Cardiac EHD2-mediated mechanisms: intersection between inflammation and lipophagy and implications for diabetes-linked heart failure

Abstract

Type 2 diabetes mellitus and obesity commonly occur together in a pathology known as diabesity. Diabesity is a significant risk factor for the development of cardiovascular diseases (CVDs) and ultimately heart failure (HF), a major cause of worldwide morbidity and mortality. In diabesity and CVDs, lipid accumulation in organelles called lipid droplets (LDs) is associated with cardiomyocyte dysfunction and death via a process called lipotoxicity. Our group have reported that Eps Homology Domain protein 2 (EHD2) is downregulated in diabesity and CVD in the heart; significantly, this protein has been shown to be important in LD breakdown in hepatic lipophagy. However, prior to this thesis research, the basis for EHD2 downregulation and whether it plays a role in cardiac lipophagy was unknown. Here, a rodent cardiac cell model, H9C2 cardiomyoblasts, and a human cardiomyocyte model, human induced pluripotent stem cell derived cardiomyocytes (hiPSC-CMs) were employed. Diabesity-linked cardiomyopathies are chronic inflammatory conditions and therefore a link between increased levels of cytokines and EHD2 expression was explored. This thesis determined that tumour necrosis factor alpha (TNF-α), reduces EHD2 at the protein and mRNA levels in rodent and human cardiac cells. Two key outputs were the discovery that TNF-α induced EHD2 downregulation is linked to calpain activity at the protein level and miR-29b-3p at the transcript level. Exploring the putative link between EHD2 and cardiac lipophagy fluorescence and electron microscopy was employed with molecular biology and imaging flow cytometry methods that led to several key outcomes: (i) EHD2 depletion increases LD volume. (ii) EHD2 colocalises with LDs. (iii) Upon induction of lipophagy, EHD2: LD colocalization events are observed to increase in both these cell types, suggesting a key role for EHD2 in cardiac lipophagy. (iv) Additionally, this study demonstrated a novel role for EHD2 in the fusion of autophagosomes and lysosomes, a critical step in LD breakdown. (v) Importantly, several key mechanisms explored here were shown to be relevant in hiPSC-CMs and thus the outputs from this thesis have translational potential. This thesis research has demonstrated that inflammation associated with diabesity and CVD drives the EHD2 downregulation observed in these pathologies. Furthermore, this inflammation induced downregulation may drive inappropriate lipid handling in cardiomyocytes resulting in cardiac lipid accumulation and lipotoxicity. These novel data provide insight into cardiac lipophagy, laying a foundation that can be built upon in future studies targeting this process therapeutically.

Date of Award	28 Jun 2024
Original language	English
Awarding Institution	The University of Manchester
Supervisor	Ashraf Kitmitto (Main Supervisor), Richard Collins (Co Supervisor) & Tao Wang (Co Supervisor)