In vitro study of heat shock protein 60 expression and mitochondrial morphology in response to mitochondrial stress in HeLa cells

Abstract

Diabetes mellitus ( DM) is a metabolic disorder and affecting worldwide as stated by WHO that diabetes will be seventh leading cause of death in 2030. Diabetes mellitus is characterized by hyperglycaemia and the production of reactive oxygen species that damages proteins, lipids and DNA. This further elicits mitochondrial specific stress, resulting in increased production of HSP60 which is a mitochondrial stress protein. The aim of this study was to investigate the mitochondrial morphology changes and heat shock protein responses of HeLa cells subjected to physiological levels of hyperglycaemia (25mM). HeLa cell growth was found to be slightly reduced with increasing glucose concentrations (10mM – 25mM ) as compared to the control (5mM). Furthermore, high glucose conditions (25mM) also affected mitochondrial activity as statistically significantly decreased mitochondrial dehydrogenase activity was observed as compared to the control. These results altogether conclude that high glucose (25mM) acted as a mitochondrial stressor. Mitochondria is a dynamic organelle that fuses and divides according to environmental stimuli and energy requirements. As a result the shape of mitochondria varies from small ovals to tubules and reticular networks. These events are mainly controlled by fission and fusion proteins and regulatory machinery. Therefore mitochondrial morphology was also analyzed by using confocal microscopy to determine the effects of high glucose (25mM) on mitochondrial morphology as compared to the control. It was found that the majority of mitochondria in the control (5mM) was tubular as compared to high glucose which showed increased mitochondrial fragmentation suggesting that growing cells in the presence of hyperglycemic conditions lead to mitochondrial stress. Mitochondrial specific stress results in selective induction of molecular chaperone, HSP60 which is mainly localized in the matrix of mitochondria. So furthermore , the expression of HSP60 was investigated. Consistent western blots results showed upregulation of HSP60 with heat shocked protein set as a positive control but not with high glucose concentration (25mM). Interestingly, HSP70 expression was found to be upregulated with high glucose (25mM) as compared to control therefore it can be concluded that there were more general cellular stress as compared to mitochondrial specific stress. This study concluded that high glucose (25mM) effects HeLa cell growth and mitochondrial activity as well as mitochondrial morphology . There are various evidence which links oxidative stress and mitochondrial dynamics and observed increased production of ROS along with short and fragmented mitochondria in high glucose conditions.