| Heat stress causes harmful changes in the cells, and prolonged duration leads to loss of function in cells, either by necrosis or by apoptosis. Heat shock proteins (Hsps) play an important role to maintain the homeostasis of the cell and help it to survive even during stressful period. Hsp60is one the main protein of Heat shock proteins’s family and it is mainly called as mitochondrial protein. The main purpose of this study was to investigate the expression and transcription of Hsp60and HSF-1and their corresponding mRNA in primary myocardial cells of neonatal rats in vitro, to correlate the relationship of Hsp60and HSF-1at various durations of heat stress, to observe the cytoprotective mechanism of Hsp60in response to heat stress.For this purpose the primary myocardial cells were cultured and divided into9groups including control group which is kept at37℃at5%CO2and95%humidity. Rest of groups was heat stressed for various durations from10,20,40,60,120,240,360and480min respectively at42℃at5%CO2and95%humidity.The heat stress model of primary myocardial cells of neonatal rats was developed through detection of myocardial enzyme elevation including AST, LDH, CK, CK-MB, as these are considered as myocardial markers and by observing the cytopathological lesions in control as well as heat stressed myocardial cells. The enzymes showed varying degrees of elevations. Level of AST was found to be significantly high throughout the stress period as compared to control group, whereas LDH displayed the significant elevation at20and60min of heat stress. CK was found significantly higher at120min and CK-MB activity was observed60and120min of heat stress. The cytopathological lesions were observed at the beginning of heat stress i.e10min with granular and vacuolar degeneration and were found persistent throughout the heat stress period, with obvious pyknotic nucleus after120min of heat stress treatment. These findings revealed that myocardial cells of neonatal rats are damaged by heat stress.The western blot results showed that Hsp60expression was decreased significantly after20and40min and then increased after120min of heat stress and was found stable till240min of heat stress. Meanwhile the immunocytochemical results showed the most prominent signals with thick Hsp60density after240min of heat stress at42℃as compared to that of60min. This change in Hsp60density indicated that due to consumption of Hsp60, the cells started to produce sufficient Hsp60after240min of heat stress. Whereas, the cytopathological lesions showed the vacuolar degeneration after60 min of heat stress and pyknosis occurred at240min of heat stress. These findings suggest that myocardial cells were protected to certain extent from heat stress until and unless Hsp60was produced to help myocardial cellsThe in vitro relationship between Hsf-1and Hsp60and their corresponding mRNA was observed to find whether Hsf-1production influence Hsp60formation or not. Hsp60expression showed fluctuations during heat stress and observed significantly low after20min of heat stress and then increased at120min and remained elevated up to240min of heat stress. After that point, Hsp60expression was reduced as compared to control group. However, hsp60mRNA transcription levels increased significantly after20min and followed the same pattern up to240min of heat stress and then decreased significantly after360min of heat stress. The levels of Hsf-1expression were declined till120min of heat stress but increased significantly after that point and remained elevated till480min of heat stress, whereas Hsf-1mRNA levels increased significantly after10min and sustained same pattern up to the end of heat treatment that is480min. These results indicate that Hsf-1is not the only factor involved in regulating the hsp60mRNA transcription and is involved in the formation of other Hsps too.It was also observed that Hsp60production could not prevent the apoptosis, if the heat stress duration is too long because the levels of Hsp60expression decreased after20min and then increased at120min and then decreased at360min. But the expression of Caspase-3and Cytochrome-c increased significantly after40min of heat stress, suggesting that myocardial cells were unable to cope with the persistent heat stress and going to apoptosis. The changes in levels of Cytochrome-c are similar to that of cleaved Caspase-3, suggesting the two proteins are closely related during heat stress. However, Caspase8and9found relatively stable during the stress period. |
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