Skip Navigation

European Journal of Heart Failure 2004 6(6):693-703; doi:10.1016/j.ejheart.2003.11.014
This Article
Right arrow Abstract Freely available
Right arrow FREE Full Text (PDF) Freely available
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to My Personal Archive
Right arrow Download to citation manager
Right arrowRequest Permissions
Right arrow Disclaimer
Google Scholar
Right arrow Articles by Dai, R.P.
Right arrow Articles by Tay, S.S.W.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Dai, R.P.
Right arrow Articles by Tay, S.S.W.
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?

© 2004 European Society of Cardiology

Differential expression of cytokines in the rat heart in response to sustained volume overload

R.P. Dai, S.T. Dheen, B.P. He and S.S.W. Tay*

Department of Anatomy, Faculty of Medicine National University of Singapore, Lower Kent Ridge Road, Singapore 117597, Singapore

* Corresponding author. E-mail address: anttaysw{at}nus.edu.sg


   Abstract
Top
 Abstract
1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
 References
 
Objective: The present study aimed to investigate whether sustained volume overload is capable of inducing persistent upregulation of cardiac cytokines including tumor necrosis factor alpha (TNF)-{alpha}, interleukin (IL)-1β, interleukin (IL)-6 and transforming growth factor (TGF)-β1.

Methods and Results: Volume overload-induced heart hypertrophy in rats was established by aortacaval fistula, and the cardiac cytokines were measured in the myocardium from 1 to 4 weeks after operation. In the post-fistula rats, cardiac IL-1β and IL-6 gene and protein levels were upregulated throughout the time of measurement. Immunohistochemistry demonstrated that IL-1β and IL-6 immunoreactive cells were widely distributed in the myocardium in the earlier time intervals, and mainly localized in the regions close to the endocardium in the later time intervals. The cardiac IL-1β immunoreactive cells were mainly localized in the blood vessels whereas the IL-6 positive cells were composed of non-myocytes and cardiomyocytes. TGF-β1 positive staining was increased in the myocardium up to 3 weeks after aortacaval fistula and then decreased to basal levels thereafter. In contrast to the activation of cardiac IL-1β and IL-6 in response to volume overload, TNF-{alpha} expression appeared unaltered in response to sustained volume overload in the transcription and protein levels.

Conclusion: The results of the present study indicate that sustained volume overload is capable of inducing persistent upregulation of some cardiac cytokines. In addition, the differential expressions of TNF-{alpha}, IL-1β and IL-6 suggest that the induction of IL-6 and IL-1β is independent of TNF-{alpha} mediated pathways in this animal model.

Key Words: Hypertrophy • Remodeling • Cytokines • Volume overload

Received May 1, 2003; Revised June 9, 2003; Accepted November 25, 2003


   1. Introduction
Top
 Abstract
 1. Introduction
2. Materials and methods
 3. Results
 4. Discussion
 References
 
Cytokines are highly potent, pleiotrophic and bioactive endogenous peptides produced by a variety of cell types. These molecules are primarily considered to be responsible for mediating inflammatory response with their coordinated production and action which is also called ‘cytokine network’ and ‘cytokine cascade’ [1,2]. Accumulating evidence suggests that the pro-inflammatory cytokines, in particular, TNF-{alpha}, IL-1β and IL-6, are important mediators of cardiovascular disease. It has been reported that TNF-{alpha}, IL-1β and IL-6 could exert negative inotropic effects on the myocardium in various experimental models [3,4]. However, recent evidence has shown that loss of TNF-{alpha} or IL-6 mediated signaling was associated with accelerated cardiac myocyte apoptosis and augmented cardiac injury, suggesting that cytokines might possess an endogenous cardiac protective role [5,6]. Although the role of transient over-expression of the pro-inflammatory cytokines in response to acute injury in the heart is controversial, it is well established that continuous over-expression of pro-inflammatory cytokines within the myocardium would contribute to the progression of cardiac remodeling, the hallmark of heart failure by inducing cardiac hypertrophy, cardiac fibrosis and cell death [7].

Cardiac remodeling, changes in the myocardium that result in rearrangement of normal structure, is generally triggered by hemodynamic overload, such as pressure and volume overload [8]. It is not known whether pro-inflammatory cytokines, when persistently activated within the myocardium in response to hemodynamic overload, are involved in the progression of cardiac remodeling. A recent study demonstrated that cardiac cytokines were only transiently activated in response to sustained pressure overload, and it was suggested that cytokines might play an important but not obligatory role in the development of pressure overload heart failure [9]. However, it is still unclear whether cardiac cytokines are involved in the development of cardiac remodeling induced by volume overload (e.g. secondary to mitral regurgitation in clinic). In contrast to pressure overload, volume overload mainly increases diastolic wall stress [10], and chronic volume overload induces ‘eccentric’ myocardial hypertrophy [11]. Early studies have shown that morphological and functional differences between pressure and volume overload were associated with some molecular phenotypes and patterns of growth factor induction [12–15]. Furthermore, recent studies have demonstrated that cytokines were continuously expressed in the non-infarct zone of the heart after chronic myocardial infarction [16–19]. Pathologically, the myocardium in the non-infarct zone will be subjected, partially at least, to volume overload and undergo cardiac remodeling. All of these factors strongly suggest that volume overload may induce a different cytokine expression pattern within the myocardium, compared with pressure overload.

In order to understand the role of pro-inflammatory cytokines in the progression of volume overload-induced cardiac remodeling, the present study attempts to investigate the expression pattern of the pro-inflammatory cytokines including TNF-{alpha}, IL-1β and IL-6 in the myocardium in response to sustained volume overload. Since it is known that similar spatial expression of cytokines is a prerequisite for their action on the heart, the present study is specifically focused on the spatial expression of cardiac cytokines.

Transforming growth factor (TGF)-β1, another important regulatory cytokine, has been shown to be associated with cardiac fibrosis and cardiomyocyte hypertrophy and contributes to the progression of heart failure when it is expressed continuously in the myocardium [20]. Previous studies have demonstrated persistent upregulation of TGF-β1 expression induced by pressure overload, and at 7 days after short-term volume overload [21–23]. However, it is not known whether TGF-β1 could be continuously activated in volume overload-induced heart failure, beyond short-term activation. Therefore, the present study also aimed to investigate whether sustained volume overload is capable of up-regulating TGF-β1 persistently within the myocardium.


   2. Materials and methods
Top
 Abstract
 1. Introduction
 2. Materials and methods
3. Results
 4. Discussion
 References
 
All experiments were performed on adult male Tokyo–Wistar rats housed under standard environmental conditions and maintained on commercial rat chow and tap water ad libitum. All studies conformed with the principles of the National Institute of Health Guide for the Care and Use of Laboratory Animals.

2.1. Surgical preparation and experimental protocol
An infrarenal abdominal aorta–vena cava fistula was created in the rat by the modification of the method described previously [24]. In brief, adult male Tokyo–Wistar rats, weighing approximately 250 g and generally in good health, were anesthetized with a dose of 0.5 ml/100 g of 7% chloral hydrate. Under sterile conditions, a ventral abdominal laparotomy was performed to expose the aorta and caudal vena cava approximately 1.5 cm below the renal arteries. Blunt dissection was employed to remove the overlying adventitia and expose the blood vessels without disrupting the tissue connecting the vessels. Both blood vessels were then occluded proximal and distal to the intended puncture site, and an 18-gauge needle was inserted into the exposed abdominal aorta and advanced through the medial wall into the vena cava to create the fistula. The needle was withdrawn, and the ventral aortic puncture was sealed with cyanoacrylate. Creation of an aortacaval fistula was visually evident by the pulsatile flow of arterial blood into the vena cava. The abdominal musculature and skin incisions were closed by 3/0 suture. The sham-operated animals underwent the same procedure, except that the puncture was not carried out.

After the operations, the experimental rats were randomly divided and killed at 1, 2, 3 and 4 weeks (n=4–7 at each time point). Age-matched, sham-operated rats were used as controls (n=4 at each time point). Under anesthesia, the body weight was measured, and the heart was rapidly excised, and right and left atria were dissected from the ventricles. The cardiac tissues were rinsed in cold physiological saline solution, blotted dry, and weighed. Transverse tissue pieces were snap frozen in liquid nitrogen for RNA and protein isolation. The left ventricle tissue was fixed in 4% paraformaldehyde for immunohistochemistry.

2.2. RNA extraction and reverse transcription
Total RNA was isolated from the tissue using TRIzol® reagent based on the company protocol (Invitrogen, USA). RNA concentration was determined by spectrophotometry at 260 nm. RNA integrity was electrophoretically verified. For reverse transcription, the reaction mixture, containing 2 µg of RNA, 2.5 µM of oligo(dT) primer, and five units of Molony Murine Leukemia Virus Reverse Transcriptase (M-MLV, Promega, USA) in a total volume of 25 µl, was incubated for 1 h at 42 °C, and stopped by heating for 5 min at 95 °C. The cDNA was quantified using spectrophotometry and diluted to the appropriate concentrations for real-time PCR assay.

2.3. Real-time PCR assay
PCR amplification efficiency analysis was performed using the LightCycler instrument (Roche, Germany). SYBR Green I, a DNA binding dye, which binds in the minor groove of double-stranded DNA, was used to perform the PCR amplification assay. Q-Gene [25], the Microsoft® Excel®-based software coded in Visual Basic was used to calculate the gene expression in the present study. In brief, PCR amplification conditions for the specific gene were optimized for the LightCycler instrument. cDNAs were amplified in the reaction mixture (20 µl) containing LightCyclerTM-FastStart DNA Master SYBR Green I, 0.5 µM of each primer, and appropriate concentration of MgCl2 using LightCyclerTM. The level of specific gene was quantified, expressed as Ct, the cycle number at which the LightCycler System detected the upstroke of the exponential phase of PCR product accumulation. The amplified product after 40 cycles of PCR showed a single prominent band in the expected size for the specific gene. The primers corresponding to the rat TNF-{alpha}, IL-1β, and TGF-β1, and the reference gene, glyceraldehyde-3-phophate dehydrogenase (GAPDH) are listed in Table 1, and the optimal PCR conditions for different genes are listed in Table 2. Representative amplification tracing, melting curve and standard curve for the amplification of TGF-β1 and GAPDH are shown in Fig. 1. The efficiencies of the test cytokines and GAPDH are listed in Table 3. All the samples were diluted to appropriate concentrations to achieve the same efficiency for the specific gene and performed in triplicate. The specific genes were normalized to the level of GAPDH expression in each individual sample. All data were entered into the data analysis software mentioned above. The mean normalized expression was used for statistical analysis.


View this table:
[in this window]
[in a new window]

  		Table 1 PCR primers of the RT-PCR assay

 


View this table:
[in this window]
[in a new window]

  		Table 2 Optimal PCR conditions for specific genes

 


Figure 1
View larger version (34K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 1 Representative amplification tracing, melting curve and standard curve for the TGF-β1 and GAPDH. (a) the amplification plots and the melting curves (the top left) obtained for TGF-β1 and GAPDH cDNA serial dilutions (200–2 µg); (b) the corresponding standard curve showing a slope of –3.6869 and –3.7341 respectively.

 


View this table:
[in this window]
[in a new window]

  		Table 3 Equations describing the standard curve obtained for specific genes and the house keeping gene (GAPDH)

 
2.4. Immunohistochemistry
To investigate the expression of cytokines in the myocardium, the ventricles were fixed for 24 h with 4% paraformaldehyde, and cryoprotected by immersion in 20% sucrose in phosphate buffer. Transverse sections of the ventricles were cut at 10 µm with a cryostat and mounted on 3-aminopropyl triethoxy-silane-coated slides. To quench endogenous peroxidase, all the sections were incubated with 3% H2O2 in methanol for 10 min. Then, non-specific binding was blocked using 5% normal serum for 1 h. Goat anti-TNF-{alpha} (dilution 1:200; R & D systems Inc., Germany), and rabbit anti-TGF-β1 (dilution 1:200), rabbit anti-IL-6 (dilution 1:200) and rabbit anti-IL-1β (dilution 1:200; Santa Cruz Biotechnology, USA) were applied overnight at 4 °C. After 30 min of washing, the sections were incubated with biotinylated secondary antibody (dilution 1:200; Chemicon, USA) for 1 h at room temperature. The slides were sequentially treated with Vectastain Elite ABC Kit (Avidin/Biotin/Horseradish Perosidase-System; Vector Laboratories, USA) for 1 h. The peroxidase reaction was visualized using 3,3'-diaminobenzidine tetrahydrochloride (DAB, Sigma) as a peroxidase substrate. Finally, the slides were counterstained with Hematoxylin. Negative controls were prepared by leaving out the primary antibodies.

2.5. Western blotting
Frozen cardiac specimens of sham-operated, and experimental animals at each time interval point were separately homogenized in a lysis buffer containing 50 mM Tris–HCl (pH 7.5), 1% NP-40, 5 mM EDTA, 1% Triton X100, and protease inhibitor cocktail (Sigma, USA). The protein concentration was determined with Bio-Rad protein assay. Samples of 40 µg total protein were separated by 15% SDS-PAGE and transferred to nitrocellulose membranes. Membranes were incubated with polyclonal rabbit anti-rat TNF-{alpha} antibody (dilution 1:5000; Chemicon, USA), rabbit anti-TGF-β1 rabbit anti-IL-1β, and rabbit anti-IL-6 (all dilutions 1:2000; Santa Cruz Biotechnology, USA) overnight at 4 °C, followed by incubation with horseradish peroxidase-conjugated anti-rabbit IgG secondary antibody (dilution 1:5000; Chemicon, USA), and detection by use of enhanced chemiluminescence (ECL) detection system (Amersham Biosciences, USA). Laser densitometry was used to quantify intensity of the bands, and data were expressed as arbitrary unit at each time point. Positive control was established from the protein extracted from the myocardium in the rat at 6 h after myocardial infarction.

2.6. Statistical analysis
Statistical analysis was performed using SPSS 10.0 software (SPSS Inc.). The data were expressed as mean±S.E.M. For evaluation of statistical significance, one-way ANOVA was performed with multiple-comparison procedure. When appropriate, non-paired t-tests were used to compare experimental groups and the sham group. A value of P<0.05 was considered significant.


   3. Results
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
4. Discussion
 References
 
3.1. Morphometric data of cardiac hypertrophy in response to persistent volume overload
Average body weight (BW), heart weight (HW), left ventricle weight (LVW), right ventricle weight (RVW), HW-to-BW ratio, LVW-to-BW ratio and RVW-to-BW ratio for volume groups, and their specific age-matched sham-operated groups are presented in Table 4. There was a progressive increase in HW, LVW, RVW, HW/BW, LVW/BW and RVW/BW in the post-fistula groups during the course of this study. Seven days after operation, HW/BW, the index of cardiac hypertrophy, was slightly increased compared with age-matched sham-operated rats. However, at the later time points, there was a significant increase in HW/BW index and LVW/BW (P<0.001) in the post-fistula groups in relation to the age-matched controls. There was no significant difference in body weight between the post-fistula groups and the age-matched sham-operated rats.


View this table:
[in this window]
[in a new window]

  		Table 4 Control and aortacaval fistula group values for morphometric parameters

 
3.2. Real-time PCR analysis of cytokine expression after operation
The expression of TGF-β1 mRNA was increased in the ventricles of the post-fistula rats when compared with aged-matched sham-operated rats. The upregulation of the TGF-β1 mRNA expression 7 days after operation was approximately 1.8 times greater (n=4) than that of the controls, and the increase was maintained for up to 3 weeks (n=4, 1.7 times greater than the control). Thereafter, levels returned to baseline values 4 weeks after operation (n=4) (Fig. 2a). Similar to the sustained upregulation of mRNA levels of TGF-β1 after volume overload, IL-1β mRNA levels were also found to be increased in response to sustained volume overload at 7 days, this increase was maintained for up to 28 days (Fig. 2b) (n=4 at each time point after operation).


Figure 2
View larger version (33K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 2 Relative expression of mRNA levels of TGF-β1 (a), IL-1β (b) and TNF-{alpha} (c) in different groups following aortacaval fistula. (a) Cardiac TGF-β1 mRNA levels were increased greatly at 1 week and maintained to 3 weeks after aortacaval fistula compared with age-matched sham-operated rats. At 4 weeks, expression of TGF-β1 mRNA levels was not distinctly different compared with sham groups. (b) Cardiac IL-β mRNA levels were up-regulated compared with sham-operated rats during all the time of measurement. (c) No significant difference in TNF-{alpha} mRNA expression within the myocardium between the experimental and sham-operated rats. Values are mean±S.E.M. (n=8 for sham, n=4 for each experimental group). *P<0.05 vs. sham, and **P<0.01 vs. sham.

 
In contrast to the upregulation of expression of TGF-β1 and IL-1β after operation, there was no significant change in TNF-{alpha} mRNA levels (Fig. 2c) in response to sustained volume overload.

3.3. Localization of cytokine expression in the heart following aortacaval fistula
In sham-operated rats, weak staining of TGF-β1 could be observed in the myocardium (Fig. 3a). After operation, the immunoreactivity of TGF-β1 within the myocardium was increased and persisted up to 3 weeks after operation (Fig. 3b–e). Higher magnification showed that the cardiomyocytes and non-myocytes (Fig. 3d) were the main cell sources of TGF-β1. In the 28-day post-fistula rats, the intensity of TGF-β1 staining was shown to be similar to that of the sham-operated rats (Fig. 3f).


Figure 3
View larger version (171K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 3 Immunohistochemistry of TGF-β1 in the sham-operated and post-fistula rats. (a) Weak staining of TGF-β1 is detected in the myocardium in the sham-operated rat. (b) At 1 week after aortacaval fistula, TGF-β1 positive staining is increased greatly in the myocardium. (c) Intense immunohistochemical staining of TGF-β1 in the myocardium in 2-week post-fistula rat. (d) Higher magnification of (c) shows that TGF-β1 is intensely stained in the cytoplasm of the cardiomyocyte. (e) TGF-β1 is moderately expressed in the myocardium in 3-week post-fistula rat. (f) Weak staining of TGF-β1 in the myocardium of the 4-week post-fistula rat. Scale Bars=25 µm in (a–c, e–f); 10 µm in (d).

 
The staining intensity of IL-1β was upregulated within the myocardium in post-fistula rats compared with that of sham and negative controls (Fig. 4a,b). Unlike TGF-β1, IL-1β was mainly localized to the blood vessels including the arterioles and venules, in spite of the fact that very weak expression was found in the cardiomyocytes within the myocardium (Fig. 4c–e). The expression of IL-1β in blood vessels was observed persistently in all the time intervals studied. In the late time interval of chronic volume overload, IL-1β was predominantly expressed in the region close to the endocardium (Fig. 4f,g).


Figure 4
View larger version (107K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 4 Immunohistochemistry of IL-1β in the myocardium in the sham-operated and post-fistula rats. (a) and (b) Representative micrographs of IL-1β positive staining in the negative control (a) and sham-operated rat (b). (c–e) Intense staining of IL-1β is localized in the blood vessels (arrows) whereas weak staining of IL-1β is present in the myocardium in 1-week (c), 2-week (d) and 3-week (e) post-fistula rats. (f) In 4-week post-fistula rat, IL-1β positive staining appears to exist in the region close to endocardium. (g) Under the higher magnification, intense staining of IL-1β is detected in the blood vessel (arrows) and weak staining of IL-1β is present in cardiomyocytes. Bars=25 µm in (a–e, and g); 100 µm in (f).

 
Almost no IL-6 positive cells were detected within the myocardium of the negative (Fig. 5a) and control (Fig. 5b) animals. However, an intense IL-6 staining was observed in the whole layer of myocardium in 1-week and 2-week post-fistula rats (Fig. 5c,d). In 3-week and 4-week post-fistula rats, IL-6 positive staining was mainly localized in the regions close to the endocardium (Fig. 5e). Higher magnification showed that IL-6 immunoreactivity was mainly localized in the non-myocytes but weakly stained in cardiomyocytes in the post-fistula rat heart (Fig. 5f,g). There were no TNF-{alpha} positive cells detected in the sham-operated and aortacaval fistula rats (data not shown).


Figure 5
View larger version (109K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 5 Immunohistochemistry of IL-6 in control and post-fistula rats. (a and b). No staining of IL-6 in the negative control and sham-operated rat; (c, d) Presence of IL-6 in the cardiomyocytes (arrowhead) and non-myocytes (arrows) in the 1-week (c) and 2-week (d) post-fistula rats. Note that the intense staining of IL-6 in the non-myocytes. (e) IL-6 positive staining is mainly localized in the region close to endocardium in 3-week post-fistula rat. (f) Higher magnification of (e) shows that IL-6 is mainly stained in the non-myocytes as well as cardiomyocytes. (g) IL-6 positive staining is present in the region close to the endocardium in the 4-week post-fistula rat. Bars=25 µm in (a–d, e and g); 100 µm in (f).

 
3.4. Western blot analysis of expression of cardiac cytokines in response to volume overload
A very weak expression of IL-1β protein was detected in the myocardium in the sham-operated rats. However, the expression was found to be significantly upregulated in the myocardium from 7 to 28 days after operation, compared with the age-matched sham-operated rats (Fig. 6a). Similarly, the expression level of IL-6 protein within the myocardium was also upregulated at all the time points after operation (Fig. 6b).


Figure 6
View larger version (35K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 6 Representative photographs of immunoblot (left panel) and corresponding densitometry unit (right panel) of IL-1β (a), TGF-β1 (b), IL-6 (c) protein in the myocardium from the sham-operated, 1-week, 2-week, 3-week and 4-week post-fistula rats. The bands were semiquantified and represented as mean±S.E.M. of arbitrary units. *P<0.05 vs. sham, and **P<0.01 vs. sham. (d) Immunoblot of TNF-{alpha} protein in the myocardium from the 6-h post-myocardial infarcted rat (positive control) and post-fistula rats at different time points.

 
The expression of TGF-β1 protein was also upregulated markedly in the myocardium after 7 days of aortacaval fistula, and the increase maintained up to 3 weeks. In 28-day post-fistula rats, there was a decline in the expression level of TGF-β1 protein in the myocardium towards the basal line (Fig. 6c). The expression of TNF-{alpha} protein was undetectable in the control and experimental rats although the myocardium from the positive control (rats subjected to myocardial infarction) showed strong expression of TNF-{alpha} protein (Fig. 6d).


   4. Discussion
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
References
 
Cardiac remodeling, the hallmark of cardiac failure, can be initiated by hemodynamic overload, but the underlying molecular mechanisms remain unclear. It is believed that the over-expression of pro-inflammatory cytokines, in particular TNF-{alpha}, IL-1β and IL-6, are involved in the progression of cardiac remodeling although the short-term effects of the cardiac cytokines in the myocardium are controversial [2]. Pressure overload, one type of hemodynamic overload, has been reported to be capable of inducing transient upregulation of cardiac cytokines in the murine heart, indicating that pro-inflammatory cytokines might not contribute to cardiac remodeling in this animal model [9]. The present study, which aimed to understand whether cytokines are involved in the pathogenesis of hemodynamic overload-induced cardiac remodeling, demonstrated that continuous volume overload, another type of hemodynamic overload, could induce long-term over-expression of cardiac IL-1β and IL-6 within the myocardium. Unlike the transient upregulation of cytokine expression, observed in pressure overload-induced heart hypertrophy, expression of IL-1β and IL-6 gene and protein levels was upregulated persistently in volume overload-induced heart hypertrophy. The discrepant expression of IL-1 and IL-6 between volume overload-induced and pressure overload-induced cardiac hypertrophy, might be due to the different stimuli induced by volume overload (mainly increasing diastolic wall stress) and pressure overload (mainly increasing wall thickness). In the post-fistula rats, cardiac remodeling, secondary to sustained volume overload, a progressive procedure, was characterized by double ventricle dilation and increased left ventricle compliance. In the present study, both HW/BW and LVW/BW, the indices of cardiac hypertrophy were significantly increased in the later time intervals following aortacaval fistula compared with age-matched sham-operated rats. This supports the hypothesis that sustained volume overload could induce progressive cardiac hypertrophy. Although the mechanisms by which volume overload induces the over-expression of cardiac IL-1β and IL-6 are not clear, the over-expression of cytokines might contribute to the progression of heart failure.

TNF-{alpha}, an upstream cytokine in the cytokine network, may induce the expression of other cytokines in the myocardium including IL-1β and IL-6. TNF-{alpha} has also been shown to be associated with cardiomyocytes hypertrophy and apoptosis [26]. Over-expression of TNF-{alpha} in the hearts of transgenic mice results in the phenotype of heart failure [27]. In the present study, unaltered expression of cardiac TNF-{alpha} in the post-fistula rats, both at the transcriptional and protein levels, is consistent with a recent study, in which cardiac TNF-{alpha} was not upregulated in response to sustained pressure overload [9]. The lack of upregulation of TNF-{alpha}, in response to volume overload, might be due to the homeostatic mechanism for downregulating the inflammatory response in the myocardium. In this connection, pressure overload could induce the activation of Tristetraprolin [TTP, (NUP, TIS11, G0S 24)], a zinc protein which could destabilize TNF-{alpha} mRNA in various cell types, and might be responsible for the downregulation of TNF-{alpha} activation within the myocardium in response to sustained pressure overload [9]. Despite the uncertain mechanism, the lack of change in expression of TNF-{alpha} suggests that TNF-{alpha} may not have a significant role in the development of volume overload-induced cardiac hypertrophy. It also supports the hypothesis that TNF-{alpha} plays an important, but not an indispensable role in the hemodynamic overload that induces the expression of the cardiac IL-1β and IL-6 [9].

Expression of IL-1β and IL-6 was found to be upregulated as a result of persistent volume overload. Previous studies have shown that IL-1β could induce IL-6 expression in various cell types, including cardiomyocytes and cardiac fibroblasts [28]. Although there was no direct evidence in the present study to indicate that IL-1β may induce IL-6 expression, both IL-1β and IL-6 immunoreactivities were mainly distributed in the region close to the endocardium at the late time intervals studied, implying that IL-1β might induce the expression of IL-6 in this animal model. The activation of both cytokines, subsequently, may have a co-ordinated action, which may contribute to disease progression [9]. In addition, the expression of IL-1β within the blood vessels suggests that it may have a role beyond cardiac remodeling. In this regard, it has been reported that TNF-{alpha} and IL-1β could induce the expression of monocyte chemotactic protein (MCP-1), a member of the chemokine family, in endothelial cells. MCP-1 has also been reported to be upregulated in the cardiac venules in volume overload-induced heart failure [29,30]. Although TNF-{alpha} could induce the activation of MCP-1 in other systems, it might be IL-1β but not TNF-{alpha} that induces the expression of MCP-1β in this animal model. The activation of MCP-1 in turn might result in the recruitment of monocytes and angiogenic response based on a recent study [31].

TGF-β1, an important regulatory cytokine, is involved in the progression of cardiac hypertrophy and cardiac fibrosis. Increased expression of TGF-β1 gene and protein has been shown in rats subjected to pressure overload and myocardial infarction [21,23]. The observation of elevated expression of TGF-β1 in the heart with volume overload, observed in the present study, pressure overload and myocardial infarction indicate that TGF-β1 might be an important common mediator during the progression of heart failure. It is not known whether cardiac TGF-β1 was induced by hemodynamic overload directly or indirectly in the present study, that is, TGF-β1 could be induced by other factors which are firstly activated by volume overload. For example, angiotensin II, which could stimulate the release of TGF-β1 in the cardiac fibroblasts [32], was shown to be activated in the heart in the rats with volume overload [33]. Therefore, it is possible that the activation of angiotensin II, induced by volume overload, is responsible for the upregulation of TGF-β1 in the present study. It is important to note that the cytokine expression decreases at 3 weeks following aortacaval fistula. This might be due to the increased left ventricle compliance and the decreased left ventricle end-diastolic pressure as reported previously [34].

In summary, the findings of the present study indicate that unlike pressure overload, persistent volume overload in the rat heart induces the over-expression of IL-1β, IL-6 and TGF-β1, but not TNF-{alpha} within the myocardium for an extended duration. In addition, the spatial expression of IL-1β and IL-6 is different within the myocardium. It is suggested that different hemodynamic overloads have different effects on the persistent expression of the cytokines in the myocardium. Furthermore, the induction of IL-6 and IL-1β is independent of TNF-{alpha} mediated pathway in this animal model.


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

  1. Blackwell T.S., Christman J.W. The role of nuclear factor-kappa B in cytokine gene regulation. Am J Respir Cell Mol Biol (1997) 17:3–9.[Abstract/Free Full Text]
  2. Mann D.L. Inflammatory mediators and the failing heart: past, present, and the foreseeable future. Circ Res (2002) 91:988–998.[Abstract/Free Full Text]
  3. Hosenpud J.D., Campbell S.M., Mendelson D.J. Interleukin-1-induced myocardial depression in an isolated beating heart preparation. J Heart Transplant (1989) 8:460–464.[Web of Science][Medline]
  4. Kinugawa K., Takahashi T., Kohmoto O., Yao A., Aoyagi T., Momomura S., et al. Nitric oxide-mediated effects of interleukin-6 on [Ca2+]i and cell contraction in cultured chick ventricular myocytes. Circ Res (1994) 75:285–295.[Abstract/Free Full Text]
  5. Kurrelmeyer K.M., Michael L.H., Baumgarten G., Taffet G.E., Peschon J.J., Sivasubramanian N., et al. Endogenous tumor necrosis factor protects the adult cardiac myocyte against ischemic-induced apoptosis in a murine model of acute myocardial infarction. Proc Natl Acad Sci USA (2000) 97:5456–5461.[Abstract/Free Full Text]
  6. Hirota H., Chen J., Betz U.A., Rajewsky K., Gu Y., Ross J. Jr, et al. Loss of a gp130 cardiac muscle cell survival pathway is a critical event in the onset of heart failure during biomechanical stress. Cell (1999) 97:189–198.[CrossRef][Web of Science][Medline]
  7. Sivasubramanian N., Coker M.L., Kurrelmeyer K.M., MacLellan W.R., DeMayo F.J., Spinale F.G., et al. Left ventricular remodeling in transgenic mice with cardiac restricted overexpression of tumor necrosis factor. Circulation (2001) 104:826–831.[Abstract/Free Full Text]
  8. Swynghedauw B. Molecular mechanisms of myocardial remodeling. Physiol Rev (1999) 79:215–262.[Abstract/Free Full Text]
  9. Baumgarten G., Knuefermann P., Kalra D., Gao F., Taffet G.E., Michael L., et al. Load-dependent and -independent regulation of proinflammatory cytokine and cytokine receptor gene expression in the adult mammalian heart. Circulation (2002) 105:2192–2197.[Abstract/Free Full Text]
  10. Grossman W., Jones D., McLaurin L.P. Wall stress and patterns of hypertrophy in the human left ventricle. J Clin Invest (1975) 56:56–64.[Web of Science][Medline]
  11. Gerdes A.M., Campbell S.E., Hilbelink D.R. Structural remodeling of cardiac myocytes in rats with arteriovenous fistulas. Lab Invest (1988) 59:857–861.[Web of Science][Medline]
  12. Mann D.L., Urabe Y., Kent R.L., Vinciguerra S., Cooper G. Cellular vs. myocardial basis for the contractile dysfunction of hypertrophied myocardium. Circ Res (1991) 68:402–415.[Abstract/Free Full Text]
  13. Urabe Y., Hamada Y., Spinale F.G., Carabello B.A., Kent R.L., Cooper G., et al. Cardiocyte contractile performance in experimental biventricular volume-overload hypertrophy. Am J Physiol (1993) 264:H1615–H1623.[Web of Science][Medline]
  14. Tsutsui H., Ishihara K., Cooper G. Cytoskeletal role in the contractile dysfunction of hypertrophied myocardium. Science (1993) 260:682–687.[Abstract/Free Full Text]
  15. Marino T.A., Kent R.L., Uboh C.E., Fernandez E., Thompson E.W., Cooper G. Structural analysis of pressure vs. volume overload hypertrophy of cat right ventricle. Am J Physiol (1985) 249:H371–H379.[Web of Science][Medline]
  16. Blum A., Miller H. Pathophysiological role of cytokines in congestive heart failure. Annu Rev Med (2001) 52:15–27.[CrossRef][Web of Science][Medline]
  17. Yue P., Massie B.M., Simpson P.C., Long C.S. Cytokine expression increases in non-myocytes from rats with postinfarction heart failure. Am J Physiol (1998) 275:H250–H258.[Web of Science][Medline]
  18. Prabhu S.D., Freeman G.L. Altered LV inotropic reserve and mechanoenergetics early in the development of heart failure. Am J Physiol Heart Circ Physiol (2000) 278:H698–H705.[Abstract/Free Full Text]
  19. Prabhu S.D., Chandrasekar B., Murray D.R., Freeman G.L. Beta-adrenergic blockade in developing heart failure: effects on myocardial inflammatory cytokines, nitric oxide, and remodeling. Circulation (2000) 101:2103–2109.[Abstract/Free Full Text]
  20. Lijnen P.J., Petrov V.V., Fagard R.H. Induction of cardiac fibrosis by transforming growth factor-beta(1). Mol Genet Metab (2000) 71:418–435.[CrossRef][Web of Science][Medline]
  21. Villarreal F.J., Dillmann W.H. Cardiac hypertrophy-induced changes in mRNA levels for TGF-beta 1, fibronectin, and collagen. Am J Physiol (1992) 262:H1861–H1866.[Web of Science][Medline]
  22. Li J.M., Brooks G. Differential protein expression and subcellular distribution of TGFbeta1, beta2 and beta3 in cardiomyocytes during pressure overload-induced hypertrophy. J Mol Cell Cardiol (1997) 29:2213–2224.[CrossRef][Web of Science][Medline]
  23. Calderone A., Takahashi N., Izzo N.J. Jr, Thaik C.M., Colucci W.S. Pressure- and volume-induced left ventricular hypertrophies are associated with distinct myocyte phenotypes and differential induction of peptide growth factor mRNAs. Circulation (1995) 92:2385–2390.[Abstract/Free Full Text]
  24. Garcia R., Diebold S. Simple, rapid, and effective method of producing aortocaval shunts in the rat. Cardiovasc Res (1990) 24:430–432.[Abstract/Free Full Text]
  25. Muller P.Y., Janovjak H., Miserez A.R., Dobbie Z. Processing of gene expression data generated by quantitative real-time RT-PCR. Biotechniques (2002) 32:1372–1379.[Web of Science][Medline]
  26. Yokoyama T., Nakano M., Bednarczyk J.L., McIntyre B.W., Entman M., Mann D.L. Tumor necrosis factor-alpha provokes a hypertrophic growth response in adult cardiac myocytes. Circulation (1997) 95:1247–1252.[Abstract/Free Full Text]
  27. Li X., Moody M.R., Engel D., Walker S., Clubb F.J. Jr, Sivasubramanian N., et al. Cardiac-specific overexpression of tumor necrosis factor-alpha causes oxidative stress and contractile dysfunction in mouse diaphragm. Circulation (2000) 102:1690–1696.[Abstract/Free Full Text]
  28. Gwechenberger M., Mendoza L.H., Youker K.A., Frangogiannis N.G., Smith C.W., Michael L.H., et al. Cardiac myocytes produce interleukin-6 in culture and in viable border zone of reperfused infarctions. Circulation (1999) 99:546–551.[Abstract/Free Full Text]
  29. Behr T.M., Wang X., Aiyar N., Coatney R.W., Li X., Koster P., et al. Monocyte chemoattractant protein-1 is upregulated in rats with volume-overload congestive heart failure. Circulation (2000) 102:1315–1322.[Abstract/Free Full Text]
  30. Saito H., Patterson C., Hu Z., Runge M.S., Tipnis U., Sinha M., et al. Expression and self-regulatory function of cardiac interleukin-6 during endotoxemia. Am J Physiol Heart Circ Physiol (2000) 279:H2241–H2248.[Abstract/Free Full Text]
  31. Ito W.D., Arras M., Winkler B., Scholz D., Schaper J., Schaper W. Monocyte chemotactic protein-1 increases collateral and peripheral conductance after femoral artery occlusion. Circ Res (1997) 80:829–837.[Abstract/Free Full Text]
  32. Gray M.O., Long C.S., Kalinyak J.E., Li H.T., Karliner J.S. Angiotensin II stimulates cardiac myocyte hypertrophy via paracrine release of TGF-beta 1 and endothelin-1 from fibroblasts. Cardiovasc Res (1998) 40:352–363.[Abstract/Free Full Text]
  33. Ruzicka M., Skarda V., Leenen F.H. Effects of ACE inhibitors on circulating vs. cardiac angiotensin II in volume overload-induced cardiac hypertrophy in rats. Circulation (1995) 92:3568–3573.[Abstract/Free Full Text]
  34. Brower G.L., Henegar J.R., Janicki J.S. Temporal evaluation of left ventricular remodeling and function in rats with chronic volume overload. Am J Physiol (1996) 271:H2071–H2078.[Web of Science][Medline]

Add to CiteULike CiteULike   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us    What's this?


This article has been cited by other articles:


Home page
 Am. J. Pathol.Home page
B. Coles, C. A. Fielding, S. Rose-John, J. Scheller, S. A. Jones, and V. B. O'Donnell
Classic Interleukin-6 Receptor Signaling and Interleukin-6 trans-Signaling Differentially Control Angiotensin II-Dependent Hypertension, Cardiac Signal Transducer and Activator of Transcription-3 Activation, and Vascular Hypertrophy in Vivo
Am. J. Pathol., July 1, 2007; 171(1): 315 - 325.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow FREE Full Text (PDF) Freely available
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to My Personal Archive
Right arrow Download to citation manager
Right arrowRequest Permissions
Right arrow Disclaimer
Google Scholar
Right arrow Articles by Dai, R.P.
Right arrow Articles by Tay, S.S.W.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Dai, R.P.
Right arrow Articles by Tay, S.S.W.
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?