Molecular mechanisms initiated within cyanotic and acyanotic infant myocardium during cardio-pulmonary bypass in vivo and ischemic-reperfusion injury in vitro

Walker, Susan (2013) Molecular mechanisms initiated within cyanotic and acyanotic infant myocardium during cardio-pulmonary bypass in vivo and ischemic-reperfusion injury in vitro. PhD thesis, University of Glasgow.

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Children with the congenital heart defect TOF require surgical repair. Their myocardium is exposed to chronic hypoxia due to insufficiently oxygenated blood shunted from the right to the left side of the heart through the VSD, resulting in cyanosis. Such corrective surgery often requires the use of CPB. During these procedures the aortic clamp is applied to allow access to the heart for the surgical correction creating an ischemic environment. When the clamp is removed and blood flow returned, the heart is reperfused. This exposes the myocardium to IR injury. Super-high oxygen (O2) levels are often used in the bypass circuit after the cross-clamp is released. This means during reperfusion the myocardium is exposed to high levels of O2. This is thought to be harmful, especially to children with cyanosis pre-operatively.

A growing body of evidence suggests that these high levels of O2 may be damaging to the infant myocardium but the mechanisms are not that well understood. The first aim of this project was to investigate the molecular changes that occur within the infant myocardium in the context of congenital heart disease by comparing myocardium from the cyanotic patient group (TOF patients) and acyanotic patient group (patients with sub-aortic stenosis or truncus arteriosus i.e. disorders requiring myocardial resection but where the myocardium is acyanotic) during the ischemic phase. This was performed to reflect as near to pre-operative gene expression levels as possible. This thesis focused on the expression of inflammatory mediators (TLRs) and stress proteins (HSPs). It was found that TLR4 (p=0.0303), TLR2 (p=0.0177) and HSP27 (0.0303) mRNA expression were significantly higher in the myocardium from the cyanotic patient group compared to the acyanotic patient group. This could indicate a consequence of the hypoxic environment within such myocardium.

HSPs are stress proteins, which are induced upon cellular stress. The Aplein receptor (APJ) is upregulated in the heart during ischemia and IR injury and thought to be cardio- protective. Additional work was performed that investigated the protein expression of HSP27 and APJ during the ischemic phase in cyanotic and non-cyanotic TOF myocardium. This expression was compared with various measurements of cardiac function. HSP27 protein expression was significantly elevated in cyanotic myocardium pre-operatively. It was also associated with improved right ventricle function and systemic perfusion. This supports a
cardio-protective effect of HSP27 in cyanotic TOF. APJ was found to be associated with 2 improved systemic perfusion when both cyanotic and non-cyanotic patient data were combined.

In order to determine if stress genes (HSPs) were inducible in the infant myocardium, they were exposed to classic heavy metal inducers sodium arsenite and cadmium chloride. Of the six HSPs investigated, all were shown to be present within the ischemic myocardium. Myocardial HSP32 (p=0.0156), HSP72 (p=0.0156) and HSP90 (p=0.0156) mRNA expression were significantly increased by exposure to heavy metals. Therefore, HSPs were shown to be inducible in infant myocardium. This was a good experimental control for the re-oxygenation experiment.

The expressions of the same genes were also examined in vitro during an attempt to mimic re-oxygenation of the ischemic myocardium when the aortic-clamp is removed. Tissue was removed during the ischemic phase from both cyanotic and acyanotic patients and exposed to differential O2 levels. It was found exposure to 20% and 60% O2 for 4 hours induced mRNA expression of HSP32 (p=0.0391 and p=0.0078 respectively) and HSP72 (p=0.0078 and p=0.0078 respectively). This could indicate cellular stress during re- oxygenation of infant myocardium during surgical correction. No difference was indicated between the cyanotic and acyanotic patient group.

The work of this thesis was focused on myocardial mRNA/ protein expression. Whether there is an alteration in circulating inflammatory mediators was not the focus of the present study but the results within this thesis highlighted the need for future studies to look at the effect of circulating factors on myocardial inflammation.

This project is novel because it allowed a better understanding of the molecular mechanisms at work within infant myocardium and how they are influenced by re- oxygenation injury. It also examined how they are affected by cyanosis. It has highlighted the best ways in which such investigations could be extended. It has contributed important molecular data to the area of research and could potentially help lead to an improvement in myocardial protection during cardiac surgery with CPB.

Item Type: Thesis (PhD)
Qualification Level: Doctoral
Keywords: Ischemic-reperfusion injury, pediatric heart surgery, heat shock proteins, toll like receptors.
Subjects: R Medicine > RJ Pediatrics
Colleges/Schools: College of Medical Veterinary and Life Sciences > School of Medicine, Dentistry & Nursing
Supervisor's Name: Lyall, Prof. Fiona and Danton, Mr Mark
Date of Award: 2013
Depositing User: Miss Susan Walker
Unique ID: glathesis:2013-4169
Copyright: Copyright of this thesis is held by the author.
Date Deposited: 12 Apr 2013 11:11
Last Modified: 21 Aug 2018 08:07

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