| Exposure to high temperature without proper cooling could be dangerous and even fatal,as high temperatures can damage the brain and other internal organs;sudden exposure to heat is reported to lead to heart attack,stroke and cardiac arrest.Several factors influence the induction of heat stress,such as medication and age-related physiological changes,which can predispose individuals to heat stroke.Heat stress induces the expression of heat shock proteins(Hsps).All Hsp families share their chaperoning function.It is generally accepted that Hsps prevent cells from lethal thermal damage.Hsf-1 is a major transcriptional regulator of Hsps,existing as a trimer with constitutive DNA binding activity.In this study,relationships between Small heat shock proteins(sHsps)expression patterns and pathological changes of myocardial cells after heat stress were examined in vitro and in vivo by using the H9C2 cell line and Sprague-Dawley rats,respectively.Furthurmore,as a model to investigate the relationship between Hsf-1 and Hsps in heat stressed rat myocardial cells,480 min heat-stressed primary rat myocardial cells were also used in present study.Sixty-day-old Sprague-Dawley(SD)rats(n = 60)were used for in vivo research and maintained at room temperature(RT)at 25℃ for 5 days.Thereafter,the rats were randomly divided into six groups(n = 10 per group)and subjected to different periods of heat stress(control,20 min,40 min,60 min,80 min,100 min).While the control rats were kept at RT,animals of the other five groups were immediately transferred into a controlled climate chamber pre-heated to 42℃,with certified fresh air and relative humidity between 55-65%.During the course of heat stress treatment,water and food were supplied,and the mental state and activities of rats were observed and noted.Within 3 min of the end of the designated heat stress period,blood was collected from the rats,which were sacrificed immediately thereafter.Heart samples were collected and fixed in formalin for pathological observation or stored in liquid nitrogen for Western blot analysis.Histopathological lesions,characterized by acute degeneration,karyopyknosis and loss of a defined nucleus,became more severe in rat hearts over the course of heat stress treatment from 20 min to 100 min.The expression ofαB-Crystallin in rat hearts showed a significant decrease(P<0.05)throughout the heat stress treatment period,except at the 40 min time point.αB-Crystallin was localized in the cytoplasm of cardiac cells both before and after heat stress.However,the densities ofαB-Crystallin in different heat-stressed groups varied.Near the beginning of the heat stress treatment(20 min),positiveαB-Crystallin signals in the cytoplasm of heart cells were detected,but the density was decreased compared to that of the control group.Except for its strong expression in the cytoplasm of myocardial cells at 40 min of heat stress,weakerαB-Crystallin signals were observed in the cytoplasm after 60 min of heat stress compared to the earlier time points.After 80 min of heat stress,even weaker positiveαB-Crystallin signals were observed compared to those of the 60 min group.The weakestαB-Crystallin signal was observed at 100 min of heat stress.In the control group,αB-Crystallin was evenly distributed in the cytoplasm of heart cells at a relatively high density.Hsp27 presents same trends withαB-Crystallin both in mRNA and protein level in vivo,In conclusion,along with lower Hsp27 andαB-Crystallin indicate severe pathological changes in rat heart in vivo.Suggesting Hsp27 andαB-Crystallin may play a protective role in heat stressed rat heart.In heat stressed H9C2 cell line in vitro,decreasedαB-Crystallin expression was also observed exposed to a high temperature,although its expression recovered to normal levels at later time points(80 and 100 min)and the cellular damage was less severe.The protein and mRNA levels of both Hsp27 andαB-crystallin decreased after 40 and 60 min of heat stress in vitro,whereas after 100 min they increased.We investigated these apparently conflicting results(Hsp27 andαB-crystallin protein and mRNA levels)between the cell line and the whole body system.Our results showed that Hsp27 andαB-crystallin(protein and mRNA levels)showed similar trends in vivo and in vitro.This finding was confirmed by analysis with STRING 9.1 software,which indicated that Hsp27 andαB-crystallin are co-expressed in rat myocardial cells before and after heat stress.To investigate further the heat induced heart damage,and the expression pattern of small heat shock proteins in heat stressed myocardial cells.Neonatal rat primary myocardial cells were subjected to heat stress in vitro.After exposure to heat stress at 42℃for different durations,the activities of enzymes expressed during cell damage increased in the supernatant of the heat-stressed myocardial cells from 10 min,and the pathological lesions were characterized by karyopyknosis and acute degeneration.Thus,cell damage was induced at the onset of heat stress.According to the Western blot results,during the480 min of heat stress,no significant variation was found in Hsp27 andαB-Crystallin expression;however,significant differences were found in the induction of their corresponding mRNAs.Hsfl expression increased significantly after 120 min compared to that at the beginning of the heat stress.The trend for Hsp90 was similar to that of Hsfl,whereas that for Hsp70 was the virtual opposite.However,there were no significant changes toαB-Crystallin during 480 min of heat stress.The interaction between the Hsps and Hsf-1 were analyzed and found that Hsf-1 interacted with Hsp90 and Hsp70,and played no role in regulatingαB-Crystallin expression.Based on our findings,Hsp70 may repress Hsf-1 in rat myocardial cells under heat stress.Hsf-1 was not the key factor for any Hsp,particularlyαB-Crystallin. |
PDF Full Text Request