Effects of Heat Shock Proteins on Protein Aggregation Induced by Transient Myocardial Ischemia

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1 CN / R [ ] (2003) ,,,,, (, ) [ ] ; ; ; ; ; B2 ; [ ] 70 B2 Wistar 70 B2 : 15 min 30 min 4 h,24 h,72 h, 15 min 4 h 24 h, 24 h 70 70, 15 min 4 h 70, B2 15 min 4 h, B2 Z ; [ ] R363 [ ] A Effects of Heat Shock Proteins on Protein Aggregation Induced by Transient Myocardial Ischemia TAN Xiao2Jun, WU Xiao2Ying, ZHANG Hua2Li, DENG Gong2Hua, TU Zi2Zhi, and XIAO Xian2Zhong ( Department of Pathophysiology, Xiang Ya School of Medicine, Central South University, Changsha, Hunan , China) [ KEY WORDS] Myocardial Ischemia2reperfusion Injury ; Protein Aggregation ; B2cystallin ; Immunoelectron Mi2 croscopy ; Heat Shock Proteins ; Ethanolic Phosphotungstic Acid Electron Microscopy [ ABSTRACT] Aim To investigate whether transient myocardial ischemia might cause protein aggregation, and to observe whether heat shock pretreatment and ischemic preconditioning could alleviate protein aggregation. Methods Myocardial is2 chemial2reperfusion injury model was prepared by ligation of left descending anterior coronary artery for 15 minutes then released for various durations in rats. Protein aggregates induced by ischemia2reperfusion injury in cardiomyocytes were observed by using ethanolic phosphotungstic acid ( EPTA) electron microscopy ( EM). The influence of heat shock protein 70 ( HSP70) and B2 crystallin on above protein damage was further investigated by immunoelectron microscopy. Results Myocardial ischemia2 reperfusion resulted in protein aggregation, which appeared at 30 min of reperfusion, peaked at 4 h of reperfusion, decreased from 24 h of reperfusion and restored to normal at 72 h of reperfusion after 15 min of ischemia. Heat shock pretreatment ( rectal temperature 42 for 15 min then recovery for 24 h) and ischemic preconditioning (ischemia for 3 min then reperfusion for 10 min ) significantly decreased myocardial protein aggregates induced by 15 min of ischemia and 4 h of reperfusion and facilitated the restoration of myocardial protein damage. HSPs played key roles in the myocardial protection of heat shock pretreatment and ischemic preconditioning. In the present study, we found that HSP70 and B2cystallin co2localized with protein aggregates in heat shock pretreatment and ischemic preconditioning groups. Conclusion This study, for the first time, demonstrated the formation, intracellular distribution and dynamic patterns of myocardial protein aggregates induced by ischemia2reperfusion injury, and found that heat shock pretreatment and ischemic preconditioning alleviated the formation of myocardial protein aggregates., [ ] [ ] [ ] (973) ( G ) ( ) [ ],,1972 : , E2mail sina. com,,1968,,,1956 : , E2mail : xi2 com

2 392 ISSN Chin J Arterioscler, Vol 11, No 5 Wistar ( g) 0. 3 % (50 mg/ kg) DNA [1 ] : : (heat shock proteins,hsps),,15 min : 15 min, (30 (molecular chaperone), min h) [2,3 ] 90 : 0. 3 % 70 B2 42, 15 min, 24 h [4,5 ] 15 min 70 4 h 24 h B2 : 3 min 10 min, min, 4 h 24 h [6 ] HSP 1. 3 ( h) (25 mmol/ L Tris HCl,26 mmol/ L,5 mmol/ L ) [ = 1 5 ( ) ], r/ min 10 min,, g /, 10 % (10 70 B2 mmol/ L Tris HCl, ph 7. 5 ;100 mmol/ L NaCl ;0. 1 % 20 ;2 % ) h,tbs (1 000 ml, ph 7. 4, Tris HCl 3 g, NaCl 8 g, KCl IgG 1 h,tbs , 15 min, 2 5 min Wistar ( g) IgG B mol/ L (Stressgen ) ; IgG (15 nm) ( 2 % 2. 5 % ) ; 200 m 0. 1 mol/ L ( ) 4 % (ph 7. 4) 1. 2 g) 3, 15 min h ( 30 % 50 % 70 % 90 %

3 CN / R %), (10 ml 100 % 5 min 1 h ( PBS 0. 1 g, 4 95 % ) 50 min, 25 min, 5 min 3, 5 min Durcupan ACM, 500! % 4 % 2. 1 ( ), 100 % 0. 1 m, mol/ L PBS(pH 7. 2) 5 min,1 % 10 min, PBS 1 % 1 40 IgG,15 nm), PBS 3, Satoh, J EOL 100CX 15 min 30 min 20 min PBS B2 2 h,pbs, 4 h,24 h 3, 5 min, PBS 3 %, 72 h ( 1, Figure 1) 1. Sham 30 min 4 h 24 h 72 h 15 min 30 min 4 h 24 h 72 h N : ; M: : Figure 1. Electron micrographs of ethanolic phosphotungstic acid staining in cardiomyocytes in sham2operated controls and ischemia reperfusion groups ,15 min, 15 min 4 h, 4 h 24 h 70, 70, 24 h ( 3, Figure 3) ( 2, Figure 2) ( ) (42,15 min), 70,15 min 24 h B2 6 h min,24 h 48 h,72 h 4 h, B2 ; B2 24 h ( 24 h ( ) ) :, B2

4 394 ISSN Chin J Arterioscler, Vol 11, No 5 Z ( 4, Figure 4) 2. A B 4 h 24 h ;C D 4 h 24 h ; E F 4 h 24 h N : ;M: : Figure 2. Ethanolic phosphotungstic acid electron microscopy showing the effect of heat shock pretreatment and ischemic precondi2 tioning on protein aggregates induced by ischemia2reperfusion injury A B C D 4 h 4 h 4 h HSP70 N : ;M: : Figure 3. HSP70 immunogold labeling in the myocardium ,, 15 min 30 min,

5 CN / R B2 A B C D 4 h 4 h 4 h N : ;M: : Figure 4. B2cystallin immunogold labeling in cardiomyocytes [7 ],, HSP ,Bloom Aghaja2 man h CA Hu [8 ], CA1 24 h ( ) HSP [9 ] (15 min) 70 B (, 42, 15 min, 24 h) ( 3 min, 10 min),15 min 4 h 24 h [10 ], 30 min,, 24 h h, 72 h 15 min,, ( 3. 3 ) [11 ] [12 ], B2, HSP

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