ABSTRACT Pulmonary arterial hypertension (PAH) is a rare and progressive disease characterised by increased pulmonary vascular resistance and elevated pulmonary artery pressure, leading to right ventricular failure and eventually death. The monoamine 5-hydroxytryptamine (5HT) has been implicated in the processes of pulmonary vasoconstriction and pulmonary artery remodelling that contribute to the development of the PAH. However, the signalling mechanisms utilised by 5HT that contribute to pulmonary vascular remodelling are still unclear and appear to be cell-type specific, with much of the work having been carried out in pulmonary artery smooth muscle cells (PASMCs). Fibroblasts also contribute significantly to the pulmonary vascular remodelling that occurs during PAH, however little is known of the role 5HT plays in this cell type. Using Chinese Hamster Lung Fibroblast (CCL-39) cells as a model system to investigate the mitogenic effects of 5HT, this study has characterised potential 5HT-mediated signalling pathways in fibroblasts that may contribute to pulmonary vascular remodelling. 5HT was found to induce a rapid and transient activation of extracellular regulated mitogen-activated protein kinase (ERK), a process central to the mitogenic effects of 5HT in CCL-39 cells. Furthermore, the 5HT transporter (5HTT), 5HT1B and 5HT2A receptors were all required for optimal ERK-dependent proliferation. Pharmacological inhibition of the Rho/ROCK (Rho-associated kinase) pathway significantly inhibited 5HT-stimulated ERK activation, cyclin D1 accumulation and proliferation. Inhibition of ROCK had no effect on the translocation of active ERK to the nucleus, but did however selectively inhibit 5HT-induced activation of a cytoplasmic pool of ERK. Additionally, ROCK inhibition had no effect on the ability of 5HT to activate mitogen-activated protein kinase kinase (MEK), suggesting ROCK is required for maintaining functional interactions between MEK and ERK. Sensitivity to ROCK inhibition is restricted to 5HT1B receptor activation of the ERK pathway. Moreover, the role of ROCK in maintaining cytoskeletal integrity is important in mediating 5HT-induced ERK activation, as disruption of the actin cytoskeleton markedly and specifically reduces 5HT- stimulated ERK activation. Using a model of PAH, arising from overexpression of 5HTT (5HTT+), the effects of ROCK inhibition in vivo were investigated. ROCK 1 and ROCK 2 transcripts were upregulated in response to chronic hypoxia, with the upregulation of ROCK 1 potentiated in 5HTT+ mice. Administration of the ROCK inhibitor Y27632 had significantly greater effects in 5HTT+ mice compared to WT, highlighting the functional importance of the increase in ROCK 1 transcript. Hypoxia-induced pulmonary vascular remodelling and elevated right ventricular pressure were attenuated more significantly by ROCK inhibition in 5HTT+ mice than in WT. Furthermore, ROCK inhibition only reduced hypoxia-derived right ventricular hypertrophy significantly in 5HTT+ animals and not WT. In conclusion, this study highlights a role for ROCK in the pulmonary vascular changes that occur during PAH and proposes a new mechanism by which cross-talk between ROCK and 5HT signalling systems occurs.

In 1979, Donald Zilversmit published a landmark paper suggesting a role for postprandial metabolism in the development and progression of atherosclerosis, and in subsequent years, a number of postprandial metabolic perturbations have been highlighted as potential risk factors for atherosclerotic development. Interventions inducing favourable changes in postprandial metabolism may, therefore, have an important role in the management and control of cardiovascular disease and its associated risk factors. Acute exercise is one such intervention, successfully attenuating postprandial lipaemia and insulinaemia, enhancing endothelial function, reducing arterial stiffness and increasing postprandial fat oxidation. It also appears that exercise might facilitate a tighter coupling between energy intake and expenditure, mediating more stable and healthy body weights, perhaps by inducing changes in appetite, energy intake and circulating concentrations of 'hunger' hormones. It does, however, remain unclear as to what extent these changes are mediated by exercise per se, or the associated energy deficit and thus what effects exercise coupled with a state of energy balance would have on postprandial metabolism, appetite control and feeding behaviour. The aim of this thesis was, therefore, to investigate the immediate and delayed effects of exercise, coupled with a state of energy balance, on postprandial metabolism and appetite regulation. Thirteen overweight, pre-menopausal women were recruited to compare the immediate effects of a state of energy balance coupled with a high (exercise) or low (rest) energy turnover. Using a one-day model, each volunteer completed a 60-minute, moderate intensity treadmill walk prior to ingesting a test breakfast and a 6-hour metabolic assessment period and a control trial during which no exercise was performed before breakfast. During the 6-hour observation period postprandial lipaemia, insulinaemia and glycaemia, total ghrelin and acylated ghrelin concentrations, pulse wave velocity and energy substrate utilisation were determined. Subjective ratings of appetite and food palatability and energy and macronutrient intakes at an ad libitum buffet meal were also recorded. Thirteen overweight and obese, middle-aged men were recruited to investigate the delayed effects of exercise withe energy balance, using a two-day model, on postprandial metabolism and appetite control. On the afternoon of day-one, each volunteer completed one of three intervention trials, a moderate intensity treadmill walk inducing a net energy expenditure of 17kJ.kg[supercript -1]. body mass, exactly the same walk with the net energy expenditure replaced or they performed no exercise. The following day, an 8.5-hour metabolic observation period was completed during which time postprandial lipaemia, insulinaemia and glycaemia, letpin and total gherin concentrations, pulse wave velocity, energy substrate utilisation and subjective ratings of appetite were recorded in response to two separate oral fat tolerance tests. The findings from these studies showed exercise, coupled with a state of energy balance, to have no significant effect, immediate or delayed, on postprandial lipaemia suggesting and exercise-induced deficit was required to lower postprandial triglyceride concentrations. A significant attenuation was, however, observed in postprandial insulin concentrations on the day following exercise with energy balance, indicitive of enhanced insulin sensitivity. Both immediate and delayed increases in postprandial fat oxidation were observed following exercise, which led to lower (ie. more negative) fat balances. This could potentially have important implications for the future management and regulation of the overweight and obese state. The role for exercise with energy balance in attenuating pulse wave velocity speeds remains equivocal with no immediate effect observed on postprandial central or peripheral pulse wave velocity, whilst significantly lower peripheral pulse wave velocity speeds were observed after a delay of some hours post-exercise. With regard to appetite regulation, exercise coupled with a state of energy balance does appear to reduce postprandial leptin concentrations on the following day, whilst total gherlin remains resistant to any exercise-induced changes either immediatley or some hours later. An exciting find does, however, show acylated ghrelin concentrations to be significantly lower following exercise with energy balance, a response that may, to some extent, mediate subsequent changes in appetite. Changes in subjective ratings of appetite were transient, lasting just one to two hours after exercise with energy balance and energy and macronutrient intake at an ad libitum buffet meal consumed six hours after exercise with energy balance was not significantly altered. The combined findings of this thesis suggest that exercise, coupled with a state of energy balance, induces favourable changes in postprandial insulinaemia, fat oxidation and peripheral pulse wave velocity, all of which may have important implications for lowering future risk of cardiovascular disease. Exercise performed in a state of energy balance may also contribute to the improved regulation of body weight, through effects on fat oxidation and balance and potentially via changes in 'hunger' hormones and appetite ratings, although the latter, in particular, requires further investigation.

Microvolt T-wave alternans is thought to predict the risk of ventricular arrhythmias in patients with heart disease, although recent clinical studies have conflicting results. Understanding the cellular basis for alternans may not only inform more effective utilisation of the clinical test, but also provide new insights into the causes of lethal arrhythmias in man. Cellular repolarisation alternans is thought to underlie T-wave alternans and in recent years, the concept of discordant repolarisation alternans has emerged as a new paradigm for the induction of re-entrant ventricular arrhythmia. This experimental observation has not been examined in clinically relevant models of pathology and so the aim of this study was to investigate whether increased transmural heterogeneity of repolarisation as a result of heart failure following myocardial infarction in the rabbit would predispose to the development of arrhythmogenic discordant alternans. A rabbit ventricular wedge preparation was developed and the transmural electrophysiology of intact rabbit ventricle was characterised using optical imaging techniques. This revealed transmural gradients of repolarisation in intact rabbit myocardium, which appeared to be influenced by electrotonic load, rather than purely being a reflection of intrinsic cellular differences. Interestingly, repolarisation alternans also appeared in transmural patterns, which were also modified by activation sequence, underlining the role of conduction and electrotonic influences in dictating the spatial patterns of alternans, which may be crucial in determining spatially discordant alternans. In this study, similar baseline electrophysiological characteristics were apparent in the remodelled myocardium of failing hearts compared with normal hearts, underlining the possible importance of dynamic factors in producing the increased vulnerability to re-entrant arrhythmias observed in failing hearts. Repolarisation alternans, elicited by low temperature and rapid pacing, occurred at lower heart rates in failing hearts. At physiological temperature, repolarisation alternans was also more common in failing hearts. Spatially discordant alternans was not consistently observed on the transmural surface and did not appear to be directly related to the development of arrhythmia. Failing hearts displayed an increased vulnerability to ventricular arrhythmia. Although heart failure was associated with both alternans and ventricular arrhythmia, there was no demonstrable mechanistic link between alternans and ventricular arrhythmias in failing hearts. These data establish the occurrence of repolarisation alternans in a clinically relevant pathology, and so constitute an important step forward in our understanding of the experimental paradigm. However, a definitive mechanistic link between alternans and arrhythmia in heart failure is yet to be shown.

Cerebral Palsy (CP) is the most common childhood disability, with an incidence around 2-2.5 in every 1,000 live births in Europe. It results from damage to the developing brain and adversely affects motor control. The limitations in motor control range from an inability to even hold the head erect and an inability to self-feed, to cases where for example walking is hampered by spasticity in one limb. The cornerstone of current treatment is physiotherapy in which the aims are to maintain and improve mobility and to prevent limitation of the range of joint movement. Specific forms of physical therapy include Conductive Education and Bobath treatment. Other interventions include botulinum toxin injections, intrathecal baclofen, selective dorsal rhizotomy and multi-level orthopaedic surgery. Despite these varied and concerted inputs, improvements in motor skills are very limited – motor skills tend to plateau around the age of seven and in fact deteriorate in adolescence. Of the four classifications of CP (Spastic, Athetoid or Dyskinetic, Ataxic and Mixed), the spastic type is the most common. Around 75% of all cases are spastic and around 60% of these are diplegic (meaning it affects both limbs, usually the legs). Spastic diplegia results from periventricular leucomalacia, where oligodendrocytes are damaged by hypo-perfusion of the periventriucular areas predominantly affecting the corticospinal tracts supplying the legs. This results in a deficit in the development of the white matter forming the insulation around those nerves and consequently compromises the signal transduction to the legs. As spastic diplegia is the most common type of CP, and the presenting symptoms are considered less complex than the other types, patients with spastic diplegia were chosen for participation in the current studies. The main symptoms presenting in spastic diplegia are reduced gross motor function, increased reflex response to muscle stretch, reduced range of ankle movement and shortened calf muscle/tendon units, evident in equinus. Whilst the main cause of spasticity in conditions other than CP is considered to be the neuropathology, altered muscle properties are considered to be the main problem in CP. Masseurs contend that they bring about a healthy response in damaged muscle by altering the resting state of the muscle, although this has not been scientifically proven until now. The initial aim of the present series of studies was to test if a specific massage sequence could increase the range of movement at the ankle joint by altering the mechanical properties of the muscle in adolescents with spastic diplegia. However the investigations indicated that instead, this type of massage changed sensory feedback from the spastic muscles, which led to significant improvements in motor skills. The physical limitations of the 12 participants with CP range from habitual wheelchair users to one participant who is able to run. Their abilities classified by the Gross Motor Function Classification System (GMFCS) ranged from level I to IV. The investigation involved the use of goniometry to measure change in the active and passive range of movement at the ankle joint and EMGs to measure incidence of stretch reflex contractions. Motor skills were assessed by an independent physiotherapist, using the Gross Motor Function Measure-66 (GMFM-66). In chapter 3, three passive ankle dorsiflexions at a controlled rate were carried out before and after massage which was given twice weekly for 5 weeks. The incidence of stretch reflex contractions during passive dorsiflexion was reduced from 40% in the first 5 massage sessions to 22% in the last 5 sessions, in the 5 participants tested. After massage the resistance of the calf muscle to stretch was not reduced as expected; in fact the muscles were stiffer (more force was needed to take the ankle through the same range of movement). However, the resting angles of the ankles often changed, indicating alteration of the resting length of the calf muscles. The change was not always in the one direction, although, on the whole, muscles lengthened after massage (shown by an average increase in dorsiflexion of 1.4º). It is argued that thixotropic properties of muscles were responsible and that the massage changed the mechanical properties of the calf muscles. In chapter 4, Gross Motor Function Measure-66 scores for all 12 adolescent participants who received the specialised massage were shown to be improved by an average of 5.8. Five of the 7 participants showed improvements in their ability to descend stairs, which is recognised to be particularly difficult in spastic diplegia. The range of voluntary ankle movement improved in some participants in some tests. Despite a lack of scientific evidence, masseurs also contend that their intervention brings about change by altering the blood flow. In the current studies, near infrared spectroscopy was used to measure oxygenation of the muscles and changes in the skin temperatures were also recorded. In chapter 5, temperature recorded from the skin over the calf muscle after massage was increased in both the CP group and the controls. Both finished with comparable temperatures although the CP group’s temperatures started 1-1.5°C below those of the control group. Contralateral effects of raised skin temperature were also observed. It was confirmed that the extent of change in skin temperature over the massaged muscles could be used to determine the effectiveness of a trainee using the massage technique. Additionally, the oxygenation of the tissue was altered significantly at some stage during massage for all participants. It is proposed that spastic muscles in CP may sometimes operate in oxygen debt, particularly in cold conditions. The improvements in GMFM-66 with massage are at least as effective as other current therapies and the massage has none of the adverse side effects of surgery and drug interventions. The mean improvement in GMFM-66 score after massage was 5.8, whereas treatments using selective dorsal rhizotomy and baclofen showed improvements of only 2.7 and 3.8 respectively. It is proposed that the mechanical properties and the feedback from spastic muscles are altered by the massage and that the CNS is able to accommodate the change in feedback to produce improved motor function. It is recommended that the massage used here be incorporated into the physiotherapy regime for individuals with CP. Author’s contact details: e-mail r.macgregor.1@research.gla.ac.uk

Pulmonary arterial hypertension (PAH) is a debilitating disease of small pulmonary resistance arteries with vasoconstriction and vascular remodelling contributing to the disease pathology. A genetic basis for the disease was linked to heterozygous loss of function mutations in the bone morphogenetic protein receptor 2 (BMPR2) gene. The mutation is found in the majority of familial PAH cases and a significant number of apparently sporadic cases. The low penetrance of the disease in families carrying BMPR2 mutations and the absence of mutations in the majority of idiopathic patients indicates that BMPR2 deficiency alone is insufficient to induce PAH. It is generally accepted PAH has a multi-factorial pathology with endogenous and environmental factors acting in concert with genetic pre-disposition to create the disease phenotype. Enhancement of the serotonin (5-HT) system has been implicated in PAH with the 5-HT transporter (5-HTT) receiving the most attention as a modifying gene in the development of PAH and there is compelling animal and human data implicating a role for increased expression of the 5-HTT as a modulating factor. The aim of this study was to investigate if genetic pre-disposition interacts with other additional modifying factors to create the symptoms of PAH. Transgenic mice overexpressing the 5-HTT (5-HTT+), deficient in BMPR2 (BMPR2+/-) or a double transgenic (5-HTT+/BMPR2+/-) were employed in addition to mice lacking tryptophan hydroxylase 1 (Tph1), the rate limiting enzyme for the synthesis of 5-HT, and therefore lacking peripheral 5-HT (Tph1-/-). Additional known or suspected modifying factors assessed in these genetic models were hypoxia, dexfenfluramine (Dfen) and its major metabolite nordexfenfluramine (NDfen), 5-HT, bone morphogenetic protein-2 (BMP-2), KCNQ channels and the role of gender. Mice were examined in vivo for evidence of a pulmonary hypertensive phenotype following exposure to hypoxia and Dfen. Female 5-HTT+ mice were the only group tohave a rise in two indices of PAH - namely right ventricular pressure (RVP) and vascular remodelling - in room air. Female 5-HTT+ mice also had an exaggerated pulmonary hypertensive phenotype in hypoxia. BMPR2+/- mice, were, unexpectedly least susceptible to hypoxic induced increases in RVP although female mice deficient in BMPR2 (both BMPR2+/- and 5-HTT+/BMPR2+/-) had more extensive vascular remodelling under hypoxia compared with WT and 5-HTT+ mice. Male mice did not express the phenotypic changes just outlined. No synergistic effect was observed between 5-HTT+ and BMPR2+/- that resulted in a more pronounced pulmonary hypertensive phenotype. WT and BMPR2+/- mice were chronically oral-dosed with Dfen. Female mice from both genotypes developed similar degrees of PAH. Male mice did not develop elevated RVP but BMPR2+/- males did have evidence of vascular remodelling, although at a lower level than the females. Female Tph1-/- mice did not develop PAH following Dfen indicating Dfen associated PAH is dependent on peripheral 5-HT synthesis. The presence of intact 5-HT synthesis was also associated with an increased vasoconstrictor response to 5-HT in isolated intralobar pulmonary arteries (IPAs), a situation not paralleled with the other serotonergic vasoconstrictors, Dfen and NDfen, indicating differing mechanisms of action underlying the respective vasoconstrictor responses. The vasoconstrictor action of 5-HT, Dfen, NDfen and the KCNQ potassium channel blocker linopirdine were all assessed in IPAs. Pulmonary arteries from BMPR2+/- mice showed enhanced vasoconstriction to 5-HT and NDfen. 5-HTT+ and 5-HTT+/BMPR2+/- mice showed enhanced vasoconstriction to NDfen but decreased vasoconstriction to 5-HT. Female 5-HTT+/BMPR2+/- mice were the only group tested to show significantly greater vasoconstriction to Dfen compared with WT. The vasoconstrictor response to linopirdine was significantly reduced in BMPR2+/- mice but neither linopirdine nor BMP-2 affected 5- HT induced vasoconstriction. Female gender is an established risk factor for PAH. To investigate possible events that may underlie this risk, male (testosterone) and female (estradiol and 2-methoxyestradiol (2-ME)) sex hormones were assessed for their vasoactive properties in IPAs. All three hormones relaxed pre-constricted vessels but only at supraphysiological (>0.1 µM) concentrations. Each hormone also reduced the vasoconstriction exerted by 5-HT at 10-5 M in male mice but not in females. No such effect, however, was observed in either gender at a physiological (10-9 M) concentration. NDfen induced vasoconstriction was also unaffected by 10-9 M estradiol. Finally, male and female mouse lungs were assessed for protein expression of 5-HT and BMPR2 signalling compounds (p-Smad1/5/8, p-ERK1/2 and p-p38 MAPK). Female mouse lungs displayed higher expression of the mitogenic mediator p-ERK1/2 than male mouse lungs with the other proteins unchanged. In conclusion, this study confirms overexpression of the 5-HTT as a trigger for elevated RVP and vascular remodelling in mice and a cause of more severe hypoxic PAH. BMPR2+/- mice are phenotypically normal in room air and show divergent pulmonary effects following hypoxia with loss of BMPR2 seemingly attenuating hypoxic induced increases in RVP but causing a simultaneous worsening of vascular remodelling, this latter effect consistent with the important role BMPR2 has in maintaining vascular integrity. Dfen induced PAH in mice was found to be dependent on peripheral 5-HT synthesis with BMPR2 mutation not acting as a risk factor. Loss of BMPR2 can enhance vasoconstriction to serotonergic agonists and when combined with overexpression of the 5-HTT, leads to a dramatic increase in sensitivity to Dfen induced vasoconstriction. Evidence was also found for altered KCNQ channel function in transgenic animals. Unexpectedly, female gender emerged as the most crucial risk factor for PAH in this thesis.

This thesis describes the use of intrinsic fluorescence measurements as a means for examining mitochondrial function in different cardiac preparations and phenotypes. Cardiac myocytes are intrinsically fluorescent and spectroscopic analysis of rabbit ventricular myocytes indicated that the majority of this fluorescence arises from the metabolic coenzymes nicotinamide adenine dinucleotide in the reduced state (NADH) and flavin adenine dinucleotide (FAD) in the oxidised state. Calibration of the NADH and FAD fluorescence signal with the mitochondrial inhibitors sodium cyanide (NaCN) and carbonyl cyanide p- (trifluoromethoxy) phenylhydrazone (FCCP) enabled calculation of mitochondrial redox states. Redox measurements reflect the balance between reduced and oxidised forms of the NAD and FAD pools and provide an index for assessing mitochondrial function in cells and tissue. The major advantage of this technique is that the intrinsically fluorescent nature of these metabolites obviates the need for exogenous indicators of mitochondrial function, which can themselves influence mitochondrial behaviour. Mitochondrial redox state was established using a variety of fluorescence techniques. Values for NADstate represent the proportion of the NADH/NAD+ redox couple in the reduced state. Calculation of NADstate using single photon, two photon and wide-field epifluorescence microscopy revealed very similar values ranging from 0.57±0.18 to 0.59±0.17 (mean±SD). FAD fluorescence measurements were used to establish FADstate (the proportion of the FADH2/FAD redox couple in the oxidised state). However, FAD fluorescence could only be detected by epifluorescence and single photon excitation fluorescence microscopy. Once again, comparable values of 0.17±0.10 and 0.18±0.07 respectively were obtained, thus demonstrating the reproducibility of the technique. Attempts were made to perform these measurements in intact cardiac tissue preparations. However, difficulties encountered with the delivery of mitochondrial inhibitor to specific areas of tissue and problems with inner filter effects complicating the interpretation of fluorescence recordings meant that this was not possible. Measurements of intrinsic fluorescence were utilised in order to assess the mitochondrial redox response of cardiac cells to increased energy demand. Isolated rabbit ventricular myocytes were field stimulated and fractional shortening was simultaneously recorded with epifluorescence measurements of NADH and FAD. Cells were paced at 0.5Hz and the stimulation frequency step increased to 1Hz, 2Hz and 3Hz in order to increase work intensity and energy demand. Step increasing stimulation frequency resulted in a decrease in NADH fluorescence and an increase in FAD fluorescence before reaching an essentially steady state. This indicated oxidation of the cell environment, suggesting a transient mismatch between metabolite supply and demand. The magnitude of this response was related to stimulation frequency, with the biggest responses taking place at the highest work intensity. Reducing work intensity back to 0.5Hz pacing resulted in immediate recovery of metabolite fluorescence. Investigation into the redox response to increased work intensity in the stroke prone spontaneously hypertensive rat (SHRSP) model of cardiac hypertrophy found that energy supply and demand matching was in fact improved in these cardiomyocytes compared to Wistar Kyoto (WKY) control myocytes. Work intensity was increased from 1Hz to 2, 4 and 6Hz pacing and the oxidative response to increased workload was found to be significantly less in SHRSP cardiomyocytes compared to WKY myocytes (p<0.01). This was despite similar levels of contractile work being performed by the two groups and may be related to the young age of the animals (16 weeks). At this age, hypertrophy of the SHRSP hearts is likely to still be in the compensated state and mitochondrial function may indeed be improved rather than detrimentally affected at this stage.

In this thesis, a method for studying the electrophysiological consequences of acute regional ischaemia in rabbit heart was established using a combination of a novel snare technique and optical mapping. The purpose of this approach was to discover the mechanistic link between acute coronary infarction and the occurrence of arrhythmias. The electrophysiology of the epicardial surface of isolated hearts was examined using the voltage sensitive dye RH237 and optical action potentials were recorded from a 13x13mm area of left ventricular epicardium using a 16x16 element Hamamatsu photodiode array. Contraction motion artefacts were practically eliminated with blebbistatin (5µM). An alternative mechanical uncoupler, BDM, was found to be not suitable for the study of arrhythmic behaviour associated with ischaemia. After occlusion of the left coronary artery, a progressive reduction in action potential duration (APD), and slowing of upstroke was observed in an area of the left ventricle anterior surface, accompanied by ECG S-T segment elevation. These effects were reversed when the coronary artery occlusion was released. Ligation (duration 12-15mins) caused a decrease in APD50 (APD at 50% repolarisation), in the zone of reduced perfusion, from 141±5.2ms to 53.3±9.3ms (mean±SEM, n=10 hearts, P<0.001). After ligation was reversed and full perfusion restored, APD50 returned to normal values (149±7.0ms, n.s.). Trise (action potential rise time from 10-90% depolarisation) increased from 7.2±1.0ms to 15.8±2.8ms (P<0.01). In the non-infarcted area of myocardium, no significant changes in APD50 (147±7.0ms vs. 147±8.1ms) or Trise (6.4±0.4ms vs 8.8±1.4ms) were observed during occlusion. T-wave alternans behaviour was observed frequently during local ischaemia and associated with alternans of optical action potentials (OAPs) in the ischaemic border zone (BZ) and in ischaemic zone (IZ). T-wave alternans amplitude was not maintained during local ischaemia but OAPs continued to show alternating behaviour. Arrhythmias (VT and VF) were common when conduction block occurred at the interface between the normal and ischaemic zone, but arrhythmias were absent when conduction into the IZ was retained. This observation suggests that the conduction block was the crucial precipitating event for the generation of arrhythmias. Acute local ischaemia was also imposed in a heart with an existing infarct scar to examine the effects of pre-existing ischaemic damage. The incidence of arrhythmias was similar to that observed in the absence of an infarct scar indicating that pre-existing damage did not predispose the heart to arrhythmias. Global ischaemic challenges, both low flow and zero flow produced similar reductions in APD and rise time and were followed by arrhythmias, but the associated changes in the ECG were complex and could not be easily interpreted. Significant temporal variability in electrophysiology was observed in global ischaemia, but absent in the local ischaemic challenge. The underlying mechanisms of these temporal flucuations in cardiac electrophysiology may be dictated by either cellular metabolism or fluctuations in coronary flow. Long-term local ischaemia (~60mins) did not reveal a second phase of arrhythmias after 40-45mins as observed in other animal models, and nor were there signs of significant further electrophysiological changes as a consequence of the additional period of local ischaemia.

1.Previous research in the bovine intraocular long posterior ciliary artery (LPCA) has suggested that its response to electrical field stimulation (EFS) is a uniphasic vasodilatation, mediated jointly by the neurotransmitters nitric oxide (NO) and calcitonin gene related-peptide (CGRP). 2.This study of the bovine intraocular LPCA, where a short period of EFS (10 s) was employed, demonstrated that the vasodilator response to EFS was in fact biphasic. The first and second components of the response peaked separately at 10 s and 50 s following the onset of stimulation. 3.Both components of vasodilatation to EFS were abolished by tetrodotoxin (TTX), confirming their neurogenic origin. 4.Inhibition of the first component of the vasodilatation to EFS by the nitric oxide synthase (NOS) inhibitor, L-NAME, and the inhibitor of soluble guanylate cyclase, ODQ, confirmed the involvement of NO from nitrergic nerves. Experiments involving treatment with capsaicin and the CGRP antagonist, CGRP8-37, failed to produce any evidence of involvement of CGRP in the second component of neurogenic vasodilatation. 5.A number of other potential neurotransmitter candidates including substance P, vasoactive intestinal polypeptide (VIP) and adenosine triphosphate (ATP) were investigated, but there was no convincing evidence to suggest that they play a role in mediating the second component of neurogenic vasodilatation. 6.As the transmitter responsible for mediating the second component of neurogenic vasodilatation could not be identified, the mechanism by which it operated was also investigated. 7.Although the first component of neurogenic vasodilatation was clearly mediated by NO, inhibitors of PDE5, PKG and the soluble guanylate cyclase/cGMP/NO pathway had no effect on the second component of vasodilatation. 8.Furthermore, various K+ channel blockers, or inhibitors of the PKA/cAMP pathway did not inhibit the second component of neurogenic vasodilatation. Therefore, neither the identity of the neurotransmitter responsible for the second component, nor its mechanism of action could be determined. 9.NG-substituted analogues of L-arginine are routinely used to inhibit the NOS family of enzymes. The first of these to be introduced, NG-methyl-L-arginine (L-NMMA), is generally reported to inhibit all three isoforms of NOS. Despite this, however, L-NMMA does not inhibit nitrergic nerve-mediated relaxation in other bovine tissues including the retractor penis muscle or penile artery. 10.In this study of the bovine intraocular LPCA, L-NMMA was found to inhibit the endothelium-dependent dilatation mediated by eNOS, but not the nitrergic vasodilatation by nNOS. Indeed, the ability of L-NMMA to protect nitrergic dilatation against blockade by L-NAME in a manner similar to L-arginine, suggests it might act as an alternative substrate for nNOS in this tissue. 11.Isoform-selective inhibitors of NOS are of great interest, both as investigational tools and potential therapeutic agents. Two nNOS-specific inhibitors have recently been identified: N-[(4S)-4-amino-5-[(2-aminoethyl)amino]pentyl]-N'-nitroguanidine tris (trifluoroacetate) salt (AAAN) and NG-propyl-L-arginine. 12.AAAN failed to affect vasodilatation induced either by nitrergic nerves or by bradykinin-induced, endothelium-derived NO in the bovine intraocular LPCA. 13.Although NG-propyl-L-arginine did inhibit nitrergic vasodilatation, it also blocked the bradykinin-induced, endothelium-dependent vasodilatation mediated by NO. It thus failed to exhibit the expected selectivity for nNOS over eNOS in the bovine intraocular LPCA. 14.These findings with AAAN and NG-propyl-L-arginine are a reminder that it is not always possible to extrapolate findings in biochemical assays to functional responses in intact tissues. 15.In conclusion, these findings show that neurogenic vasodilatation in the bovine LPCA involves two components: a fast, transient component mediated by NO from nitrergic nerves, and a second slower, more sustained component mediated by an as yet unidentified second neurotransmitter.

Functional, electrically stimulated (FES) cycle training can improve the cardiorespiratory fitness of spinal cord injured (SCI) individuals, but the extent to which this can occur following high volume FES cycle endurance training is not known. The effect of training on aerobic endurance capacity, as determined by the appearance of respiratory gas exchange thresholds, is also unknown. The oxygen cost (O2 cost) of this type of exercise is about 3.5 times higher than that of volitional cycling, but the source of this inefficiency, and of the variation between subjects, has not yet been investigated. The electrical cost of FES cycling, measured as the stimulation charge required per Watt of power produced (stim/Pt), has neither been calculated nor investigated before. It is also not known whether a period of FES cycling can alter the O2 cost or the stim/Pt of this unique form of exercise. Additionally, the acute metabolic responses to prolonged, high intensity FES cycling after a 12-month period of high-volume training have not yet been characterised for this subject group. Accordingly, these parameters were investigated over the course of a 12-month homebased FES cycle training programme (up to 5 x 60 min per week) in 9 male and 2 female individuals with paraplegia. Outcomes were investigated using a novel, sensitive test bed that accounted for both internal and external power production (Pt). The test protocol permitted high resolution analyses of cycling power and metabolic thresholds, and a sensitive training dose-response analysis, to be performed for the first time in FES cycling. Efficiency estimates were calculated within a new theoretical framework that was developed for those with severe disability, and the stim/Pt was determined using a novel measure designed for this study. The current training programme resulted in significant improvements in cardiorespiratory fitness and peak cycling power, but only over the first 6 months when training was progressive. These improvements were positively related to the number of training hours completed during this time. It is not known whether the plateau in training response that was found after this time was due to a physiological limitation within the muscles, or to limitations in the current stimulation strategy and of the training protocol used. The efficiency of FES cycling was not significantly altered by any period of training. However, the stim/Pt of cycling had reduced over the first 6 months, probably as a result of a fibre hypertrophy within the stimulated motor units. The relationship that was found between variables after this time suggest that differences in the efficiency of FES cycling ii between subjects and over time related primarily to the stim/Pt, which determined the number of motor units recruited per unit of power produced, rather than to metabolic changes within the muscle itself. The aerobic gas exchange threshold (GET) was detected at an oxygen uptake (˙VO2) equivalent to that normally elicited by very gentle volitional exercise, even after training. This provided metabolic evidence of anaerobic fibre recruitment from the outset, as a consequence of the non-physiological motor unit recruitment pattern normally found during FES. The cardiorespiratory stress of training was found to be significantly higher than that elicited by the incremental work rate tests, calling into question the validity of using traditional, continuous incremental work rate tests for establishing the peak oxygen uptake (˙VO2peak) of FES cycling. The respiratory exchange dynamics observed over a 60 min training session were characterised and provide a unique insight into the remarkable aerobic and anaerobic capacity of trained paralytic muscles. For this particular highly motivated subject group, training for 60 min per day on more than 4 days of the week was demonstrated to be feasible, but not able to be sustained. Further work is therefore recommended to develop and to evaluate different stimulation patterns and parameters, loading strategies and training protocols. The aim would be to determine the optimal combination of training parameters that would maximise favourable training responses within a more viable and sustainable lower volume, training programme for this subject group. In conclusion, the outcomes of this multi-centre study have demonstrated the clinical significance of using otherwise redundant, paralytic leg muscles to perform functional, regular physical exercise to improve cardiorespiratory and musculoskeletal health after SCI. Additionally, the significant increases in cycling power and endurance that were achieved opened up new mobility and recreational possibilities for this group of individuals. These findings highlight the clinical and social relevance of regular FES cycle training, and the importance of integrating FES cycling into the lives of those affected by SCI. The early and judicious implementation of this form of exercise is strongly recommended for the maintenance of a healthy body, wellbeing, and of an active lifestyle after SCI.

Pulmonary arterial hypertension (PAH) is a progressive disease which results from increases in mean pulmonary artery pressure and pulmonary vascular resistance. If untreated it leads to right ventricular failure and death. 5-Hydroxytryptamine (5-HT) has been implicated in the disease process and is thought to promote both vasoconstriction and remodelling of the pulmonary vasculature. The activity of potassium ion (K+) channels plays a major role in influencing pulmonary artery tone by regulating resting membrane potential, intracellular Ca2+ concentration and contraction of vascular smooth muscle. This study aimed to investigate possible interactions between 5-HT and K+ channels in the development of PAH in the mouse. The actions of 5-HT and a range of K+ channel blockers were investigated on isolated intralobar pulmonary arteries (IPA) from wild type (WT) mice and mice over-expressing the serotonin transporter (5HTT), which spontaneously develop PAH. Both 5-HT and linopirdine, a KCNQ K+ channel inhibitor, were found to induce contraction of IPA, but were more potent in IPA from WT mice than 5-HTT+ mice. The 5-HT induced vasoconstriction was found to involve influx of Ca2+ from the extracellular space, Ca2+ release from the sacroplasmic reticulum and a rho kinase–dependent increase in the sensitivity of the contractile machinery of pulmonary artery smooth muscle cells (PASMC) to intracellular Ca2+. Ca2+ entered the cell via both voltage operated calcium channels (VOCC), activated by membrane depolarisation, and a separate Ca2+ entry pathway, the latter appearing to contribute more in 5-HTT+ mice. The effects of linopirdine were shown to be due entirely to the entry of Ca2+ through VOCC in both WT and 5-HTT+ mice IPA. The difference in vasoconstrictor potency between WT and 5HTT+ mice was not seen with any other K+ channel blocker, suggesting a selective loss of KCNQ channels and/or VOCC in PAH resulting from 5HTT over expression. KCNQ channel activity was further investigated using the KCNQ channel openers, flupirtine and retigabine. These agents were more potent in dilating IPA from WT mice compared to 5-HTT+ mice, consistent with the loss of expression or activity of KCNQ channels in 5-HTT+ mice. Despite this, orally administered flupirtine was shown to reverse two indices of established PAH in the 5HTT+ mice; right ventricular pressure and right ventricular hypertrophy. This action of flupirtine was also seen in chronic hypoxic mice, where it prevented the development of PAH. In conclusion, this study provides evidence of an interaction between KCNQ channels and the 5-HT system in the development of PAH. By showing that a KCNQ channel opener can attenuate PAH, both in the developing and established disease situation, this study proposes a new potential therapeutic target in the treatment of PAH.