Obesity has reached worldwide epidemic proportions and with this increased incidence of obesity, comes an increase in incidence of the comorbidities associated with obesity such as diabetes and cardiovascular disease (CVD). The underlying mechanisms which connect these diseases are still poorly understood. One system which has been shown to be up-regulated in the setting of obesity and diabetes is that of the G-protein coupled receptor-55/Lysophosphatidylinositol (GPR55/LPI). Despite being upregulated in the setting of obesity, the function of GPR55 in obesity and other disease states remains elusive. Therefore, the present study aimed to 1) investigate the role of GPR55 in obesity by characterising the phenotype of the GPR55 knockout (GPR55-/-) mouse when challenged with a high fat diet (HFD) intervention, 2) elucidate any effect of the GPR55 knockout and HFD intervention on the myocardial infarct size sustained following a period of ischaemia/reperfusion (I/R) and 3) make use of an in vitro model to elucidate the mechanisms by which changes occur in the adipose tissue of mice fed a HFD. GPR55-/- mice fed a HFD for 12-weeks gained significantly more weight in the form of fat mass, compared to wild-type (WT) controls and consequently become obese. Obese GPR55-/- mice displayed hypertrophic adipose tissue concurrent with the significant dysregulation of plasma lipids, increases in specific circulating LPI species, increased lipid deposition within the liver and a change in adipose tissue gene expression profile. These changes were not observed in GPR55-/- mice fed a standard diet or WT mice fed a HFD. Following a period of I/R, the myocardial infarct size in hearts from WT HFD fed mice was significantly smaller than in hearts from WT standard diet fed mice. This reduction in infarct size due to HFD intervention was not dependent on RISK-pathway activation and was not observed in hearts from GPR55-/- mice, therefore demonstrating that the cardio-protective effect of a HFD on infarct size is dependent on GPR55. In vitro studies using 3T3-L1 cells determined that the changes in adipose tissue gene expression of HFD fed mice was not due to enhanced stimulation with LPI or via hypoxic mechanisms. The results of these studies demonstrate that GPR55 has an anti-obesity function in vivo and also mediates the cardio-protective effect of a HFD on myocardial infarct size, through currently unknown mechanisms.

The Slit family of secreted proteins act as axonal repellents during embryogenesis. Slit2 via its receptor, Roundabout-1, also inhibits chemotaxis of multiple leukocyte subsets. Using static and microfluidic shear assays, we found that Slit2 inhibited multiple steps required to recruit circulating neutrophils. Slit2 blocked capture and firm adhesion of human neutrophils to and transmigration across inflamed primary vascular endothelial cells. To determine the response of Slit2 in renal ischemia reperfsuion injury, Slit2 was administered prior to bilateral renal pedicle clamping in mice. This led to significant decreases in both renal tubular necrosis score and neutrophil infiltration. Administration of Slit2 also prevented elevation of plasma creatinine following injury in a dose-dependent manner. Furthermore, administration of Slit2 did not increase hepatic bacterial load in mice infected with L.monocytogenes infection. Collectively, these data demonstrate Slit2 as an exciting therapeutic molecule to combat renal ischemia reperfusion injury without compromising protective host innate immune functions.

The Slit family of secreted proteins act as axonal repellents during embryogenesis. Slit2 via its receptor, Roundabout-1, also inhibits chemotaxis of multiple leukocyte subsets. Using static and microfluidic shear assays, we found that Slit2 inhibited multiple steps required to recruit circulating neutrophils. Slit2 blocked capture and firm adhesion of human neutrophils to and transmigration across inflamed primary vascular endothelial cells. To determine the response of Slit2 in renal ischemia reperfsuion injury, Slit2 was administered prior to bilateral renal pedicle clamping in mice. This led to significant decreases in both renal tubular necrosis score and neutrophil infiltration. Administration of Slit2 also prevented elevation of plasma creatinine following injury in a dose-dependent manner. Furthermore, administration of Slit2 did not increase hepatic bacterial load in mice infected with L.monocytogenes infection. Collectively, these data demonstrate Slit2 as an exciting therapeutic molecule to combat renal ischemia reperfusion injury without compromising protective host innate immune functions.

Thesis (Ph. D.)--University of Hong Kong, 2008. / Includes bibliographical references (leaves 172-238) Also available in print.

Artemisinin is herbal drug with a wide range of biological and physiological function. It is currently administered in the treatment against uncomplicated F.Palcifarum infections. It has also been shown to be cytotoxic against a variety of cancer cells. Despite the promise of many anti cancer drugs, drug induced cardiotoxicity has constantly threatened drug applicability especially in patients with co-morbities. Artemisinin has been shown to be cardioprotective, although the intracellular pathways remain to be elucidated. In this study, isolated perfused rat hearts were subjected to 35 minutes of ischaemia and 120 minutes reperfusion or primary cardiac myocytes subjected to 120 minutes hypoxia and 120 minutes reoxygenation where artemisinin (4.3μM) was administered in presence and absence of the PI3K inhibitor (wortmannin) (0.1μM), p70S6K inhibitor (rapamycin) (0.1μM), non selective nitric oxide synthase inhibitor (L-NAME) (100μM) and inducible nitric oxide synthase inhibitor (aminoguanidine) (100μM). At the end of the experiment, hearts underwent infarct size to risk ratio assessment via tri-phenyltetrazolium chloride staining or western blot analysis for p-Akt and p70S6K. Cardiac myocytes were assessed for either MTT analysis, cleaved-caspase 3 or for eNOS/iNOS or p-BAD activity using flow cytometry. In isolated hearts, artemisinin (0.1μM-100μM) showed a significant dose dependent decrease in infarct size (P<0.01-0.001 vs. I/R control). It was also shown to significantly improve cellular viability (66.5±6.3% vs. 29.3±6.1% in H/R, P<0.01) and decrease the levels of cleaved caspase-3 compared to the H/R control group (17.1±2.0% vs. 26.8±2.0% in H/R, P<0.001). Artemisinin was shown to confer protection via the activation of the PI3K-Akt-p70S6k cell survival pathway and presented an upregulation in p-eNOS and iNOS expression. Furthermore, co-administering artemisinin with doxorubicin showed artemisinin reverses I/R or H/R injury as well as doxorubicin-induced injury via the nitric oxide signalling pathway. Additionally, in HL-60 cells, the co-administration doubled artemisinins cytotoxicity while also implicating the nitric oxide pathway. This is the first study to shows that artemisinin ameliorates doxorubicin mediated cardiac injury whilst enhancing its cytotoxicity in HL-60 in a nitric oxide dependent manner. This study concluded that artemisinin was both anti apoptotic and protective against myocardial I/R injury via the PI3K-Akt-BAD/P70S6K and via the nitric oxide cell survival pathway as well as pro-apoptotic against HL-60 in a nitric oxide dependent manner.

No / Lower limb ulcers (LLU) are a common manifestation of long-standing vascular disease and may be exacerbated by trauma or dependency. Ulcers can be grouped according to the underlying aetiology with approximately 76% due to venous disease, 22% due to arterial and 5% occurring as a complication of diabetes. In up to 20% of patients there is some crossover where a mixed picture is present. / BioMonde

Ischaemic heart disease is a major contributor to premature death and heart failure in the Westernised world. Ischaemic injury within the heart may be beneficially modulated by the nucleoside adenosine. Derived from catabolism of ATP, adenosine was initially known as a potent bradycardic and hypotensive agent. However, more recently the protective function of adenosine has been investigated. The protective effects of adenosine are mediated via activation of adenosine receptors: A1, A2A, A2B, and A3 receptors. Activation of these potentially protective (or retaliatory) adenosine receptors hinges upon accumulation of adenosine during ischaemia-reperfusion. This Thesis examines the role and mechanisms of adenosine mediated cardioprotection in young and aged hearts, exploring endogenous and exogenous adenosine receptor activation, genetic manipulation of A1 receptors and adenosine deaminase and pharmacological manipulation of adenosine metabolism. The effects of age on ischaemic responses and adenosine handling and protection are also assessed. The core approach to assess each of the above issues involved the Langendorff isolated mouse heart preparation. Experiments within Chapter 3 focuses on the contractile effects of adenosine receptors in normoxic hearts. This study indicates A2A receptors have no direct effect on contractility, while adenosine exerts positive inotropy independently of coronary flow and perfusion pressure (i.e. Independent of the Gregg phenomenon). In addition, investigations in genetically modified hearts hint at positive inotropy in response to A1 receptors. Since the latter is only evidenced in modified lines, it is possible A1-mediated inotropy may be abnormal or supraphysiological. In Chapter 4 the impact of genetic 'deletion' of A1ARs and/or adenosine deaminase (ADA) on intrinsic tolerance to ischaemia were studied. Data demonstrate that genetic deletion of A1 receptors significantly limits the ability of the mouse myocardium to withstand injury during ischaemic insult. Thus, providing strong support for a role of A1ARs in determining intrinsic tolerance to ischaemia-reperfusion. ADA KO mice confirm protection afforded by endogenous adenosine and the notion of adenosine metabolism modification as a protective strategy. Interestingly, effects of A1AR KO differ from A1AR overexpression or A1AR agonism in that the latter decrease contractile diastolic dysfunction while A1AR KO selectively increase systolic dysfunction and increase oncosis without altering diastolic injury. This challenges current dogma regarding the action of A1 adenosine receptors on ischaemic injury. In Chapter 5 the effects of adenosine metabolism inhibition (via adenosine deaminase (ADA) and adenosine kinase (AK) inhibitors) were studied. Inhibition of adenosine deaminase with the drug EHNA, and adenosine phosphorylation with iodotubercidin significantly protected mouse hearts from ischaemia-reperfusion, reducing contractile dysfunction and cardiac enzyme efflux. However, inhibitors failed to improve the outcome of the aged myocardium. 8-SPT and 5-HD reduced the protective effects of EHNA and iodotubercidin demonstrating thus; cardioprotection via ADA and AK appears to rely on adenosine receptor activation and involves a mitoK ATP dependent mechanism. Since aging is of considerable importance with regard to outcomes of ischaemic heart disease, experiments in Chapter 6 focused on effects of aging (and gender) on cardiovascular function and injury during ischaemia-reperfusion. In assessing post ischaemic outcomes in hearts from young adult (2-4 months), mature adult (8 months), middle aged (12 months), aged (18 months) and senescent (24-28 months) C57/BL/6J mice, data reveal a substantial age-related decline in ischaemic tolerance (which appears selective for myocardial vs. vascular injury). The decline in ischaemic tolerance is expressed primarily within the initial 12 months in both males and females with relatively little further decline with continued aging. There is evidence of a modest improvement in tolerance in senescence vs. aged hearts possibly reflecting selection of a protected phenotype in senescent populations. In addition, mature and middle-aged female hearts showed improved tolerance to ischaemia-reperfusion compared to males, supporting a role for hormonal changes. Effects of aging and purine metabolism were studied in Chapter 7. Data suggest impaired tolerance to ischaemia-reperfusion in older hearts may stem in part from shifts in myocardial purine catabolism. Data reveal reduced accumulation of salvageable and cardioprotective adenosine and enhanced accumulation of poorly salvaged (and potentially injurious) hypoxanthine and xanthine. These changes may potentially predispose the aged myocardium to ischaemic injury and radical generation via the xanthine oxidase reaction. The final data Chapter of this Thesis describes preliminary data regarding aging, signalling and adenosine mediated protection. It was found that protein kinase C (PKC) and A1 receptors mediate protection in young hearts and also that A1 receptors appear to mediate protection via a PKC LindependentLi signalling cascade. In addition, experiments in aged hearts (attempting to elucidate mechanisms behind impaired adenosinergic protection with age) suggest a PKC-independent A1-mediated protection path may be preserved with aging, since A1 receptors continue to offer some protection while PKC activation does not. It is possible the failure of exogenous adenosine to offer protection in aged hearts may result from a lesion at or downstream of PKC.

published_or_final_version / Physiology / Doctoral / Doctor of Philosophy

Atherosclerosis is a multifactorial, chronic inflammatory condition characterised by endothelial dysfunction, hyperlipidaemia and the accumulation of fatty deposits within the tunica intima of medium-to-large sized muscular arteries. This disease can prove fatal with patients suffering lethal myocardial infarction or stroke. Recently, two studies investigating the role of G-protein-coupled receptor 55 (GPR55) in atherosclerosis reported conflicting results; one reported a pro-atherogenic role for GPR55 and the other, an anti-atherogenic role for this receptor. Interestingly, another study demonstrated that the activation of GPR55 by lysophosphatidylinositol (LPI) in cultured rat neonatal ventricular cardiomyocytes provokes distinct cellular functions that are dependent on the location of GPR55, leading to suggestions that GPR55 may regulate cardiomyocyte function at two cellular sites and be a potential therapeutic target for cardiac disorders. While it has been demonstrated that GPR55 is important in the maintenance of cardiac function of healthy mice, what is currently unknown is if GPR55 has a role in the cardiovascular remodelling and cardiac function of atherosclerosis prone mice. To address this, the present studies were conducted to investigate 1) the role of GPR55 in atherogenesis, 2) if GPR55 has a role in the cardiac function of mice suffering from atherosclerosis, 3) the signalling pathway by which LPI activates cardiomyocytes, 4) the impact of GPR55 activation on the outcome of myocardial ischaemia/reperfusion (I/R) injury and, 5) the signalling mechanisms by which GPR55 elicits any observed effects on the myocardium in response to such injury. Using C57BL/6 (wildtype; WT), apolipoprotein E knockout (ApoE-/-; mouse model of atherosclerosis), GPR55 knockout (GPR55-/-) and novel ApoE-/-/GPR55-/- mice, this study has established that in the presence of high fat feeding (to accelerate atherosclerosis), GPR55 has a complex role whereby it both regulates risk factors associated with atherosclerosis (i.e. body weight and fat mass) yet promotes the development of fatty streaks within the vasculature, via a lipid independent mechanism. In terms of cardiac function, GPR55 exerted a protective role by maintaining the systolic function of high fat fed ApoE-/- mice, yet negatively affected the contractile reserve of these mice. With regard to infarct size, the present study established that LPI-induced activation of GPR55 (pre-global ischaemia) exacerbates myocardial tissue injury via a Rho-associated protein kinase (ROCK) dependent mechanism. Finally, this study established that LPI signals through the same signalling pathway as it did in the isolated heart, in both mouse and human-induced pluripotent stem cell-derived cardiomyocytes thus suggesting a translational role for GPR55 in the human heart. In conclusion, despite further research being required, the data presented within this thesis provides evidence that GPR55 may have the potential to be targeted for therapeutic gains in atherosclerosis and myocardial I/R injury.

The overall aim of this Thesis was to characterise the energetic properties of the mouse papillary muscle as this preparation could become a useful model to study alterations of energetic aspects of cardiac pathologies and heart-focussed genetic changes. Measurements of resting and active metabolism of the papillary muscles were made in vitro using the myothermic technique. In the first study the mechanism underlying impaired contractility of post-ischaemic rat papillary muscle was investigated. The rat preparation is well established and was used to develop protocols and approaches that could later be used as the basis for studies with mouse papillary muscle. The muscles were exposed to simulated ischaemia for 60 min and change in energetics was studied 30 min into the reperfusion phase. The work output was reduced to 66 ± 3% of the pre-ischaemia value and the enthalpy output decreased to 71 ± 3% of pre-ischaemia value. However, there was no change in either initial, 19 ± 3%, or net mechanical efficiency, 9.0 ± 0.9%. These data, in combination with studies of Ca2+ handling, suggests that the reduced work output was caused by attachment of fewer cross-bridges in each twitch, but with no change in work generated by each cross-bridge. The following two studies involved characterisation of the energetics of the mouse papillary muscle and included measurements of resting and active metabolism. The resting metabolic rate varied with muscle size but the mean initial value was tilda 25 mW g-1 and the estimated steady value tilda 5 mW g-1 . The resting metabolic rate declined exponentially with time towards a steady value, with a time constant of 18 ± 2 min. There was no alteration in isometric force output during this time. The magnitude of resting metabolism depended inversely on muscle mass, more than doubled following a change in substrate from glucose to pyruvate and was increased 2.5-fold when the osmolarity of the bathing solution was increased by addition of 300 mM sucrose. Addition of 30 mM BDM affected neither the time course of the decline in metabolic rate nor the eventual steady value. The energy requirements associated with contractile activity were tilda7 mJ g-1 twitch-1 at a contraction frequency of 1 Hz. The enthalpy output was not affected by changing substrate from glucose to pyruvate but did decrease with an increase in temperature. The enthalpy output was partitioned into force-dependent and force-independent components using BDM to selectively inhibit cross-bridge cycling. The force-independent enthalpy output was 18.6 ± 1.9% of the initial enthalpy output. Muscle initial efficiency was &tilda32% and net efficiency tilda 17% when shortening at a realistic velocity. The enthalpy output decreased with increased contraction frequency but was independent of shortening velocity. On the basis of these values, it was calculated that the twitch energetics were consistent with ATP splitting by half the cross-bridges and the pumping of one Ca 2+ into the SR for every three cross-bridge cycles. The lack of influence of shortening velocity on energy cost supports the idea that the amount of energy to be used is determined early in a twitch and is not greatly influenced by events that occur during the contraction. The suitability of the mouse papillary muscle as a model to study ischaemia and reperfusion damage was also assessed. This preparation is excellent for studying muscle specific changes in work and enthalpy output; however, due to the long-term instability and variability amongst preparations, the suitability of this preparation in prolonged experiments remains uncertain.