Obesity is linked to remodelling of mitochondrial dynamics and the inflammasome in the heart

Abstract

Background: Obesity affects 890 million adults worldwide, with the number of obese people predicted to rise to 1 billion by 2030. Obesity is a common precursor to type 2 diabetes (T2DM) both of which are driving the prevalence of Heart Failure with preserved Ejection Fraction (HFpEF). HFpEF has few treatment options in part due to a limited understanding of the pathophysiological processes. This thesis took a combined in-vivo and in-vitro approach to characterise the intersection between changes to cardiac function, mitochondrial dynamics and the inflammasome resulting from diet-induced obesity (DIO) (a longitudinal study), and the effects of associated stressors. The experimental findings additionally led to investigations of the protein MIRO1 (a regulator of mitochondrial motility in neuronal cells), as little is known about the role of this protein in heart health. Methods and Results: 8-week-old C57BL/6J male mice were fed either a 60% High Fat Diet (HFD) or normal chow diet for 16 or 19 weeks. After 16 weeks, the HFD mice developed hyperglycaemia and hyperinsulinemia. Cardiac structure and function, as assessed by echocardiography and electrocardiography (ECG), revealed mild impairment of systolic function, associated with eccentric hypertrophy with no changes to the ECG. Western blotting and RT-qPCR showed a shift towards fission, with a reduction in mitochondrial fusion proteins MFN1 and MFN 2 (~0.6-fold, p = 0.04 and p = 0.05, respectively). Levels of MIRO1 also fell ~2-fold (p=0.0005). In contrast to expectations, extending the HFD protocol to 19-weeks did not affect cardiac function relative to control mice. Furthermore, at 19 weeks there was a shift towards increased fusion (up-regulation of the fusion proteins MFN1 and OPA1 and down-regulation of the fission protein DRP1). Interestingly, the mitophagy proteins PINK1 and PARKIN mirrored changes detected in the 16 week model (up and down-regulation respectively). Protein levels for the NLRP3 inflammasome components increased. Proteomics analysis of isolated cardiac mitochondria (19 weeks) identified increased expression of proteins regulating ketogenic activity. In-vitro (H9C2) cytokine treatments had mixed effects on cardiomyocytes. IL-1β treatment did not affect the mitochondrial and inflammasome proteins, whereas IL-6 and TNF-ɑ affected expression level changes to inflammasome related proteins (NLRP3 and Caspase 1) and proteins linked to mitophagy (PARKIN) and mitochondrial motility (MIRO1 and MIRO2). A cardiac specific MIRO1 knockout mouse (Cre-loxP) was next developed. Partial deletion of MIRO1, Cre+Het (heterozygous), mice exhibited mild diastolic dysfunction, which was exacerbated in the MIRO1cKO (homozygous) model. Mitochondrial function of MIRO1cKO mice was assessed using a high-resolution respirometer (Oxygraph) and displayed impaired oxidative phosphorylation and increased levels of mitochondrial reactive oxygen species (ROS) relative to wild type (WT) mice. Tissue from the apex of MIRO1cKO hearts was fixed for Transmission electron microscopy (EM) revealing more interfibrillar (IFM) and subsarcolemma (SSM) mitochondria with disrupted cristae, consistent with increased Cytochrome C expression compared to WT mice. Cre+Het mice when given a combination of a 60% HFD and L-NAME, when compared to control mice, exhibited a rapid onset of cardiac dysfunction consistent with HFpEF pathophysiology. Conclusion: This Thesis work developed and characterised a DIO mouse model that reiterated features of HFrEF, identifying a shift towards mitochondrial fission. Surprisingly, extending the HFD duration resulted in a reversion of the cardiac dysfunction to a healthy cardiac phenotype. This finding afforded the opportunity to compare the 16 and 19 week models at the molecular level to identify mitochondrial proteins that exhibited plasticity and thus may be linked to the pathogenesis of HF. For example, the shift from fission to fusion could suggest that promoting fusion improves cardiac outcomes. Additionally, upregulation of the ketogenesis pathway proteins was also identified at 19 weeks, suggesting HMGCS2 and BDH1 linked processes may represent intervention pathways. The link between obesity and inflammation status emerged as complex and further studies are required for clear stratification to HFrEF progression. This thesis also generated novel data revealing that the protein MIRO1 plays a crucial role in cardiac function and loss of MIRO1 leads to cardiac and mitochondrial dysfunction and altered the mitochondrial morphology. When combined, the novel results from this Thesis research have identified potential new target candidates/directions for developing novel approaches to preventing/delaying obesity-linked HF and serve as a platform for future investigation.