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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.

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.

The phytocannabinoid cannabidiol (CBD) has a complex pharmacology which is thought to include, but is not limited to, an ability to act as an inverse agonist at the CB1 and CB2 receptors and an antagonist of GPR55. Moreover, is has been shown to reduce infarct size and ameliorate reductions in left ventricular function in vivo. These improvements in the pathogenesis of experimental MI are accompanied by a reduction in inflammatory cell migration to the area at risk. More recently it has been shown that CBD is anti-arrhythmic in acute experimental MI. Thus, it was suggested that the cardioprotective effects of CBD might be due to an anti-inflammatory action. In addition, GPR55 receptor activation is acknowledged to mediate mobilisation of intracellular Ca2+ (Ca2+i) which could potentially be pro-arrhythmic and so CBD, as an antagonist may confer cardioprotection via GPR55. However, the receptors and/or mechanisms responsible for mediating the cardioprotective effects of CBD are get to be determined. The present studies were therefore performed to; (1) better understand the pharmacology of CBD by assessing haemodynamic responses to CBD and other cannabinoids ligands in anaesthetised rats, (2) investigate the receptors involved in the anti-arrhythmic effect of CBD in a rat model of coronary artery occlusion (CAO), and (3) investigate if CBD can alter [Ca2+]i in isolated rat cardiomyocytes. The characterisation of the pharmacology of CBD in vivo showed that; firstly, CB1 receptor activation causes a hypotensive response which can be dose-dependently inhibited by AM251; secondly, both CBD and AM251 alone (a CB1 receptor antagonist and GPR55 agonist) can induce vasodepressor responses and finally, CBD can potentiate the AM251-mediated hypotension when co-administered, suggesting possible cross-talk between the CB1 and GPR55. Results from CAO studies showed that CBD and AM251 each have the capacity to reduce arrhythmias. Moreover, when CBD and AM251 were co-administered the anti-arrhythmic capacity of either alone was potentiated. However, the degree of potentiation was dependent on the order of administration, suggesting that more than one receptor is involved in the summative anti-arrhythmic effects. The investigation of cardiomyocyte [Ca2+]i suggested that AM251 can modulate [Ca2+]i at the level of the cardiomyocyte, while CBD cannot. These data give novel insight into the anti-arrhythmic effects of CBD and, moreover, for the first time demonstrate that AM251 is anti-arrhythmic. In addition, these data suggest a role for GPR55 in increasing [Ca2+]i via AM251.

Electrical excitation of the dorsal aspect of the rostral thoracic spinal cord imparts long-term therapeutic benefits to patients with angina pectoris. Such spinal cord stimulation also induces short-term suppressor effects on the intrinsic cardiac nervous system. The purpose of this study was to determine whether spinal cord stimulation (SCS) induces long-term effects on the intrinsic nervous system, particularly in the presence of myocardial ischaemia. The activity generated by right atrial neurons was recorded in 10 anesthetized dogs during basal states, during prolonged (15 min) occlusion of the left anterior descending coronary artery, and during the subsequent reperfusion phase. Neuronal activity and cardiovascular indices were also monitored when the dorsal T1-T4 segments of the spinal cord were stimulated electrically (50 Hz; 0.2 ms) at an intensity 90% of motor threshold (mean 0.32 mA) for 17 min. SCS was performed before, during and after 15-min periods of regional ventricular ischaemia. Occlusion of a major coronary artery, one that did not perfuse investigated neurons, resulted in their excitation. Ischaemia-induced neuronal excitatory effects were suppressed (-76% from baseline) by SCS. SCS suppression of intrinsic cardiac neuronal activity persisted during the subsequent reperfusion period; after terminating 17 min of SCS, at least 20 min elapsed before intrinsic cardiac neuronal activity returned to baseline values. It is concluded that populations of intrinsic cardiac neurons are activated by inputs arising from the ischaemic myocardium. Ischaemia-induced activation of these neurons is nullified by SCS. The neuronal suppressor effects that SCS induces persist not only during reperfusion, but also for an extended period of time thereafter. These long-term effects may account, in part, for the fact that SCS imparts clinical benefit to patients with angina of cardiac origin not only during its application, but also for a time thereafter.

Aims Toll-like receptor (TLR)-mediated signalling pathways have been implicated in myocardial ischaemia/reperfusion (I/R) injury. Activation of the phosphoinositide 3-kinase (PI3K)/Akt pathway protects the myocardium from ischaemic injury. We hypothesized that the modulation of TLR2 would induce cardioprotection against I/R injury via activation of the PI3K/Akt signalling. Methods and results Mice were treated with TLR2 ligands, peptidoglycan (PGN) or Pam3CSK4, respectively, 1 h before the hearts were subjected to ischaemia (1 h), followed by reperfusion (4 h). Infarct size was determined by triphenyltetrazolium chloride staining. Cardiac function and haemodynamic performance were evaluated. Infarct size was significantly reduced in PGN-or Pam3CSK4-treated mice compared with untreated I/R mice. Administration of TLR2 ligands improved cardiac function following I/R. PGN treatment increased the levels of phospho-Akt and phospho-GSK-3β (glycogen synthase kinase-3β), compared with untreated I/R hearts. PGN stimulation increased TLR2 tyrosine phosphorylation and association of the p85 subunit of PI3K with TLR2. To investigate the role of PI3K/Akt signalling in PGN-induced cardioprotection, we administered the PI3K inhibitor, Wortmannin, to the mice 15 min before PGN treatment. We also administered PGN to kinase-deficient Akt (kdAkt) transgenic mice 1 h before myocardial I/R. Both PI3K inhibition and kdAkt mice abolished the cardioprotection induced by PGN. To examine the role of TLR2 in PGN-induced cardioprotection, we administrated PGN to TLR2 knockout mice 1 h before the hearts were subjected to I/R. PGN-induced cardioprotection was lost in TLR2-deficient mice. Conclusion These results demonstrate that TLR2 ligands induced cardioprotection, which is mediated through a TLR2/PI3K/Akt-dependent mechanism.

Intraaortic balloon counterpulsation (IABP) is the most widely used mechanical support in the assistance of a failing heart.1 Despite extensive research in this field no experimental or clinical studies have been undertaken to evaluate the most effective manner to wean IABP.2 The research reported in this thesis examines early recognition of cardiac decompensation and the management of IABP weaning. Conducted in three phases, the aim of this research programme was to determine the best manner by which to wean IABP. Phase 1 utilised a comparative descriptive design to examine IABP practice at a single cardiothoracic tertiary referral hospital. The majority of data collection was prospective, however, the required sample size saw inclusion of some retrospective data. This single centre data were than compared with an international registry to contrast IABP management and outcome. Phase 2 utilised a questionnaire survey to audit all Australasian intensive care units. Survey results were combined and statistically analysed to describe Australasian IABP management, weaning and outcome. Phase 3 utilised a quasi-experimental, one-group, posttest-only design to clinically validate a tool designed to monitor a patient's cardiac function - the 'cardiac decompensation tool'. Phase 1 saw data collected for 669 IABP insertions over an 11 year period at a single Australian hospital. This cohort was compared against the 38,606 patient dataset of The Benchmark Counterpulsation Outcomes Registry. Australian IABP practice saw later application of the device in a higher acuity patient. Australian practice demonstrated a prejudice toward intraoperative use (34.2% versus 16.6%; p=< 0.0001) and an aversion to catheter laboratory support (10.6% versus 19%; p=< 0.0001). Australian mortality while slightly higher, remained comparable (22% versus 20.8%; p=ns). Phase 2 response rate was 60%. The most common Australasian method of IABP support withdrawal was ratio reduction only (61%). Units with a documented weaning policy were less likely to require balloon reinsertion or pharmacologic escalation following IABP removal (p=0.06). Indicators most likely to demonstrate a patient's readiness for IABP weaning were blood pressure (92%), heart rate (76%) and wedge pressure (59%). Phase 3 revealed cardiac decompensation tool scores to increase immediately prior to a treatment escalation (p=0.022) and decrease immediately following this escalation in therapy (p=0.0096). There was also some indication of decreasing scores prior to treatment minimisation (p=0.005). Tool scores demonstrated a corresponding treatment fluctuation up to three hours prior to the treatment intervention. With Phase 1 and 2 revealing many aspects of IABP practice to vary, the need for some direction regarding weaning is evident. Timely recognition of cardiac decompensation during IABP weaning allows an opportunity for the earlier escalation of treatment and consequent provision of increased cardiac support. Application of the Phase 3 cardiac decompensation tool can only assist in ensuring the best manner by which to support IABP weaning.