Characterisation of the role of RUNX1 in the context of myocardial ischaemia-reperfusion injury

Riddell, Alexandra Helen (2020) Characterisation of the role of RUNX1 in the context of myocardial ischaemia-reperfusion injury. PhD thesis, University of Glasgow.

Abstract

Coronary artery disease is the leading cause of death and disability worldwide and is typically caused by atherosclerotic narrowing of the coronary arteries, which impairs blood flow (ischaemia) to heart. In severe cases atherosclerotic plaques destabilise and rupture, resulting in complete occlusion of the coronary artery and progressive ischaemic necrosis (myocardial infarction, MI). The optimal treatment is to restore the patency of the coronary artery using primary percutaneous coronary intervention (PPCI) to reperfuse the ischaemic myocardium and limit necrosis. However, paradoxically, the full efficacy of PPCI is limited by additional injury initiated by reperfusion that causes cell death and contributes to contractile dysfunction. The collective injury caused by ischaemia and reperfusion, termed ischaemia-reperfusion (I/R) injury, leaves patients vulnerable to adverse cardiac remodelling and the development of heart failure, a hugely debilitating condition with high mortality rates. As such, there is a pressing, unmet need for novel therapeutic strategies that can be used as an adjunctive to reperfusion therapy to limit contractile dysfunction and adverse remodelling post-MI and prevent heart failure development.
Previous work has highlighted RUNX1 as a promising therapeutic target for the management of I/R injury and adverse cardiac remodelling. RUNX1 belongs to the RUNX family of transcription factors and is upregulated in border zone cardiomyocytes following MI. Using mice with a cardiomyocyte specific deficiency in Runx1 (Runx1∆/∆ mice) it was previously shown that RUNX1 is detrimental to contractility and contributes to adverse cardiac remodelling in experimental models of MI and I/R injury. It was subsequently demonstrated that increased contractility of Runx1∆/∆ hearts at 2 weeks post-MI was explained by improved cardiomyocyte calcium handling, caused by a decrease in protein phosphatase 1 (PP1) expression, increased phosphorylation of phospholamban (PLB) and the relief of sarcoplasmic reticulum calcium ATPase (SERCA) inhibition.
This thesis aimed to further investigate the role of RUNX1 in the context of myocardial I/R injury. Specifically, it aimed to study potential triggers for Runx1 mRNA that may arise in the infarct and border zone including I/R injury and stretch. It also aimed to explore whether Runx1 deficiency offers acute (<24 h) protection against contractile dysfunction in response to I/R injury and whether this was linked to altered calcium handling protein expression.
To fulfil these aims, a mouse Langendorff model was established to allow evaluation of the acute responses of isolated hearts to I/R injury and myocardial stretch in the ex vivo setting. Two separate models of I/R injury were characterised: i) no-flow I/R injury where perfusion was completely halted during ischaemia and ii) low-flow I/R injury where residual perfusion was provided during ischaemia to mimic perfusion via collateral vessels. Isolated hearts demonstrated a robust and consistent response to these protocols that paralleled findings reported by others in the field. In addition, the results demonstrated that lengthening the duration of no-flow ischaemia increased the final infarct size, corroborating the work of others and further validating the I/R protocol used.
Using the Langendorff set up, it was shown that perfusion was sufficient to induce rapid Runx1 mRNA upregulation, indicating that changes in Runx1 mRNA expression can occur within hours in the isolated heart, independently of neuroendocrine or systemic inflammatory interactions. No-flow I/R injury but not low-flow I/R injury, decreased Runx1 mRNA expression relative to time-matched continuously perfused control hearts. At the same time, the mRNA expression of Runx2 and Runx3 was also measured. Runx2 expression did not change in any of the conditions tested; however, Runx3 mRNA expression was decreased by perfusion, and this downregulation could be offset by no-flow I/R injury but not low-flow I/R injury. Using an intraventricular balloon to stretch the left ventricle of isolated hearts, we investigated whether stretch stimuli affected Runx1 mRNA. Interestingly, intermittent stretch (cycles of balloon inflation and deflation) but not sustained stretch was able to offset the upregulation of Runx1 mRNA by perfusion, indicating that stretch can regulate Runx1 expression when present as a dynamic stimulus.
Using the Langendorff model of no-flow I/R injury, it was next demonstrated that Runx1∆/∆ hearts had improved post-ischaemic contractile recovery compared to control hearts, indicating, for the first time, that the protective effects of Runx1 deficiency manifest within an hour of I/R injury and occur in isolation from systemic stimuli. There was no significant difference between PLB, phosphorylated PLB at the serine 16 residue or PP1 expression between Runx1∆/∆ and control hearts either at the beginning or end of the reperfusion period. Taken together, this suggests that the effect of Runx1 deficiency on contractility post-Langendorff I/R injury occurs via a distinct mechanism from that at 2 weeks post-MI.
In the final part of this project, the role of Runx1 in hypoxic responses in neonatal rat cardiomyocytes (NRCMs) was investigated. To explore whether Runx mRNAs are upregulated alongside a hypoxic gene profile, NRCMs were treated with hypoxic mimetics. Dimethyloxalyglycine (DMOG) did not affect the expression of Runx mRNAs, whereas cobalt chloride (CoCl2) and deferoxamine (DFO) selectively upregulated Runx1 and Runx3 mRNA, respectively. Further work is necessary to understand the different effects of these mimetics on the Runx family.
In non-cardiac tissue, physical and functional interactions exist between hypoxia inducible factor 1α (HIF-1α) and RUNX1. As HIF1-α directs the transcription of a large set of genes (including VEGF and GLUT1) as part of the cellular response to ischaemia, we explored whether RUNX1 expression levels modulate expression of HIF-1α target transcripts in NRCMs. However, RUNX1 overexpression did not affect the mRNA expression of HIF-1α target genes VEGF or GLUT1 under baseline conditions or in the presence of CoCl2.
Overall, the work in this thesis has confirmed that the beneficial effects of Runx1 deficiency previously found in vivo extend to the ex vivo setting and manifest acutely during reperfusion. It was demonstrated that a range of stimuli can alter Runx1 mRNA in the isolated heart, including intermittent stretch, which has not previously been linked to Runx1 mRNA regulation in the heart. Developing these findings further will be key to understanding myocardial RUNX1 function and the translational potential of targeting RUNX1 as a therapeutic strategy in the future.

• Item Type: Thesis (PhD)
• Qualification Level: Doctoral
• Keywords: Ischaemia, ischaemia reperfusion injury, cardiac remodelling, Runx1, myocardial infarction.
• Subjects: Q Science > Q Science (General); Q Science > QP Physiology
• Colleges/Schools: College of Medical Veterinary and Life Sciences > School of Cardiovascular & Metabolic Health
• Funder's Name: British Heart Foundation
• Supervisor's Name: Loughrey, Professor Christopher, Nicklin, Professor Stuart and Cameron, Professor Ewan
• Date of Award: 2020
• Embargo Date: 19 November 2023
• Depositing User: Dr Alexandra Riddell
• Unique ID: glathesis:2020-81815
• Copyright: Copyright of this thesis is held by the author.
• Date Deposited: 30 Nov 2020 12:00
• Last Modified: 08 Apr 2022 17:03
• Thesis DOI: 10.5525/gla.thesis.81815
• URI: https://theses.gla.ac.uk/id/eprint/81815