Background Highest rates of exertion related cardiac death occur during cold air inhalation (CAI): e.g. shovelling snow, but the pathophysiology is unclear. Coronary micro-vascular resistance (MVR) is the major factor regulating coronary blood flow and subsequent myocardial perfusion. Patients with significant coronary artery disease may be more prone to adverse events due to a reduced vasodilator reserve. Novel intracoronary wires, that simultaneously measure coronary artery pressure (Pd) and coronary blood flow (CBF) allow quantification of MVR, enabling physiological investigation of the effects of CAI during exercise. Study 1. hMR versus IMR at predicting microvascular dysfunction. We compared headto- head, the diagnostic accuracy of the two available invasive indices of MVR, Dopplerderived hyperaemic microvascular resistance (hMR) versus thermo-dilution-derived index of microcirculatory resistance (IMR), at predicting microvascular dysfunction. We then used the most accurate measure of MVR in Study 2. Study 2. Cold air with and without exercise on MVR in CAD patients. We explored the effects of CAI alone and during exercise on MVR and CBF in patients with significant coronary artery disease. Methods Study 1. 56 patients (61 ± 10 years) undergoing cardiac catheterisation for stable coronary artery disease or acute myocardial infarctions (AMI) were recruited. Simultaneous intracoronary pressure, Doppler flow velocity and thermodilution were carried out in 74 vessels without obstructive epicardial disease, at rest and during hyperaemia. In the absence of a gold-standard, the following three measures of microvascular dysfunction were used, using a pre-defined dichotomous threshold for each parameter: 1) Mean coronary flow reserve (CFRmean), defined as the average value of Doppler and thermodilution derived coronary flow reserve 2) Cardiac Magnetic Resonance (CMR) defined myocardial perfusion reserve index (MPRI) 3) CMR defined extensive microvascular obstruction (MVO). Study 2. 35 patients (62 ± 9 years) with significant coronary artery stenoses who were undergoing coronary angiography were allocated to 5 minutes of either: 1. CAI (-15oC), n=10 2. Exercise (Incremental supine ergometry), n=24 3. Exercise with CAI, n=13. (12 patients did both conditions 2. and 3., and for these patients the order was randomised). Forty seven datasets were obtained in total. We compared rest and peak measurements of Doppler-derived MVR (Pd/CBF) and CBF. We also used wave intensity analysis to identify waves that accelerate and decelerate CBF, and calculated the proportional contribution of accelerating waves as a coronary perfusion efficiency index. Results Study 1. hMR had better diagnostic accuracy than IMR to predict CFRmean (area under curve, (AUC) 0.82 versus 0.58, p < 0.001, sensitivity/specificity 77/77% versus 51/71%) and MPRI (AUC 0.85 versus 0.72, p=0.19, sensitivity/specificity 82/80% versus 64/75%). In AMI patients, the AUCs of hMR and IMR at predicting extensive MVO were 0.83 and 0.72 respectively (p=0.22, sensitivity/specificity 78/74% versus 44/91%). Study 2. MVR increased during CAI alone, whereas MVR decreased during exercise. Exercise with CAI was associated with less decrease in MVR. The increase in CBF was similarly less during exercise with CAI versus without. Coronary perfusion efficiency increased during exercise. However the addition of CAI during exercise abolished this. Conclusion Study 1. In our study cohort Doppler-derived hMR had superior diagnostic accuracy over IMR at predicting several invasive and non-invasive measures of microvascular function. This measure was therefore used to measure MVR in Study 2. Study 2. In CAD patients CAI substantially attenuated the reduction in MVR and the increase in CBF that normally occur during exercise. Moreover, while the heart has improved coronary perfusion efficiency during exercise, this may be attenuated when exercise is combined with CAI. This suggests that CAI during exercise may impede coronary vasodilatation and ventricular relaxation, rendering the heart more susceptible to ischaemia. Complementary studies (Studies 3 and 4) were performed in the absence of invasive measures of central and coronary haemodynamics to examine the differential effects of isometric and dynamic exercise (Study 3) and that of first and second exercise efforts (Study 4).

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We now report that thrombostasis is potentially maintained by the relative level of two opposing and different cell death programs within megakaryocytes and platelets. Firstly, platelet formation from megakaryocytes occurs by a unique, compartmentalised form of caspase-dependent apoptosis that results in the formation of multiple functional anucleate progeny. However, whilst the megakaryocyte cell body contains active caspases and displays nuclear condensation and fragmentation typical of apoptosis, the forming platelets retain functional mitochondria, do not contain active caspases, and are not phagocytosed, emphasising an atypical compartmentalised mitochondrial-independent apoptotic program. Platelet formation from megakaryocytes could be significantly augmented by ligation of the Fas death receptor, and reduced by treatment with caspase inhibitors. Secondly, we report a constitutive but caspase-independent program for the specific phagocytic clearance of intact effete platelets. Platelets aged <i>in vitro </i>exhibited increased expression of proapoptotic Bak and Bax, underwent diminution of function, and displayed cytoplasmic condensation and plasma membrane changes that lead to recognition by phagocyte scavenger receptors. In addition, evidence of platelets with an identical morphology was found in patients having sustained a non-Q-wave myocardial infarction. However, although platelets contained the effector caspase-3 they lacked caspase-9, a key component of the apoptosis initiator complex the apoptosome. Intriguingly, megakaryocytes contained both caspase-3 and -9, suggesting sequestration of the enzyme by the progenitors during platelet formation, again underscoring the compartmentalised death of megakaryocytes and thus explaining the inability of platelets to activate caspases during constitutive death, and hence their commitment to undergo a caspase-independent death.

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In an attempt to determine the influence of cardiorespiratory events on sway behaviour. a series of four experiments were undertake.n on a total of 95 subjects, all young healthy adults. Sway tiehaviour, defined as the corrective force recorded between the soles of the feet and the surface of a biomechanical measuring platform (Kistler, 9261A), was first examined to determine the extent to which it is a function.of sex and physique. Height, weight and obesity measurements were taken from 58 subjects (29 male, 29 female) and their influence on sway behaviour analysed. The second experiment was an extended ideographic study designed to test the constancy of sway behaviour over a six-week period for ten subjects (six male, four female) in an attempt to identify the personal characteristics of postural sway. This led to the formulation of a dynamic model of postural sway behaviour based on cardiorespiratory events. In the third experiment the magnitude of the cardiac forces and stroke volume,by transcutaneous aortovelography, were measured on 18 subjects (eight male, ten female), and used to establish the direct effect of cardiac action on sway behaviour. In the final experiment the role of 18 antigravity muscles of the lower limbs and trunk in postural maintenance was examined in nine subjects (five male, four female) to test the widely held hypothesis that sway is a direct outcome of the dynamic equilibrium that exists between gravitatiqnal forces and the myotatic reflex responses. The validity of the model was tested by comparing the predicted sway based on cardiorespiratory events with actual sway behaviour.

ERG is a crucial regulator of endothelial gene expression and controls endothelial functions including cell survival and monolayer permeability. Previous studies indicate a role for ERG in angiogenesis and vascular development, however the pathways through which ERG controls angiogenesis are unclear. Transcriptome profiling comparing ERG-positive and ERG-deficient endothelial cells has previously shown that ERG controls a network of genes that are essential to angiogenesis. This analysis identified genes involved in the Wnt, Notch and Angiopoietin1/Tie2 signalling pathways as candidate ERG targets. ERG has been shown to drive expression of the junction molecule vascular endothelial (VE)-cadherin, which binds β-catenin, a crucial mediator of Wnt signalling, at the cell membrane. Here, I show that ERG controls β-catenin stability, by driving expression of both VE-cadherin and the Wnt receptor Frizzled-4- the balance of which regulates β-catenin localisation and activity. ERG promotes angiogenesis via Wnt/β-catenin signalling, since activation of Wnt signalling with lithium chloride, which stabilises β-catenin, corrects the angiogenic defect in ERG-deficient endothelial cells. The Notch signalling pathway is critical for promoting vascular quiescence and I demonstrate that ERG controls Notch signalling by regulating the levels of two Notch ligands, Delta like ligand (Dll)-4 and Jagged-1, with reported opposing roles in the vasculature. ERG simultaneously drives expression of pro-quiescent Dll4 and represses expression of pro-angiogenic Jagged-1, which has been shown to antagonize Dll4-mediated signalling. The Angiopoietin1/Tie2 pathway, also connected to the Wnt and Notch pathways, is a regulatory growth factor system essential for vessel maturation and quiescence. The results from this thesis suggest that ERG mediates growth factor Angiopoietin-1-dependent signals and ERG is required for Angiopoietin-1-induced Notch and Wnt signalling. Thus, ERG is able to integrate with three signalling pathways controlling vascular growth and stability - Wnt, Notch, Angiopoietin1/Tie2- which may function downstream of ERG to regulate blood vessel patterning during angiogenesis.

The contractile function of the heart depends on efficient β adrenergic receptor (βAR) signalling which involves cycling nucleotides as second messengers. Correct secondary messenger signalling is only possible in healthy, well structured cardiac myocytes. Of the three βAR subtypes present in human cardiomyocytes β1AR and β2AR classically signal via 3'-5' cyclic adenosine monophosphate (cAMP) to regulate contraction after catecholamine administration, whereby the second isoform may also be cardioprotective. The far less characterised β3AR has been controversially associated to both increasing contraction through cAMP and protecting the heart through 3'-5' cyclic guanosine monophosphate (cGMP) signalling. During the progression of heart failure following myocardial infarction (MI) both the normal cell structure and the regulation of cAMP and cGMP signalling are changed. This happens in part due to changes in catecholaminergic stimulation of the βARs and in mechanical load, as well as due to a progressive development of hypertrophy. Some of the alterations initially appear to be of a compensatory nature but escalate into HF by worsening cardiomyocyte function and cell survival. The work presented here (1) investigates the structural integrity of healthy, isolated, single cardiomyocytes by looking at the surface topography via Scanning Ion Conductance Microscopy (SICM) imaging and by examining the internal Transverse Axial Tubule (TAT) network via confocal imaging; (2) elucidates the cyclic nucleotide response to catecholamine stimulation following either global (in the solution) or local (in the SICM pipette) stimulation of either β2ARs or β3ARs and measuring either cAMP or cGMP levels via Förster Resonance Energy Transfer (FRET) sensors in a combined FRET/SICM imaging setup; (3) determines how both the structure and β2AR and β3AR dependent second messenger signalling change in a progressive rat model of HF 4, 8 and 16 weeks after the induction of chronic MI. The major findings of the presented work are as follows: In control cardiomyocytes the structure is highly intricate with regular Z-grooves and crest areas. In MI cells the normal suface topography progressively deteriorates, with the eventual disappearance of Z-grooves by week 16, which correlates with the disorganisation of the cardiomyocyte's internal transverse axial tubule (TAT) network of T-tubules emanating from the cell surface and traversing into the cell centre. This is accompanied by the gradual redistribution of β2ARs from their normal position inside the T-tubules to the unstructured areas on the cardiomyocyte membrane. The regularity and density of the TAT network is already severely compromised at 4 weeks post MI; at the same time a significant drop in the expression of the structural protein Junctophilin 2 (JPH2) occurs. At 4 and 8 weeks post MI a potentially compensatory increase in the number of longitudinal elements takes place which was no longer detectable at 16 weeks. The production of cAMP following local stimulation of β2ARs in the T-tubule openings was already suppressed at 4 weeks post MI and a β2AR response becomes detectable after local stimulation at the cell crests (areas between Z-grooves) at 8 weeks post MI. At 16 weeks post MI the β2AR-dependent cAMP level following both global and local stimulations was reduced due to an overall decrease in the adenylate cyclase (AC) activity. The production of the second cyclic nucleotide, cGMP, following β3AR stimulation is evident in control cells and to a significantly lesser extent in myocytes isolated from hearts at the end stage of HF. These β3AR-cGMP levels were degraded mainly by phosphodiesterases (PDE) 2 and 5. Local stimulation through the SICM pipette reveals that functional β3ARs are primarily localized inside T-tubules in control cells but redistribute equally in between T-tubules and crests in cells isolated from failing hearts. To improve the accuracy and reliability of local application of agonists via the SICM nanopipette voltage was applied to the pipette, as opposed to previously employed displacement of the liquid in the pipette via air pressure. Mathematical modelling served to determine the correct settings for this voltage driven application. It shows that the SICM nanopipette can reliably and precisely unload the βAR agonist ISO onto the nanoscale structure of cardiomyocytes via voltage.