Deciphering a potential cytoprotective role of novel heat shock responsive proteins using a proteomic approach
Myocardial infarction, commonly known as a heart attack, is a condition where the blood supply to the heart tissue is cut off, starving the tissue from oxygen and nutrient supply, with consequent lethal damage to the heart tissue. This damage is as a result of the death of cardiomyocytes. Numerous studies demonstrated that the death of these cells is as a result of programmed cell death or apoptosis. Heat shock proteins can protect cardiomyocytes against cell death by inhibiting apoptosis. For this reason heat shock responsive proteins are emerging as therapeutic targets to suppress cell death in cardiomyocytes during myocardial infarction. RhoE and TIP41 are also amongst the genes that are upregulated in cardiomyocytes after heat stress. These genes do not encode classical heat shock proteins. The question that arises is whether the induction of RhoE during heat stress in cardiomyocytes has any cytoprotective role. This research project aims to investigate the potential cytoprotective role of RhoE and TIP41 in rat cardiomyocytes. Mutant cell lines that stably over-express RhoE and TIP41 were generated by transfecting H9c2 cells with the pcDNA-3.1-TOPO vector containing these genes. DNA transfections were performed using the metafectene transfection reagent. Over-expression was investigated using Western blot analysis. The mutant cell lines were treated with ceramide and camptothecin for a period of 24 hours and cell viability was assessed by the MTT assay. Two dimensional proteomic analysis was carried out to compare the proteomes of H9c2 and H9c2 cells that over-express RhoE. This research demonstrates that both RhoE and TIP41 are induced in response to heat stress and that the over-expression of RhoE is able to protect H9c2 against camptothecin induced cell death. Furthermore a proteomic 2D analysis demonstrates differential protein expression between H9c2 cells and H9c2 that over-express RhoE. Proteomic analysis demonstrates that the over-expression of RhoE leads to the down-regulation of Rho-GDI α. It can be concluded from this study that the expression of RhoE in response to heat shock is a cytoprotective event. The mechanism of cytoprotection is likely to involve Rho-GDI α.