Natural and Synthetic Factors Which Influence the Calcium Sensitivity of Chemically-Skinned Rat Cardiac Muscle

The work which comprises this thesis is concerned with natural and synthetic substances which influence the Ca2+ -sensitivity of the intracellular systems in chemically-skinned cardiac muscle. Preparations skinned with Triton-X100 retain only traces of membrane structure while the contractile proteins remain functional. This form of treatment is ideal for investigating factors which influence the Ca2+-sensitivity of the contractile proteins. Selectively-(saponin-) skinned trabeculae retain the intracellular membranes associated with the s. r. and mitochondria, while the surface membrane is rendered permeable to the bathing solution. This type of preparation has been used to investigate interventions which influence the functioning of the s. r. In chapter 3 the experimental protocol employed to study the functioning of the s. r. is described. This involved producing trains of caffeine contractures in selectively-treated preparations. Between exposures to caffeine, the muscle was 'Ca2+-loaded' in a solution with a [Ca2+] subthreshold for tension production. The contracture amplitude increased as the duration of Ca2+-loading or the [Ca2+] of the Ca2+-loading solution was increased. Such trains of contractures proved reproducible over prolonged periods of time. Using this method, it was possible to study the effects of various substances which influence the functioning of the s. r. under strictly controlled Ca2+-loading conditions. However, adequate interpretation of these results requires an understanding caffeine's action on the s. r. The reported effects of caffeine on a variety of preparations are discussed during the introduction of this chapter. Following this, a method is described which allows a direct assessment of the ability of the s. r. to effect relaxation in saponin-skinned preparations. The action of caffeine on net s. r. Ca2+-accumulation was studied using this method. The results suggest that millimolar concentrations of caffeine render the s. r. incapable of net Ca2+-accumulation under these experimental conditions. This led to the suggestion that the s. r. is unlikely to contribute significantly to the relaxation of the caffeine contracture in selectively-skinned trabeculae. The possible involvement of the mitochondria in the caffeine-induced response was also studied. However, a mitochondrial contribution was excluded as the caffeine contractures were unaffected by addition of azide or changing the [Na] of the bathing solutions. Chapter 4 addresses the possibility that enzymes which influence the intracellular levels of cAMP may persist following saponin treatment. This could potentially influence the results presented in the following chapters as many of the substances studied are known to affect phosphodiesterase (PDE) activity and other cellular systems which alter the levels of cyclic nucleotides in intact cells. The Effects of PDE inhibition, and stimulation of adenylate cyclase were studied. In addition, an attempt was made to characterise the response of the s. r. and contractile proteins to exogenous cAMP. Caffeine-induced contractures were potentiated in the presence of cAMP. This finding is consistent with previous studies in a variety of preparations. However, the contractile proteins were unaffected by exogenous cAMP unless the duration of skinning was reduced to an unacceptable level. Addition of forskolin toxin potentiated the caffeine contracture. This introduced the possibility that (i) cAMP production may continue following saponin-skinning and (ii) interventions which influence the adenylate cyclase activity may potentiate the caffeine response via a cAMP mediated mechanism. The results presented in the following chapters were interpreted with reference to these findings. Chapter 5 is a comparative study of the effects of caffeine and sulmazole on the contractile proteins and s. r. Both substances induce s. r. Ca2+-release and increase myofilament Ca2+-sensitivity. The contribution of increased Ca2+-sensitivity to transient sulmazole and caffeine contractures was assessed. The opposing effects of sulmazole and caffeine on Cmax were also studied. In chapter 6, the intracellular actions of ORG30029 (N-hydroxy-5,6-dimethoxy-benzo[b]thiophene -2-carboximidamide) were investigated. Chapter 7 considers the effects of various natural and synthetic modulators of Ca2+-sensitivity on the development of rigor tension. Chapter 8 investigates the effect of taurine on s. r. Ca2+-accumulation and myofilament Ca2+-sensitivity. Taurine was found to produce a small increase in Ca2+-sensitivity but a marked increase in the ability of the s. r to accumulate Ca2+. An attempt was made to correlate the intracellular actions of taurine described in this chapter with the reported effects of the amino acid on intact cardiac preparations. The possible physiological role of taurine in the heart is discussed

Automated detection and analysis of Ca(2+) sparks in x-y image stacks using a thresholding algorithm implemented within the open-source image analysis platform ImageJ.

Previous studies have used analysis of Ca(2+) sparks extensively to investigate both normal and pathological Ca(2+) regulation in cardiac myocytes. The great majority of these studies used line-scan confocal imaging. In part, this is because the development of open-source software for automatic detection of Ca(2+) sparks in line-scan images has greatly simplified data analysis. A disadvantage of line-scan imaging is that data are collected from a single row of pixels, representing only a small fraction of the cell, and in many instances x-y confocal imaging is preferable. However, the limited availability of software for Ca(2+) spark analysis in two-dimensional x-y image stacks presents an obstacle to its wider application. This study describes the development and characterization of software to enable automatic detection and analysis of Ca(2+) sparks within x-y image stacks, implemented as a plugin within the open-source image analysis platform ImageJ. The program includes methods to enable precise identification of cells within confocal fluorescence images, compensation for changes in background fluorescence, and options that allow exclusion of events based on spatial characteristics

A key role for peroxynitrite-mediated inhibition of cardiac ERG (Kv11.1) K+ channels in carbon monoxide–induced proarrhythmic early afterdepolarizations

Exposure to carbon monoxide (CO) causes early afterdepolarization arrhythmias. Previous studies in rats indicated arrhythmias arose due to augmentation of the late Na+ current. The purpose of the present study was to examine the basis of CO-induced arrhythmias in guinea pig myocytes in which action potentials more closely resemble those of human myocytes. Whole-cell current- and voltage-clamp recordings were made from isolated guinea pig myocytes and also from HEK293 cells expressing wild-type or a C723S mutant form of Kv11.1 (ERG). We also monitored formation of peroxynitrite (ONOO-) in HEK293 cells fluorimetrically. CO, applied as the CO releasing molecule, CORM-2, prolonged action potentials and induced early after-depolarizations (EADs) in guinea pig myocytes. In HEK293 cells CO inhibited wild-type but not C723S mutant Kv11.1 K+ currents. Inhibition was prevented by an antioxidant, mitochondrial inhibitors or inhibition of nitric oxide formation. CO also raised ONOO- levels, an effect reversed by the ONOO- scavenger, FeTPPS which also prevented CO inhibition of Kv11.1 currents, and abolished the effects of CO on Kv11.1 tail currents and action potentials in guinea pig myocytes. Our data suggest that CO induces arrhythmias in guinea pig cardiac myocytes via ONOO--mediated inhibition of Kv11.1 K+ channel

Energy Metabolism in the Failing Right Ventricle: Limitations of Oxygen Delivery and the Creatine Kinase System

Pulmonary arterial hypertension (PAH) results in hypertrophic remodeling of the right ventricle (RV) to overcome increased pulmonary pressure. This increases the O₂ consumption of the myocardium, and without a concomitant increase in energy generation, a mismatch with demand may occur. Eventually, RV function can no longer be sustained, and RV failure occurs. Beta-adrenergic blockers (BB) are thought to improve survival in left heart failure, in part by reducing energy expenditure and hypertrophy, however they are not currently a therapy for PAH. The monocrotaline (MCT) rat model of PAH was used to investigate the consequence of RV failure on myocardial oxygenation and mitochondrial function. A second group of MCT rats was treated daily with the beta-1 blocker metoprolol (MCT + BB). Histology confirmed reduced capillary density and increased capillary supply area without indications of capillary rarefaction in MCT rats. A computer model of O₂ flux was applied to the experimentally recorded capillary locations and predicted a reduction in mean tissue Po₂ in MCT rats. The fraction of hypoxic tissue (defined as Po₂ < 0.5 mmHg) was reduced following beta-1 blocker (BB) treatment. The functionality of the creatine kinase (CK) energy shuttle was measured in permeabilized RV myocytes by sequential ADP titrations in the presence and absence of creatine. Creatine significantly decreased the KmADP in cells from saline-injected control (CON) rats, but not MCT rats. The difference in KmADP with or without creatine was not different in MCT + BB cells compared to CON or MCT cells. Improved myocardial energetics could contribute to improved survival of PAH with chronic BB treatment

Complexity in decision making: Determining university library opening hours

Making decisions on academic library opening hours is complex with many pressures on managers. This research surveys senior academic library managers from the UK, using a questionnaire to reveal views on library opening hours, the decision making process, and the pressures which influenced their decisions. A variety of factors were found, in particular satisfying undergraduate demands. The research also revealed the sources of information important in making decisions on opening hours and the influence of ‘political’ issues in the decision making process. Some institutions remove complexity by utilising 24/7 opening, though this is not an option for many

DHPR activation underlies SR Ca2+ release induced by osmotic stress in isolated rat skeletal muscle fibers

Changes in skeletal muscle volume induce localized sarcoplasmic reticulum (SR) Ca2+ release (LCR) events, which are sustained for many minutes, suggesting a possible signaling role in plasticity or pathology. However, the mechanism by which cell volume influences SR Ca2+ release is uncertain. In the present study, rat flexor digitorum brevis fibers were superfused with isoosmotic Tyrode's solution before exposure to either hyperosmotic (404 mOsm) or hypoosmotic (254 mOsm) solutions, and the effects on cell volume, membrane potential (Em), and intracellular Ca2+ ([Ca2+]i) were determined. To allow comparison with previous studies, solutions were made hyperosmotic by the addition of sugars or divalent cations, or they were made hypoosmotic by reducing [NaCl]o. All hyperosmotic solutions induced a sustained decrease in cell volume, which was accompanied by membrane depolarization (by 14–18 mV; n = 40) and SR Ca2+ release. However, sugar solutions caused a global increase in [Ca2+]i, whereas solutions made hyperosmotic by the addition of divalent cations only induced LCR. Decreasing osmolarity induced an increase in cell volume and a negative shift in Em (by 15.04 ± 1.85 mV; n = 8), whereas [Ca2+]i was unaffected. However, on return to the isoosmotic solution, restoration of cell volume and Em was associated with LCR. Both global and localized SR Ca2+ release were abolished by the dihydropyridine receptor inhibitor nifedipine by sustained depolarization of the sarcolemmal or by the addition of the ryanodine receptor 1 inhibitor tetracaine. Inhibitors of the Na-K-2Cl (NKCC) cotransporter markedly inhibited the depolarization associated with hyperosmotic shrinkage and the associated SR Ca2+ release. These findings suggest (1) that the depolarization that accompanies a decrease in cell volume is the primary event leading to SR Ca2+ release, and (2) that volume-dependent regulation of the NKCC cotransporter contributes to the observed changes in Em. The differing effects of the osmotic agents can be explained by the screening of fixed charges by divalent ions

“I have no clue what I drunk last night” Using Smartphone technology to compare in-vivo and retrospective self-reports of alcohol consumption.

This research compared real-time measurements of alcohol consumption with retrospective accounts of alcohol consumption to examine possible discrepancies between, and contextual influences on, the different accounts.Building on previous investigations, a specifically designed Smartphone technology was utilized to measure alcohol consumption and contextual influences in de facto real-time. Real-time data (a total of 10,560 data points relating to type and number of drinks and current social / environmental context) were compared with daily and weekly retrospective accounts of alcohol consumption.Participants reported consuming more alcoholic drinks during real-time assessment than retrospectively. For daily accounts a higher number of drinks consumed in real-time was related to a higher discrepancy between real-time and retrospective accounts. This effect was found across all drink types but was not shaped by social and environmental contexts. Higher in-vivo alcohol consumption appeared to be related to a higher discrepancy in retrospectively reported weekly consumption for alcohol beverage types other than wine. When including contextual factors into the statistical models, being with two or more friends (as opposed to being alone) decreased the discrepancy between real-time and retrospective reports, whilst being in the pub (relative to being at home) was associated with greater discrepancies.Overall, retrospective accounts may underestimate the amount of actual, real-time alcohol consumed. Increased consumption may also exacerbate differences between real-time and retrospective accounts. Nonetheless, this is not a global effect as environmental and social contexts interact with the type of alcohol consumed and the time frame given for reporting (weekly vs. daily retrospective). A degree of caution therefore appears warranted with regards to the use of retrospective self-report methods of recording alcohol consumption. Whilst real-time sampling is unlikely to be completely error free, it may be better able to account for social and environmental influences on self-reported consumption

Twenty years of stereotype threat research: A review of psychological mediators

This systematic literature review appraises critically the mediating variables of stereotype threat. A bibliographic search was conducted across electronic databases between 1995 and 2015. The search identified 45 experiments from 38 articles and 17 unique proposed mediators that were categorized into affective/subjective (n = 6), cognitive (n = 7) and motivational mechanisms (n = 4). Empirical support was accrued for mediators such as anxiety, negative thinking, and mind-wandering, which are suggested to co-opt working memory resources under stereotype threat. Other research points to the assertion that stereotype threatened individuals may be motivated to disconfirm negative stereotypes, which can have a paradoxical effect of hampering performance. However, stereotype threat appears to affect diverse social groups in different ways, with no one mediator providing unequivocal empirical support. Underpinned by the multi-threat framework, the discussion postulates that different forms of stereotype threat may be mediated by distinct mechanisms

Comparative effects of RRR-alpha- and RRR-gamma-tocopherol on proliferation and apoptosis in human colon cancer cell lines

BACKGROUND: Mediterranean societies, with diets rich in vitamin E isoforms, have a lower risk for colon cancer than those of northern Europe and the Americas. Vitamin E rich diets may neutralize free radicals generated by fecal bacteria in the gut and prevent DNA damage, but signal transduction activities can occur independent of the antioxidant function. The term vitamin E represents eight structurally related compounds, each differing in their potency and mechanisms of chemoprevention. The RRR-γ-tocopherol isoform is found primarily in the US diet, while RRR-α-tocopherol is highest in the plasma. METHODS: The effectiveness of RRR-α- and RRR-γ-tocopherol at inhibiting cell growth and inducing apoptosis in colon cancer cell lines with varying molecular characteristics (SW480, HCT-15, HCT-116 and HT-29) and primary colon cells (CCD-112CoN, nontransformed normal phenotype) was studied. Colon cells were treated with and without RRR-α- or RRR-γ-tocopherol using varying tocopherol concentrations and time intervals. Cell proliferation and apoptosis were measured using the trypan blue assay, annexin V staining, DNA laddering and caspase activation. RESULTS: Treatment with RRR-γ-tocopherol resulted in significant cell death for all cancer cell lines tested, while RRR-α-tocopherol did not. Further, RRR-γ-tocopherol treatment showed no cytotoxicity to normal colon cells CCD-112CoN at the highest concentration and time point tested. RRR-γ-tocopherol treatment resulted in cleavage of PARP, caspase 3, 7, and 8, but not caspase 9. Differences in the percentage cell death and apoptosis were observed in different cell lines suggesting that molecular differences in these cell lines may influence the ability of RRR-γ-tocopherol to induce cell death. CONCLUSION: This is the first study to demonstrate that multiple colon cancer cell lines containing varying genetic alterations will under go growth reduction and apoptosis in the presence of RRR-γ-tocopherol without damage to normal colon cells. The amount growth reduction was dependent upon the molecular signatures of the cell lines. Since RRR-γ-tocopherol is effective at inhibition of cell proliferation at both physiological and pharmacological concentrations dietary RRR-γ-tocopherol may be chemopreventive, while pharmacological concentrations of RRR-γ-tocopherol may aid chemotherapy without toxic effects to normal cells demonstrated by most chemotherapeutic agents

Effects of antiplatelet therapy on stroke risk by brain imaging features of intracerebral haemorrhage and cerebral small vessel diseases: subgroup analyses of the RESTART randomised, open-label trial

Background Findings from the RESTART trial suggest that starting antiplatelet therapy might reduce the risk of recurrent symptomatic intracerebral haemorrhage compared with avoiding antiplatelet therapy. Brain imaging features of intracerebral haemorrhage and cerebral small vessel diseases (such as cerebral microbleeds) are associated with greater risks of recurrent intracerebral haemorrhage. We did subgroup analyses of the RESTART trial to explore whether these brain imaging features modify the effects of antiplatelet therapy