Abstract
Transplantation of resident cardiac stem cells (CSCs) to the injured myocardium presents the potential to improve functioning of the diabetic heart in patients post-myocardial infarction. However, this therapy is not effective in people with diabetes due to reduced number and impaired functionality of CSCs and the molecular mechanisms underlying this impairment remain obscure. Recent studies have shown marked modulation of microRNAs (miRs) before the development of structural and functional changes in the diabetic heart. Among several miRs, microRNA-34a (miR-34a) is highly expressed in the diseased heart and differentially expressed in various stem cells. The aim of this study is to determine the role of miR-34a in diabetic CSCs in both acute and chronic diabetic states. We hypothesised an increase in miR-34a expression under both conditions and detrimental effect of miR-34a activation on CSC function.
CSCs isolated from Type 2 diabetic (BKS.Cg-m+Leprdb/J) db/db mice demonstrated a 3 fold increase in miR-34a expression (p<0.05). The functional effects were confirmed with western blotting analysis which showed a 90% downregulation of miR-34a target protein pro-survival deacetylase SIRT1 (p<0.05 vs. non-diabetic). We also observed a significant upregulation of p53, a transcription factor negatively regulated by SIRT1 which is involved in apoptosis and senescence. The acute diabetic model comprised of CSCs isolated from non-diabetic patients incubated in 30mmol high glucose media for 24hours while the chronic diabetic model was composed of CSCs isolated from people with diabetes and cultured in 30mmol high glucose media indefinitely. The acute diabetic model (n=8) demonstrated a significant 1.2 fold increase in miR-34a expression compared to non-diabetic CSCs (n=8) incubated in 20mmol mannitol (p<0.05). Consistent to this, the chronic diabetic model (n=5) demonstrated a significant 5 fold increase in mean miR-34a expression (p<0.05). Interestingly, we also identified a significant inverse correlation between miR-34a expression and diabetes duration (r=-0.912; p<0.05).
Surprisingly, after miR-34a inhibition, CSCs in the acute and chronic diabetic models demonstrated a trend towards a 3.5% and 13% increase in apoptosis respectively. Furthermore, the acute and diabetic models demonstrated a trend towards a decrease in proliferation following miR-34a inhibition. Interestingly, expression of senescent gene marker TP53, a gene encoding for p53 protein, showed a trend towards a 15% decrease in TP53 expression in the non-diabetic model following miR-34a inhibition. Unexpectedly, the acute and chronic diabetic models demonstrated trend towards a 30% and 90% increase in TP53 respectively. However, we observed a decrease in p53 protein expression in the non-diabetic and diabetic models following miR-34a inhibition. Interestingly, these findings suggest miR-34a plays a dual role in the regulation of TP53 in non-diabetic and diabetic conditions.
Findings from this study suggest a protective role of miR-34a in CSCs of the diabetic heart and modulation of miR-34a expression may improve diabetic CSC function.