SR. It is shown that Ca²⁺ release stimulated by anthraquinones is inhibited by preincubating the quinone with dithionite, a strong reducing agent. Spectrophotometric measurements show that the dithionite treated quinone is in a reduced state. Previous work in this lab has shown that the photooxidizing xanthene dye rose bengal stimulates rapid Ca²⁺ release from skeletal muscle SR vesicles. In this thesis, it is shown that following fusion of vesicles to a bilayer lipid membrane (BLM), Ca²⁺ channel activity is stimulated by nanomolar concentrations of rose bengal in the presence of a broad-spectrum light source. This stimulation is shown to be independent of the Ca²⁺ concentration but is inhibited by μM ruthenium red. The photooxidation of rose bengal is shown to not affect either the K+ or Cl- channels which are present in the SR. Exposure of the Ca²⁺ release channel to 500 nM rose bengal in the presence of light is shown to reverse the modification to the channel induced by μM ryanodine. This apparent displacement of bound ryanodine by nanomolar concentrations of rose bengal is directly observed upon measurement of [³H]ryanodine binding to TSR vesicles. Evidence is presented which suggests that Ca²⁺ release is mediated by singlet oxygen. Micromolar concentrations of the porphyrin meso-Tetra(4-N-methylpyridyl)porphine tetraiodide (TMPyP) is shown to induce the rapid release of Ca²⁺ from skeletal muscle SR vesicles. Porphyrin-induced Ca²⁺ release is stimulated by adenine nucleotides and μM Ca²⁺, and is inhibited by mM Mg²⁺ and μM ruthenium red. High-affinity [³H]ryanodine binding is also enhanced in the presence of the porphyrin. The presence of 1 mM Mg²⁺ in the assay medium sensitizes ryanodine binding to activation by ca²⁺. Porphyrin stimulated single channel activity is also sensitized to activation by Ca²⁺ in the presence of Mg²⁺. Reduction of the porphyrin by dithionite, a strong reducing agent, prior to exposure to the Ca²⁺ release channel inhibited the ability of TMPyP to stimulate Ca²⁺ release. These observations indicate that anthraquinones, rose bengal , and porphyrins induce a stimulation of the Ca²⁺ release protein from skeletal muscle SR by interacting with the ryanodine binding site. In addition, the mechanism of interaction for these compounds appears to be via an oxidation reaction. Nanomolar to micromolar concentrations of ryanodine are shown to alter the gating kinetics of the Ca²⁺ release channel from skeletal muscle SR fused with bilayer lipid membranes. In the presence of asymmetric CsCl, 5 to 40 nM concentrations of ryanodine are shown to activate the channel by increasing the open probability (P₀) without changing the conductance. Statistical analysis of gating kinetics reveal that the open and closed dwell times exhibit bi-exponential distributions that are significantly modified by nM ryanodine. The altered channel gating kinetics seen with low nM ryanodine is reversible and is shown to correlate with the binding kinetics of [³H]ryanodine with its highest affinity site under identical ionic conditions. Ryanodine concentrations between 20 and 50 nM are observed to induce occasional 1/2 conductance fluctuations while ryanodine concentrations greater than 50 nM stabilize the channel into a ½ conductance state which is not reversible. These results are shown to correlate with [³H]ryanodine binding to a second site having lower affinity than the first site. Ryanodine at concentrations greater than 70 μM from the 1/2 to a 1/4 conductance fluctuation , whereas ryanodine concentrations greater than 200 μM cause complete closure of the channel. The concentration of ryanodine required to stabilize either the 1/4 conductance transitions or channel closure do not directly correlate with the measured [³H]ryanodine equilibrium binding constants. However, these results can be explained by considering the association kinetics of ryanodine concentrations greater than 200 nM in the presence of 500 mM CsCl. These results indicate that ryanodine stabilizes four discrete states of the SR release channel and supports the existence of multiple interacting ryanodine binding sites on the channel protein.

A dissertation submitted to the Faculty of Science, University of the Witwatersrand, Johannesburg, in fulfilment of the requirements for the degree of Master of Science. Johannesburg, 2013 / The chloride intracellular channel CLIC1 is an anion channel protein that has been implicated in a number of physiological processes. It is fascinating in that it is synthesised as a soluble monomer that is able to reversibly bind membranes without the aid of a membrane-targeting tag or receptor. CLIC1 membrane binding is promoted by low pH and involves separation of the N- and C-domains and subsequent refolding of the N-domain, which traverses the membrane as an α-helix. At the low pH of a membrane surface, pH 5.5, soluble CLIC1 demonstrates decreased conformational stability and forms a partially unfolded intermediate state under mild denaturing conditions. In this study, these pH-effects are proposed to occur as a result of low pH-induced protonation of two conserved glutamate residues, Glu85 and Glu228. Both are involved in domain-maintaining interactions and are proposed to form part of an electrostatic network of pH-sensitive residues. At low pH, protonation of these glutamates would break their electrostatic interactions, allowing separation of the domains. To investigate this possibility, Glu228 and Glu85 were mutated to leucine residues. Each variant protein was then investigated at pH 7.0 and pH 5.5 and results were compared to the wild-type. Secondary and tertiary structures were examined using far-UV circular dichroism and fluorescence spectroscopy, respectively. Conformational flexibility was investigated with limited thermolysin proteolysis. Stability was studied using thermal and urea-induced equilibrium unfolding. The unfolding intermediate state was detected using ANS binding and its structure was characterised. While neither residue substitution caused global structural perturbations, both destabilised the structure and promoted intermediate formation at pH 5.5. This was particularly evident for the E85L variant, which also formed a significant intermediate population at pH 7.0. It was concluded that the interactions of Glu228 and Glu85 are involved in maintaining the CLIC1 native state. Additionally, the lack of pH-dependence of intermediate formation in the E85L variant suggested that Glu85 is likely to function as a pH-sensor. It is thus involved in the „priming‟ of the CLIC1 structure for the conformational changes that may lead to membrane binding.

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Cardiovascular disease is the leading cause of death world-wide, with 42% of sudden cardiac death in young adults caused by myocarditis. Viruses represent the main cause of myocarditis, with adenovirus being a leading pathogen. However, it is not understood how adenoviruses cause sudden cardiac arrest. Myocarditis is defined by two phases, acute and chronic. The acute phase involves viral-mediated remodeling of subcellular structures in the myocardium, which is thought to contribute to arrhythmogenesis. The chronic phase is immune response-mediated, where the host immune system causes damage that induces gross remodeling of the heart, which can result in cardiac arrest or heart failure. Electrical impulses of the heart are propagated by cardiomyocytes, via gap junctions, ion channels, and intracellular junctions, creating the healthy heartbeat. Cx43, the primary gap junction protein in the myocardium, not only propagates electrical signals, but also anti-viral molecules. Viral targeting of gap junction function leads to reduced anti-viral responses in neighboring cells. However, reduced cellular communication would dangerously alter cardiac conduction. Using a cardiotropic adenovirus, MAdV-3, we find that viral genomes are significantly enriched in the heart, with a decrease of gap junction and ion channel mRNA in infected hearts, however, their protein levels were unchanged. Phosphorylation of Cx43 at serine 368, known to reduce gap junction open probability, was increased in infected hearts. Ex vivo optical mapping illustrated decreased conduction velocity in the infected heart and patch clamping of isolated cardiomyocytes revealed prolonged action potential duration, along with decreased potassium current density during infection. Pairing mouse work with human induced pluripotent stem cell-derived cardiomyocytes, we found that human adenovirus type-5 infection increased pCx43-Ser368 and perturbation of intercellular coupling, as we observed with in vivo MAdV-3 infection. Allowing adenovirus infection to progress in vivo, we find myocardium remodeling and immune cell infiltration. Together, these data demonstrate the complexity of cardiac infection from viral-infection induced subcellular alterations in electrophysiology to immune-mediated cardiomyopathy of cardiac adenoviral infection. Our data describe virally induced mechanisms of arrhythmogenesis, which could lead to the development of new diagnostic tools and therapies, to help protect patients from arrhythmia following infection. / Doctor of Philosophy / Viral infection has long thought to be a cause of unexplained sudden cardiac death, especially in young adults. Viruses have been identified to cause many cases of deleterious remodeling of the heart, which can result in heart failure. The heart relies on electrical signaling that moves in a coordinated fashion to contract and pump blood throughout the body. The cells within the heart that do this are called cardiomyocytes, and they join end-to-end to communicate with each other via gap junctions. Gap junctions are tunnels that allow for ions that create electrical impulses to pass, and molecules, such as ones that are important in immune responses to infection. In addition to gap junctions in the heart, ion channels, which are highly selective to allow only one ion flow, unlike gap junctions, create the healthy heartbeat. The most common gap junction in the heart comprises Cx43 proteins. If a virus were to alter how Cx43 connects to a neighboring cell, this would cause a better environment for the virus, as this would keep anti-viral surveillance low, however, this would change how the electrical signal moves throughout the heart, creating arrhythmias. Adenoviruses are a common cold virus, but have been found in the hearts of many cardiac arrest patients. However, little is known on how adenoviruses may cause cardiac arrest, because human adenoviruses are only successful in humans, and mouse adenoviruses are only successful in mice. This creates a challenge when studying the dynamic heart, which does not translate well to cells in a dish. A mouse adenovirus, called Mouse Adenovirus Type-3 (MAdV-3) was reported to favor infecting the heart in mice, but no research has been published on if this virus can answer how adenoviruses change the heart. Because of this virus, and our prior research that adenoviruses can decrease Cx43 within skin cells in a dish, we used MAdV-3 to understand if, how adenoviruses could cause sudden cardiac arrest, and if longer infection could change the overall structure of the heart. We find that MAdV-3 infection prefers the heart to other organs, and that early stages, reduce both the speed of the electrical signal moves through heart and, looking within a cardiomyocyte, how it creates that electrical signal. These changes are arrhythmogenic and accompany modification of Cx43 that would close the gap junction between two cells, changing how ions and molecules move between cells. Using a human adenovirus infection in human cardiomyocytes created from stem cells, this result is also observed. If infection is allowed to continue in the mouse to cause chronic infection, the heart itself changes shape and is diseased. Together, this work shows that adenoviruses create a diseased heart, first the virus changes how the electrical signal moves and then later, causes thinning of the heart muscle. These data illustrate the role viruses play in causing cardiac arrest and could lead to diagnostic or drug targets.

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<p> This thesis launches some new inquires and makes significant progress in the active research areas of joint source-channel coding and multiple description coding. Two interesting but previously untreated problems are investigated and partially settled: 1) can index assignment of source codewords be optimized with respect to a given joint source-channel decoding scheme, and if so, how? 2) can joint source-channel coding be optimized with respect to a given multiple description code, and if so, how?</p> <p> The first problem is formulated as one of quadratic assignment. Although quadratic assignment is NP-hard in general, we are able to develop a near-optimum index assignment algorithm for joint source-channel (JSC) maximum a posteriori (MAP) decoding, if the input is a Gaussian Markov sequence of high correlation. For general cases, good heuristic solutions are proposed. Convincing empirical evidence is presented to demonstrate the performance improvement of the index assignments optimized for MAP decoding over those designed for hard-decision decoding.</p> <p> The second problem is motivated by applications of signal communication and estimation in resource-constrained lossy networks. To keep the encoder complexity at a minimum, a signal is coded by a multiple description quantizer (MDQ) without channel coding. The code diversity of MDQ and the path diversity of the network are exploited by decoders to combat transmission errors. A key design objective is resource scalability: powerful nodes in the network can perform JSC-MD estimation under the criteria of maximum a posteriori probability or minimum mean-square error (MMSE), while primitive nodes resort to simpler multiple description (MD) decoding, all working with the same MDQ code. The application of JSC-MD to distributed estimation of hidden Markov models in a sensor network is demonstrated. The proposed JSC-MD MAP estimator is an algorithm of the longest path in a weighted directed acyclic graph, while the JSC-MD MMSE decoder is an extension of the well-known forward-backward algorithm to multiple descriptions. They outperform the existing hard-decision MDQ decoders by large margins.</p> / Thesis / Doctor of Philosophy (PhD)

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