Investigating the role of toll-like receptor 4 in myocardial ischaemic-preconditioning and ischaemic-reperfusion injury

Abstract

Ischaemic heart disease remains a significant cause of death throughout the developed world. Although technological advancements and improved health care have significantly reduced mortality rates in the last century, additional research is required. Recently myocardial inflammation has drawn increasing interest as a potential therapeutic against cardiovascular disease. Because inflammation is activated by a broad range of pro-inflammatory mediators, the innate immune response can be suppressed at multiple locations. Currently, most antiinflammatory agents in phase III trials target specific pro-inflammatory cytokines such as TNF- α, IL-1β and IL-6. These mediators have various roles ranging from the recruitment of white blood cells to triggering cell death. In 2017 CANTOS (Canakinumab Anti-inflammatory Thrombosis Outcomes Study), which investigated the therapeutic benefits of Canakinumab (an IL-1β antagonist) in the cardiovascular setting, was completed. Although the study reported that patients had a reduced risk of inheriting cardiovascular diseases, Canakinumab did not reduce mortality rates. Considering the large number of inflammatory mediators involved in myocardial ischaemic-reperfusion injury, blockade of a single cytokine may be insufficient within the clinical setting. To date, no study has investigated whether the suppression of upstream inflammatory receptors is more effective in attenuating myocardial inflammation during ischaemic-reperfusion injury. This thesis investigates the role of toll-like receptor 4 (TLR4), an immunosurveillance receptor, in both myocardial ischaemic-reperfusion injury and ischaemic-preconditioning. Receptor activity is triggered when DAMPs (danger associated molecular pattern molecules) are released from necrotic cells and bind onto the TLR4 receptor complex. Evidence in the last two decades suggests that TLR4 can either exacerbate ischaemicreperfusion injury or trigger a preconditioning response under certain conditions. Genetic or pharmacological blockade of TLR4 has been reported limit infarct size, improve survival rates, and suppress myocardial inflammation in in-vivo infarct animal studies. On the other hand, evidence also suggests that low levels of TLR4 ligands can trigger preconditioning. In the 1990s, studies showed that pretreating animals with lipopolysaccharide, a bacterial ligand recognised by TLR4, could protect animals against ischaemic-reperfusion injury. Additionally, evidence also suggests that DAMPs can also elicit a preconditioning response when administered prior ischaemia. Considering these findings TLR4 signalling may be regulated in a biphasic manner which is dependent on the degree of TLR4 stimulation and the timing of TLR4 activation. Study 1 and 2 investigated whether the direct administration of a TLR4 antagonist during ischaemic-preconditioning can influence contractile recovery after irreversible ischaemic injury. The isolated heart technique was used to determine whether contractile function was directly influenced by the blockade of TLR4. Study 3 and 4 examined if novel TLR4 antagonists could protect against ischaemic-reperfusion injury in the in-vitro and in-vivo setting. To date, no study has investigated whether the rapid administration of a TLR4 antagonist during ischaemia can protect against myocardial ischaemic-reperfusion injury. In brief, study 1 and 2’s results revealed that the suppression of TLR4 signalling in ischaemic-preconditioned hearts depressed contractile recovery after ischaemic insult. Although protein analyses revealed that cardiac fatty acid binding protein (cFABP) and high mobility group box one (HMGB1) were influenced by the suppression of TLR4 the data collected was conflicting. Study 3 showed that (+)-naloxone and (+)-naltrexone suppressed LPS induced inflammation but did not improve cell viability after simulated ischaemic-reperfusion injury. Finally (+)- naloxone and TAK242 showed that both compounds could reduce myocardial infarct size and inflammation in an in-vivo left anterior descending artery ligation model. The findings from this thesis highlight the bivalent nature of TLR4 in ischaemic-preconditioning and ischaemicreperfusion injury in both the acute and chronic setting.