Novel mechanisms of diastolic dysfunction in diabetes

Abstract

Diabetic cardiomyopathy is primarily characterised by diastolic dysfunction and deranged cardiomyocyte performance. The underlying mechanisms are unknown, and specific treatment targets are lacking. Recent findings (Mellor et al, publication pending) have shown that glycogen accumulation is a consistent observation in the myocardium in numerous model types of diabetes and is correlated with poor functional outcomes. The glycogen phenotype could not be explained by activation changes in the well described glycogen handling enzymes, glycogen synthase and glycogen phosphorylase. Related findings from the Mellor-Delbridge labs have identified that glycogen-selective autophagy (glycophagy) is a process of lysosomal glycogen degradation that may operate in the myocardium in parallel with conventional macro-protein autophagy, and involves selective molecular partners to tag glycogen cargo (STBD1) and to localize glycogen cargo to the engulfing autophagosome (GABARAPL1). Arising from these observations, a role for disrupted glycophagy in the aetiology of diabetic heart disease was hypothesized. Thus, the goal of this Thesis was to address the following questions: (1) What are the molecular alterations underlying increased glycogen deposition in the diabetic heart? (Chapter 3) (2) What is the role of the key glycogen autophagy (glycophagy) protein, GABARAPL1, in regulating cardiac function and glycogen content? (Chapter 4) (3) Can glycophagy-targeting interventions rescue cardiac function and provide therapeutic potential for diabetic cardiomyopathy? (Chapter 5) Cardiac RNA from 3 rodent models of diabetes was analysed using a custom-designed PCR arrays to evaluate gene expression characteristics. Functional network analysis (STRING) was performed to identify molecular signalling pathways involved in glycogen handling disruption providing evidence of GABARAPL1 involvement. To investigate the role of disrupted cardiac glycophagy in cardiac pathophysiology, Gabarapl1 gene deletion in vivo was achieved via CRISPR-Cas9 gene editing. Cardiac function in Gabarapl1-KO mice was assessed via transthoracic echocardiography and glycogen content was measured (calorimetric assay) in hearts extracted ex vivo. To determine whether a glycophagy-augmenting intervention could rescue cardiac function, an AAV9 Gabarapl1- expressing virus was used to achieve cardiac-specific Gabarapl1 upregulation in a type 2 diabetic (T2D) model induced by high fat/sugar diet feeding. Key findings: (1) Molecular discovery: The diabetic heart is characterised by glycophagy disturbance involving Gabarapl1 downregulation. (2) Glycophagy proof of concept: Reduction in GABARAPL1 availability is sufficient to induce cardiac glycogen accumulation and diastolic dysfunction in vivo. (3) Glycophagy disease intervention: Gabarapl1 upregulation rescues diabetes-induced cardiac glycogen accumulation and diastolic dysfunction in vivo in a T2D mouse model. This Thesis presents original findings which link diabetic cardiac glycogen accumulation with disturbances in the novel glycogen autophagy pathway, glycophagy, and in particular identifies Gabarapl1 downregulation as a pathogenic state in diabetes. Further, this Thesis provides the first demonstration that GABARAPL1 plays a central role in maintaining cardiac glycogen levels and is crucial for the preservation of cardiac contractile function. Finally, this study presents new evidence that upregulation of glycophagy by Gabarapl1 gene delivery produces favourable functional outcomes for the diabetic heart. Collectively these investigations identify glycophagy disturbance as a novel mechanism of diastolic dysfunction in the diabetic heart. In overview these findings provide an evidence base for targeting glycophagy as a potential therapeutic strategy for treatment of diabetic heart disease.