Oxidant stress increases heat shock protein 70 mRNA in isolated perfused rat heart.
Kukreja R C,Kontos M C,Loesser K E,Batra S K,Qian Y Z,Gbur C J,Naseem S A,Jesse R L,Hess M L
The American journal of physiology
Ischemia/reperfusion (I/R) and preconditioning of the heart by coronary artery occlusions increase expression of heat shock protein 70 (HSP 70). Because free radicals are generated during I/R, we hypothesized that the oxidant stress might contribute to an increased expression of HSP 70. Isolated rat hearts were perfused with free radical-generating systems such as xanthine/xanthine oxidase (X/XO), irradiated rose bengal (RB) generating singlet oxygen, and H2O2 for 15 min followed by 30 min of recovery period. Significant decrease in developed pressure and coronary flow occurred after perfusion with X/XO, H2O2, and RB. During I/R, the developed pressure and coronary flow were 60 +/- 8 and 80 +/- 5%, respectively, of control, which improved significantly with superoxide dismutase. The expression of HSP 70 mRNA increased over 13-fold in hearts perfused with X/XO, 6- to 7-fold with RB, and over 5-fold with H2O2. With I/R, an over 10-fold increase in HSP 70 mRNA was observed, which decreased significantly in the presence of superoxide dismutase. These results demonstrate that oxidant stress directly increases HSP 70 mRNA in the rat heart. It is concluded that one of the potential mechanisms of expression of HSP 70 by I/R may be oxygen radicals.
Heat shock proteins, end effectors of myocardium ischemic preconditioning?
Guisasola María Concepcion,Desco Maria del Mar,Gonzalez Fernanda Silvana,Asensio Fernando,Dulin Elena,Suarez Antonio,Garcia Barreno Pedro
Cell stress & chaperones
The purpose of this study was to investigate (1) whether ischemia-reperfusion increased the content of heat shock protein 72 (Hsp72) transcripts and (2) whether myocardial content of Hsp72 is increased by ischemic preconditioning so that they can be considered as end effectors of preconditioning. Twelve male minipigs (8 protocol, 4 sham) were used, with the following ischemic preconditioning protocol: 3 ischemia and reperfusion 5-minute alternative cycles and last reperfusion cycle of 3 hours. Initial and final transmural biopsies (both in healthy and ischemic areas) were taken in all animals. Heat shock protein 72 messenger ribonucleic acid (mRNA) expression was measured by a semiquantitative reverse transcriptase-polymerase chain reaction (RT-PCR) method using complementary DNA normalized against the housekeeping gene cyclophilin. The identification of heat shock protein 72 was performed by immunoblot. In our "classic" preconditioning model, we found no changes in mRNA hsp72 levels or heat shock protein 72 content in the myocardium after 3 hours of reperfusion. Our experimental model is valid and the experimental techniques are appropriate, but the induction of heat shock proteins 72 as end effectors of cardioprotection in ischemic preconditioning does not occur in the first hours after ischemia, but probably at least 24 hours after it, in the so-called "second protection window."
Reactive oxygen species play an important role in the activation of heat shock factor 1 in ischemic-reperfused heart.
Nishizawa J,Nakai A,Matsuda K,Komeda M,Ban T,Nagata K
BACKGROUND:The myocardial protective role of heat shock protein (HSP) has been demonstrated. Recently, we reported that ischemia/reperfusion induced a significant activation of heat shock factor (HSF) 1 and an accumulation of mRNA for HSP70 and HSP90. We examined the role of reactive oxygen species (ROSs) in the induction of stress response in the ischemic-reperfused heart. METHODS AND RESULTS:Rat hearts were isolated and perfused with Krebs-Henseleit buffer by the Langendorff method. Whole-cell extracts were prepared for gel mobility shift assay using oligonucleotides containing the heat shock element. Induction of mRNA for HSP70 and HSP90 was examined by Northern blot analysis. Repetitive ischemia/reperfusion, which causes recurrent bursts of free radical generation, resulted in burst activation of HSF1, and this burst activation was significantly reduced with either allopurinol 1 mmol/L (an inhibitor of xanthine oxidase) or catalase 2x10(5) U/L (a scavenger of H2O2). Significant activation of HSF1 was observed on perfusion with buffer containing H2O2 150 micromol/L or xanthine 1 mmol/L plus xanthine oxidase 5 U/L. The accumulation of mRNA for HSP70 or HSP90 after repetitive ischemia/reperfusion was reduced with either allopurinol or catalase. CONCLUSIONS:Our findings demonstrate that ROSs play an important role in the activation of HSF1 and the accumulation of mRNA for HSP70 and HSP90 in the ischemic-reperfused heart.
Melatonin provides neuroprotection by reducing oxidative stress and HSP70 expression during chronic cerebral hypoperfusion in ovariectomized rats.
Ozacmak Veysel H,Barut Figen,Ozacmak Hale S
Journal of pineal research
Oxidative stress is believed to contribute to functional and histopathologic disturbances associated with chronic cerebral hypoperfusion (CCH) in rats. Melatonin has protective effects against cerebral ischemia/reperfusion injury. This effect has mainly been attributed to its antioxidant properties. In the present study, we evaluate the effects of melatonin on chronic cerebral hypoperfused rats and examined its possible influence on oxidative stress, superoxide dismutase (SOD) activity, reduced glutathione (GSH) levels, and heat shock protein (HSP) 70 induction. CCH was induced by permanent bilateral common carotid artery occlusion in ovariectomized female rats. Extensive neuronal loss in the hippocampus at day 14 following CCH was observed. The ischemic changes were preceded by increases in malondialdehyde (MDA) concentration and HSP70 induction as well as reductions in GSH and SOD. Melatonin treatment restored the levels of MDA, SOD, GSH, and HSP70 induction as compared to the ischemic group. Histopathologic analysis confirmed the protective effect of melatonin against CCH-induced morphologic alterations. Taken together, our results document that melatonin provides neuroprotective effects in CCH by attenuating oxidative stress and stress protein expression in neurons. This suggests melatonin may be helpful for the treatment of vascular dementia and cerebrovascular insufficiency.
Cardioprotection by CaMKII-deltaB is mediated by phosphorylation of heat shock factor 1 and subsequent expression of inducible heat shock protein 70.
Peng Wei,Zhang Yan,Zheng Ming,Cheng Heping,Zhu Weizhong,Cao Chun-Mei,Xiao Rui-Ping
RATIONALE:Ca2+/calmodulin-dependent protein kinase (CaMK)II is a multifunctional kinase involved in vital cellular processes such as Ca(2+) handling and cell fate regulation. In mammalian heart, 2 primary CaMKII isoforms, deltaB and deltaC, localize in nuclear and cytosolic compartments, respectively. Although previous studies have established an essential role of CaMKII-deltaC in cardiomyocyte apoptosis, the functional role of the more abundant isoform, CaMKII-deltaB, remains elusive. OBJECTIVE:Here, we determined the potential role of CaMKII-deltaB in regulating cardiomyocyte viability and explored the underlying mechanism. METHODS AND RESULTS:In cultured neonatal rat cardiomyocytes, the expression of CaMKII-deltaB and CaMKII-deltaC was inversely regulated in response to H2O2-induced oxidative stress with a profound reduction of the former and an increase of the later. Similarly, in vivo ischemia/reperfusion (IR) led to an opposite regulation of these CaMKII isoforms in a rat myocardial IR model. Notably, overexpression of CaMKII-deltaB protected cardiomyocytes against oxidative stress-, hypoxia-, and angiotensin II-induced apoptosis, whereas overexpression of its cytosolic counterpart promoted apoptosis. Using cDNA microarray, real-time PCR and Western blotting, we demonstrated that overexpression of CaMKII-deltaB but not CaMKII-deltaC elevated expression of heat shock protein (HSP)70 family members, including inducible (i)HSP70 and its homolog (Hst70). Moreover, overexpression of CaMKII-deltaB led to phosphorylation and activation of heat shock factor (HSF)1, the primary transcription factor responsible for HSP70 gene regulation. Importantly, gene silencing of iHSP70, but not Hst70, abolished CaMKII-deltaB-mediated protective effect, indicating that only iHSP70 was required for CaMKII-deltaB elicited antiapoptotic signaling. CONCLUSIONS:We conclude that cardiac CaMKII-deltaB and CaMKII-deltaC were inversely regulated in response to oxidative stress and IR injury, and that in contrast to CaMKII-deltaC, CaMKII-deltaB serves as a potent suppressor of cardiomyocyte apoptosis triggered by multiple death-inducing stimuli via phosphorylation of HSF1 and subsequent induction of iHSP70, marking both CaMKII-delta isoforms as promising therapeutic targets for the treatment of ischemic heart disease.
A novel role of microRNA in late preconditioning: upregulation of endothelial nitric oxide synthase and heat shock protein 70.
Yin Chang,Salloum Fadi N,Kukreja Rakesh C
MicroRNAs (miRNAs) are noncoding RNAs of 18 to 24 nucleotides that are involved in posttranscriptional regulation of protein expression. Their role in ischemic preconditioning (IPC) is currently unknown. We hypothesized that miRNAs induced after IPC in the heart may create a preconditioned phenotype through upregulating proteins including endothelial nitric oxide synthase (eNOS)/inducible nitric oxide synthase (iNOS) and heat shock protein (HSP)70, which are implicated in the late-phase protection of IPC. miRNAs were extracted from hearts of ICR mice following IPC. The purified miRNAs were injected in vivo into the left ventricular wall of mice, and, 48 hours later, the hearts were subjected to regional ischemia/reperfusion injury by left anterior descending artery ligation for 30 minutes followed by reperfusion for 24 hour. IPC caused no changes in miRNA-23b and miRNA-483 whereas miRNA-1, miRNA-21and miRNA-24 were significantly increased. The IPC-miRNA treatment caused an increase in eNOS mRNA and protein, whereas iNOS was not changed. HSF-1 (heat shock transcription factor 1) and HSP70 were also increased with IPC-miRNA treatment versus control. Moreover, injection of IPC-miRNA protected the hearts against ischemia/reperfusion injury, as shown by a reduction of infarct size as compared with saline or non-IPC miRNA-treated control. We conclude that IPC-induced miRNAs trigger cardioprotection similar to the delayed phase of IPC, possibly through upregulating eNOS, HSP70, and the HSP70 transcription factor HSF-1.
Heat preconditioning attenuates renal injury in ischemic ARF in rats: role of heat-shock protein 70 on NF-kappaB-mediated inflammation and on tubular cell injury.
Jo Sang-Kyung,Ko Gang Jee,Boo Chang Su,Cho Won Yong,Kim Hyoung Kyu
Journal of the American Society of Nephrology : JASN
Although heat preconditioning has been known to be protective in various types of injury, the precise molecular mechanism for this is unclear. Recent observations that indicate that previous heat shock has an anti-inflammatory, antiapoptotic effect led to this investigation of the in vivo effect of heat preconditioning on NF-kappaB activation and inflammation and also on tubular cell injury in ischemic acute renal failure (ARF). Heat preconditioning provided marked functional protection and also reduced histologic evidence of tubular necrosis. Ischemia/reperfusion-induced NF-kappaB activation was suppressed by heat preconditioning with a subsequent decrease in monocyte chemoattractant protein-1 expression and inflammatory cell infiltration. Heat preconditioning also suppressed the accumulation of phosphorylated inhibitory kappaBalpha (IkappaBalpha) with a resultant depletion of cytoplasmic IkappaBalpha, indicating that heat preconditioning blocked the activation of the IkappaB kinase complex. Tubular cell apoptosis, determined by terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling staining, also was decreased by heat preconditioning, and this was accompanied by decreased caspase 3 activation. Among several heat-shock proteins (HSP), HSP-70 was induced primarily by heat preconditioning. Inhibition of HSP-70 by quercetin almost completely reversed the functional protection that was provided by heat preconditioning. These data provide evidence that HSP-70 affords protection via inhibition of NF-kappaB-mediated inflammation and also inhibition of the cell death pathway in ischemic ARF. Further elucidation of the cytoprotective mechanism of stress proteins could facilitate new target or drug development in the treatment of ARF.
Therapeutic perspectives of heat shock proteins and their protein-protein interactions in myocardial infarction.
Wu Junxuan,Chen Sixuan,Liu Yuntao,Liu Zhongqiu,Wang Dawei,Cheng Yuanyuan
Myocardial infarction (MI) is one of major causes of human death around the world. Heat shock proteins (HSPs) are a large family of conserved proteins, which can promote correct protein folding, maintain protein stability, and regulate cell metabolism, cellular homeostasis and other biological processes as molecular chaperones. Notable, HSPs are involved in MI-related pathophysiology, such as apoptosis, inflammatory response and oxidative stress. Here, we review recent studies and systematically summarize the role of HSPs in MI and myocardial ischemia/reperfusion injury (MIRI) and discuss the role of direct and indirect protein-protein interactions (PPI) of HSP complexes in the pathophysiology and therapeutic strategies of myocardial infarction. A comprehensive understanding of the cardioprotective role of PPIs of HSP complexes in myocardial infarction can provide new insights for MI or MIRI therapy.
Heat shock protein 70 gene transfection protects rat myocardium cell against anoxia-reoxygeneration injury.
Liu Ji-chun,He Ming,Wan Li,Cheng Xiao-shu
Chinese medical journal
BACKGROUND:A number of studies suggest that the expression of heat shock protein 70 (HSP(70)) induced by heat stress are associated with protection against ischemia-reperfusion injury. But the protective effects may be contaminated by other factors in the same stress. This study was conducted to explore the protective role of HSP(70) expression in acute myocardial anoxia/reoxygeneration (A/R) injury with a liposome-mediated gene transfer technique for the introduction of pCDNA HSP(70) into the neonatal rat myocardial cells. In addition, heat shock stress cytoprotection was also investigated for comparison. METHODS:The cultured primary neonatal rat myocardiocytes with an acute myocardial A/R injury model and the HS-treated rat myocardiocyte model were used. Three-day cultured myocardiocytes were randomly divided into four groups (n = 8): control group, A/R group, HS + A/R group and pCDNA HSP(70) + A/R group. A liposome-coated HSP(70) pCDNA plasmid was transfected into the primary neonatal rat myocardiocytes; HSP(70) mRNA and its protein were confirmed by reverse transcriptase polymerase chain reaction (RT-PCR) and Western blotting. The cell viability was assayed by monotetrazolium (MTT) and the lactate dehydrogenase (LDH) and creatine phosphokinase (CPK) activity of cells during incubation and the changes in cells ultrastructure were examined. NF-kappaB activity in the primary neonatal rat myocardiocytes was measured with flow cytometry. RESULTS:Compared with viability in the A/R group ((35.4 +/- 6.9)%) the cell viability in the HS + A/R group ((72.8 +/- 11.6)%) and the pCDNA HSP(70) + A/R group ((76.3 +/- 12.2)%) was improved significantly (P < 0.05). The activity of LDH and CPK was significantly elevated in the A/R group. However, in the HS + A/R group and pCDNA HSP(70) + A/R group, significant decreases in activity were observed. The cell ultrastructure of the A/R group cells was abnormal, whereas nearly normal ultrastructure was observed in HS + A/R group and pCDNA HSP(70) + A/R group. HSP(70) mRNA and protein were slightly expressed in the myocardiocytes of the A/R group. However, obvious overexpression was observed in the HS + A/R group and in the pCDNA HSP(70) + A/R group (P < 0.01). And there was a significant difference between the HS + A/R group and the pCDNA HSP(70) + A/R group in the expression of HSP(70) mRNA and protein (P < 0.01). A high activity of NF-kappaB (5.76 +/- 0.64) was detected in the A/R group. But in the HS + A/R group there was a statistically significant decrease in the activity of NF-kappaB compared with the A/R group (3.11 +/- 0.52 vs 5.76 +/- 0.64, P < 0.01). The same statistically significant difference was also observed in the pCDNA HSP(70) + A/R group and A/R group (2.83 +/- 0.49 vs 5.76 +/- 0.64, P < 0.01). CONCLUSIONS:Overexpression of HSP(70) alone by gene transfection leads to protection for cardiac myocyte against anoxia-reoxygeneration. These cardioprotective effects were related to the reduction in activation of NF-kappaB.
Gene transfer of heat-shock protein 70 reduces infarct size in vivo after ischemia/reperfusion in the rabbit heart.
Okubo S,Wildner O,Shah M R,Chelliah J C,Hess M L,Kukreja R C
BACKGROUND:Heat-shock protein 70 (HSP 70) plays a role in myocardial protection. No studies are available, however, to show that direct gene transfer of HSP 70 reduces myocardial infarction in vivo. METHODS AND RESULTS:Rabbit hearts were injected with vehicle or Ad.HSP70 at 3 sites (1.5x10(9) pfu, 50 microL/site) in the left ventricle (LV). Four days later, hearts were removed, and expression of inducible (HSP 70) and constitutive (HSC 70) proteins was measured in the LV and right ventricle (RV). Subsets of 5 to 7 animals in the vehicle-, Ad.lacZ-, and Ad.HSP70-treated groups were subjected to 30 minutes of ischemia and 3 hours of reperfusion. Infarct size was measured by tetrazolium staining. Increased expression of HSP 70 was observed in LV injected with Ad.HSP70 compared with vehicle-treated hearts. HSP 70 was undetectable in RV, the noninjected region of the heart. The expression of HSC 70 remained unchanged in hearts treated with vehicle or Ad.HSP70. Infarct size (% risk area) decreased to 24.5+/-2.8 in Ad.HSP70-injected hearts compared with 41.9+/-2.8 and 42.7+/-2.5 in the vehicle- and Ad.LacZ-treated hearts (P<0.01). The infarct size was not different between the vehicle- and Ad.LacZ-treated hearts (P>0.05). The risk areas (% of LV) were not different among the 3 groups, ie, 50.1+/-5.2, 47.7+/-3.5, and 53.3+/-2.9 in vehicle-, Ad.lacZ-, and Ad.HSP70-treated groups (P>0.05). CONCLUSIONS:Direct gene delivery of HSP 70 in vivo reduces the severity of ischemic injury in the heart.
Gene therapy for myocardial protection: transfection of donor hearts with heat shock protein 70 gene protects cardiac function against ischemia-reperfusion injury.
Jayakumar J,Suzuki K,Khan M,Smolenski R T,Farrell A,Latif N,Raisky O,Abunasra H,Sammut I A,Murtuza B,Amrani M,Yacoub M H
BACKGROUND:Heat shock protein 70 (HSP70) gene transfection has been shown to enhance myocardial tolerance after normothermic ischemia-reperfusion. We investigated the effect of HSP70 gene transfection on mechanical and endothelial function in a protocol mimicking clinical heart preservation. METHODS AND RESULTS:Rat hearts were infused ex vivo with Hemagglutinating Virus of Japan-liposome complex containing HSP70 gene (HSP, n=8) or no gene (CON, n=8), and heterotopically transplanted into recipient rats. Four days after surgery, transfected hearts were perfused on a Langendorff apparatus for 45 minutes, arrested with St Thomas' No. 1 cardioplegia for 4 hours at 4 degrees C, and reperfused for 1 hour. Mechanical and endothelial function was studied before and after ischemia. Creatine kinase was measured in reperfusion effluent. Hearts underwent Western blotting and immunohistochemistry to confirm HSP70 overexpression. Postischemic recovery of mechanical function (% preischemic+/-SEM) was greater in HSP versus CON: Left ventricular developed pressure recovery was 76.7+/-3.9% versus 60. 5+/-3.1% (P:<0.05); dP/dtmax recovery was 79.4+/-4.9% versus 56. 2+/-3.2% (P:<0.05); dP/dtmin recovery was 74.8+/-4.6% versus 57. 3+/-3.6% (P:<0.05). Creatine kinase release was attenuated in HSP versus CON: 0.22+/-0.02 versus 0.32+/-0.04 IU/min/g wet wt. (P:<0. 05). Recovery of coronary flow was greater in HSP versus CON: 76. 5+/-3.8% versus 59.2+/-3.2% (P:<0.05). Recovery of coronary response to 5-hydroxytryptamine (5 x 10(-)(5) mol/L) was 55.6+/-4.7% versus 23. 9+/-3.2% (P:<0.05); recovery of coronary response to glyceryltrinitrate (15 mg/L) was not different between HSP and CON: 87.4+/-6.9% versus 84.3+/-5.8% (NS). CONCLUSIONS:In a clinically relevant donor heart preservation protocol, HSP70 gene transfection protects both mechanical and endothelial function.
Heat shock protein 70 gene transfection protects mitochondrial and ventricular function against ischemia-reperfusion injury.
Jayakumar J,Suzuki K,Sammut I A,Smolenski R T,Khan M,Latif N,Abunasra H,Murtuza B,Amrani M,Yacoub M H
BACKGROUND:Upregulation of heat shock protein 70 (HSP70) is beneficial in cardioprotection against ischemia-reperfusion injury, but the mechanism of action is unclear. We studied the role of HSP70 overexpression through gene therapy on mitochondrial function and ventricular recovery in a protocol that mimics clinical donor heart preservation. METHODS AND RESULTS:Hemagglutinating virus of Japan (HVJ)-liposome technique was used to transfect isolated rat hearts via intracoronary infusion of either the HSP70 gene (HSP group, n=16) or no gene (CON group, n=16), which was heterotopically transplanted into recipient rats. Four days after surgery, hearts were either perfused on a Langendorff apparatus for 30 minutes at 37 degrees C (preischemia studies [n=8/group]) or perfused for 30 minutes at 37 degrees C, cardioplegically arrested for 4 hours at 4 degrees C, and reperfused for 30 minutes at 37 degrees C (postischemia studies [n=8/group]). Western blotting and immunohistochemistry confirmed HSP70 upregulation in the HSP group. Postischemic mitochondrial respiratory control indices (RCIs) were significantly better preserved in HSP than in CON hearts: NAD(+)-linked RCI values were 9.54+/-1.1 versus 10.62+/-0.46 before ischemia (NS) but 7.98+/-0.69 versus 1.28+/-0.15 after ischemia (P<0.05), and FAD-linked RCI values were 6.87+/-0.88 versus 6.73+/-0.93 before ischemia (NS) but 4.26+/-0.41 versus 1.34+/-0.13 after ischemia (P<0.05). Postischemic recovery of mechanical function was greater in HSP than in CON hearts: left ventricular developed pressure recovery was 72.4+/-6.4% versus 59.7+/-5.3% (P<0.05), maximum dP/dt recovery was 77.9+/-6.6% versus 52.3+/-5.2% (P<0.05), and minimum dP/dt recovery was 72.4+/-7.2% versus 54.8+/-6.9% (P<0.05). Creatine kinase release in coronary effluent after reperfusion was 0.20+/-0.04 versus 0.34+/-0.06 IU. min(-1). g wet wt(-1) (P<0.05) in HSP versus in CON hearts. CONCLUSIONS:HSP70 upregulation protects mitochondrial function after ischemia-reperfusion injury; this was associated with improved preservation of ventricular function. Protection of mitochondrial function may be important in the development of future cardioprotective strategies.
Effects of ulinastatin on renal ischemia-reperfusion injury in rats.
Chen Cong-Cong,Liu Zi-Ming,Wang Hui-Hua,He Wei,Wang Yi,Wu Wei-Dong
Acta pharmacologica Sinica
AIM:To investigate the effect and possible mechanism of ulinastatin on renal ischemia-reperfusion injury in rats. METHODS:Male Sprague-Dawley rats were subjected to 45-min bilateral renal ischemia, treated with intravenously 12,500 U ulinastatin at 30 min prior to ischemia and at the beginning of reperfusion, compared with a nontreated group without ulinastatin and a sham-operation group without bilateral renal ischemia. After 0 h, 2 h, 6 h, 12 h, and 24 h of reperfusion, serum creatinine and blood urea nitrogen were measured for the assessment of renal function, renal sections were used for histologic grading of renal injury, for immunohistochemical localization of Bcl-2 and heat shock protein 70. Renal ultrastructure was observed through a transmission electron microscope. RESULTS:Ulinastatin significantly reduced the increase in blood urea nitrogen and creatinine produced by renal ischemia-reperfusion, suggesting an improvement in renal function. Ulinastatin reduced the histologic evidence of renal damage associated with ischemia-reperfusion and accompanied with an up-regulation in the expression of Bcl-2 protein, but it had no significant effect on the expression of HSP 70. Ulinastatin also significantly reduced kidney ultrastructure damage caused by renal ischemia-reperfusion. CONCLUSION:The protease inhibitor, ulinastatin, reduced the renal dysfunction and injury associated with ischemia-reperfusion of the kidney. The protective effect of ulinastatin might be associated with the up-regulation of Bcl-2 expression and the effect on membrane fragility.
Role of heat shock protein 70 in hepatic ischemia-reperfusion injury in mice.
Kuboki Satoshi,Schuster Rebecca,Blanchard John,Pritts Timothy A,Wong Hector R,Lentsch Alex B
American journal of physiology. Gastrointestinal and liver physiology
It is well established that liver ischemia-reperfusion induces the expression of heat shock protein (HSP) 70. However, the biological function of HSP70 in this injury is unclear. In this study, we sought to determine the role of HSP70 in hepatic ischemia-reperfusion injury in mice. Male mice were subjected to 90 min of partial hepatic ischemia followed by up to 8 h of reperfusion. HSP70 was rapidly upregulated after reperfusion. To explore the function of HSP70, sodium arsenite (8 mg/kg iv) was injected before surgery. We found that this dose induced HSP70 expression within 6 h of treatment. Induction of HSP70 with arsenite resulted in a >50% reduction in liver injury as determined by serum transaminases and histology. In addition, arsenite similarly reduced liver neutrophil recruitment and liver nuclear factor-kappaB activation, and attenuated serum levels of tumor necrosis factor-alpha and macrophage inflammatory protein-2, but increased levels of interleukin (IL)-6. In HSP70 knockout mice, arsenite did not protect against liver injury but did reduce liver neutrophil accumulation. Arsenite-induced reductions in neutrophil accumulation in HSP70 knockout mice were found to be mediated by IL-6. To determine whether extracellular HSP70 contributed to the injury, recombinant HSP70 was injected before surgery. Intravenous injection of 10 microg of recombinant HSP70 had no effect on liver injury after ischemia-reperfusion. The data suggest that intracellular HSP70 is directly hepatoprotective during ischemia-reperfusion injury and that extracellular HSP70 is not a significant contributor to the injury response in this model. Targeted induction of HSP70 may represent a potential therapeutic option for postischemic liver injury.
Carboxy terminus of heat shock protein (HSP) 70-interacting protein (CHIP) inhibits HSP70 in the heart.
Zhao Bijun,Sun Guocheng,Feng Guanli,Duan Weixun,Zhu Xiaoling,Chen Shaoyang,Hou Lichao,Jin Zhenxiao,Yi Dinghua
Journal of physiology and biochemistry
Heat shock protein (HSP) 70 plays a critical role in protecting the heart from various stressor-induced cell injuries; the mechanism remains to be further understood. The present study aims to elucidate the effect of a probiotics-derived protein, LGG-derived protein p75 (LGP), in alleviating the ischemia/reperfusion (I/R)-induced heart injury. We treated rats with the I/R with or without preadministration with LGP. The levels of HSP70 and carboxy terminus of HSP70-interacting protein (CHIP) in the heart tissue were assessed by enzyme-linked immunosorbent assay (ELISA) and Western blotting. The effect of CHIP on suppression of HSP70 and the effect of LGP on suppression of CHIP were investigated with an I/R rat model and a cell culture model. The results showed that I/R-induced infarction in the heart could be alleviated by pretreatment with LGP. HSP70 was detected in naïve rat heart tissue extracts. I/R treatment significantly suppressed the level of HSP70 and increased the levels of CHIP in the heart. A complex of CHIP/HSP70 was detected in heart tissue extracts. The addition of recombinant CHIP to culture inhibited HSP70 in heart cells. LGP was bound CHIP in heart cells and prevented the CHIP from binding HSP70. In summary, I/R can suppress HSP70 and increase CHIP in heart cells. CHIP can suppress HSP70 that can be prevented by pretreatment with LGP. The results imply that CHIP may be a potential target in the prevention of I/R-induced heart cell injury.
Blockade of Hsp20 phosphorylation exacerbates cardiac ischemia/reperfusion injury by suppressed autophagy and increased cell death.
Qian Jiang,Ren Xiaoping,Wang Xiaohong,Zhang Pengyuan,Jones W Keith,Molkentin Jeffery D,Fan Guo-Chang,Kranias Evangelia G
RATIONALE:The levels of a small heat shock protein (Hsp)20 and its phosphorylation are increased on ischemic insults, and overexpression of Hsp20 protects the heart against ischemia/reperfusion injury. However, the mechanism underlying cardioprotection of Hsp20 and especially the role of its phosphorylation in regulating ischemia/reperfusion-induced autophagy, apoptosis, and necrosis remain to be clarified. OBJECTIVE:Herein, we generated a cardiac-specific overexpression model, carrying nonphosphorylatable Hsp20, where serine 16 was substituted with alanine (Hsp20(S16A)). By subjecting this model to ischemia/reperfusion, we addressed whether: (1) the cardioprotective effects of Hsp20 are associated with serine 16 phosphorylation; (2) blockade of Hsp20 phosphorylation influences the balance between autophagy and cell death; and (3) the aggregation pattern of Hsp20 is altered by its phosphorylation. METHODS AND RESULTS:Our results demonstrated that Hsp20(S16A) hearts were more sensitive to ischemia/reperfusion injury, evidenced by lower recovery of contractile function and increased necrosis and apoptosis, compared with non-TG hearts. Interestingly, autophagy was activated in non-TG hearts but significantly inhibited in Hsp20(S16A) hearts following ischemia/reperfusion. Accordingly, pretreatment of Hsp20(S16A) hearts with rapamycin, an activator of autophagy, resulted in improvement of functional recovery, compared with saline-treated Hsp20(S16A) hearts. Furthermore, on ischemia/reperfusion, the oligomerization pattern of Hsp20 appeared to shift to higher aggregates in Hsp20(S16A) hearts. CONCLUSIONS:Collectively, these data indicate that blockade of Ser16-Hsp20 phosphorylation attenuates the cardioprotective effects of Hsp20 against ischemia/reperfusion injury, which may be attributable to suppressed autophagy and increased cell death. Therefore, phosphorylation of Hsp20 at serine 16 may represent a potential therapeutic target in ischemic heart disease.
Matrine Protects Cardiomyocytes From Ischemia/Reperfusion Injury by Regulating HSP70 Expression Via Activation of the JAK2/STAT3 Pathway.
Guo Suping,Gao Chuanyu,Xiao Wentao,Zhang Jing,Qu Yongsheng,Li Jiang,Ye Famin
Shock (Augusta, Ga.)
Studies have shown that matrine showed cardiovascular protective effects; however, its role and mechanism in myocardial ischemia/reperfusion (I/R) injury remain unknown. The Janus kinase 2/signal transducer and activator of transcription 3 (JAK2/STAT3) pathway activation and elevated heat shock protein (HSP) 70 are closely related to the prevention of myocardial I/R injury. The cardioprotective effects of matrine were determined in hypoxia/reoxygenation (H/R)-treated primary rat cardiomyocytes and left anterior descending coronary artery ligation and reperfusion animal models. The molecular mechanisms of matrine in myocardial I/R injury were focused on JAK2/STAT3 pathway activation and HSP70 expression. We found that matrine significantly increased H/R-induced the suppression of cell viability, decreased lactate dehydrogenase release, creatine kinase activity, and cardiomyocytes apoptosis in vitro. Moreover, matrine notably reduced the serum levels of creatine kinase-myocardial band (CK-MB) and cardiac troponin I, lessened the infarcted area of the heart, and decreased the apoptotic index of cardiomyocytes induced by I/R in vivo. Matrine activated the JAK2/STAT3 signaling, upregulated HSP70 expression both in vitro and in vivo. The cardioprotective effects of matrine were abrogated by AG490, a JAK2 inhibitor, and HSP70 siRNA. In addition, AG490 reduced HSP70 expression increased by matrine. In conclusion, matrine attenuates myocardial I/R injury by upregulating HSP70 expression via the activation of the JAK2/STAT3 pathway.
Hepatocellular iNOS protects liver from ischemia/reperfusion injury through HSF1-dependent activation of HSP70.
Qiao Yingli,Zhang Xueli,Zhao Guimei,Liu Zhiheng,Yu Mingyong,Fang Zheping,Li Xuehua
Biochemical and biophysical research communications
Although the role of inducible nitric oxide synthase (iNOS) in hepatic ischemia/reperfusion (I/R) injury remains controversial and confusing, with both harmful and beneficial effects in animal studies, the mechanism of these incongruous actions remains unclear. In the current study, we generated bone marrow chimeric mice with hepatocyte-restricted expression of iNOS. Chimeric mice and primary hepatocytes were subjected to I/R or anoxia/reoxygenation stimulation, respectively. The role of iNOS in liver I/R injury and the underlying molecular mechanisms were investigated. Hepatocyte-derived iNOS resulted in hepatoprotection from I/R injury, as well as in vitro experiments. Mechanistically, iNOS upregulates Heat shock protein (HSP) 70 by augmenting heat shock factor 1 (HSF1) binding to the HSP70 gene promoter. Importantly, inhibition of HSP70 partly reversed the iNOS overexpression-mediated hepatoprotection. The present findings demonstrate that hepatocellular iNOS protects from hepatic I/R injury through the HSF1-dependent activation of the HSP70. The upregulation of hepatocellular iNOS may offer a promising strategy for protecting against I/R injury.
Hydrogen sulfide treatment protects against renal ischemia-reperfusion injury via induction of heat shock proteins in rats.
Du Yang,Liu Xiu-Heng,Zhu Heng-Cheng,Wang Lei,Wang Zhi-Shun,Ning Jin-Zhuo,Xiao Cheng-Cheng
Iranian journal of basic medical sciences
Objectives:Hydrogen sulfide (HS) attenuates ischemia-reperfusion injury (IRI) in different organs. However, its mechanism of action in renal IRI remains unclear. The present study investigated the hypothesis that HS attenuates renal IRI via the induction of heat shock proteins (HSPs). Materials and Methods:Adult Wistar rats were subjected to unilateral renal ischemia for 45 min followed by reperfusion for 6 hr. One group of rats underwent I/R without treatment, one group was administered 150 μmol/l sodium hydrosulfide (NaHS) prior to I/R, one group was injected with 100 mg/kg quercetin (an HSP inhibitor) intraperitoneally prior to I/R, and another group received quercetin prior to I/R and treatment with NaHS following I/R. Two other groups underwent a sham operation and one of them received 150 μmol/l NaHS following the sham operation whereas the other received no treatment. Renal function and histological changes were compared and relevant indices of oxidative stress, apoptosis, and inflammation were examined. Results:IRI increased serum creatinine and blood urea nitrogen concentrations, promoted lipid peroxidation by elevating malondialdehyde levels, suppressed superoxide dismutase activity, stimulated inflammation by inducing NF-kB, IL-2, and TLR-4 expression, and increased renal apoptosis. Levels of HSP 70, heme-oxygenase-1 (HO-1) and HSP 27 were increased following IRI and reversed following HS treatment. HS attenuated changes observed in pathology, lipid peroxidation, inflammation, and apoptosis following IRI. The administration of quercetin reversed all protective effects of HS. Conclusion:The present study indicated that HS protected renal tissue against IRI induced lipid peroxidation, inflammation, and apoptosis, which may be attributed to the upregulation of HSP 70, HO-1, and HSP 27.
Morphine Reduces Myocardial Infarct Size via Heat Shock Protein 90 in Rodents.
Small Bryce A,Lu Yao,Hsu Anna K,Gross Garrett J,Gross Eric R
BioMed research international
Opioids reduce injury from myocardial ischemia-reperfusion in humans. In experimental models, this mechanism involves GSK3β inhibition. HSP90 regulates mitochondrial protein import, with GSK3β inhibition increasing HSP90 mitochondrial content. Therefore, we determined whether morphine-induced cardioprotection is mediated by HSP90 and if the protective effect is downstream of GSK3β inhibition. Male Sprague-Dawley rats, aged 8-10 weeks, were subjected to an in vivo myocardial ischemia-reperfusion injury protocol involving 30 minutes of ischemia followed by 2 hours of reperfusion. Hemodynamics were continually monitored and myocardial infarct size determined. Rats received morphine (0.3 mg/kg), the GSK3β inhibitor, SB216763 (0.6 mg/kg), or saline, 10 minutes prior to ischemia. Some rats received selective HSP90 inhibitors, radicicol (0.3 mg/kg), or deoxyspergualin (DSG, 0.6 mg/kg) alone or 5 minutes prior to morphine or SB216763. Morphine reduced myocardial infarct size when compared to control (42 ± 2% versus 60 ± 1%). This protection was abolished by prior treatment of radicicol or DSG (59 ± 1%, 56 ± 2%). GSK3β inhibition also reduced myocardial infarct size (41 ± 2%) with HSP90 inhibition by radicicol or DSG partially inhibiting SB216763-induced infarct size reduction (54 ± 3%, 47 ± 1%, resp.). These data suggest that opioid-induced cardioprotection is mediated by HSP90. Part of this protection afforded by HSP90 is downstream of GSK3β, potentially via the HSP-TOM mitochondrial import pathway.
The role of heat shock protein 90 in modulating ischemia-reperfusion injury in the kidney.
O'Neill Stephen,Ross James A,Wigmore Stephen J,Harrison Ewen M
Expert opinion on investigational drugs
INTRODUCTION:Kidney transplantation is the gold standard treatment for end-stage renal disease. Ischemia-reperfusion injury (IRI) is an unavoidable consequence of the transplantation procedure and is responsible for delayed graft function and poorer long-term outcomes. AREAS COVERED:Pharmacological induction of heat shock protein (Hsp) expression is an emerging pre-conditioning strategy aimed at reducing IRI following renal transplantation. Hsp90 inhibition up-regulates protective Hsps (especially Hsp70) and potentially down-regulates NF-κB by disruption of the IκB kinase (IKK) complex. However, the clinical application of Hsp90 inhibitors is currently limited by their toxicity profile and the exact mechanism of protection conferred is unknown. Toll-like receptor 4 (TLR4) is a further regulator of NF-κB and recent studies suggest TLR4 plays a dominant role in mediating kidney damage following IRI. The full interaction of Hsps with TLRs is yet to be delineated and whether TLR4 signalling can be targeted by Hsp90 inhibition in IRI remains uncertain. EXPERT OPINION:Pharmacological pre-conditioning by Hsp90 inhibition involves direct treatment to the kidney donor and/or organ, which aims to reduce injury prior to the onset of ischemia. The major challenges going forward are to establish the exact mechanism of protection offered by these drugs and the investgiation of less toxic analogues that could be safely translated into human studies.
Heat shock protein 90 inhibition and multi-target approach to maximize cardioprotection in ischaemic injury.
Der Sarkissian Shant,Aceros Henry,Williams Pierre-Marc,Scalabrini Catherine,Borie Mélanie,Noiseux Nicolas
British journal of pharmacology
Despite several advances in medicine, ischaemic heart disease remains a major cause of morbidity and mortality. The unravelling of molecular mechanisms underlying disease pathophysiology has revealed targets for pharmacological interventions. However, transfer of these pharmcological possibilities to clinical use has been disappointing. Considering the complexity of ischaemic disease at the cellular and molecular levels, an equally multifaceted treatment approach may be envisioned. The pharmacological principle of 'one target, one key' may fall short in such contexts, and optimal treatment may involve one or many agents directed against complementary targets. Here, we introduce a 'multi-target approach to cardioprotection' and propose heat shock protein 90 (HSP90) as a target of interest. We report on a member of a distinct class of HSP90 inhibitor possessing pleiotropic activity, which we found to exhibit potent infarct-sparing effects.
Urocortin increases the expression of heat shock protein 90 in rat cardiac myocytes in a MEK1/2-dependent manner.
Brar B K,Railson J,Stephanou A,Knight R A,Latchman D S
The Journal of endocrinology
We have previously demonstrated that urocortin protects cultured cardiac myocytes from ischaemic and reoxygenation injury and decreases the infarct size in the rat heart exposed to regional ischaemia and reperfusion. Urocortin-mediated cardioprotection is via activation of the mitogen-activated protein kinase (MAP kinase, MEK1/2) pathway. In addition, it is well documented that heat shock protein (hsp) 70 and hsp90 are cardioprotective against lethal stress. In this study we show, for the first time, that urocortin induces the expression of hsp90 but not hsp70 in primary cultures of rat neonatal cardiac myocytes. Levels of hsp90 protein increase by 1.5-fold over untreated cells within 10 min of urocortin treatment and are sustained for 24 h with a maximal increase of 2.5-fold at 60 min (P<0.05 at all time points). The increase in hsp90 expression by urocortin was not inhibited by actinomycin D, and urocortin failed to increase hsp90 promoter activity. Urocortin induction of hsp90 was inhibited by the MEK1/2 inhibitor PD98059 (P<0.001) and by cycloheximide, and both inhibitors abrogate urocortin-mediated cardioprotection (P<0.05 for cycloheximide, P<0.001 for PD98059). Hence, MEK1/2 and protein synthesis are involved in the cardioprotective effect of urocortin against hypoxic-mediated cell death, possibly due to an increase in expression of hsp90 protein. This is the first report of heat shock protein induction by urocortin or any other member of the corticotrophin-releasing hormone family.