title: Peri-transplant cardiovascular dynamics in an ovine model of heart transplantation following 24-hrs donor brain stem death
Creators: name: Wells, Matthew A
Publisher: name: Griffith University
Year: 2021
Subjects: subject: Heart transplantation
subject: heart failure

description: Background: Heart transplantation (HTx) for end-stage heart failure is one of the most challenging complex health problems faced in intensive care units across the globe. This is partly due to the shortage of viable donor hearts and primary graft failure post-transplant. The mechanisms behind these shortfalls are diverse and arise from donor brain stem death (BSD), a significant stressor on the heart and the main pool from which donor hearts are sourced. A compromised myocardium is then preserved in cold preservation solution on ice (CSS), as per the current clinical standard, initiating significant ischaemia. The final stage of HTx is implantation in the recipient, which induces both warm ischaemia during the procedure and reperfusion injury. These multifarious injuries contribute to both non-viable donor hearts, reducing the number of available hearts, and graft dysfunction post-HTx. This doctoral project aims to utilise a clinically relevant model of transplantation to investigate 3 main aspects of cardiovascular function, 1) cardiac adrenergic excitation-contraction-coupling and perfusion, 2) mitochondrial function and 3) metabolic regulation. Methods: Sheep were semi-randomised, influenced by blood group matching, into 2 main groups. Group A consisted of heart donors with confirmed BSD or sham operated controls (SHAM). Group B consisted of a separate group of heart donors both BSD (BSD-Tx) and SHAM (SH-Tx), which progressed through to CSS and consecutive HTx in a healthy recipient animal. The healthy recipient (HR) heart was excised and used as a healthy control following the establishment of cardio-pulmonary bypass (CPB). Heart collection occurred at end-points, defined as 24 hrs after BSD confirmation for Group A and 6 hrs after weaning the healthy recipient off CPB for Group B. Heart tissue was collected in ice cold oxygenated Krebs solution for in-vitro analyses, mitochondrial respiration and metabolic assessment. In-vitro analyses: Briefly, left and right ventricular trabeculae were dissected and mounted on Blinks tissue blocks. Trabeculae were stimulated at 1 Hz intervals and the force of contraction was recorded over a concentration-effect curve to -(-)Noradrenaline. Potency at the β1-adrenoceptor was determined and expressed as the concentration required to elicit a 50% maximum response in log units (pEC50). Mitochondrial respiration: Tissue homogenate was injected into an Oroboros Oxygraph and respiration was measured using both carbohydrate and fatty acid substrates. Mitochondrial membrane potential (MMP) was measured fluorometrically. Respiration was measured during 3 respiratory states; LEAK state, non-phosphorylative oxygen utilisation, ATP producing Complex I oxidative phosphorylative state (OXPHOS) and Complex II OXPHOS. Tissue levels of 3-nitrotyrosine and the glutathione to reduced glutathione ratio (GSH:GSSG) were assessed using plate based assays. Metabolomics: Biopsies were collected in a similar manner from similar regions as those used for mitochondrial respiration. Biopsies were pooled per ventricle prior to the detection of polar metabolites via LC/MS. Metabolites were extracted via MassHunter and statistical analyses were performed using MetaboAnalyst 4.0. Results: Twenty-four sheep weighing on average 47 ± 6 kg were semi-randomised into the donor and transplant groups. There were 6 animals in each donor group, SHAM and BSD and 12 separate donors continued through to transplantation, 6 in each group, SH-Tx and BSD-Tx. In-vitro analyses: Donor BSD caused a significant reduction in RV contractility, however β1 sensitivity was unchanged. Post-transplanted hearts, regardless of donor BSD injury were characterised by bi-ventricular reductions in both contractility and pEC50 (SH-Tx: 0.59 p=0.04, and BSD-Tx: 0.62 p=0.02, mean difference). Mitochondrial respiration: Following donor BSD, LEAK respiration in the RV was significantly elevated compared to HR (BSD: 0.11 ± 0.03 FCR, p&lt;0.01). LV levels of 3-NT also trended upward, with a significant reduction in the GSH:GSSG ratio. CII OXPHOS in BSD using fatty acid substrates was significantly lower than HR hearts. Post-transplantation however, there were bi-ventricular elevations in LEAK respiration and reductions in CII OXPHOS for both SH-Tx and BSD-Tx groups. These data were also in the presence of reduced MMP in LEAK and CII OXPHOS states. Transplantation was also characterised by significant increases in RV 3-NT levels and reduced LV GSH:GSSG ratios. Metabolomics: Metabolically, BSD increased accumulation of myocardial amino-acids and glycolytic metabolites involved in oxidative and osmotic stress. Post-HTx, particularly in those exposed to donor BSD, there was a significant decrease in metabolites involved in mitochondrial respiration (eg. NAD, Acetyl-CoA) and accumulation of fatty acids and xanthine. Conclusion: Using a clinically relevant model of HTx following donor BSD, this study focussed on cardiac contractility, mitochondrial function and metabolism in an effort to explain peri-transplant cardiac compromise. Based on these results it appears that donor oxidative stress, mitochondrial function and glycolytic metabolite build up are important determinants in cardiovascular dysfunction. Post-transplantation however, the mitochondrial, oxidative, metabolic and adrenergic systems are significantly impaired. Further research should investigate how best to support the donor heart outside of adrenergic agents, reduce oxidative stress and support metabolism. Whereas strategies to improve heart preservation and targetable mitochondrial/metabolic therapeutics may reduce reperfusion injury. Collectively, these approaches have the potential to increase the quality and quantity of HTx.