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Experimental Physiology 92.2 pp 409-416
DOI: 10.1113/expphysiol.2006.036590
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Heart/Cardiac Muscle

Eccentric cardiac hypertrophy was induced by long-term intermittent hypoxia in rats

 Li-Mien Chen1,2, Wei-Wen Kuo3, Jaw-Ji Yang4, Shyi-Gang P. Wang5, Yu-Lan Yeh6, Fuu-Jen Tsai7, Ying-Jui Ho8, Mu-Hsin Chang9, Chih-Yang Huang10,11 and Shin-Da Lee12

1 Center of General Education, Central Taiwan University of Science & Technology, Taichung, Taiwan 2 Departments of Internal Medicine9 Division of Cardiology, Armed Forces Taichung General Hospital, Taichung, Taiwan 3 Department of Biological Science and Technology, China Medical University, Taichung, Taiwan Schools of 4 Dentistry8 Psychology, Chung-Shan Medical University, Taichung, Taiwan 5 Department of Physiology, National Yang-Ming University, Taipei, Taiwan 6 Department of Pathology, Changhua Christian Hospital, Changua, Taiwan 7 Department of Pediatrics, Medical Research and Medical Genetics, China Medical University Hospital, Taichung, Taiwan 10 Graduate Institute of Chinese Medical Science11 Institute of Medical Science12 Department of Physical Therapy, China Medical University, Taichung, Taiwan


   Abstract
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 Abstract
Introduction
 Methods
 Results
 Discussion
 References
 
It is unclear whether cardiac hypertrophy and hypertrophy-related pathways will be induced by long-term intermittent hypoxia. Thirty-six Sprague–Dawley rats were randomly assigned into three groups: normoxia, and long-term intermittent hypoxia (12% O2, 8 h per day) for 4 weeks (4WLTIH) or for 8 weeks (8WLTIH). Myocardial morphology, trophic factors and signalling pathways in the three groups were determined by heart weight index, histological analysis, Western blotting and reverse transcriptase-polymerase chain reaction from the excised left ventricle. The ratio of whole heart weight to body weight, the ratio of left ventricular weight to body weight, the gross vertical cross-section of the heart and myocardial morphological changes were increased in the 4WLTIH group and were further augmented in the 8WLTIH group. In the 4WLTIH group, tumour necrosis factor-{alpha}(TNF{alpha}), insulin-like growth factor (IGF)-II, phosphorylated p38 mitogen-activated protein kinase (P38), signal transducers and activators of transcription (STAT)-1 and STAT-3 were significantly increased in the cardiac tissues. However, in the 8WLTIH group, in addition to the above factors, interleukin-6, mitogen-activated protein kinase (MEK)5 and extracellular signal-regulated kinase (ERK)5 were significantly increased compared with the normoxia group. We conclude that cardiac hypertrophy associated with TNF{alpha} and IGF-II was induced by intermittent hypoxia. The longer duration of intermittent hypoxia further activated the eccentric hypertrophy-related pathway, as well as the interleukin 6-related MEK5–ERK5 and STAT-3 pathways, which could result in the development of cardiac dilatation and pathology.

(Received 11 November 2006; accepted after revision 20 December 2006; first published online 21 December 2006)
Corresponding author Shin-Da Lee: Department of Physical Therapy, China Medical University, 91 Hsueh-Shih Road, Taichung 40202, Taiwan. Email: shinda{at}mail.cmu.edu.tw


   Introduction
Top
 Abstract
 Introduction
Methods
 Results
 Discussion
 References
 
Intermittent hypoxaemia or nocturnal hypoxaemia during sleeping hours, implicated in the pathogenesis of cardiac complications, has often been reported in a range of clinical hypoxic conditions, such as neuromuscular disorders with abnormal breathing control (Kopelman et al. 1986), some obese patients with respiratory abnormalities (Ohtake et al. 1990) and most patients with sleep-related breathing disorders (Ostadal et al. 1981). Our previous studies showed that short-term intermittent hypoxia for 1 or 3 days appeared to exert protective effects on the heart, whereas long-term intermittent hypoxia for 1 and 2 weeks appeared to exert deleterious effects (Lee et al. 2006d,e). However, the mechanism of cardiac hypertrophy resulting from long-term intermittent hypoxia has not been well established.

Cardiac hypertrophy, a cardiac adaptive response to any stress, can exist in a state of compensation or progress to a decompensated state over time (Dorn & Hahn, 2004). Eccentric cardiac hypertrophy, in which chamber volume enlarges without a relative increase or even with a relative decrease in its wall thickness, is known to progress to dilated cardiomyopathy, heart failure and sudden death (Rossi & Carillo, 1991; Nicol et al. 2001). Accumulating evidence indicates that pro-inflammatory cytokines and growth factors play pathogenic roles in myocardial remodelling processes, including cardiac hypertrophy and apoptosis, in various cardiac diseases (Neyses & Pelzer, 1995; Hirano, 1998; Lee et al. 2006a,c). Tumour necrosis factor-{alpha} (TNF{alpha}) and insulin-like growth factor (IGF)-II have also been implicated in the pathophysiology of cardiac hypertrophy (Lee et al. 2006b). Tumour necrosis factor-{alpha} is a potent pro-inflammatory cytokine with a broad range of concentration-dependent pleiotropic effects, found in a diverse array of cell types, including cardiac myocytes (Bazzoni & Beutler, 1996; Yokoyama et al. 1997). Tumour necrosis factor-{alpha} provokes a hypertrophic growth response in the adult mammalian cardiac myocyte (Yokoyama et al. 1997). Insulin-like growth factor-II, acting on the IGF-II receptor, is known to induce hypertrophy of adult rat ventricular cardiomyocytes (Huang et al. 2002b,c; Lee et al. 2006b). However, the roles of TNF{alpha} and IGF-II in cardiac hypertrophy resulting from long-term intermittent hypoxia are still unclear.

Interleukin-6 (IL-6), a typical cytokine, was found to have a potent hypertrophic effect on cardiomyocytes (Kanda & Takahashi, 2004). The IL-6 receptor system consists of an IL-6 specific binding molecule, IL-6R, and a signal transducer, gp130. Following gp130 dimerization, the multiple intracellular signalling pathways evoked by IL-6 and gp130 include P38 mitogen-activated protein kinase (p38), signal transducer and activator of transcription (STAT)–1–STAT-3 herterodimer pathway, STAT-3 homodimer pathway, and MAPK extracellular signal-regulated kinase (ERK) pathway (Hirota et al. 1995; Kodama et al. 1997; Hirano, 1998; Ogata et al. 1999). In addition, the STAT-3-dependent signalling pathway was reported to promote cardiac myocyte hypertrophy (Kunisada et al. 1998), and both STAT-1 and STAT-3 were shown to be chronically phosphorylated in the dilated cardiomyopathy of failing hearts (Ng et al. 2003). The ERK5, also known as big MAPK1 (BMK1), plays a critical role in postnatal eccentric hypertrophy of the heart (Nicol et al. 2001; Cameron et al. 2004). ERK5 and its upstream MAPK-kinase 5 (MEK5) reveal a specific role in transduction of cytokine signals that regulate serial sarcomere assembly and also play a role in the induction of eccentric cardiac hypertrophy that progresses to dilated cardiomyopathy and sudden death (Nicol et al. 2001). Therefore, it is crucial to investigate the pathological role of the IL-6–MEK5–ERK5 signalling pathway resulting from long-term intermittent hypoxia.

In the present study, to understand whether long-term intermittent hypoxia leads to pathological hypertrophy in rat hearts, the myocardial hytertrophic effects, morphological changes, levels of trophic factors such as IL-6, IGF-II and TNF{alpha}, and signalling pathways such as p38, STAT-1, STAT-3, MEK5 and ERK5 in three groups, i.e. normoxia, 4 week long-term intermittent hypoxia (4WLTIH) and 8 week long-term intermittent hypoxia (8WLTIH), were determined by heart weight index, histological analysis, Western blotting and reverse transcriptase-polymerase chain reaction (RT-PCR) from the excised left ventricle.


   Methods
Top
 Abstract
 Introduction
 Methods
Results
 Discussion
 References
 
Animal model

Male Sprague–Dawley rats, 12 weeks old and weighing about 350–400 g, were purchased from National Science Council Animal Center, Taiwan. These animals were housed, three per cage, in an environmentally controlled animal room. Ambient temperature was maintained at 25°C and the animals were kept on an artificial 12 h–12 h light–dark cycle. The light period began at 7.00 am. Rats were provided with standard laboratory chow (Laboratory Diet 5001; PMI Nutrition International Inc., Brentwood, MO, USA) and water ad libitum. All protocols were approved by the Institutional Animal Care and Use Committee of Chang Shan Medical University, Taichung, Taiwan, and the principles of laboratory animal care (NIH publication no. 86-23, revised 1985) were followed.

Exposure to hypoxia

A total of 36 rats were randomly divided into three groups (n = 12, 12, 12), i.e. normoxia, 4WLTIH and 8WLTIH. The normoxia group were exposed to room air for 4 (n = 6) or 8 weeks (n = 6), the 4WLTIH group were placed in a chamber which exposed them to long-term intermittent hypoxia (LTIN, 12% O2 and 88% N2, for 8 h day–1, n = 12) for 4 weeks, and the 8WLTIH group were placed in a chamber which exposed them to LTIH for 8 weeks (n = 12). After 4WLTIH and 8WLTIH, rats were weighed and decapitated. The hearts of animals were excised and cleaned with distilled H2O. The left and right atrium and ventricle were separated and weighed. The ratios of the total heart weight and the left ventricle weight to body weight were calculated.

Heart cross-section and Haematoxylin–Eosin staining

After the heart was removed, it was soaked in formalin and covered with wax. Cross-sections of whole hearts were sliced, and maximal cross-sections were selected. Slides were prepared by deparaffination and dehydration. They were passed through a series of graded alcohols (100, 95 and 75%) for 15 min each. The slides were then dyed with Mayer Haematoxylin for 5–10 min, followed by washing with tap water for 10–20 min. Each slide was then soaked in mild warm water until it turned bright violet before putting it into Eosin solution for 3–5 min. After gently rinsing with water, each slide was then soaked with 85% alcohol, 100% alcohol twice for 15 min each. Finally, it was soaked with xylene twice. Photomicrographs were obtained using Zeiss Axiophot microscopes.

Tissue extraction

Cardiac tissue extracts were obtained by homogenizing the left ventricle samples in a phosphate-buffered saline (PBS) buffer (0.14 M NaCl, 3 mM KCl, 1.4 mM KH2PO4 and 14 mM K2HPO4), at a concentration of 100 mg tissue per 0.5 ml PBS, for 5 min. The homogenates were placed on ice for 10 min and then centrifuged at 12 000 g for 30 min. The supernatant was collected and stored at –70°C for further experiments.

Electrophoresis and Western blot

The tissue extract samples were prepared as described by homogenizing with buffer. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis was done with 10% polyacrylamide gels. The samples were electrophoresed at 140 V for 3.5 h and equilibrated for 15 min in 25 mM Tris-HCl, pH 8.3, containing 192 mM glycine and 20% (v/v) methanol. Electrophoresed proteins were transferred to nitrocellulose paper (Amersham, Hybond-C Extra Supported, 0.45 µm pore size) using a Bio-Rad Scientific Instruments Transphor Unit at 100 mA for 14 h. Nitrocellulose papers were incubated at room temperature for 2 h in blocking buffer containing 100 mM Tris-HCl, pH 7.5, 0.9% (w/v) NaCl and 0.1% (v/v) fetal bovine serum. Monoclonal antibodies were diluted 1:200 in antibody binding buffer containing 100 mM Tris-HCl, pH 7.5, 0.9% (w/v) NaCl, 0.1% (v/v) Tween-20 and 1% (v/v) fetal bovine serum. Incubations were performed at room temperature for 3.5 h. The immunoblots were washed three times in 50 ml blotting buffer for 10 min and then immersed in the second antibody solution containing alkaline phosphatase goat antirat IgG (Promega) for 1 h and diluted 1000-fold in binding buffer. The immunoblots were then washed in blotting buffer for 10 min three times. Colour development was presented in a 20 ml mixture consisting of 7 mg nitro blue tetrazolium, 5 mg 5-bromo-4-chloro-3-indolyl phosphate, 100 mM NaCl and 5 mM MgCl2 in 100 mM Tris-HCl, pH 9.5.

RNA extraction

Total RNA was extracted using the Ultraspec RNA Isolation System (Biotecx Laboratories, Inc., Houston, TX, USA) according to the manufacturer's instructions. Each heart was thoroughly homogenized in 1 ml Ultraspec reagent per 100 mg tissue using a Polytron homogenizer. The homogenates were washed twice with 70% ethanol by gentle vortexing. RNA precipitates were then collected by centrifugation at 12 000g and dried under vacuum for 5–10 min before dissolving in 50 µl diethylpyrocarbonate-treated water, and then incubated at 55–60°C for 10–15 min.

Reverse transcriptase-polymerase chain reaction (RT-PCR)

Total RNA was reverse transcribed and then amplified by the polymerase chain reaction using a Super Script Preamplification System for first strand cDNA Synthesis and Taq DNA polymerase (Life Technologies [Gibco BRL], Rockville, MD, USA). RT-PCR products (45 µl) were separated on a 1.25% agarose gel (Life Technologies [Gibco BRL]). Amplimers were synthesized based on cDNA sequences from GenBank. Rat glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as an internal standard.

Statistical analysis

The data were compared among groups of animals in normoxia, 4WLTIH and 8WLTIH using one-way ANOVA with preplanned contrast comparison against the control (normoxia) group. In all cases, a difference at P < 0.05 was considered statistically significant.


   Results
Top
 Abstract
 Introduction
 Methods
 Results
Discussion
 References
 
Cardiac architecture changes

The heart weight and ventricular weight, expressed as the ratio of body weight, were significantly higher in the 4WLTIH group, and the increase of heart weight to body weight ratio was more obvious in the 8WLTIH group than in the 4WLTIH group (Table 1). To further define the possible cause of the cardiac hypertrophy, we made a cross-section of whole heart and carried out histopathological analysis of ventricular tissue stained with Haematoxylin and Eosin. We found that ventricular wall thickness significantly increased in the 4WLTIH group but significantly decreased in the 8WLTIH group (Fig. 1A). The ratio of wall thickness to cavity diameter significantly decreased and dilated hearts were observed in the 8WLTIH group (Fig. 1A). The ventricular myocardium in the normoxia control group showed normal architecture with normal interstitial space. In contrast, an abnormal myocardial architecture and increased interstitial space were observed in the 4WLTIH group and became more obvious in the 8WLTIH group, which even showed structural disorganization and cardiomyocyte disarray in x400 magnification images (Fig. 1B).


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  		Table 1.  The heart weight index following long-term intermittent hypoxia
 

Figure 1
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  		Figure 1.  Cardiac cross-sections and cardiomyopathic changes of rats exposed to normoxia, 4WLTIH and 8WLTIH
More detail is provided in the main text. A, representative cross-sections of whole hearts from the 3 groups. Arrows indicate that the left ventricular lumen diameter increased in the 4WLTIH group and further increased in the 8WLTIH group. B, cardiac tissue sections stained with Haematoxylin and Eosin for histopathological analysis. The images were magnified by 400 times. Scale bar = 15 µm

 
Changes in gene expression of TNF{alpha}

To investigate the trophic factor TNF{alpha} associated with the cardiac hypertrophy induced by 4WLTIH and 8WLTIH, the mRNA expression of TNF{alpha} was measured by RT-PCR. The mRNA expression of TNF{alpha} was significantly increased in the 4WLTIH group and the 8WLTIH group compared with the normoxia group (Fig. 2).


Figure 2
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  		Figure 2.  The mRNA expressions of TNF{alpha} extracted from the left ventricles of excised hearts in 24 of 36 rats randomly divided into 3 groups, i.e normoxia, 4WLTIH and 8WLTIH, measured by RT-PCR analysis
Bars in B represent the relative quantification on the basis of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and indicate mean values ± S.D. (n = 8, 8, 8). *P < 0.05, **P < 0.01, significant differences between 4WLTIH and normoxia groups or between 8WLTIH and normoxia groups.

 
Changes in IGF-II protein levels

To investigate the trophic factor IGF-II associated with the cardiac hypertrophy induced by 4WLTIH and 8WLTIH, the protein products of IGF-II were measured by Western Blotting. The protein products of IGF-II were significantly increased in the 4WLTIH group and further increased in the 8WLTIH group, compared with the normoxia group (Fig. 3).


Figure 3
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  		Figure 3.  The protein products of IGF-II (Fig. 5) extracted from the left ventricles of excised hearts in 24 of 27 rats randomly divided into 3 groups, i.e normoxia, 4WLTIH and 8WLTIH, measured by Western blotting analysis
Bars in the bar graph represent the relative quantification on the basis of {alpha}-tubulin and indicate mean values ± S.D. (n = 8, 8, 8). *P < 0.05, **P < 0.01, significant differences between 4WLTIH and normoxia groups or between 8WLTIH and normoxia groups.

 
Changes in IL-6, MEK5 and ERK5 protein levels

In order to identify the trophic factor IL-6 and mitogen-activated protein kinase/ERK (MEK) signalling pathways associated with the cardiac hypertrophy induced by 4WLTIH and 8WLTIH, the protein products of IL-6, MEK5 and ERK5 were measured by Western blotting. After 4 weeks hypoxic exposure, the protein products of IL-6 showed a non-significant increase, but MEK5 and ERK5 were not increased in the cardiac tissues. All IL-6, MEK5 and ERK5 protein products were significantly increased in the cardiac tissues after 8WLTIH (Fig. 4).


Figure 4
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  		Figure 4.  The protein products of IL-6, MEK5 and ERK5 extracted from the left ventricles of excised hearts in 24 of 27 rats randomly divided into 3 groups, i.e. normoxia, 4WLTIH and 8WLTIH, measured by Western blotting analysis
Bars in the bar graph represent the relative quantification on the basis of {alpha}-tubulin and indicate mean values ± S.D. (n = 8, 8, 8). *P < 0.05, **P < 0.01, significant differences between 4WLTIH and normoxia groups or between 8WLTIH and normoxia groups.

 
Changes in p38, STAT-1 and STAT-3 protein levels

To investigate the signalling pathways associated with the cardiac hypertrophy induced by 4WLTIH and 8WLTIH, we examined the levels of the components of phosphorylated p38 mitogen-activated protein kinase, signal transducers and activators of transcription (STAT)-1 and STAT-3 pathways. Compared with the normoxia group, phosphorylated p38 and STAT-1 levels rose in the 4WLTIH group and the 8WLTIH group. Additionally, STAT-3 in the 8WLTIH group was significantly higher than in the 4WLTIH group and much higher than in the normoxia group (Fig. 5).


Figure 5
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  		Figure 5.  The protein products of phosphorylated p38 mitogen-activated protein kinase (*P-p38), STAT-1 and STAT-3 extracted from the left ventricles of excised hearts in 24 of 27 rats randomly divided into 3 groups, i.e normoxia, 4WLTIH and 8WLTIH, measured by Western blotting analysis
Bars in the bar graph represent the relative quantification on the basis of {alpha}-tubulin and indicate mean values ± S.D. (n = 8, 8, 8). *P < 0.05, **P < 0.01, significant differences between 4WLTIH and normoxia groups or between 8WLTIH and normoxia groups.

 

   Discussion
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
References
 
Major findings

Our main findings can be summarized as follows: (1) the hypertrophic architecture of the myocardium and the increased interstitial space observed in the 4WLTIH group were associated with significant increases in the expression of the TNF{alpha}, IGF-II, phosphorylated p38, STAT-1 and STAT-3; and (2) the 8WLTIH group showed eccentric dilatation and myocardial disarray, with further increases in the expression of TNF{alpha}, IGF-II, phosphorylated p38, STAT-1 and STAT-3, as well as IL-6, MEK5 and ERK5 levels. Integrating our present findings into previously proposed hypertrophic theory, this confirms our proposed hypothesis that cardiac hypertrophy caused by long-term intermittent hypoxia is associated with the trophic factors TNF{alpha} and IGF-II, and the signalling pathway involving p38, STAT-1 and STAT-3. However, the longer duration stress of intermittent hypoxia also induced eccentric dilated cardiac hypertrophy, which is mediated by the IL-6-related MEK5–ERK5 pathway (Fig. 6).


Figure 6
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  		Figure 6.  Hypothesis
After integrating our present findings into the previous theory, our proposed hypothesis is that cardiac hypertrophy caused by 4 week long-term intermittent hypoxia is associated with the increased TNF{alpha} and IGF-II, and increased phosphorylated p38, STAT-1 and STAT-3 signalling pathways. In addition, 8 week long-term intermittent hypoxia also induces dilated eccentric cardiac hypertrophy, which is mediated by the IL-6-related MEK5–ERK5 pathway and is associated with increased STAT-3 signalling pathway.

 
Cardiac architecture changes

Hypoxia and oxidative stress were shown to be potential inducers of cardiac hypertrophy. In the present study, abnormal myocardial architecture and increased interstitial space were observed after 4WLTIH, and became more obvious with 8WLTIH. In addition, a decreased ratio of wall thickness to cavity diameter and cardiac dilatation were observed after 8WLTIH. Therefore, we speculate that a longer duration of intermittent hypoxia could induce abnormal myocardial architecture and even lead to eccentric and dilated cardiac hypertrophy.

TNF{alpha}

The biological effects of TNF{alpha} may ultimately be beneficial or, alternatively, detrimental, depending on the quantity released and the specific microenvironment in which this cytokine exerts its effects (Yokoyama et al. 1997). Expression and peptide production of TNF{alpha} are upregulated in the adult heart in response to pressure overload and in response to myocardial infarction or ischaemia (Sack, 2002). An increased production of TNF{alpha} and IL-6 in cultured rat cardiac myocytes (H9c2) was demonstrated under hypoxic conditions (Mandi et al. 2000). No other study has directly observed TNF{alpha}-related cardiac hypertrophy induced by hypoxia. Our findings have demonstrated that the rat left ventricle produces TNF{alpha} in response to the stress of intermittent hypoxia leading to cardiac hypertrophy. Tumour necrosis factor-{alpha} provokes a hypertrophic growth response in a dose-dependent manner and increases net actin and myosin heavy chain synthesis in cardiac myocytes (Yokoyama et al. 1997). However, in our study, the challenge of 8WLTIH did not result in a further increase of TNF{alpha} compared with 4WLTIH; in contrast, the longer hypoxia challenge appeared to be associated with downregulation of the TNF{alpha} gene expression previously induced by hypoxia. We have no idea how a longer duration of hypoxia and TNF{alpha} interact with each other.

Insulin-like growth factor-II

Our previous studies showed that IGF II directly induces hypertrophy of adult rat ventricular cardiomyocytes in serum-free medium, as demonstrated by their increased size, total protein synthesis and transcription of muscle-specific genes (Huang et al. 2002a), and suggested that IGF-II and IGF-II-mediated pathways probably involve the IGF-IIR and stimulate hypertrophy of the cardiomyocytes (Huang et al. 2002c; Lee et al. 2006b). In the present study, cardiac IGF-II was significantly increased in the 4WLTIH and 8WLTIH groups compared with the normoxia group. Our findings suggest that IGF-II-mediated cardiac hypertrophy might be induced by long-term intermittent hypoxia. However, it is still controversial whether IGF-II-mediated cardiac hypertrophy plays a beneficial or detrimental role in longer-term hypoxia. Further studies are required to clarify the protective or detrimental role of IGF-II in eccentric dilated cardiac hypertrophy, as demonstrated in the 8WLTIH group.

Interleukin-6 and related pathways

Interleukin-6, a typical cytokine, was found to have a potent hypertrophic effect on cardiomyocytes (Kanda & Takahashi, 2004). Overexpression of IL-6 was confirmed in the injured hypertrophic myocardium of patients who died within 7 days after the onset of acute myocardial infarction (Kaneko et al. 1997). In the present study, increased IL-6 protein products in the cardiac tissue were found after 8WLTIH, suggesting that 8WLTIH-related oxidative stress may induce IL-6 protein production. The IL-6 overexpressed in the myocardium as a result of acute myocardial infarction or other stressors appears to play a central role in the pathogenesis of cardiac hypertrophy (Kaneko et al. 1997; Kanda & Takahashi, 2004). Interleukin-6 is involved in multiple intracellular signalling pathways, including p38 MAPK, STAT-1–STAT-3 heterodimer pathway, STAT-3 homodimer pathway, and ERKs pathway (Hirota et al. 1995; Hirano, 1998; Kodama et al. 1997; Ogata et al. 1999). The MEK5–ERK5 pathway plays a critical role in the induction of eccentric cardiac hypertrophy that progresses to dilated cardiomyopathy and sudden death (Nicol et al. 2001; Cameron et al. 2004). In the present study, IL-6, MEK5, ERK5 and STAT-3 levels were significantly increased in the 8WLTIH group compared with the normoxia group. Therefore, we suggest that eccentric dilated cardiac hypertrophy caused by 8WLTIH is mostly mediated by the IL-6-related MEK5–ERK5 pathway and probably partly by the IL-6 related STAT-3 pathway.


   Footnotes
 
C.-H. Huang and S.-D. Lee contributed equally to this work.


   References
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   Acknowledgements
 
The research is supported by grant NSC 93-2314-B-040-011 from the National Science Council, and CMU95-262 Taiwan.




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