Dysregulation of Mitofusin-2 in the diabetic heart

UoM administered thesis: Phd

  • Authors:
  • Lucy Murfitt

Abstract

Diabetes is associated with an increased risk of cardiovascular disease, which accounts for approximately half of the fatalities in diabetic patients. Mitochondrial dysfunction is closely linked to diabetic cardiomyopathy, however the pathophysiological mechanisms responsible remains elusive. Maintenance of mitochondrial function relies on mitochondrial dynamics, including fusion governed by Mitofusin (Mfn) proteins. Emerging evidence implicates Mfn2 in the pathogenesis of diabetes. Nonetheless, the role of Mfn2 in the heart is poorly understood. Therefore, this study investigated the hypothesis that diabetic stimuli alters mitochondrial dynamics in the heart, promoting changes to mitochondrial structure and function.Diabetes was induced in male Wistar rats via administration of streptozotocin (STZ) and investigations carried out 16 weeks' post-injection. Left ventricle tissue was isolated and protein expression profiles examined. Mitochondrial OXPHOS function was investigated using enzymatic assay. Mitochondrial morphology was assessed using serial block face scanning electron microscopy. Mfn1 and Mfn2 expression were upregulated in the STZ myocardium concurrent with a downwards trend in PINK1 expression and increased JNK activation, supporting a mechanism of Mfn2 regulation by PINK1 and not JNK as previously described. Additionally, mitochondrial function appeared impaired. Structurally, there was no change to mitochondrial density or number but there was a significant increase in mitochondrial volume in the STZ myocardium. These results suggest that decreased mitophagy may inhibit the removal of dysfunctional mitochondria. Quantitative mass spectrometry confirmed increased levels of Mfn2, mitochondrial impairment and activation of the β oxidation and oxidative stress pathways in the diabetic myocardium. Cell-based studies were employed to investigate Mfn2 regulation. Protein expression profiles were first examined in response to various diabetic stimuli. Hyperglycaemia and oxidative stress stimulated increased Mfn2 expression. Conversely, palmitate (a saturated free fatty acid) appeared to inhibit Mfn2 expression. Investigations into Mfn2 regulation revealed that PPARdelta was activated in the STZ myocardium. To test the relationship between Mfn2 and PPARdelta, cells were treated with the PPARdelta agonist GW0742, causing increased Mfn2 expression concurrent with a downwards trend in PINK1 expression. These findings suggest PPARdelta activation may be responsible for the molecular changes displayed in the STZ myocardium. Finally, in order to gain a better understanding of Mfn2 function, recombinant Mfn2 constructs were developed in Escherichia coli and Mfn2 function assessed using a GTPase assay. This work involved extensive methodology development as GST-Mfn2 constructs were largely insoluble. However, small yields of soluble protein were obtainable after various buffer screens Both the full-length GST-Mfn2 and N-terminal construct GST-Mfn2(2-604) displayed GTPase activity indicating functionality. Furthermore, investigations into the impact of oxidative stress on Mfn2 function indicated that ROS induces a binomial response in GTPase activity, suggesting that oxidative status may regulate mitochondrial fusion.This work has demonstrated for the first time, an increase in Mfn1/Mfn2 in response to diabetic insult, in association with mitochondrial impairment and altered morphology. Furthermore, investigations into Mfn2 regulation demonstrated that PPARdelta may indirectly affect Mfn2 protein expression and that Mfn2 function may be influenced by ROS. These new data advance current understanding of the role of Mfn2 in the heart at a structural, functional and molecular level and how mitochondrial dynamics are perturbed in the diabetic heart. Furthermore, this work has identified PPARdelta agonists/antagonists as potential therapeutic drugs to regulate mitochondrial dynamics by targeting Mfn2.

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Original languageEnglish
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Award date1 Aug 2017