Faculty of Health Sciences & Medicine

Future Research Projects

DEVELOPMENT OF GENE KNOCKDOWN APPROACHES AS A THERAPEUTIC FOR ISCHAEMIC HEART DISEASE

Supervisor

Dr Kevin Ashton
Assistant Professor Cell Biology

BACKGROUND

The aged heart differs substantially from the young heart and, importantly, is both less resistant to the damaging effects of heart attack/infarction and less responsive to protective therapeutic strategies. We have extensively studied the mouse heart as a model of cardiac ageing and revealed that the development of >70% of loss of resistance to heart attack occurs prior to development of hypertrophy, fibrosis and necrosis. This suggests that these changes in heart structure may be secondary to prior molecular changes, and appear not to be critical in development of intolerance to injurious stress. Using microarray analysis we have identified >100 genes that are significantly down regulated in the aged heart before and/or after an ischaemic episode (i.e. heart attack). Restoring relevant gene expression levels to those observed in the young heart may be a potential therapeutic approach to improving the tolerance of the aged heart to the damaging effects of a heart attack.

AIMS OF THE PROJECT

To assess the effects of decreasing specific gene expression (the exact target genes are under final consideration) on ischaemic tolerance in cultured cardiac cells. This will confirm that reducing the expression of these genes makes cardiac cells less tolerant to ischaemic stress leading to apoptosis/cell death.

METHODS

Briefly, short hairpin RNAs (shRNAs) will be introduced into HL-1 cardiac cells; this RNAi approach achieves long-term, stable knockdown by interfering with gene expression of the target gene. Gene knockdown consequently leads to the decrease in translation of the corresponding protein encoded by the gene. Reduction in gene and protein expression levels will be confirmed using real-time PCR and Western blotting respectively. Once stable gene/protein knockdown is achieved HL-1 cells will be exposed to simulated ischaemia-reperfusion to mimic the conditions of a heart attack. The loss of tolerance to ischaemia will be measured by evaluating the extent of apoptosis/cell death (via flow cytometry and fluorescent microscopy) in HL-1 cells with and without gene knockdown. An increase in apoptosis/cell death in cells with decreased expression of target genes will demonstrate the importance of that gene/protein in maintaining the heart’s resistant to heart attack/ischaemic episode.

By gaining insights into these molecular mechanisms, we will begin to understand more about the failure in both tolerance to ischaemic stress and failure of cardioprotective approaches in older hearts. We may also be in a position to selectively modify the way young and aged hearts respond and adapt to changes in energy supply and demand, and to injurious stimuli such as ischaemia.