| As a powerful animal raising country,animal industry is often suffering great economic losses because of the seasonal climate in the most parts of China.Therefore,it is very important to study the properties of heat stress damages for reducing the economic losses caused by heat stress.Heat stress is a systematical response,which takes an important effect on animal behavior,ingestation,production trait,blood circulation,neuroendocrine,immunization and reproduction and so on.However,a group of highly conserved proteins known as heat shock proteins(Hsps),which play important roles in the protection of cells and stabilization of internal environment,are rapidly transcribed and synthesis when living organisms are exposed to heat stress.Hsp60 was one of the important Hsps and has been demonstrated to involve in generation and development of cardiovascular diseases and immune system disorder.The objective of this study was to investigate the expressions and localizations of Hsp60 and its corresponding mRNA in the myocardial cells from the heat stressed rats in vivo and heat stressed H9C2 in vitro,and to correlate the Hsp60 levels with myocardial cell damages at various stressing times and to investigate the cell protection mechanism induced by Hsp60.To study the cellular injures and its damage degree induced by heat stress in rat myocardial cells in vivo and in vitro,the histopathological changes and the fatality rate of heat stressed rat myocardial cells in vivo and in the cell supernatant of heat stressed H9C2 cell line in vitro were studied.The results showed that the breathing rates of heat stressed rats were accelerated,the clothing hair was crude,and the rats became dysphoria at the beginning of heat stress.After 60 min of heat stress,the rat death caused by heat was firstly observed.And half of the rats were dead after 100 min of heat stress and all experimental rats died of heat within 2 h of heat stress;The activities of H9C2 myocardial cells in vitro started to decrease obviously after 60 min of heat stress and half of H9C2 myocardial cells died of heat stress at 240 min,indicating that H9C2 myocardial cells have longer heat stress tolerance in vitro than that in vivo;Histopathologically,After 20 min of heat stress,Granular degeneration characterized by cloudy cytoplasm in the myocardial fibers and obvious hyperemia in the blood capillaries was observed.After 40 min,marked acute degeneration characterized by light pink staining of tiny granular particles in the myocardial fibers.After 100 min,necrosis recognized by karyolysis in the myocardial fibers and loss of striation in the cytoplasm of swollen myocardial cells were occasionally observed.Cytopathologically,the cells had become rounder in shape and enlarged in size after 20 min of heat stress,with the appearance of granular particles in the cytoplasm of some swollen cells.After 40 min,acute granular degeneration characterized by light pink staining of tiny particles in the cytoplasm was observed.After 60 min,the cells had become irregular in shape,obvious acute degeneration characterized by light pink staining of tiny particles in the cytoplasm.In a word,with the increasing of heat stress,the tendency of histopathological damage of myocardial cells,the levels of enzyme activities and the fatality rate became serious both in vivo and in vitro.However,the H9C2 myocardial cells in vitro displayed longer heat stress tolerance than that of heart cells in vivo.To investigate the protective role of Hsp60 against stress damage and its role in the sudden death caused by stress,the expression levels of Hsp60 protein and its corresponding hsp60 mRNA,the localization and the distribution of Hsp60 in the myocardial cells in vivo and in vitro were studied.The relationship between Hsp60 expressions and the heat-induced damages of myocardial cells were also studied.The results showed that the levels of Hsp60 protein and its corresponding hsp60 mRNA in the rat myocardial cells significantly increased in both in vivo and in vitro with increasing of heat stress;Although Hsp60 expression were different at the same time points of the heat stress in vivo and in vitro,the levels of Hsp60 expression were consistent with the histopathological lesions observed in the myocardial cells both in vivo and in vitro.Differences of Hsp60 expression may reflect the degree of injury sustained by myocardial cells in vivo and in vitro.Hsp60 positive signals were only detected in the cytoplasm of rat myocardial cells both in vitro and in vivo.With prolong of heat stress,the signals of Hsp60 were scattered and gradually diffused to the cell membrane both in vivo and in vitro.It indicated that Hsp60 in the myocardial cells maybe transmembrane during heat stress.To research the relationship between the expression of Hsp60,and its corresponding protein Hsp10,and the mechanism of environmental stress tolerance,the expressions of Hsp60 and its corresponding protein Hsp10 in the myocardial cells of rats under different heat stress time were studied.The results showed that the expression levels of Hsp60 and Hsp10 in the H9C2 myocardial cells in vitro displayed the induction tendency after heat stress although the expression levels of Hsp10 showed no significant difference compared with control group.The transcriptions levels of hsp60 mRNA and hsp10 mRNA showed a temporary reduction and then increase in the rat myocardial cells both in vivo and in vitro.At the early stage of heat stress,the transcription of hsp10 mRNA decreased more significantly in vivo than that in vitro.Unlike the distribution of Hsp60,the signals of Hsp10 in the heat stressed H9C2 myocardial cells displayed the gathering tendency towards the direction of the nucleus after heat stress.It is presumed that Hsp60 and Hsp10 maybe play a protective role in the process of heat stress as a community.The localization and distribution of Hsp60 in the H9C2 rat myocardial cells during heat stress will gradually spread towards to the cell membrane.With the heat stress,the density of the positive Hsp60 can be detected using immunofluorescence on the cellular membranes,indicating that Hsp60 may play a protective role in the extracellular environment by passing across the cellular membrane out of cells.In order to further verify the inference,the excess Hsp60 antibody was added into the cell culture medium to neutralize the Hsp60 which across the cell membrane and spread to the extracellular environment.The results showed that the ratio of necrosis and apoptosis cells were increased significantly compare to that did not treated with Hsp60 antibody,after add excess Hsp60 antibody into the cell culture medium,indicating that extracellular Hsp60 play an important role in protecting cells from heat stress.Further study showed that the JNK phosphorylation may promote the realization of protection and the ERK phosphorylation may inhibit extracellular Hsp60 to protect the heat stressed cells.After inhibition of extracellular Hsp60,the transcriptions of hsp60 and hsp10 and expressions of Hsp10 in the heat stressed H9C2 myocardial cells were increased significantly,but the expressions of Hsp60 were decreased significantly,this maybe because of the Hsp60 antibody which was added into the supernatant and combined with the Hsp60 on the cell membrane.The study further indicated that Hsp60 and Hsp10 maybe play a protective role in the process of heat stress both as a community and individual. |
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