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European Journal of Heart Failure 2007 9(8):762-769; doi:10.1016/j.ejheart.2007.03.007
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© 2007 European Society of Cardiology

Over-expression of heat shock protein 27 attenuates doxorubicin-induced cardiac dysfunction in mice

Li Liua,d, Xiaojin Zhanga, Bo Qiana, Xiaoyan Mina, Xiang Gaob, Chuanfu Lic, Yunlin Chenga and Jun Huangd,*

a Department of Geriatrics, First Affiliated Hospital with Nanjing Medical University Nanjing, China
b National Resource Center of Mutant mice, Nanjing University Nanjing, China
c Department of Surgery, East Tennessee State University, Johnson City TN, USA
d Department of Cardiology, First Affiliated Hospital with Nanjing Medical University Guang Zhou Rd 300, Nanjing 210029, China

* Corresponding author. Tel.: +86 25 83718836 6315; fax: +86 25 83724440. E-mail address: huangjun{at}njmu.edu.cn


   Abstract
Top
 Abstract
1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
 Acknowledgments
 References
 
Background: Oxidative stress and myocyte apoptosis are thought to play an important role in the pathogenesis, progression and prognosis of heart failure (HF). Heat shock protein 27 (Hsp27) has been found to confer resistance to oxidative stress in cultured cells; however, the role of Hsp27 in in-vivo hearts remains to be determined.

Aim: To investigate the effects of Hsp27 over-expression on doxorubicin-induced HF.

Methods and Results: Transgenic mice (TG) with cardiac specific over-expression of Hsp27 and their wild type littermates (WT) were challenged with doxorubicin (25 mg/kg, IP) to induce HF. At day 5, TG mice had significantly improved cardiac function and viability and decreased loss of heart weight following doxorubicin exposure compared with WT. In another parallel experiment, doxorubicin-induced increased levels of reactive oxygen species, protein carbonylation, apoptosis and morphologic changes were detected in the mitochondria in WT hearts, whereas these effects were markedly attenuated in TG hearts. In addition, upregulation of heat shock protein 70 and heme oxygenase-1 was present in the TG hearts after doxorubicin stimulation in comparison to WT hearts.

Conclusion: These findings indicate that Hsp27 may play a key role in resistance to doxorubicin-induced cardiac dysfunction.

Key Words: Heart failure • Small heat shock protein • Free radical • Apoptosis • Doxorubicin • Haemodynamics

Received June 30, 2006; Revised January 19, 2007; Accepted March 21, 2007


   1. Introduction
Top
 Abstract
 1. Introduction
2. Materials and methods
 3. Results
 4. Discussion
 Acknowledgments
 References
 
Heart failure is a severe cardiac disorder characterized by decreased cardiac output and increased filling pressure. Though it has yet to be definitively explained, mounting evidence from animal and human studies suggests that oxidative stress and apoptosis may play important roles in the pathogenesis, progression and poor prognosis of HF [1]. This theory is supported by a series of recent studies, which demonstrate that HF induced by myocardial infarction or doxorubicin can be attenuated by antioxidants or anti-apoptosis molecules such as glutathione peroxidase and Bcl-xl [2,3].

Doxorubicin (Dox) is an effective antitumour agent in a variety of cancers [4]; however, side effects of irreversible cardiomyopathy and HF limit its clinical use [5]. The cardiotoxicity of Dox involves increased oxidative stress, apoptosis and a direct effect on DNA synthesis in cardiomyocytes [5-7].

Heat shock proteins (Hsps) can be induced acutely by environmental stress including heat shock and ischaemia/reperfusion (I/R) [8]. Some Hsps have been shown to be effective in cell survival and adaptation. But interestingly, different Hsps can exert different levels of cardiac protection. Over-expression of Hsp70, HO-1 and Hsp20 has been shown to result in potent cardiac protection, whereas Hsp56 was not protective [9-12]. Hsp27, with a molecular weight of 27 kDa in humans and 25 kDa in rodents, has emerged recently as an anti-apoptosis molecule [13]. Studies based on various non-myogenesis cell culture systems in vitro, suggest that the anti-apoptotic effect of Hsp27 may involve regulating redox status, cytochrome c release and a function as a chaperone molecule [13-15]. Using an intact Hsp27 transgenic animal, Akbar et al. showed that Hsp27 could protect mice from Kainate induced hippocampal cell death [16].

Cardiac protection by Hsp27 against ischaemia/reperfusion injury has been shown in isolated hearts in vitro. Using isolated and Langendorff perfused hearts, over-expression of Hsp27 minimized infarct size and improved contractile function in mice [17], and enhanced tolerance to ischaemia in short-term type 1 diabetic rats [18]. However, two important issues still remain unresolved: (1) the biological role of Hsp27 in intact animal hearts in vivo, especially in HF, and (2) the potential molecular events associated with the cardiac protection by Hsp27. To address these questions, we generated transgenic mice with cardiac-specific over-expression of Hsp27, to investigate the effect of Hsp27 on Dox-induced HF.


   2. Materials and methods
Top
 Abstract
 1. Introduction
 2. Materials and methods
3. Results
 4. Discussion
 Acknowledgments
 References
 
2.1. Reagents
Doxorubicin, nicotinamide, anti-{alpha}-tubulin antibody, 2, 4-dinitrophenylhydrazine (DNPH) and anti-DNP antibodies were purchased from Sigma (St. Louis, MO). 2', 7'-dichlorofluorescin (DCFH) was obtained from Merck (Darmstadt, Germany). Primary antibodies against Hsp70, HO-1, Hsp27, Hsp25 and {alpha}B-crystallin ({alpha}BC) were obtained from Stressgen (Victoria BC, Canada). Protease inhibitor cocktail was purchased from Roche (Mannheim, Germany). The TUNEL assay kit was from Promega (Madison, WI). The BCA protein assay kit and supersignal west pico chemiluminescent substrate were obtained from Pierce (Rockford, IL).

2.2. Generation of transgenic mice
A cDNA encoding human Hsp27 was subcloned into pBSII-SK+ containing the {alpha}-myosin heavy chain ({alpha}-MHC) promoter (generously provided by Dr. J. Robbins, Children's Hospital of Cincinnati, Ohio). BamHI-digested linear transgene consisting of the {alpha}-MHC promoter, Hsp27 cDNA, and poly (A) of human growth hormone (hGH) was microinjected into the fertilized eggs from CBA/BL6 mice. Following successful transmission, a range of tissues including heart, lung, liver, brain, skeletal muscle, spleen and kidney were screened by Western blot to confirm the cardiac specific expression of the transgene.

2.3. Experimental design
Eight-week old WT and TG mice were given a single dose of Dox (25 mg/kg) or saline (control) administered intra-peritoneally (IP) on day 0. Two independent experiments were performed.

2.3.1. Experiment 1
The first experiment was conducted to monitor mortality and to measure left ventricular (LV) function. After administration of Dox (n=34/WT, n=32/TG) or saline (n=11/group), mortality was monitored and recorded for 5 days. At day 5, the obviously dying mice were sacrificed. Of the survivors, 13 animals were randomly selected from the DOX-treated, WT and TG mice for measurement of LV performance. Eleven of the control animals treated with saline also underwent assessment of LV performance. Autopsies were performed on the dead mice immediately after death, and the lungs and hearts were removed for paraffin section and HE staining.

2.3.2. Experiment 2
The second experiment was a biochemical and histological study. Three days after treatment with Dox or saline, hearts were harvested for the following studies. (A) Both right and left ventricles were removed and snap-frozen in liquid nitrogen for Western blot (n=3/group) or DCFH assay (n=6/group). (B) Hearts were removed and fixed in 10% formalin for paraffin sections (n=3/group). (C) 1 mm3 of left ventricular tissue was fixed in 4% glutaraldehyde for ultrathin sections (n=3/group).

Animal welfare and experimental procedures were carried out strictly in accordance to the care and use of laboratory animals (National Research Council, 1996) and the related ethical regulations of Nanjing University.

2.4. Cardiac haemodynamic measurements
LV performance was analyzed in mice anaesthetized with chloral hydrate (400 mg/kg IP), a micro-tip® catheter transducer (SPR671; Millar Instruments) was inserted into the left ventricle with the chest opened and mechanically ventilated. After stabilization for several minutes, the signals were recorded continuously with an AcqKnowledge® pressure conductance system (Version 3.8.1, BIOPAC system) coupled with a transducer control unit (TC-510, Millar Instruments), stored, and displayed on a computer. The heart rate (HR), maximal LV systolic pressure (LVSP), maximal slope of systolic pressure increment (+dP/dt) and maximal slope of diastolic pressure decrement (–dP/dt), were calculated.

2.5. Electron microscopy
Ultrathin sections (60-70 nm) were cut with an ultramicrotome. The sections were collected on 200 mesh copper grids (Ernest F. Fullam, Inc.), contrast-stained with uranyl acetate and lead citrate, and examined using a JEOL 100-CX transmission electron microscope.

2.6. Histological study
To examine histological changes at day 3, hearts were harvested and fixed in 10% formalin. Then the tissues were paraffin-embedded, and cut into 4 µM serial sections. Counterstaining of haematoxylin and eosin (H&E) was performed. Apoptosis in cardiomyocytes was determined in three steps. Step one was the TUNEL assay. This was performed according to the manufacturer's instructions. Briefly, tissue sections were deparaffinized, rehydrated, fixed and permeabilized, and then incubated with FITC-labelled TdT incubation buffer at 37 °C for 1 h. Step two was anti-{alpha}-actinin immunohistochemistry. Sections were blocked, incubated with primary antibody against {alpha}-actinin for 1 h followed by incubation with Cy3-conjugated anti-IgG for 1 h. The final step was to use Hoechst 33342 reagent to counter stain the nuclei. The fluorescence images were observed and captured using a confocal microscope (2-Photon, LSM 510, Zeiss).

2.7. Western blot analysis
2.7.1. Protein sample preparation
Samples were prepared as described previously [19]. Briefly, the frozen ventricular tissues were homogenized in lysis buffer (Tris-HCl 20 mM pH 7.4, EDTA 5 mM, EDTA-free protease inhibitor cocktail 2 mM), centrifuged at 10,000 xg for 10 min at 4 °C. The supernatant solution was recovered as the cytosolic fraction for western blot analysis.

2.7.2. Western blot
Equal quantities of protein samples were separated by SDS-PAGE, and then transferred onto immobilon-P membrane (Milipore) followed by incubation with the primary antibodies overnight at 4 °C. After further incubation of the membrane with HRP-labelled anti-IgG for 1.5 h at room temperature, the signals were developed using a chemiluminescent substrate and exposure to films (Kodak). Quantification of the developed target bands was determined by densitometric evaluation.

2.8. Determination of the total level of reactive oxygen species (ROS)
ROS levels were measured using the fluorescence indicator DCFH, as previously described [19]. When DCFH is added to the tissue homogenate, ROS in the homogenate will lead to the oxidation of DCFH, producing the fluorescent product DCF. In our experiment, 1 µM DCFH was incubated with 1 ml of 100 µg cardiac cytosolic proteins, and fluorescence was recorded following 1 h incubation using a fluorometer (Synergy HT, BIO-TEK) at an excitation wavelength of 485 nm, and an emission wavelength of 535 nm.

2.9. Detection of protein carbonyls
Frozen ventricular tissue was minced and homogenized in cold lysis buffer (0.3 M sucrose, 0.03 M nicotinamide and 0.02 M EDTA pH 7.4) as described previously [20]. The homogenate was centrifuged at 10,000 xg for 5 min at 4 °C. The supernatant solution was recovered as the cytosolic fraction for protein carbonylation assay. A volume of 500 µL containing 100 µg protein was incubated with 2 mM DNPH in the dark for 1 h. The DNPH-treated proteins were used for immunochemical detection by western blot.

2.10. Statistics
Results are reported as mean±SD. For tests of significance between two groups within multiple groups, one-way analysis of variance (ANOVA) and post-hoc test was performed. Survival curves were compared by the log-rank test. Probability values of P<0.05 were considered significant.


   3. Results
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
4. Discussion
 Acknowledgments
 References
 
3.1. Generation of mice expressing human Hsp27 in cardiac myocytes
Cardiac tissues from three independent TG lines expressed Hsp27 (Fig. 1A). To test whether Hsp27 was expressed in the heart specifically, we analyzed Hsp27 expression in different tissues from the TG mice. As expected, Hsp27 was only expressed in cardiac tissues, and not in the liver, spleen, lung, kidney, brain or skeletal muscle. Surprisingly, Hsp25, the endogenous isoform of Hsp27 in mice, was down-regulated by Hsp27 over-expression (Fig. 1B).


Figure 01
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  		Fig. 1 Over-expression of Hsp27. (A) Western blot shows differential Hsp27 expression in WT hearts and TG hearts. Note that Hsp25 was down regulated by Hsp27 transgene. Equal protein loadings were documented by western blot anti-{alpha}-tubulin. (B) Western blot anti-Hsp27 was performed on 2 µg lysates of 7 tissues from all 3 lines of transgenic mice. Hsp27 expressed specifically in the hearts. TG85=TG line 85, TG94=TG line 94, and TG112=TG line 112.

 
3.2. Over-expression of Hsp27 attenuated Dox-induced mice mortality
Mortality was evident in both groups (WT&TG) by day 4 post-Dox injection. However, there was a statistically significant increase in mortality in the WT compared to the TG groups (9/34 vs. 1/32, P<0.01), and this trend lasted to day 5 (13/34 vs. 3/32, P<0.001). Autopsy revealed considerable pleural effusion and pulmonary congestion in all dead animals. HE staining demonstrated that alveoli collapse and red blood cell leakage were present in the lungs of all mice which were dead at day 4. In the heart sections of the mice which died at day 4 and day 5, pronounced myocyte damage and inhomogeneous HE staining were observed in the ventricles of all mice except for one TG mouse which died at day 4, which showed homogeneous HE staining and only slight myocyte damage. Interestingly, fibrosis was exhibited in the ventricles of 50% of the WT mice (2/4) which died at day 5, but not in the TG mice.

3.3. Hsp27 decreased the loss of heart weight induced by Dox
Hearts were removed and weighed after haemodynamic measurements at day 5. In all experimental groups receiving Dox, heart weight (HW) declined compared to control. HW normalized to tibia length (TL), deceased by 20.53% in WT (64.77±6.96 vs. 51.47±6.53, P<0.001. n=9 and 15, respectively) and by 8.51% in TG (71.88±11.42 vs. 65.76 ± 13.4, P>0.05. n=17 and 18, respectively). The Dox-induced reduction in HW to TL ratio was significantly attenuated in TG compared to WT mice (P<0.001).

3.4. Alleviated cardiac dysfunction induced by Dox in TG
Haemodynamic measurements were recorded to determine the protective effect of Hsp27 over-expression on LV function in Dox-treated animals. Our analysis demonstrated no significant difference in haemodynamic indices between WT and TG controls (Fig. 2). In contrast, both TG and WT animals treated with Dox had a significant decline in +dP/dt and –dP/dt compared to their respective controls (P<0.05, P<0.01). However, the Dox-induced degeneration of +dP/dt and –dP/dt was significantly attenuated by Hsp27 over-expression in the TG animals, compared to the WT Dox-treated group (P<0.05, Fig. 2). Similar results were observed for HR and LVSP (data not shown).


Figure 02
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  		Fig. 2 Effects of Hsp27 over-expression on Dox-induced cardiac dysfunction. Haemodynamic parameters in the left ventricle were measured at day 5 after Dox injection. Hsp27 over-expression significantly attenuated the decline in + dP/dt and –dP/dt after Dox treatment. +dP/dt=Maximal slope of systolic pressure increment, –dP/dt=maximal slope of diastolic pressure decrement. Results are mean±SD of 11 to 13 experiments. *P<0.01, #P<0.05.

 
3.5. Less apoptotic cells in TG than WT in response to Dox
HE staining demonstrated no obvious interstitial fibrosis in both WT and TG hearts 3 days after Dox injection. It is known that apoptosis is one of the critical events during Dox-induced heart failure [3]. On day 3, myocardial sections from Dox-treated WT and TG animals demonstrated an increase in apoptosis compared to their saline controls, but much fewer apoptotic cells were seen in TG than in WT hearts (Fig. 3). These results imply that over-expression of Hsp27 in cardiomyocytes may trigger stronger resistance to Dox toxicity, resulting in less Dox-induced apoptosis in the heart.


Figure 03
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  		Fig. 3 Dox-induced apoptosis determined by TUNEL assay. Hearts were removed at day 3 and fixed and cut into 4 µm serial paraffin sections. The TUNEL assay demonstrated that apoptosis in myocytes mostly appeared clustered in WT hearts while it was scattered in TG hearts. {alpha}-actinin was used to document myocytes. The florescence images were captured from left ventricular free walls. Representative images were from three independent experiments. Scale bar=10 µm. -> indicates positive nuclei of TUNEL staining.

 
3.6. Hsp27 over-expression suppressed Dox-induced oxidative stress
Previous studies have shown that down-regulation of Hsp 25, {alpha}B-Crystallin and Hsp70 simultaneously caused by heat shock factor 1 (hsf1) deficiency led to significant higher ROS generation in the murine heart [19,20]. If Hsp 25/27 plays a critical role during this procedure, we postulate that Hsp27 over-expression would decrease ROS levels in the heart in vivo following Dox administration. To test this possibility, total heart cellular ROS was measured. At day 3, ROS content increased significantly in the hearts of WT (P<0.01) and TG mice (P<0.05) compared to controls. However, the level of ROS was significantly lower in the Dox-treated TG group compared to the Dox-treated WT group (P<0.05) (Fig. 4A).


Figure 04
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  		Fig. 4 Over-expression of Hsp27 attenuates Dox-induced oxidative stress and protein oxidative modification. (A) Intracellular total ROS content measured by DCFH assay at day 3. Dox led to a significant increase of ROS in both WT and TG, but with much more increase in WT than in TG. n=6. (B) Protein carbonylation (a marker of protein oxidative damage) determined by western blot at day 3. Note that though significantly more protein carbonylation existed in both TG and WT after Dox injection, markedly higher levels of protein carbonylation were observed in WT than in TG mice. (C) The intensity quantification of (B). *P<0.01, #P<0.05, n=3.

 
To determine whether Hsp27 over-expression affects protein carbonylation in response to Dox administration, we examined the carbonylation of cardiac cytosolic proteins. Fig. 4B and C show that Dox induced more protein carbonyl modification in hearts of both WT (P<0.01) and TG (P<0.05) compared to control, with much lower levels of carbonylation in TG than in WT (P<0.01).

3.7. Effects of Hsp27 over-expression on Dox-induced ultrastructural changes in cardiomyocytes
Several morphologic changes in the mitochondria, including increased density, swollen cristae and loss of cristae definition, were seen in the examined myocytes of the LV myocardial samples from the Dox-treated mice. Additionally, vacuolation of the sarcoplasmic reticulum was also observed in the samples from Dox-treated mice. The observed morphological alterations in the sarcoplasmic reticulum were similar between WT and TG hearts, however, the morphological alterations in the mitochondria were more pronounced in WT than in TG hearts (Fig. 5), implying that preservation of mitochondrial structure may be associated with the cardiac protection provided by Hsp27 over-expression in the current study.


Figure 05
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  		Fig. 5 Representative electron micrographs in cardiac sections from three independent analysis. Control mice showed normal morphology, but Dox-administered mice showed abnormal morphology in the mitochondria and sarcoplasmic reticulum. Note that more severe changes appeared in the mitochondria of WT (vacuolation, increased density of membrane and loss of cristae definition in the mitochondria), only a few mitochondria with increased density were present in TG. However, morphological changes in the sarcoplasmic reticulum (vacuolation) were comparable between Dox-treated WT and TG hearts. -> abnormal mitochondria; -> abnormal sarcoplasmic reticulum (x8000).

 
3.8. Expression profile of other Hsps
Some other inducible Hsps (iHsps), including Hsp70, {alpha}B-Crystallin and HO-1 may also be effective in protection against cardiac injury. To determine whether these Hsps were involved in our model, their expression was assayed. As shown in Fig. 6, in control mice, over-expression of Hsp27 did not change the expression of Hsp70, {alpha}B-Crystallin and HO-1. But at day 3 after Dox injection, upregulation of Hsp70 and enhanced upregulation of HO-1 appeared in TG compared with WT mice (P<0.01 or P<0.05), whereas {alpha}B-Crystallin remained unchanged.


Figure 06
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  		Fig. 6 Over-expression of Hsp27 altered the expression of inducible Hsps in hearts after Dox administration. In control mice, there was no difference in the expression level of {alpha}BC, HO-1 and Hsp70 between WT and TG. At the 3rd day after Dox injection, Hsp27 over-expression upregulated Hsp70 and enhanced the upregulation of HO-1, with no effects on {alpha}BC expression. #P<0.01, vs. respective saline controls; *P<0.01, vs. Dox-treated WT. n=3.

 

   4. Discussion
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
Acknowledgments
 References
 
The work presented in this paper tests the hypothesis that Hsp27 plays a critical role in improving LV contractile and diastolic function by suppressing oxidative status and apoptosis following Dox administration in transgenic mice with cardiac-specific over-expression of Hsp27.

We created TG mice with cardiac specific over-expression of human Hsp27 using {alpha}MHC-promotor. Three lines were generated which all expressed Hsp27 cardio-specifically, this provided a firm basis for in vivo studies of the effect of Hsp27 on Dox cardiotoxicity. In Hsp27 expression experiments, the primary antibody only recognizes human Hsp27 and has no cross-reaction with Hsp25. Therefore, we cannot calculate how much Hsp27 increased compared with Hsp25 in TG hearts. Interestingly and unexpectedly, Hsp27 over-expression caused down-regulation of Hsp25, which is in contrast to the result of Hollander [17]. In transgenic mice with non-tissue specific expression of Hsp27, Hollander and colleagues demonstrated that Hsp27 over-expression forced Hsp25 upregulation in the heart. We cannot explain this disparity between our findings, but it may be related to the Hsp27 over-expression profile.

We have demonstrated that Hsp27 over-expression mice were more resistant to Dox-induced heart failure in vivo, and accordingly, mortality was also significantly decreased. The mechanisms of Dox-induced LV dysfunction may involve mitochondrial dysfunction, free radical production and myocyte death [5-7], and some of these are also features of HF caused by ischemia/reperfusion or cardiomyopathy. Our studies suggest that target expression of Hsp27 could be an alternative approach in the gene therapy of human HF.

We demonstrated that Hsp27 over-expression inhibited cardiomyocytes apoptosis markedly at day 3 after Dox injection. Apoptosis is known to be involved in Dox-induced heart failure and other pathological conditions including cardiomyopathy and transplant rejection [21-23]. The observations made by Cesselli et al. [24] added further insight into the activation of proapoptotic signalling cascades during HF development. We found that Hsp27 over-expression resulted in the attenuation of both LV dysfunction and cardiomyocyte apoptosis following doxorubicin administration. Since apoptosis and LV dysfunction in response to Dox exposure was inhibited by Hsp27, it is likely that, regulation of oxidative stress may be associated with HF attenuation by Hsp27. We observed a better morphology maintenance of mitochondria, less ROS generation and lower protein carbonylation in the heart after DOX injection in TG mice. As the mitochondrion is the main source of ROS, maintaining mitochondrial structure may be beneficial for inhibiting ROS production and cell death.

Cardio-protection by Hsp70 and HO-1 has been reported previously. For example, over-expression of Hsp70 in rat neonatal cardiomyocytes and the murine heart has been shown to effectively attenuate ischaemia/reperfusion injury [9,25]. Similarly, cardiac over-expression of HO-1 has been shown to protect the heart from ischaemia/reperfusion and prevent chronic deterioration of heart allografts [26,27].

iHsps increase acutely in response to a wide variety of environmental stresses; however, in states of chronic stress, such as in advanced heart failure, the response may be different [3,28]. Indeed, upregulated Hsp70 and enhanced upregulated HO-1 were observed in the current study in TG hearts after Dox injection. Interestingly, by using hyperthermia to challenge isolated HF murine hearts with differential cardiac dysfunction, Tanonaka et al. [29] found that with greater deterioration of LV function there was less induction of Hsp70 in hearts. We speculate that more severe HF is responsible for desensitized synthesis of Hsp70 and HO-1 in WT compared with TG mice following Dox exposure in the current study, and the depressed induction Hsp70 and HO-1 may be not helpful in maintaining cardiac function.

4.1. Implications of this study
Our study demonstrates that Hsp27 over-expression effectively attenuates cardiac dysfunction following acute doxorubicin exposure. Beyond its importance in understanding the oxidative events in the signalling of Hsp27-dependent cardiac protection, our work raises new questions, such as, whether there are cross-talks between Hsp27 and other anti-apoptotic molecules. Our current model suggests that the cardiac-specific Hsp27 over-expression transgenic mouse is a robust genetic tool for dissecting molecular and genetic events involving Hsp27, which could be a therapeutic target in heart failure.


   Acknowledgments
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
 Acknowledgments
References
 
This work was supported by the Jiangsu "135" Key Medical Project of China. We are grateful to Dr. J. Robbins and colleagues (Children's Hospital of Cincinnati, Ohio, USA) for supplying the vector containing {alpha}-MHC promoter, and to Dr. J. Kelley (East Tennessee State University, USA) and Dr. LJ. Yan (North Texas University, USA) for language correction, and Dr. ZN. Ding (First Affiliated Hospital with Nanjing Medical University, China) for helpful discussion.


   References
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
 Acknowledgments
 References
 

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