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.