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Effects of carvedilol on cardiac cytokines expression and remodeling in rat with acute myocardial infarction
International Journal of Cardiology 111 (2006) 247 – 255 www.elsevier.com/locate/ijcard
Effects of carvedilol on cardiac cytokines expression and remodeling in rat with acute myocardial infarction Bin Li, Yu-Hua Liao *, Xiang Cheng, Hongxia Ge, Heping Guo, Min Wang Laboratory of Cardiovascular Immunology, Institute of Cardiology, Union Hospital, Tongji Medical College of Huazhong University of Science and Technology, Wuhan 430022, China Received 16 September 2004; received in revised form 15 May 2005; accepted 19 August 2005 Available online 23 November 2005
Abstract Objective: A number of observations suggest that cytokines may be important modulators in the ventricular remodeling process. It is unclear whether carvedilol modulates myocardial pro-inflammatory and anti-inflammatory cytokines expression. We hypothesized that carvedilol could improve ventricular remodeling partly through the modulation of cytokines. The goal of this study was to evaluate the effects of carvedilol on cardiac cytokines expression as well as on myocardial and extracellular matrix remodeling in rats with acute myocardial infarction. Methods: Rats with AMI induced by left anterior descending branch ligation were randomized to carvedilol and control group which were further compared to sham-operated group. We studied the effects of 4-weeks therapy with carvedilol starting 24 h after infarction on 1) hemodynamics, 2) tissue weights, 3) myocardial cytokines (TNF-a, IL-1h, IL-6, IL-10 and TGF-h1) expression by semi-quantitative RTPCR and immunoblotting, 4) matrix metalloproteinases activity by gelatin zymography, 5) collagen expression by immunohistochemistry, 6) myocardium fetal gene (a and h myosin heavy chain) expression. Results: Treatment with carvedilol 1) reduced the pro-inflammatory cytokines and fibrogenic cytokine TGF-h1 levels in myocardium and was associated with the amelioration of the elevated left ventricular diastolic pressure. 2) increased anti-inflammatory cytokine, IL-10 protein expression. 3) reduced matrix metalloproteinases-2 and matrix metalloproteinases-9 activity 4) reduced myocardial collagens 5) did not modify fetal gene re-expression. Conclusion: Pro-inflammatory, anti-inflammatory and fibrogenic cytokines are all involved in the process of post-infarction myocardial remodeling. One mechanism underlying the beneficial effects of carvedilol on post-infarction myocardial remodeling may be modulation of the balance between pro- and anti-inflammatory cytokines as well as fibrogenic cytokines and extracellular matrix (ECM) remodeling. D 2005 Elsevier Ireland Ltd. All rights reserved. Keywords: Cytokines; Myocardial infarction; Remodeling; Matrix metalloproteinase; Fibrosis
1. Introduction Ventricular remodeling is the basic mechanism of heart failure following acute myocardial infarction(MI). It contributes significantly to ventricular dilation and dysfunction, disability and death. Ventricular remodeling is a complex dynamic process by which ventricular size, shape, and function are regulated by mechanics, neurohormonal
* Corresponding author. Tel.: +86 27 85726376; fax: +86 27 85727140. E-mail address: [email protected] (Y.-H. Liao). 0167-5273/$ - see front matter D 2005 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.ijcard.2005.08.065
mechanisms, cytokines, oxidative stress and genetic factors [1]. The process of remodeling includes surviving myocyte hypertrophy and extracellular matrix remodeling. Evidences have shown that several pro-inflammatory cytokines, such as tumor necrosis factor-a (TNF-a), interleukin-1h (IL-1h) and interleukin-6 (IL-6), are involved in the remodeling process. On the other hand, anti-inflammatory cytokine such as interleukin-10 (IL-10) may neutralize the effect of pro-inflammatory cytokines [2,3]. In addition, myocardial matrix remodeling has been proposed to participate in the development of ventricular dilation and heart failure. Matrix metalloproteinases have been found to
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play a significant role in the development of myocardial remodeling and congestive heart failure. Tissue inhibitors of matrix metalloproteinases regulate the expression and activity of matrix metalloproteinases. Tissue inhibitors of matrix metalloproteinases are endogenous physiological inhibitors of matrix metalloproteinases and their concomitant downregulation in heart failure suggests the critical balance that exists between matrix metalloproteinases and tissue inhibitors of matrix metalloproteinases in the maintenance of a normal myocardial interstitial homeostasis. Several cytokines modulate the synthesis and secretion of prosoma of matrix metalloproteinases and tissue inhibitors of matrix metalloproteinases [4]. Numerous clinical trials have documented the beneficial effects of beta-blockers on left ventricular performance and mortality in patients with heart failure. Carvedilol, a nonselective beta-adrenergic blocker with alpha1-blocking and antioxidant properties, prevents the progression of heart failure leading to improvement in left ventricular function, reduction in heart size, and improved survival in patients with New York Heart Association functional Class II and III symptoms [5]. But the effect of it on cytokines, extracellular matrix (ECM) expression and fetal gene re-expression in myocardial remodeling is unclear. We hypothesized that carvedilol could lead to improved post-MI remodeling which is associated with the modulation of myocardial cytokines and extracellular matrix. So in this study, we observed the effect of carvedilol on hemodynamics, total cardiac collagen, type I and III collagens, alpha- and beta-myosin heavy chain (MHC) and cytokines (TNF-a, IL-1h, IL-6, IL-10 and TGF-h1) expression and the activity of matrix metalloproteinases.
2. Methods 2.1. Experimental infarction Myocardial infarction was induced in 126 male Wistar rats (8-week-old ) weighting 200 to 250 g (mean 222.5 T 14.8 g) through ligation of the left anterior descending coronary artery as previously described [6]. A 40% mortality rate was observed within 24 h of this procedure. The sham ligation group underwent a similar procedure except that the suture was not tightened around the coronary artery. All experimental procedures and protocols used in this investigation were reviewed and approved by the Institutional Authority for Laboratory Animal Care. 2.2. Drug administration 24 h after coronary ligation, the surviving rats with MI were randomized into two groups. The first group received carvedilol 10 mg/kg bid (MI-C group, n = 34). The second group received vehicle (soya bean oil) (MI group, n = 32). The sham-ligated rats also received vehicle as a control (SH
group, n = 16). Carvedilol (Dilatrend, provided by Roche Incorporation) was dissolved in soya bean oil. All drugs were given by gavage. The treatment was continued for 28 days. All rats were housed under identical conditions in a 12-h light/dark cycle and given food and water. 2.3. Hemodynamic measurements At the end of the fourth week following coronary ligation, after the body weights of each of the rats were obtained, the rats were anesthetized with sodium pentobarbital (30¨40 mg/kg, i.p.). The left ventricular pressures were measured via a saline-filled cannula, which was inserted through the right carotid artery and connected to a pressure transducer (Powerlab/4SP ML750, AD Instrument).The cannula was inserted into the left ventricle to monitor the left ventricular systolic pressure (LVSP), left ventricular end-diastolic pressure (LVEDP) and left ventricular +dp / dt (maximum rate of pressure rise). 2.4. Cardiac weights and sample obtainment After completing the cardiac hemodymanic measurements, all the rats had their hearts stopped in diastole by an intravenous injection of a 10% potassium chloride sodium 2¨3 ml. Then the hearts were obtained isolated from atrials and aortas. The right ventricles were separated from the hearts by the septums. After the left ventricular weights (LVW) and right ventricular weights (RVW) were determined by electronic balance, the vertical slices of the left ventricles of subdivision of rats were made along the left ventricular long-axis for morphological and immunohistochemical analyses. The myocardium of the rest rats were immediately frozen in liquid nitrogen and stored at 70 -C for biochemical and molecular biologic measurements. The pale and thin zones of ventricles represent infarcted myocardium, the zones on the opposite side of infarcted zones represent non-infarcted zones (far from infarcted zones). 2.5. RNA isolation and semi-quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) analysis of cytokines, a-and b-myosin heavy chains mRNA expression Total RNA was isolated from non-infarcted myocardium by acid guanidium thiocyanate-phenol-chloroform extraction. The RNA, stored at 70 -C before use, was quantified by measuring the absorption at 260 nm. The RT-PCR experiments were performed on total ribonucleic acid based on a method published by Dukas et al. [7]. The following oligonucleotides were used as rat sense and antisense primers, respectively. TNF-a (691 bp):5V-ATG AGC ACG GAA AGC ATG ATC CGA-3V; and 5V-CCA AAG TAG ACC TGC CCG GAC TC-3V; IL-1h(560 bp): 5VATG GCA ACT GTC CCT GAA CTC AAC T-3V; and 5VCAG GAC AGG TAT AGA TTC AAC CCC TT-3V; IL-6
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(660 bp) : 5V-CCA GTT GCC TTC TTG GGA CTG ATG3V; and 5V-ATT TTC TGA CCA CAG TGA GGA ATG-3V; IL-10 (346 bp) : 5V-TGC CTT CAG TCA AGT GAA GACT-3V; and 5V-AAA CTC ATT CAT GGC CTT GTA-3V ;TGFh1 (396 bp) : 5V-GCC TCC GCA TCC CAC CTT TG-3V; and 5V-GCG GGT GAC TTC TTT GGC GT-3V;amyosin heavy chain (81 bp) :5V-CAG AAA ATG CAC GAT GAG GA-3; and 5V-TCA AGC ATT CAT ATT TAT TGT GGG-3V; h-myosin heavy chain (100 bp): 5V-GGG CCT GAA TGA AGA GTA GAT-3V; and 5V-GTG TTT CTG CCT AAG GTG CT-3V; the housekeeping gene hactin (249 bp) was used as internal control. The RT-PCR products were visualized on 1.5% or 3% agarose gels electrophoresed in 1 TAE buffer containing 0.5 ug ml 1 ethidium bromide. 2.6. Immunoblot of TNF-a, IL-1b, IL-6, IL-10 and TGF-b1 protein The total cell plasma protein of myocardium in noninfarcted zones were extracted by 50 mmol/L Tris – HCl (pH 7.4). Samples containing 30 Ag of total protein were electrophoresed on 15% polyacrylamide gels (Bio-Rad) for 1 h (4 -C, constant current for 80 mA). The separated proteins were electrophoretically transferred onto a nitrocellulose membrane (Invitrogen, USA). The transfer time respectively was: TNF-a, 28 min; IL-1h, 28 min; IL-6, 30 min; IL-10, 35 min; TGF-h1, 30 min. Then blocked with 5% defatted milk for 2 h at 37 -C. The cytokines protein expression were detected by using a 1 : 500 dilution of a goat anti-rat TNF-a, IL-6, IL-10, TGF-h1 polyclonal antibody (RandD Systems, Minneapolis, Minnesota) and 1 : 2000 dilution of a mouse anti-rat IL-1h monoclonal antibody (Serotec Ltd, Kidlington, Oxford, UK) as the first antibody, respectively. A 1 : 5000 dilution of horseradish peroxidase-conjugated rabbit anti-goat and rabbit antimouse IgG (Santa Cruz, Santa Cruz Biotechnology Inc., California) were then used as the second antibody, respectively and developed with the enhanced chemiluminescence Western blotting detection system (Pierce Company Product). 2.7. Immunohistochemistry for TNF-a and collagens The left ventricles used for immunohistochemistry were fixed in 10% formalin phosphate buffer for 24 h, and then embedded in paraffin. Expression of TNF-a and type I and III collagen in cardiac tissues was determined on two of the cardiac cross-sections (6 Am). Two cross-sections were obtained on microtome between the base and the apex of the LV. Sections were respectively exposed to 1 : 100 dilution goat anti-rat polyclonal TNF-a, type I and III collagen antibodies. A 1 : 500 dilution of biotinylated rabbit anti-goat IgG as the second antibody, respectively. Then after incubation with avidin – biotin – horseradish peroxidase complexes, peroxidase was visualized followed by
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incubation with diaminobenzidine enhancing solution. Positive TNF-a, type I and III collagen expression were confirmed by a specific brown staining of the cells. Each section was analyzed by HMIAS Series Color Medical Image Analyze System and IBAS computerized system (Champion Image Ltd., China). 2.8. Gelatin zymography for matrix metalloproteinase-2 and matrix metalloproteinase-9 activity assay The total protein of non-infarcted myocardium was extracted by 50 mmol/L Tris – HCL but proteinase inhibitors such as ethylenediaminetetra –acetic acid and phenylmethylsulfonyl fluoride, that might inhibit the activity of matrix metalloproteinases, were not used. The media supernatants were treated with sodium dodecyl sulfatepolyacrylamide gel (SDS-PAGE) sample buffer without boiling. The levels of protein were determined by the Bradford methods. The volume was adjusted to contain the same quantity of protein (5 Ag). Samples containing 30 Ag of total protein were electrophoresed on 7.5% polyacrylamide gels containing gelatin (1 mgIml 1) by electrophoresis at 40 mA for 1 h at 4 -C. The gels were soaked in 2.5% Trixton-100 for 30 min 2 times at room temperature to remove the SDS, and incubated in a digestion buffer (50 mmol Tris –HCl, pH 7.6, 5 mmol CaCl2, 100 mmol NaCl, 2 Amol ZnCl2, and 0.01% Brij-35) at 37 -C staying overnight for more than 24 h to allow proteinase digestion of its substrate. Gels were rinsed again in distilled water, stained with 0.25% Coomassie brilliant blue R-250 for 2 h, and destained with 10% acetic acid. Gelatinolytic activity appeared as clear bands of a digested gelatin against a dark blue background of stained gelatin. The activity of matrix metalloproteinase-2 and matrix metalloproteinase-9 were determined according to the signal of the bands by the Table 1 Body weight, heart weight and hemodynamic measurements x¯ T s SH group (n = 10)
MI group (n = 12)
MI-C group (n = 11)
Body weights (g) Experiment before 221 T16.6 220 T 15.5 221 T14.5 4-week end 217 T 12.3# 219 T 14.2# 219 T 11.1# LVW / BW (mg/g) 2.19 T 0.19 2.56 T 0.20** 2.37 T 0.14g RVW / BW (mg/g) 0.53 T 0.02 1.02 T 0.03** 0.99 T 0.03g HR (bpm) 424.1 T15.18 433.2 T 14.89 387.7 T 14.11gg LVEDP (mmHg) 6.43 T 0.70 18.6 T 2.71** 10.19 T 2.8gg LVSP (mmHg) 138.48 T 5.86 119.03 T 6.28** 114.69 T 7.47 +dp / dt max (mmHg/s) 2523.2 T 186.5 1821.6T265.3** 2374.6 T 166.7gg Compared with experiment before, #P > 0.05. Compared with SH group, **P < 0.01. Compared with MI group, gP < 0.05, ggP < 0.01. BW = body weight; LVW = left ventricular weight; RVW = right ventricular weight; MI = myocardial infarction; MI-C = myocardial infarction treated with carvedilol; SH = sham-operated group; HR = heart rate; LVEDP= left ventricular end-diastolic pressure; LVSP= left ventricular systolic pressure; LV + dp / dt max = left ventricular rate of pressure development.
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GSD8000 Density Scan Analysis System (UVP Ltd. Britain) reading the area and density of bands. The activity of matrix metalloproteinases is equivalent to an area of band (the density of band the density of background). 2.9. Cardiac fibrosis assessment This procedure consisted of using cardiac tissue sections stained with picric acid – fuchsin acid, resulting in cardiac myocytes being stained yellow and collagen fibers stained red in colour. Collagen volume density fraction (CVF) was then determined by measuring the area as a proportion of the total area under observation. The collagen-rich border zone of vessels and the scar were excluded in the calculations. Ten fields were analyzed in the subendocardial layer and ten fields in the subepicardial layer in each LV.
2.10. Statistical analysis All values are expressed as mean T standard deviation. Results were analyzed by using analysis of variance (ANOVA) for multiple comparisons followed by a twosided Dunnett’s test or Student – Newman– Keuls test, when appropriate. Gels were analyzed by densitometry and the results were presented as mean (arbitrary units) T SEM. Statistical significance was assumed at p < 0.05.
3. Results 3.1. Hemodynamics, body weight and ventricular weight Before and after gavage for four weeks, there were no obvious changes of body weights of the three groups
Fig. 1. Cardiac morphology and collagens in SH, MI and MI-C group, respectively. Row A shows cardiac morphologic changes. Row B shows total collagen stained with picric acid-fuchsin acid (200) in non-infarcted region of left ventricle. The red represents total collagen. The yellow represents myocardium. Row C and D show localization of type I and III collagen immunohistochemical analysis in boundary zone, respectively (200). MI group have significant positive staining. MI-C group have weaker positive staining and SH group have very weak positive staining for type I and negative staining for type III collagen. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
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(see Table 1).It suggested that carvedilol had not caused obvious changes in body weights of rats. Compared with the sham-operated group, the ratio of left ventricular weight/body weight, right ventricular weight/body weight and left ventricular end-diastolic pressure(LVEDP) were significantly elevated whereas the left ventricular systolic pressure (LVSP) and left ventricular maximum rate of pressure rise (LV + dp / dt max) were
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obviously reduced in MI group rats at the end of the fourth week ( P < 0.01). After carvedilol treatment for 4 weeks, the ratio of left ventricular weight / body weight, right ventricular weight / body weight, heart rate and left ventricular end-diastolic pressure were significantly decreased, while the left ventricular maximum rate of pressure rise (LV + dp / dt max) was obviously increased (see Table 1).
Fig. 2. Cardiac cytokines mRNA expression (A – E) evaluated by semi-quantitative RT-PCR. *Compared with sham-operated group, P < 0.01. DCompared with MI group, P < 0.01. TNF = tumor necrosis factor; IL= interleukin; TGF = transforming growth factor.
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3.2. Cardiac morphology and collagens Compared with sham-operated group, left ventricular chamber enlarged and wall thinning in MI group, and total collagen volume fraction (CVF) increased significantly (5.87 T 0.49 vs. 2.81 T 0.20, respectively, P < 0.01). Compared to the MI group, carvedilol decreased the enlarged ventricular chamber and slightly increased the thickness of scar, and reduced the total collagen volume fraction (3.24 T 0.33 vs. 5.87 T 0.49, respectively, P < 0.01) (Fig. 1 row B) (Fig. 1 row A and row B). On immunohistochemical staining, both type I and III collagen could be found in myocardium of MI group. Carvedilol reduced both type I and III collagen immunoreactivity (Fig. 1 row C and D). 3.3. Cardiac myosin heavy chain mRNA expression a- and h-myosin heavy chain mRNA in non-infarcted zone were elevated compared with the sham-operated group at the end of the fourth week after MI. But no obvious changes of a- and h-myosin heavy chain mRNA expression were found between MI treated with carvedilol and that with vehicle (data not shown). 3.4. Cardiac cytokines gene expression Compared to the sham-operated group, TNF-a, IL-1h, IL-6, IL-10, and TGF-h1 mRNA in non-infarcted zone were all elevated at the end of the fourth week after MI. Four weeks treatment with carvedilol reduced the elevated TNF-a, IL-1h, IL-6 and TGF-h1 mRNA expression but had no obvious effect on IL-10 mRNA expression. Especially, IL-6 and IL-1h mRNA expression in noninfarcted zone were reduced near to those in shamoperated group (Fig. 2A –E). 3.5. Location of TNF-a protein By immunohistochemical staining, we found TNF-a immunoreactivity predominated in surviving cardiomyocytes except in the infiltrating inflammatory cells of the infarct zone and cardiomyocytes in non-infarct zone (Fig. 3A – D). Carvedilol decreased cardiomyocyte positive staining compared with MI group not treated with carvedilol, but had no effect on infiltrating inflammatory cells (Fig. 3E –F). 3.6. Cardiac cytokines protein expression The protein of cytokines (TNF-a, IL-1h, TGF-h1, IL-6) expression in non-infarcted zone were also elevated in MI group (13.49 T 1.44, 13.9 T 1.65, 18.09 T 0.98 and 13.26 T 0.78, respectively) compared with sham-operated group (2.89 T 0.74, 2.43 T 0.6, 5.27 T 0.37 and 3.1 T 0.69, respectively, P < 0.01 for all) at the end of the fourth week
Fig. 3. Immuntostaining of TNF-a. Magnification: 400. (A) Positive staining in surviving cardiomyocytes in the infarct zone. (B) Positive staining in infiltrating inflammatory cells in the infarct zone. (C) Positive staining in remote cardiomyocytes in non-infarct zone. (D) Negative staining in sham-operated group. (E) Positive staining in myocardium of MI group (200). (F) Positive staining in myocardium of MI-C group (200).
after MI. Treatment with carvedilol reduced TNF-a, IL-1h, TGF-h1 (8.91 T 0.28, 10.02 T 1.19, 13.01 T 0.78, respectively, P < 0.01 for all) and IL-6 (11.67 T 1.48, P = 0.018) protein expression which coincided with the change of gene expression. Particularly, carvedilol increased cardiac IL-10 protein expression from 13.81 T 0.82 to 15.19 T 1.29 ( P = 0.022) different from the result of gene expression, thereby reducing the ratio of TNF-a/IL-10 from 0.98 T 0.11 to 0.59 T 0.16 ( P < 0.01) (Fig. 4). 3.7. Cardiac matrix metalloproteinase-2 and matrix metalloproteinase-9 activity Gelatin zymography (Fig. 5) showed that treatment with carvedilol reduced the elevated matrix metalloproteinase-2 and matrix metalloproteinase-9 activity compared to those in MI group. Again, matrix metalloproteinases activity assay results confirmed the findings from zymography. As shown in the bar graphs, matrix metalloproteinase-2 and matrix metalloproteinase9 activity in MI group were 16.67 T 2.24 and 14.66 T 1.02, respectively, which were significantly higher than the sham-operated group (4.09 T 0.33 and 5.13 T 1.29, respectively, P < 0.01 for both). After carvedilol therapy for four weeks, matrix metalloproteinase-2 and matrix metalloproteinase-9 activity were reduced to 5.05 T 0.98
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Fig. 4. Western blot shows 30 Ag of total protein from concentrated supernatant were subjected to gel electrophoresis and immunoblotting. The six bar graphs showed that the level of five cytokines protein expression and TNF-a / IL-10 ratio in non-infarcted myocardium in the three groups, respectively. *Compared with SH group, P < 0.01. DCompared with MI group, P < 0.01.
and 7.73 T 0.93, respectively, which were significantly lower than MI group ( P < 0.01 for both).
4. Discussion This study indicates that carvedilol, when started 24 h after MI, markedly ameliorates cardiac hemodynamic index and ventricular weights. This was accompanied by the reduction in the mRNA and protein expression of proinflammatory cytokines (including TNF-a, IL-1h and IL-6) and fibrogenic cytokine (TGF-h1), and the increase in antiinflammatory cytokine, IL-10 protein expression. This did not appear to be the result of prevention of cardiac fetal gene expression but instead, might have been related to reduced myocardial matrix metalloproteinase-2 and matrix metal-
loproteinase-9 activity, and reduced cardiac type I and III collagens. Taken together, these results suggest that a range of cytokines including pro-inflammatory, anti-inflammatory, pro-fibrosis cytokines and the imbalance in pro-inflammatory and anti-inflammatory cytokines participate in the process of ventricular remodeling after MI, and that carvedilol can improve extracellular matrix (ECM) remodeling and modulate cardiac cytokines expression. In the recent years, more and more evidences have suggested that inflammatory cytokines are involved in myocardial remodeling. The cytokine, tumor necrosis factor-a, has been causally linked to left ventricular (LV) remodeling. Matrix metalloproteinases (MMPs) have been implicated in cardiac remodeling and can be regulated by TNF-a [8]. Dysregulation of the myocardial extracellular matrix contributes to abnormal cardiac
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Fig. 5. Zymogram for evaluating the differences of myocardium MMPs activity among the MI, MI-C and SH group rats. The bar graphs showed the results of MMP-2 and MMP-9 activity assay. *Compared with shamoperated group, P < 0.01. DCompared with MI group, P < 0.01. MW = molecular weight. MMP= matrix metalloproteinase. MI = myocardial infarction. MI-C = myocardial infarction treated with carvedilol. SH = sham-operated group.
muscle function. Changes in the balance between matrix deposition and matrix degradation by matrix metalloproteinases (MMPs) can lead to cardiac fibrosis and dilation. Inflammatory cytokines such as TNF-a and IL-1h can regulate this process [9]. In contrast, IL-10 is a pleiotropic cytokine with potent anti-inflammatory effects in many cells lines. Selzman et al. has shown that IL-10 could be used therapeutically to influence vascular remodeling by inhibiting TNF-induced vessel smooth muscle cell proliferation [10]. In addition, IL-10 downregulates the production of metalloproteinases (MMPs) and upregulates that of their tissue inhibitors (TIMPs) thereby modulating extracellular matrix remodeling. Clinical trials have shown significantly elevated levels of IL-10 in patients with acute myocardial infarction [11]. Accordingly, the imbalance of the pro-inflammatory and anti-inflammatory cytokines maybe related to the ventricular remodeling process. TGF-h1 certainly has also been proven to participate in remodeling mechanism of ventricles. Activated TGF-h1 mRNA expression is accompanied by the appearance of myocardial fibroblast and the expression of fibrillar collagens and tissue inhibitors of matrix metalloproteinases, suggesting that this fibrogenic cytokine may contribute to collagen remodeling in the rat heart after myocardial infarction [12]. Carvedilol is a new generation vasodilator which is a nonselective beta-adrenergic antagonist with additional alpha1-blocking, anti-oxidant and anti-proliferating activity that may contribute to the overall clinical benefit seen in the treatment of left ventricular dysfunction after myocardial infarction [5]. Carvedilol has been proved to reduced
myocardial IL-1h expression and myocardial collagen deposition in rats [13,14]. In the present study, carvedilol was given in dose of 10 mg/kg/d for four weeks and was demonstrated to decrease left ventricular end-diastole pressure and heart rate, and increase LV + dp / dt max while having no significant effect on left ventricular systolic pressure. Moreover, carvedilol decreased the left ventricular weight / body weight, right ventricular weight / body weight and the enlargement of the ventricular chamber, and slightly increased the thickness of scar. These results suggested that carvedilol had beneficial effect on cardiac function and cardiac remodeling. First, we found carvedilol reduced cardiac gene expression and protein production of TNF-a, IL-1h, IL-6 and TGF-h1. Carvedilol did not change the gene expression of IL-10 but increased the protein level of it in non-infarcted zone suggesting that carvedilol might affect the IL-10 protein production at post-transcriptional level. Furthermore, we found that carvedilol reduced TNF-a, / IL-10 protein ratio. Thus, carvedilol may affect the ventricular remodeling after MI by modulating the imbalance of proinflammatory and anti-inflammatory cytokines and decreasing the fibrogenic cytokine TGF-h1. Interestingly, except for the infiltrating inflammatory cells, we found significant immunoactivity of TNF-a in surviving cardiomyocytes in infarcted zone and remote myocardium suggesting cardiomyocytes as also being one of the major sources of the cytokines. Carvedilol reduced TNF-a secretion from myocardium but had no significant effect on infiltrating inflammatory cells. This suggested that carvedilol may modulate TNF-a secretion of cardiomyocytes by blocking myocardial a1- or h-adrenergic receptors. Taking account of the obvious change in heart rate but no significant change in left ventricular systolic pressure with the use of carvedilol, we think that carvedilol modulated cytokines expression at least partly by the mechanism of h-adrenergic blockade. Carvedilol might modulate cytokines production via the effect on adrenergic receptors on cardiomyocytes. It was found that h-adrenergic stimulation delayed the activation of cardiac STAT in mice thereby increasing IL-6 gene family expression of cardiac fibroblasts. In addition, the stimulation of norepinephrine induced left ventricular gene expression of IL-1h and IL-6 in rat and can be blocked by carvedilol [15]. It has been demonstrated that carvedilol inhibited cytokine production from various stimuli-activated T cell by specifically downregulating NF-kappa B activity in activated T cells [16]. Second, we found that carvedilol decreased the left ventricular matrix metalloproteinase-2 and matrix metalloproteinase-9 activity as well as total collagens and type I and III collagens. Cytokines have been proved to be the important modulators of matrix metalloproteinases gene expression. The inflammatory cytokines such as TNF-a and IL-1h are involved in ventricular remodeling both directly as well as indirectly through modulation of matrix metalloproteinases gene expression [17 –19]. It has been
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suggested that norepinephrine regulates synthesis of myocardial type I collagen via an indirect effect [20]. Activated TGF-h1 mRNA expression is accompanied by the appearance of myocardial fibroblasts and the expression of fibrillar collagens and tissue inhibitors of matrix metalloproteinases, suggesting that this fibrogenic cytokine may contribute to collagen remodeling in the rat heart after myocardial infarction [21]. So the mechanism of the effects of carvedilol on ventricular matrix metalloproteinases activity and collagens maybe partly related to the inhibition of cardiac inflammatory and fibrogenic cytokines production. But the exact mechanisms that account for the alterations of matrix metalloproteinases and collagens are complex. Unfortunately, we did not find an obvious effect of carvedilol on ventricular fetal gene (a-MHC and h-MHC) re-expression suggesting that fetal gene re-expression was not probably related to h- or a-adrenergic signaling pathways and it did not contribute to improved left ventricular function with the use of carvedilol. In summary, this study indicates that pro-inflammatory, anti-inflammatory and fibrogenic cytokines contribute to the ventricular remodeling after MI in rats. The effect of carvedilol on the modulation of the balance between the pro-inflammatory and anti-inflammatory cytokines and the reduction of fibrogenic cytokine as well as on ventricular matrix metalloproteinases activity and collagens in noninfarcted zone may be one of the salutary mechanisms on ventricular remodeling after myocardial infarction.
Acknowledgements This work was supported in part by the laboratory of pathology, immunology and molecular biology and animal center of Tongji Medical College of Huazhong University of Science and Technology.
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[5] The CAPRICORN investigators. Effect of carvedilol on outcome after myocardial infarction in patients with left ventricular dysfunction: the CAPRICORN randomized trial. Lancet 2001;357:1385 – 90. [6] Nguyen Q, Cernacek P, Calderoni A, et al. Endothelin A receptor blockade causes adverse left ventricular remodeling but improves pulmonary artery pressure after infarction in the rat. Circulation 1998;98:2323 – 30. [7] Dukas K, Sarfati P, Vaysse N, Pradayrol L. Quantitation of changes in the expression of multiple genes by simultaneous polymerase chain reaction. Ann Biochem 1993;215:66 – 72. [8] Bradham WS, Moe G, Wendt KA, et al. TNF-alpha and myocardial matrix metalloproteinases in heart failure: relationship to LV remodeling. Am J Physiol Heart Circ Physiol 2002 (Apr);282(4):H1288 – 95. [9] Siwik DA, Colucci WS. Regulation of matrix metalloproteinases by cytokines and reactive oxygen/nitrogen species in the myocardium. Heart Fail Rev 2004 (Jan);9(1):43 – 51. [10] Selzman CH, Meldrum DR, Cain BS, et al. Interleukin-10 inhibits post injury tumor necrosis factor-mediated human vascular smooth muscle proliferation. J Surg Res 1998 (Dec);80(2):352 – 6. [11] Tziakas DN, Chalikias GK, Hatzinikolaou HI. Anti-inflammatory cytokine profile in acute coronary syndromes: behavior of interleukin10 in association with serum metalloproteinases and proinflammatory cytokines. Int J Cardiol 2003 (Dec);92(2-3):169 – 75. [12] Sun Y, Zhang JQ, Zhang J, et al. Cardiac remodeling by fibrous tissue after infarction in rats. J Lab Clin Med 2000 (Apr);135(4): 316 – 23. [13] Sia YT, Parker TG, Tsoporis JN, Liu P, Adam A, Rouleau JL. Longterm effects of carvedilol on left ventricular function, remodeling, and expression of cardiac cytokines after large myocardial infarction in the rat. J Cardiovasc Pharmacol 2002 (Jan);39(1):73 – 87. [14] Wei S, Chow LT, Sandson JE. Effect of carvedilol in comparison with metoprolol on myocardial collagen postinfarction. J Am Coll Cardiol 2000 (Jul);36(1):276 – 81. [15] Yin F, Li P, Zheng M, et al. Interleukin-6 family of cytokines mediates isoproterenol-induced delayed STAT3 activation in mouse heart. J Biol Chem 2003 (Jun 6);278(23):21070 – 5; Barth W, Deten A, Bauer M, et al. Differential remodeling of the left and right heart after norepinephrine treatment in rats: studies on cytokines and collagen. J Mol Cell Cardiol 2000 (Feb);32(2): 273 – 84. [16] Yang SP, Ho LJ, Lin YL, et al. Carvedilol, a new antioxidative betablocker, blocks in vitro human peripheral blood T cell activation by downregulating NF-kappaB activity. Cardiovasc Res 2003 (Sep 1);59(3):776 – 87. [17] Bradham WS, Moe G, Wendt KA, et al. TNF-alpha and myocardial matrix metalloproteinases in heart failure: relationship to LV remodeling. Am J Physiol Heart Circ Physiol 2002 (Apr);282(4):H1288 – 95. [18] Bradham WS, Bozkurt B, Gunasinghe H, et al. Tumor necrosis factoralpha and myocardial remodeling in progression of heart failure: a current perspective. Cardiovasc Res 2002 (Mar);53(4):822 – 30. [19] Siwik DA, Chang DL, Colucci WS. Interleukin-1beta and tumor necrosis factor-alpha decrease collagen synthesis and increase matrix metalloproteinase activity in cardiac fibroblasts in vitro. Circ Res 2000 (Jun 23);86(12):1259 – 65. [20] Bhambi B, Eghbaii M. Effect of norepinephrine on myocardial collagen gene expression and response of cardiac fibroblasts after norepinephrine treatment. Am J Pathol 1991;139:1131 – 42. [21] Sun Y, Zhang JQ, Zhang J, Lamparter S. Cardiac remodeling by fibrous tissue after infarction in rats. J Lab Clin Med 2000 (Apr);135(4):316 – 23.
Effects of carvedilol on cardiac cytokines expression and remodeling in rat with acute myocardial infarction Bin Li, Yu-Hua Liao *, Xiang Cheng, Hongxia Ge, Heping Guo, Min Wang Laboratory of Cardiovascular Immunology, Institute of Cardiology, Union Hospital, Tongji Medical College of Huazhong University of Science and Technology, Wuhan 430022, China Received 16 September 2004; received in revised form 15 May 2005; accepted 19 August 2005 Available online 23 November 2005
Abstract Objective: A number of observations suggest that cytokines may be important modulators in the ventricular remodeling process. It is unclear whether carvedilol modulates myocardial pro-inflammatory and anti-inflammatory cytokines expression. We hypothesized that carvedilol could improve ventricular remodeling partly through the modulation of cytokines. The goal of this study was to evaluate the effects of carvedilol on cardiac cytokines expression as well as on myocardial and extracellular matrix remodeling in rats with acute myocardial infarction. Methods: Rats with AMI induced by left anterior descending branch ligation were randomized to carvedilol and control group which were further compared to sham-operated group. We studied the effects of 4-weeks therapy with carvedilol starting 24 h after infarction on 1) hemodynamics, 2) tissue weights, 3) myocardial cytokines (TNF-a, IL-1h, IL-6, IL-10 and TGF-h1) expression by semi-quantitative RTPCR and immunoblotting, 4) matrix metalloproteinases activity by gelatin zymography, 5) collagen expression by immunohistochemistry, 6) myocardium fetal gene (a and h myosin heavy chain) expression. Results: Treatment with carvedilol 1) reduced the pro-inflammatory cytokines and fibrogenic cytokine TGF-h1 levels in myocardium and was associated with the amelioration of the elevated left ventricular diastolic pressure. 2) increased anti-inflammatory cytokine, IL-10 protein expression. 3) reduced matrix metalloproteinases-2 and matrix metalloproteinases-9 activity 4) reduced myocardial collagens 5) did not modify fetal gene re-expression. Conclusion: Pro-inflammatory, anti-inflammatory and fibrogenic cytokines are all involved in the process of post-infarction myocardial remodeling. One mechanism underlying the beneficial effects of carvedilol on post-infarction myocardial remodeling may be modulation of the balance between pro- and anti-inflammatory cytokines as well as fibrogenic cytokines and extracellular matrix (ECM) remodeling. D 2005 Elsevier Ireland Ltd. All rights reserved. Keywords: Cytokines; Myocardial infarction; Remodeling; Matrix metalloproteinase; Fibrosis
1. Introduction Ventricular remodeling is the basic mechanism of heart failure following acute myocardial infarction(MI). It contributes significantly to ventricular dilation and dysfunction, disability and death. Ventricular remodeling is a complex dynamic process by which ventricular size, shape, and function are regulated by mechanics, neurohormonal
* Corresponding author. Tel.: +86 27 85726376; fax: +86 27 85727140. E-mail address: [email protected] (Y.-H. Liao). 0167-5273/$ - see front matter D 2005 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.ijcard.2005.08.065
mechanisms, cytokines, oxidative stress and genetic factors [1]. The process of remodeling includes surviving myocyte hypertrophy and extracellular matrix remodeling. Evidences have shown that several pro-inflammatory cytokines, such as tumor necrosis factor-a (TNF-a), interleukin-1h (IL-1h) and interleukin-6 (IL-6), are involved in the remodeling process. On the other hand, anti-inflammatory cytokine such as interleukin-10 (IL-10) may neutralize the effect of pro-inflammatory cytokines [2,3]. In addition, myocardial matrix remodeling has been proposed to participate in the development of ventricular dilation and heart failure. Matrix metalloproteinases have been found to
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play a significant role in the development of myocardial remodeling and congestive heart failure. Tissue inhibitors of matrix metalloproteinases regulate the expression and activity of matrix metalloproteinases. Tissue inhibitors of matrix metalloproteinases are endogenous physiological inhibitors of matrix metalloproteinases and their concomitant downregulation in heart failure suggests the critical balance that exists between matrix metalloproteinases and tissue inhibitors of matrix metalloproteinases in the maintenance of a normal myocardial interstitial homeostasis. Several cytokines modulate the synthesis and secretion of prosoma of matrix metalloproteinases and tissue inhibitors of matrix metalloproteinases [4]. Numerous clinical trials have documented the beneficial effects of beta-blockers on left ventricular performance and mortality in patients with heart failure. Carvedilol, a nonselective beta-adrenergic blocker with alpha1-blocking and antioxidant properties, prevents the progression of heart failure leading to improvement in left ventricular function, reduction in heart size, and improved survival in patients with New York Heart Association functional Class II and III symptoms [5]. But the effect of it on cytokines, extracellular matrix (ECM) expression and fetal gene re-expression in myocardial remodeling is unclear. We hypothesized that carvedilol could lead to improved post-MI remodeling which is associated with the modulation of myocardial cytokines and extracellular matrix. So in this study, we observed the effect of carvedilol on hemodynamics, total cardiac collagen, type I and III collagens, alpha- and beta-myosin heavy chain (MHC) and cytokines (TNF-a, IL-1h, IL-6, IL-10 and TGF-h1) expression and the activity of matrix metalloproteinases.
2. Methods 2.1. Experimental infarction Myocardial infarction was induced in 126 male Wistar rats (8-week-old ) weighting 200 to 250 g (mean 222.5 T 14.8 g) through ligation of the left anterior descending coronary artery as previously described [6]. A 40% mortality rate was observed within 24 h of this procedure. The sham ligation group underwent a similar procedure except that the suture was not tightened around the coronary artery. All experimental procedures and protocols used in this investigation were reviewed and approved by the Institutional Authority for Laboratory Animal Care. 2.2. Drug administration 24 h after coronary ligation, the surviving rats with MI were randomized into two groups. The first group received carvedilol 10 mg/kg bid (MI-C group, n = 34). The second group received vehicle (soya bean oil) (MI group, n = 32). The sham-ligated rats also received vehicle as a control (SH
group, n = 16). Carvedilol (Dilatrend, provided by Roche Incorporation) was dissolved in soya bean oil. All drugs were given by gavage. The treatment was continued for 28 days. All rats were housed under identical conditions in a 12-h light/dark cycle and given food and water. 2.3. Hemodynamic measurements At the end of the fourth week following coronary ligation, after the body weights of each of the rats were obtained, the rats were anesthetized with sodium pentobarbital (30¨40 mg/kg, i.p.). The left ventricular pressures were measured via a saline-filled cannula, which was inserted through the right carotid artery and connected to a pressure transducer (Powerlab/4SP ML750, AD Instrument).The cannula was inserted into the left ventricle to monitor the left ventricular systolic pressure (LVSP), left ventricular end-diastolic pressure (LVEDP) and left ventricular +dp / dt (maximum rate of pressure rise). 2.4. Cardiac weights and sample obtainment After completing the cardiac hemodymanic measurements, all the rats had their hearts stopped in diastole by an intravenous injection of a 10% potassium chloride sodium 2¨3 ml. Then the hearts were obtained isolated from atrials and aortas. The right ventricles were separated from the hearts by the septums. After the left ventricular weights (LVW) and right ventricular weights (RVW) were determined by electronic balance, the vertical slices of the left ventricles of subdivision of rats were made along the left ventricular long-axis for morphological and immunohistochemical analyses. The myocardium of the rest rats were immediately frozen in liquid nitrogen and stored at 70 -C for biochemical and molecular biologic measurements. The pale and thin zones of ventricles represent infarcted myocardium, the zones on the opposite side of infarcted zones represent non-infarcted zones (far from infarcted zones). 2.5. RNA isolation and semi-quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) analysis of cytokines, a-and b-myosin heavy chains mRNA expression Total RNA was isolated from non-infarcted myocardium by acid guanidium thiocyanate-phenol-chloroform extraction. The RNA, stored at 70 -C before use, was quantified by measuring the absorption at 260 nm. The RT-PCR experiments were performed on total ribonucleic acid based on a method published by Dukas et al. [7]. The following oligonucleotides were used as rat sense and antisense primers, respectively. TNF-a (691 bp):5V-ATG AGC ACG GAA AGC ATG ATC CGA-3V; and 5V-CCA AAG TAG ACC TGC CCG GAC TC-3V; IL-1h(560 bp): 5VATG GCA ACT GTC CCT GAA CTC AAC T-3V; and 5VCAG GAC AGG TAT AGA TTC AAC CCC TT-3V; IL-6
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(660 bp) : 5V-CCA GTT GCC TTC TTG GGA CTG ATG3V; and 5V-ATT TTC TGA CCA CAG TGA GGA ATG-3V; IL-10 (346 bp) : 5V-TGC CTT CAG TCA AGT GAA GACT-3V; and 5V-AAA CTC ATT CAT GGC CTT GTA-3V ;TGFh1 (396 bp) : 5V-GCC TCC GCA TCC CAC CTT TG-3V; and 5V-GCG GGT GAC TTC TTT GGC GT-3V;amyosin heavy chain (81 bp) :5V-CAG AAA ATG CAC GAT GAG GA-3; and 5V-TCA AGC ATT CAT ATT TAT TGT GGG-3V; h-myosin heavy chain (100 bp): 5V-GGG CCT GAA TGA AGA GTA GAT-3V; and 5V-GTG TTT CTG CCT AAG GTG CT-3V; the housekeeping gene hactin (249 bp) was used as internal control. The RT-PCR products were visualized on 1.5% or 3% agarose gels electrophoresed in 1 TAE buffer containing 0.5 ug ml 1 ethidium bromide. 2.6. Immunoblot of TNF-a, IL-1b, IL-6, IL-10 and TGF-b1 protein The total cell plasma protein of myocardium in noninfarcted zones were extracted by 50 mmol/L Tris – HCl (pH 7.4). Samples containing 30 Ag of total protein were electrophoresed on 15% polyacrylamide gels (Bio-Rad) for 1 h (4 -C, constant current for 80 mA). The separated proteins were electrophoretically transferred onto a nitrocellulose membrane (Invitrogen, USA). The transfer time respectively was: TNF-a, 28 min; IL-1h, 28 min; IL-6, 30 min; IL-10, 35 min; TGF-h1, 30 min. Then blocked with 5% defatted milk for 2 h at 37 -C. The cytokines protein expression were detected by using a 1 : 500 dilution of a goat anti-rat TNF-a, IL-6, IL-10, TGF-h1 polyclonal antibody (RandD Systems, Minneapolis, Minnesota) and 1 : 2000 dilution of a mouse anti-rat IL-1h monoclonal antibody (Serotec Ltd, Kidlington, Oxford, UK) as the first antibody, respectively. A 1 : 5000 dilution of horseradish peroxidase-conjugated rabbit anti-goat and rabbit antimouse IgG (Santa Cruz, Santa Cruz Biotechnology Inc., California) were then used as the second antibody, respectively and developed with the enhanced chemiluminescence Western blotting detection system (Pierce Company Product). 2.7. Immunohistochemistry for TNF-a and collagens The left ventricles used for immunohistochemistry were fixed in 10% formalin phosphate buffer for 24 h, and then embedded in paraffin. Expression of TNF-a and type I and III collagen in cardiac tissues was determined on two of the cardiac cross-sections (6 Am). Two cross-sections were obtained on microtome between the base and the apex of the LV. Sections were respectively exposed to 1 : 100 dilution goat anti-rat polyclonal TNF-a, type I and III collagen antibodies. A 1 : 500 dilution of biotinylated rabbit anti-goat IgG as the second antibody, respectively. Then after incubation with avidin – biotin – horseradish peroxidase complexes, peroxidase was visualized followed by
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incubation with diaminobenzidine enhancing solution. Positive TNF-a, type I and III collagen expression were confirmed by a specific brown staining of the cells. Each section was analyzed by HMIAS Series Color Medical Image Analyze System and IBAS computerized system (Champion Image Ltd., China). 2.8. Gelatin zymography for matrix metalloproteinase-2 and matrix metalloproteinase-9 activity assay The total protein of non-infarcted myocardium was extracted by 50 mmol/L Tris – HCL but proteinase inhibitors such as ethylenediaminetetra –acetic acid and phenylmethylsulfonyl fluoride, that might inhibit the activity of matrix metalloproteinases, were not used. The media supernatants were treated with sodium dodecyl sulfatepolyacrylamide gel (SDS-PAGE) sample buffer without boiling. The levels of protein were determined by the Bradford methods. The volume was adjusted to contain the same quantity of protein (5 Ag). Samples containing 30 Ag of total protein were electrophoresed on 7.5% polyacrylamide gels containing gelatin (1 mgIml 1) by electrophoresis at 40 mA for 1 h at 4 -C. The gels were soaked in 2.5% Trixton-100 for 30 min 2 times at room temperature to remove the SDS, and incubated in a digestion buffer (50 mmol Tris –HCl, pH 7.6, 5 mmol CaCl2, 100 mmol NaCl, 2 Amol ZnCl2, and 0.01% Brij-35) at 37 -C staying overnight for more than 24 h to allow proteinase digestion of its substrate. Gels were rinsed again in distilled water, stained with 0.25% Coomassie brilliant blue R-250 for 2 h, and destained with 10% acetic acid. Gelatinolytic activity appeared as clear bands of a digested gelatin against a dark blue background of stained gelatin. The activity of matrix metalloproteinase-2 and matrix metalloproteinase-9 were determined according to the signal of the bands by the Table 1 Body weight, heart weight and hemodynamic measurements x¯ T s SH group (n = 10)
MI group (n = 12)
MI-C group (n = 11)
Body weights (g) Experiment before 221 T16.6 220 T 15.5 221 T14.5 4-week end 217 T 12.3# 219 T 14.2# 219 T 11.1# LVW / BW (mg/g) 2.19 T 0.19 2.56 T 0.20** 2.37 T 0.14g RVW / BW (mg/g) 0.53 T 0.02 1.02 T 0.03** 0.99 T 0.03g HR (bpm) 424.1 T15.18 433.2 T 14.89 387.7 T 14.11gg LVEDP (mmHg) 6.43 T 0.70 18.6 T 2.71** 10.19 T 2.8gg LVSP (mmHg) 138.48 T 5.86 119.03 T 6.28** 114.69 T 7.47 +dp / dt max (mmHg/s) 2523.2 T 186.5 1821.6T265.3** 2374.6 T 166.7gg Compared with experiment before, #P > 0.05. Compared with SH group, **P < 0.01. Compared with MI group, gP < 0.05, ggP < 0.01. BW = body weight; LVW = left ventricular weight; RVW = right ventricular weight; MI = myocardial infarction; MI-C = myocardial infarction treated with carvedilol; SH = sham-operated group; HR = heart rate; LVEDP= left ventricular end-diastolic pressure; LVSP= left ventricular systolic pressure; LV + dp / dt max = left ventricular rate of pressure development.
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GSD8000 Density Scan Analysis System (UVP Ltd. Britain) reading the area and density of bands. The activity of matrix metalloproteinases is equivalent to an area of band (the density of band the density of background). 2.9. Cardiac fibrosis assessment This procedure consisted of using cardiac tissue sections stained with picric acid – fuchsin acid, resulting in cardiac myocytes being stained yellow and collagen fibers stained red in colour. Collagen volume density fraction (CVF) was then determined by measuring the area as a proportion of the total area under observation. The collagen-rich border zone of vessels and the scar were excluded in the calculations. Ten fields were analyzed in the subendocardial layer and ten fields in the subepicardial layer in each LV.
2.10. Statistical analysis All values are expressed as mean T standard deviation. Results were analyzed by using analysis of variance (ANOVA) for multiple comparisons followed by a twosided Dunnett’s test or Student – Newman– Keuls test, when appropriate. Gels were analyzed by densitometry and the results were presented as mean (arbitrary units) T SEM. Statistical significance was assumed at p < 0.05.
3. Results 3.1. Hemodynamics, body weight and ventricular weight Before and after gavage for four weeks, there were no obvious changes of body weights of the three groups
Fig. 1. Cardiac morphology and collagens in SH, MI and MI-C group, respectively. Row A shows cardiac morphologic changes. Row B shows total collagen stained with picric acid-fuchsin acid (200) in non-infarcted region of left ventricle. The red represents total collagen. The yellow represents myocardium. Row C and D show localization of type I and III collagen immunohistochemical analysis in boundary zone, respectively (200). MI group have significant positive staining. MI-C group have weaker positive staining and SH group have very weak positive staining for type I and negative staining for type III collagen. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
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(see Table 1).It suggested that carvedilol had not caused obvious changes in body weights of rats. Compared with the sham-operated group, the ratio of left ventricular weight/body weight, right ventricular weight/body weight and left ventricular end-diastolic pressure(LVEDP) were significantly elevated whereas the left ventricular systolic pressure (LVSP) and left ventricular maximum rate of pressure rise (LV + dp / dt max) were
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obviously reduced in MI group rats at the end of the fourth week ( P < 0.01). After carvedilol treatment for 4 weeks, the ratio of left ventricular weight / body weight, right ventricular weight / body weight, heart rate and left ventricular end-diastolic pressure were significantly decreased, while the left ventricular maximum rate of pressure rise (LV + dp / dt max) was obviously increased (see Table 1).
Fig. 2. Cardiac cytokines mRNA expression (A – E) evaluated by semi-quantitative RT-PCR. *Compared with sham-operated group, P < 0.01. DCompared with MI group, P < 0.01. TNF = tumor necrosis factor; IL= interleukin; TGF = transforming growth factor.
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3.2. Cardiac morphology and collagens Compared with sham-operated group, left ventricular chamber enlarged and wall thinning in MI group, and total collagen volume fraction (CVF) increased significantly (5.87 T 0.49 vs. 2.81 T 0.20, respectively, P < 0.01). Compared to the MI group, carvedilol decreased the enlarged ventricular chamber and slightly increased the thickness of scar, and reduced the total collagen volume fraction (3.24 T 0.33 vs. 5.87 T 0.49, respectively, P < 0.01) (Fig. 1 row B) (Fig. 1 row A and row B). On immunohistochemical staining, both type I and III collagen could be found in myocardium of MI group. Carvedilol reduced both type I and III collagen immunoreactivity (Fig. 1 row C and D). 3.3. Cardiac myosin heavy chain mRNA expression a- and h-myosin heavy chain mRNA in non-infarcted zone were elevated compared with the sham-operated group at the end of the fourth week after MI. But no obvious changes of a- and h-myosin heavy chain mRNA expression were found between MI treated with carvedilol and that with vehicle (data not shown). 3.4. Cardiac cytokines gene expression Compared to the sham-operated group, TNF-a, IL-1h, IL-6, IL-10, and TGF-h1 mRNA in non-infarcted zone were all elevated at the end of the fourth week after MI. Four weeks treatment with carvedilol reduced the elevated TNF-a, IL-1h, IL-6 and TGF-h1 mRNA expression but had no obvious effect on IL-10 mRNA expression. Especially, IL-6 and IL-1h mRNA expression in noninfarcted zone were reduced near to those in shamoperated group (Fig. 2A –E). 3.5. Location of TNF-a protein By immunohistochemical staining, we found TNF-a immunoreactivity predominated in surviving cardiomyocytes except in the infiltrating inflammatory cells of the infarct zone and cardiomyocytes in non-infarct zone (Fig. 3A – D). Carvedilol decreased cardiomyocyte positive staining compared with MI group not treated with carvedilol, but had no effect on infiltrating inflammatory cells (Fig. 3E –F). 3.6. Cardiac cytokines protein expression The protein of cytokines (TNF-a, IL-1h, TGF-h1, IL-6) expression in non-infarcted zone were also elevated in MI group (13.49 T 1.44, 13.9 T 1.65, 18.09 T 0.98 and 13.26 T 0.78, respectively) compared with sham-operated group (2.89 T 0.74, 2.43 T 0.6, 5.27 T 0.37 and 3.1 T 0.69, respectively, P < 0.01 for all) at the end of the fourth week
Fig. 3. Immuntostaining of TNF-a. Magnification: 400. (A) Positive staining in surviving cardiomyocytes in the infarct zone. (B) Positive staining in infiltrating inflammatory cells in the infarct zone. (C) Positive staining in remote cardiomyocytes in non-infarct zone. (D) Negative staining in sham-operated group. (E) Positive staining in myocardium of MI group (200). (F) Positive staining in myocardium of MI-C group (200).
after MI. Treatment with carvedilol reduced TNF-a, IL-1h, TGF-h1 (8.91 T 0.28, 10.02 T 1.19, 13.01 T 0.78, respectively, P < 0.01 for all) and IL-6 (11.67 T 1.48, P = 0.018) protein expression which coincided with the change of gene expression. Particularly, carvedilol increased cardiac IL-10 protein expression from 13.81 T 0.82 to 15.19 T 1.29 ( P = 0.022) different from the result of gene expression, thereby reducing the ratio of TNF-a/IL-10 from 0.98 T 0.11 to 0.59 T 0.16 ( P < 0.01) (Fig. 4). 3.7. Cardiac matrix metalloproteinase-2 and matrix metalloproteinase-9 activity Gelatin zymography (Fig. 5) showed that treatment with carvedilol reduced the elevated matrix metalloproteinase-2 and matrix metalloproteinase-9 activity compared to those in MI group. Again, matrix metalloproteinases activity assay results confirmed the findings from zymography. As shown in the bar graphs, matrix metalloproteinase-2 and matrix metalloproteinase9 activity in MI group were 16.67 T 2.24 and 14.66 T 1.02, respectively, which were significantly higher than the sham-operated group (4.09 T 0.33 and 5.13 T 1.29, respectively, P < 0.01 for both). After carvedilol therapy for four weeks, matrix metalloproteinase-2 and matrix metalloproteinase-9 activity were reduced to 5.05 T 0.98
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Fig. 4. Western blot shows 30 Ag of total protein from concentrated supernatant were subjected to gel electrophoresis and immunoblotting. The six bar graphs showed that the level of five cytokines protein expression and TNF-a / IL-10 ratio in non-infarcted myocardium in the three groups, respectively. *Compared with SH group, P < 0.01. DCompared with MI group, P < 0.01.
and 7.73 T 0.93, respectively, which were significantly lower than MI group ( P < 0.01 for both).
4. Discussion This study indicates that carvedilol, when started 24 h after MI, markedly ameliorates cardiac hemodynamic index and ventricular weights. This was accompanied by the reduction in the mRNA and protein expression of proinflammatory cytokines (including TNF-a, IL-1h and IL-6) and fibrogenic cytokine (TGF-h1), and the increase in antiinflammatory cytokine, IL-10 protein expression. This did not appear to be the result of prevention of cardiac fetal gene expression but instead, might have been related to reduced myocardial matrix metalloproteinase-2 and matrix metal-
loproteinase-9 activity, and reduced cardiac type I and III collagens. Taken together, these results suggest that a range of cytokines including pro-inflammatory, anti-inflammatory, pro-fibrosis cytokines and the imbalance in pro-inflammatory and anti-inflammatory cytokines participate in the process of ventricular remodeling after MI, and that carvedilol can improve extracellular matrix (ECM) remodeling and modulate cardiac cytokines expression. In the recent years, more and more evidences have suggested that inflammatory cytokines are involved in myocardial remodeling. The cytokine, tumor necrosis factor-a, has been causally linked to left ventricular (LV) remodeling. Matrix metalloproteinases (MMPs) have been implicated in cardiac remodeling and can be regulated by TNF-a [8]. Dysregulation of the myocardial extracellular matrix contributes to abnormal cardiac
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Fig. 5. Zymogram for evaluating the differences of myocardium MMPs activity among the MI, MI-C and SH group rats. The bar graphs showed the results of MMP-2 and MMP-9 activity assay. *Compared with shamoperated group, P < 0.01. DCompared with MI group, P < 0.01. MW = molecular weight. MMP= matrix metalloproteinase. MI = myocardial infarction. MI-C = myocardial infarction treated with carvedilol. SH = sham-operated group.
muscle function. Changes in the balance between matrix deposition and matrix degradation by matrix metalloproteinases (MMPs) can lead to cardiac fibrosis and dilation. Inflammatory cytokines such as TNF-a and IL-1h can regulate this process [9]. In contrast, IL-10 is a pleiotropic cytokine with potent anti-inflammatory effects in many cells lines. Selzman et al. has shown that IL-10 could be used therapeutically to influence vascular remodeling by inhibiting TNF-induced vessel smooth muscle cell proliferation [10]. In addition, IL-10 downregulates the production of metalloproteinases (MMPs) and upregulates that of their tissue inhibitors (TIMPs) thereby modulating extracellular matrix remodeling. Clinical trials have shown significantly elevated levels of IL-10 in patients with acute myocardial infarction [11]. Accordingly, the imbalance of the pro-inflammatory and anti-inflammatory cytokines maybe related to the ventricular remodeling process. TGF-h1 certainly has also been proven to participate in remodeling mechanism of ventricles. Activated TGF-h1 mRNA expression is accompanied by the appearance of myocardial fibroblast and the expression of fibrillar collagens and tissue inhibitors of matrix metalloproteinases, suggesting that this fibrogenic cytokine may contribute to collagen remodeling in the rat heart after myocardial infarction [12]. Carvedilol is a new generation vasodilator which is a nonselective beta-adrenergic antagonist with additional alpha1-blocking, anti-oxidant and anti-proliferating activity that may contribute to the overall clinical benefit seen in the treatment of left ventricular dysfunction after myocardial infarction [5]. Carvedilol has been proved to reduced
myocardial IL-1h expression and myocardial collagen deposition in rats [13,14]. In the present study, carvedilol was given in dose of 10 mg/kg/d for four weeks and was demonstrated to decrease left ventricular end-diastole pressure and heart rate, and increase LV + dp / dt max while having no significant effect on left ventricular systolic pressure. Moreover, carvedilol decreased the left ventricular weight / body weight, right ventricular weight / body weight and the enlargement of the ventricular chamber, and slightly increased the thickness of scar. These results suggested that carvedilol had beneficial effect on cardiac function and cardiac remodeling. First, we found carvedilol reduced cardiac gene expression and protein production of TNF-a, IL-1h, IL-6 and TGF-h1. Carvedilol did not change the gene expression of IL-10 but increased the protein level of it in non-infarcted zone suggesting that carvedilol might affect the IL-10 protein production at post-transcriptional level. Furthermore, we found that carvedilol reduced TNF-a, / IL-10 protein ratio. Thus, carvedilol may affect the ventricular remodeling after MI by modulating the imbalance of proinflammatory and anti-inflammatory cytokines and decreasing the fibrogenic cytokine TGF-h1. Interestingly, except for the infiltrating inflammatory cells, we found significant immunoactivity of TNF-a in surviving cardiomyocytes in infarcted zone and remote myocardium suggesting cardiomyocytes as also being one of the major sources of the cytokines. Carvedilol reduced TNF-a secretion from myocardium but had no significant effect on infiltrating inflammatory cells. This suggested that carvedilol may modulate TNF-a secretion of cardiomyocytes by blocking myocardial a1- or h-adrenergic receptors. Taking account of the obvious change in heart rate but no significant change in left ventricular systolic pressure with the use of carvedilol, we think that carvedilol modulated cytokines expression at least partly by the mechanism of h-adrenergic blockade. Carvedilol might modulate cytokines production via the effect on adrenergic receptors on cardiomyocytes. It was found that h-adrenergic stimulation delayed the activation of cardiac STAT in mice thereby increasing IL-6 gene family expression of cardiac fibroblasts. In addition, the stimulation of norepinephrine induced left ventricular gene expression of IL-1h and IL-6 in rat and can be blocked by carvedilol [15]. It has been demonstrated that carvedilol inhibited cytokine production from various stimuli-activated T cell by specifically downregulating NF-kappa B activity in activated T cells [16]. Second, we found that carvedilol decreased the left ventricular matrix metalloproteinase-2 and matrix metalloproteinase-9 activity as well as total collagens and type I and III collagens. Cytokines have been proved to be the important modulators of matrix metalloproteinases gene expression. The inflammatory cytokines such as TNF-a and IL-1h are involved in ventricular remodeling both directly as well as indirectly through modulation of matrix metalloproteinases gene expression [17 –19]. It has been
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suggested that norepinephrine regulates synthesis of myocardial type I collagen via an indirect effect [20]. Activated TGF-h1 mRNA expression is accompanied by the appearance of myocardial fibroblasts and the expression of fibrillar collagens and tissue inhibitors of matrix metalloproteinases, suggesting that this fibrogenic cytokine may contribute to collagen remodeling in the rat heart after myocardial infarction [21]. So the mechanism of the effects of carvedilol on ventricular matrix metalloproteinases activity and collagens maybe partly related to the inhibition of cardiac inflammatory and fibrogenic cytokines production. But the exact mechanisms that account for the alterations of matrix metalloproteinases and collagens are complex. Unfortunately, we did not find an obvious effect of carvedilol on ventricular fetal gene (a-MHC and h-MHC) re-expression suggesting that fetal gene re-expression was not probably related to h- or a-adrenergic signaling pathways and it did not contribute to improved left ventricular function with the use of carvedilol. In summary, this study indicates that pro-inflammatory, anti-inflammatory and fibrogenic cytokines contribute to the ventricular remodeling after MI in rats. The effect of carvedilol on the modulation of the balance between the pro-inflammatory and anti-inflammatory cytokines and the reduction of fibrogenic cytokine as well as on ventricular matrix metalloproteinases activity and collagens in noninfarcted zone may be one of the salutary mechanisms on ventricular remodeling after myocardial infarction.
Acknowledgements This work was supported in part by the laboratory of pathology, immunology and molecular biology and animal center of Tongji Medical College of Huazhong University of Science and Technology.
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