Paediatrics (RCH) - Theses
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ItemRemote ischaemic preconditioning-evoked intracellular signalling pathways in cardiac maturation and disease( 2013)Background & Aims: Children with congenital heart defects are burdened with additional stress on the myocardium, which is exacerbated by cardiopulmonary bypass (CPB) and cardiac surgery. Ischaemic preconditioning (IPC) induced by brief, intermittent periods of ischaemia and reperfusion (IR) on the coronary vasculature can activate intrinsic protective mechanisms. However, remote IPC (RIPC) induced by inflating and deflating a standard blood-pressure cuff attached to a limb is a practical, non-invasive and clinically applicable model for protection against sustained ischaemic injury. RIPC regulates phosphorylation of key intracellular proteins that are recruited by IR and propagate signalling for metabolic control of the heart. Agonism of G-protein coupled receptors promotes protein signalling via key kinases such as Akt, p38MAPK, GSK3β, and HSP27 amongst other important effectors for cell survival against IR injury. However, most RIPC studies have focussed on adult myocardium. There is a dearth of such studies in immature myocardium. Thus, the aims of this Thesis were to: • test the efficacy of RIPC in a double-blind randomised trial in patients undergoing cardiac surgery for tetralogy of Fallot (ToF) and to measure key signalling protein kinases; • determine whether these signalling pathways are developed in murine neonatal hearts, and to compare their activation and ability to functionally recover after sublethal IR compared to adults; • determine the efficacy of RIPC in immature (4 weeks) and adult (12 weeks) hearts to functionally recover and express kinase signalling proteins after sublethal IR; • examine the effects of chronic hypoxia and fentanyl on immature cardiomyocytes on modulating kinase signalling proteins; • characterise the ability for advanced age (72 weeks) murine hearts to functionally recover and express kinase signalling proteins after sublethal IR. Methods: Limb RIPC was induced by four 5 min periods of inflation (ischaemia) and deflation (reperfusion) of a pressure cuff. IR occurred in ToF neonates during cardiac surgery utilising CPB, and in murine hearts during Langendorff-mode isolated heart perfusion. Resected right ventricular outflow tract myocardium from neonates and murine heart protein homogenates were assayed for protein expression by western immunoblotting. Effects of chronic hypoxia and fentanyl were studied in immature cardiomyocytes differentiated from murine P19 cells. Results: The major findings from this Thesis are: • remotely preconditioned ToF neonates did not have different expression of pro-survival signalling proteins relative to sham controls, which had a high proportion of phospho-kinase activation masking the effects of RIPC; • complex proteomic changes and a greater ability to functionally recover from sublethal IR were evident in immature murine hearts relative to the adult; • remotely preconditioned immature murine hearts had improved left ventricular (LV) functional recovery and an increase in the total available pool of protein kinases available for phosphorylation; • immature cardiomyocytes exposed to chronic hypoxia had an increase in total abundance of protein kinases available for phosphorylation; hypoxia blunted the effect of the opioid receptor agonist fentanyl; • advanced age hearts had impaired LV functional recovery, with a greater propensity for apoptosis and equitable levels of necrosis and autophagy, relative to adults. Conclusions: A protective role exists for RIPC in immature murine hearts exposed to IR despite undetectable protection in RIPC neonates undergoing ToF surgery. An extrapolation of these findings indicates confounders related to cyanotic disease progression (adaptation to chronic hypoxia) and pharmacological intervention may limit protection by RIPC in the surgical setting.