Expression of Hsp47 in H9c2 Cells in Vitro and in Rats Myocardial Cells in Vivo under Heat Stress Conditions.pdf

1、
　　Environmental stress factors can induce cellular damages in different organs, including the heart.Heat stress is one of these factors which cause harmful changes in the tissues and the cells.When cells are exposed

2、to any stress agent, they undergo either necrosis or apoptosis, reliant to the stress inducer and level.Under extreme circumstances, the cells undergo necrosis when the stress level abolishes the capability of the cell t

3、o survive.At mild stress levels, cells activate homeostatic mechanisms which include expression of the heat shock proteins (Hsps) that protect cells.Heat shock protein 47 (Hsp47) is one of these Hsps which is expressed c

4、onstitutively in cells that synthesize collagen under stress conditions.Most of the previous studies investigated the expression of Hsp47 in different cell lines and animal models in vitro and in vivo have been concerned

5、 with its function in relation to collagen synthesis.Currently, no available studies demonstrate the relationship between Hsp47 and heat stress.The aim of the present study was to correlate the expression of Hsp47 and st

6、ress injury in heat-stressed myocardial cells in vitro and to compare variations in expression of Hsp47 in rat myocardial cells in vivo exposed to different heat stress periods, by using of different laboratory technique

7、s such as the colometric enzymesassay, cyto-histopathological techniques, irnmuno blotting, quantitative real time polymerase chain reaction (qRT-PCR), immunofluorescent assay (IFA), Enzyme-linked immunosorbent assay (EL

8、ISA) and immunohistochemistry (IHC). Although the immunofluorescence results showed that Hsp47 expression was constitutively localized in the cytoplasm of the H9c2 cells in all time points during heatstress, after ex

9、posure to heat stress at 42℃, significant myocardial cells damage was observed, accompanied with the cells damage-related enzyme aspartate aminotransferase(AST) in the supernatants of H9c2 cells, which was occurred after

10、 60 min (P＜0.01) and reaching the highest level at 100 min (P＜0.01) of heat stress.Cytopathological analysisrevealed that the acute cellular degenerations exhibited after H9c2 cells exposure to different periods of heat

11、stress.The results suggest that the increased enzyme activities of AST coupled with cellular damages in the H9c2 cells in vitro undergo cellular damage after the myocardial cells exposed to various periods of heat stress

12、 at 42℃. To correlate the localization and expression of Hsp47 and the stress injury in the heat-stressed rat heart tissue exposed to different heat stress periods, the myocardial celldamage-concerned enzymes, histop

13、athological changes and Hsp47 distribution in the myocardial cells were studied by means of the detection of the cell damage concernedenzymes, histopathological observation and immunohistochemical staining.After exposure

14、to heat stress at 42℃, the induction patterns of the heart damage related enzyme lactatedehydrogenase (LDH) were detected obviously at 60 min (P＜0.01) and 100 min (P＜0.05)of heat stress, while aspartate aminotransferase

15、(AST) reached the highest level at 100 min(P＜0.01) of heat stress, indicating that the rat heart tissue undergo cellular damage and theenzymes of AST and LDH are much more sensitive to the heat stress exposure.The findin

16、gwas also confirmed by the histopathological analysis, which revealed that heart tissuesexhibited cellular degenerations in response to different periods of heat stress.Immunohistochemical staining results showed that Hs

17、p47 was constitutively expressed inthe cytoplasm of the rat heart fibers in all time points during heat stress.The results indicatethat the rat heart tissues can be damaged after exposed to different periods of heat stre

18、ss at42℃ except for constitutive expression of Hsp47 in the myocardial cells. The variations of Hsp47 expression and its corresponding mRNA in the rat myocardialcells in vitro and in vivo were studied after exposed t

19、o different heat stress periods, bymeans of Western blotting and quantitative real time polymerase chain reaction (qRT-PCR).After kept in a 5％ CO2, 37℃ incubator in 25 cm2 cell culture flasks until 80％-90％confluency, H9c

20、2 cells were divided into six groups (group 0, 20, 40, 60, 80 and 100 min),all groups, except for control group, were suddenly subjected to different periods heat stressat 42℃.Meanwhile, 60 days old Sprague Dawley (SD) r

21、ats were randomly divided into sixgroups (group 0, 20, 40, 60, 80 and 100 min) after 3 days adaptation feeding in roomtemperature.All the rats, except for control group, were exposed to 42℃ heat stress.Western blot analy

22、sis results displayed that no significant difference was detected in theexpression of Hsp47 in H9c2 cells in vitro and in heart tissue in vivo.However, qRT-PCRresults showed the transcription ofhsp47 mRNA in response to

23、heat stress was significantlyincreased in H9c2 cells at 60 min (P＜0.01) and 100 min (P＜0.01) in vitro, and significantlyincreased at 100 man (P＜0.01) in the rat hearts in vivo.The results indicate that Hsp47 iselevated s

24、ignificantly after hyperthermia at the mRNA level, but not the protein level bothin vitro and in vivo, suggesting that the turnover of Hsp47 may be increased during heatstress or the consumption of the Hsp47 exceeds its

25、production. It is known that various cellular and environmental stresses can trigger heat shockresponse, which induces expression of highly conserved proteins that can act as molecularchaperones to protect proteins f

26、rom denaturation.These stressors activate heat shocktranscription factor-1 (HSF-1), which binds to the heat shock elements (HSEs) in thepromoter region of heat shock protein genes, leading to the expression of heat shock

27、proteins (HSPs).The correlation of heat shock transcription factor-1 (HSF-1) and Hsp47expression in the rat myocardial cells in response to the heat stress was also investigated.After 20 min of heat stress, the levels of

28、 HSF-1 in the rat myocardial cells increased(P＜0.05) and reached the highest level at 100 min (P＜0.01) in vivo.The HSF-1 expressionin the H9C2 myocardial cells exposed to different periods of heat stress decreased within

29、the first 20-60 min ofheat stress, and the levels of HSF-1 expression increasedsignificantly (P＜0.05) to reach the highest level at 100 min.The results indicate that theincreasing of the expression of HSF-1 invitro and i

30、n vivo may confirm the essential role ofHSF-1 in mediating the heat shock response and expression of Hsp47. In conclusion, this thesis suggests that myocardial cells may consume more Hsp47protein or the consumption o

31、f Hsp47 exceeds its production during heat stress.Furthermore,increased expression of Hsp47 at the transcriptional levels in the later period of exposure tohigh temperature was associated with cytopathological and/or his