Characterisation of cardiac mitochondrial dysregulation in a model of Maturity Onset diabetes in the Young (MODY2) and impact of diet

  • Bodour Rajab

Student thesis: Phd


Maturity Onset of Diabetes in the Young (MODY2) is a genetic form of diabetes with a mutation in the Gck gene. MODY affects 1-2% of people diagnosed with diabetes mellitus with the majority of MODY2 cases undiagnosed due to the absence of diabetes-like clinical symptoms. Diabetes suffers are at a higher risk of developing cardiovascular dysfunction. Despite this, treatment for cardiovascular dysfunction, particularly for MODY2 patients, is limited as the molecular mechanisms leading to dysfunction are complex and poorly understood. Molecular and cellular mechanisms are yet to be defined or examined within the heart. Here we employed a clinically relevant genetic mouse model of Gck-MODY2, GENA348, to investigate mitochondrial structural and molecular remodelling that may contribute to reduced contractile function and development of diabetic cardiomyopathy. Employing a combination of physiological measurements, biochemical and mitochondrial functional assays coupled with proteomics we characterised the cardiac phenotype of the GENA348 mouse (Chapter 2). Our results revealed that the GENA348 mice develop early LV dysfunction at 6 months of age (in agreement with previous work from the Cartwright group) and demonstrated for the first time that mitochondrial dysfunction is a feature of the GENA348 myocardium. Specifically, we determined a down-regulation of several cardiac mitochondrial protein subunits that regulate oxidative phosphorylation (OXPHOS) with impaired Complex I, II and Complex IV activity and altered rates of oxygen consumption. Further, at the molecular level we determined that proteins regulating mitochondrial fission and fusion and mitophagy were perturbed. Chapter 3 revealed that in keeping with the molecular data that the GENA348 mitochondria were enlarged compared to control, in particular the mitochondria in contact with the sarcolemma were most affected. Together, this data reveals that the GENA348 mice develop cardiovascular complications, with impaired mitochondrial function that will likely contribute to aberrant metabolic function. We further showed in Chapter 4 that obesity exacerbates mitochondrial dysfunction in the GENA348 mice through introducing a high fat (60%) feeding (HFD) regimen for 12 weeks. Here, we noted a common pathological development in the GENA348-chow mice compared to wild-type mice fed either chow or a HFD, suggesting a similar pathophysiology in obesity and MODY2 diabetes. We also recorded further decreases to mitochondrial complex activity and OXPHOS in the GENA348 HFD mice, suggesting that obesity adds a further stress impacting upon mitochondrial function. Through both biochemical and quantitative mass spectrometry analysis of isolated mitochondria from the GENA348 mouse myocardium we determined that there was a 0.2-fold decrease in Miro1 expression compared to WT control mice; a protein important for regulating mitochondrial movement and turnover. Therefore in Chapter 5 we investigated the effects of Miro1 knockdown in a cardiomyocyte cell line (H9c2). While depletion of Miro1 had no effect upon the expression of proteins mediating mitochondrial motility there was a significant increase in the fusion protein Opa1 and regulator of mitophagy PINK1 expression in si-Miro1 lines, suggesting an important role in regulating mitochondrial quality control through fission/fusion pathways and mitophagy. We also determined that levels of the inflammatory molecules IL-1β, IL-6 and TNF-α were elevated in the GENA348 heart; therefore, we developed cell-based studies to investigate how these cytokines may influence mitochondrial function and dynamics. We determined that each cytokine had differential effects upon Miro1 expression as well as proteins influencing mitochondrial fission and fusion; indicating a link between inflammation and mitochondrial function but through a diverse number of complex pathways. In conclusion, this thesis work has yielded novel data with potential translational imp
Date of Award31 Dec 2020
Original languageEnglish
Awarding Institution
  • The University of Manchester
SupervisorAshraf Kitmitto (Supervisor) & Elizabeth Cartwright (Supervisor)

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