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European Journal of Heart Failure 2007 9(11):1081-1085; doi:10.1016/j.ejheart.2007.09.004
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© 2007 European Society of Cardiology

Tobacco smoke-induced left ventricular remodelling is not associated with metalloproteinase-2 or -9 activation

Edson Castardeli, Daniella R. Duarte, Marcos F. Minicucci, Paula S. Azevedo, Beatriz B. Matsubara, Luiz S. Matsubara, Álvaro O. Campana, Sergio A.R. Paiva and Leonardo A.M. Zornoff*

Departamento de Clinica Médica, Faculdade de Medicina de Botucatu, UNESP — São Paulo State University Botucatu, Brazil

* Corresponding author. Departamento de Clínica Médica, Faculdade de Medicina de Botucatu, Rubião Júnior s/n, Botucatu, SP, Brazil, CEP: 18618-000. Tel.: +55 14 38222969; fax: +55 14 38222238. E-mail address: lzornoff{at}fmb.unesp.br


   Abstract
Top
 Abstract
1. Introduction
 2. Methods
 3. Results
 4. Discussion
 References
 
Aim: To investigate the role of MMP-2 and MMP-9 in cardiac remodelling induced by tobacco smoke exposure in rats.

Methods: Rats were allocated into two groups: C (n=9): control animals; ETS (n=9): exposed to tobacco smoke. After 4months, the animals underwent echocardiography, morphometric study and determination of MMP-2 and MMP-9 activity.

Results: ETS rats had larger diastolic (C=15.6 ±1.2mm/kg, ETS=18.0±0.9mm/kg; p < 0.001) and systolic (C=7.3±1.2mm/kg, ETS=9.2±0.9mm/kg; p=0.001) ventricular diameters adjusted for body weight. Fractional shortening (C53–4.8%, ETS+48±3.3%; p=0.031) and ejection fraction (C=0.89±0.03, ETS=0.86±0.02; p=0.030) were smaller in the ETS group. Myocyte cross-sectional area (C=245±8µm2, ETS=253±8µm2; p=0.028) was higher in ETS rats. There were no differences in MMP-2 (C=50±14%; ETS=43±11%, p=0.228) or MMP-9 (C=0.36±0.3%; ETS=0.62±0.3%, p=0.630) activity between the groups.

Conclusion: MMP-2 and MMP-9 did not participate in the remodelling process induced by tobacco smoke exposure.

Key Words: Ventricular function • Cardiac remodelling • Ventricular dilatation

Received April 26, 2007; Revised June 28, 2007; Accepted September 13, 2007


   1. Introduction
Top
 Abstract
 1. Introduction
2. Methods
 3. Results
 4. Discussion
 References
 
Heart failure is a leading cause of hospitalisation and death in the developed world [1]. The development of heart failure is often a slow sequence of events. In many cases, there is a precedent index event, for instance myocardial infarction, acute inflammation, or onset of hypertension [2]. In response to the index event, the heart undergoes a process called ventricular remodelling. This process may be defined as changes in size, geometry, shape, composition, and function of the heart after cardiac injury [2-4]. At first, ventricular remodelling is a compensatory process, since the morphological adaptations are key in preserving cardiac function in response to cardiac injury. However, chronic ventricular remodelling is now recognized as an important pathological process, which results in progressive ventricular dysfunction and the development of heart failure or sudden death [2-4].

The family of extracellular matrix degrading enzymes called metalloproteinases (MMPs) are involved in the remodelling process. To date, more than 20 MMPs have been described. Numerous MMPs, including gelatinases (MMP-2 and MMP-9), are associated with adverse remodelling following different types of cardiac injury [5-7].

Several recent studies have shown that exposure to tobacco smoke (ETS) can result in cardiac remodelling and compromised cardiac function [8-10]. However, the mechanisms involved in this phenomenon are not known. Since MMP activation may participate in both vascular and lung damage induced by ETS [11], we hypothesized that cardiac remodelling induced by ETS would be associated with activation of MMP. Therefore, the objective of this study was to investigate the role of MMP-2 and MMP-9 in the cardiac remodelling induced by tobacco smoke exposure in rats.


   2. Methods
Top
 Abstract
 1. Introduction
 2. Methods
3. Results
 4. Discussion
 References
 
2.1. Groups and treatment
All experiments and procedures were performed in accordance with the National Institute of Health's Guide for the Care and Use of Laboratory Animals and were approved by the Animal Ethics Committee of our Institution.

Male Wistar rats weighing 200-250g were allocated into two groups: Group C (n=9): control animals not exposed to tobacco smoke; Group ETS (n=9): rats exposed to tobacco smoke (40 cigarettes/day).

The ETS rats were exposed to cigarette smoke in a chamber (dimensions 95x80x65cm) connected to a smoking device based on a model published by Wang et al. [12] and adapted by Paiva et al. [8]. The smoke was drawn out of filtered commercial cigarettes (composition per unit: 1.1mg of nicotine; 14mg of tar; and 15mg of carbon monoxide) with a vacuum pump and was exhausted into the smoking chamber. During the first week, the number of cigarettes was gradually increased from 5 to 10 cigarettes over a 30-min period, twice in the afternoon. After that, 10 cigarettes were used in each smoking trial, repeated four times/day, twice in the morning and twice in the afternoon.

2.2. Echocardiographic study
After 4months, all animals were weighed and underwent transthoracic echocardiography. A commercially available echocardiography machine (Philips model TDI 5500) equipped with a 12MHz phased array transducer was used. Imaging was performed with a 60° sector angle and a 3cm imaging depth. Rats were lightly anesthetized by intramuscular injection with a mixture of ketamine (50mg/kg) and xylazine (1mg/kg). Then the chest was shaved and the rat was placed in the left lateral position. Two-dimensional targeted M-mode echocardiograms were obtained from short-axis views of the left ventricle (LV) at or just below the tip of the mitral-valve leaflets, and at the level of the aortic valve and left atrium. M-mode images of the LV, left atrium, and aorta were recorded. All measurements were obtained by the same observer, according to the leading-edge method recommended by the American Society of Echocardiography [13]. Measurements represented the mean of at least five consecutive cardiac cycles. LV end-diastolic dimension (LVEDD) and posterior wall thickness (LVWT) were measured at maximal diastolic dimension, and the end-systolic dimension (LVESD) was taken at the maximal anterior motion of the posterior wall. The left atrium (LA) was measured at its maximal diameter and the aorta at the end of diastole. LV systolic function was assessed by calculating fractional shortening {(LVDD– LVSD) / LVDD x 100}, and ejection fraction {(LVDD3 LVSD3) / LVDD3}. The E/A ratio was used as an index of LV diastolic function.

2.3. Morphometric analysis
At completion of the functional study, the right (RV) and left ventricles (including the interventricular septum) were dissected, separated, and weighed.

The morphometric analysis of the myocardium was performed as described previously [14,15]. Transverse 3-mm-thick sections of LV were fixed in 10% buffered formalin and embedded in paraffin. Five-micron-thick sections were stained with hematoxylin-eosin and with the collagen-specific stain picrosirius red (Sirius red F3BA in aqueous saturated picric acid). Myocyte cross-sectional area (CSA) was determined for at least 100 myocytes per slide stained with hematoxylin-eosin. The measurements were performed using a Leica microscope (magnification lens x 400) attached to a video camera and connected to a personal computer equipped with image analyzer software (Image-Pro Plus 3.0, Media Cybernetics, Silver Spring, MD). CSA was measured with a digitizing pad, and the selected cells were transversely cut with the nucleus clearly identified in the centre of the myocyte. Interstitial collagen volume fraction (IC) was determined for the entire picrosirius-red-stained cardiac section using an automated image analyzer (Image-Pro Plus 3.0, Media Cybernetics). Cardiac tissue components were identified according to the colour level: red for collagen fibres, yellow for myocytes, and white for interstitial space. The digitized profiles were sent to a computer that calculated collagen volume fraction as the sum of all connective tissue areas divided by the sum of all connective tissue and myocyte areas. On average, 35 microscopic fields were analyzed with an x 40 lens. Perivascular collagen was excluded from this analysis.

2.4. Metalloproteinase-2 and -9 activity
Metalloproteinase-2 and -9 activity was determined as previously reported by Tyagi et al. [16]. In brief, samples for analysis were prepared by dilution in extraction sample buffer consisting of 50 mM Tris, pH 7.4; 0.2M NaCl; 0.1% Triton-X and 10mM CaCl2. Then they were diluted in application sample buffer consisting of 0.5M Tris, pH 6.8; 100% glycerol, and 0.05% bromophenol blue. The samples were loaded into wells of 8% SDS-polyacrylamide containing 1% gelatin. Electrophoresis was performed in a Bio-Rad apparatus at 80V for 2 h, until the bromophenol blue reached the bottom of the gel. The gel was removed and washed twice with 2.5% Triton-X-100 and then washed with 50 mM Tris pH 8.4. The gel was then incubated at 37°C overnight in activation solution consisting of 50 mM Tris pH 8.4; 5 mM CaCl2 and Zn Cl2. The staining was performed for 2 h with 0.5% comassie blue and destaining in 30% methanol and 10% acetic acid until clear bands over a dark background were observed. Staining and destaining were performed at room temperature on a rotatory shaker. The gels were photographed and the intensity of gelatinolytic action (clear bands) analyzed in UVP,UV, White Darkhon image analyzer.

2.5. Statistical analysis
Comparisons between groups were made by Student's t test for variables with normal distribution. Otherwise, comparisons between groups were made using the Mann-Whitney U test. Data were expressed as mean ± SD or medians (including the lower and upper quartiles). Data analysis was carried out with SigmaStat for Windows v2.03 (SPSS Inc, Chicago, IL). A significance level of 5% was used.


   3. Results
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
4. Discussion
 References
 
Table 1 summarizes the echocardiographic data. ETS rats had higher diastolic (C = 15.6 ± 1.2mm/kg, ETS=18.0±0.9 mm/kg; p < 0.001) and systolic (C = 7.3 ± 1.2mm/kg, ETS = 9.2 ± 0.9mm/kg; p=0.001) ventricular diameters adjusted for body weight, compared to control animals. Fractional shortening (C=53±4.8%, ETS=48±3.3%; p=0.031) and ejection fraction (C=0.89±0.03, ETS=0.86±0.02; p=0.030) were smaller in ETS rats, when compared to controls. LV mass index was higher in ETS when compared to control animals (C=1.25±0.23 g/kg, ETS=1.57±0.21 g/kg; p=0.008). No differences were observed between the groups for LVWT, LVWT/LVEDD, or E/A ratio.


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  		Table 1 Echocardiographic study

 
The morphometric data are shown in Table 2. The myocyte cross-sectional area (C = 245 ± 8 µm2, ETS = 253 ± 8 µm2; p=0.028) was higher in ETS than in control animals. There were no differences in BW, LVW/BW, RVW/BW, or interstitial collagen volume fraction between the groups.


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  		Table 2 Morphological data

 
There were no differences in MMP-2 (C=50±14%; ETS=43±11%, p=0.228) or MMP-9 (C = 0.36 ± 0.3%; ETS = 0.62 ± 0.3%, p=0.630) activity between the groups (Fig. 1).


Figure 01
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  		Fig. 1 Metalloproteinase-2 (MMP-2, black bars) and metalloproteinase-9 (MMP-9, grey bars) activity. ETS: exposure to tobacco smoke. There were no differences between the groups (p>0.05). Data are expressed as mean±SD.

 

   4. Discussion
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
References
 
The objective of this study was to investigate the role of MMP-2 and MMP-9 in the cardiac remodelling induced by tobacco smoke exposure in rats. Our results show that tobacco smoke exposure resulted in morphological and functional abnormalities. However, this remodelling process was not associated with MMP-2 or MMP-9 activation.

One important finding of our study was that ETS induced myocardial hypertrophy and ventricular enlargement, which is in agreement with previous studies [8-10]. It is well established that both hypertrophy and ventricular dilation play a key role in the remodelling process. Therefore, although we did not study all variables involved in the remodelling process, such as cell constitution and tissue structure, our results identified morphological alterations induced by tobacco smoke exposure that characterize left ventricular remodelling.

One of the most striking characteristics of cardiac remodelling is that it invariably results in a progressive decrease in ventricular function. Initially, due to cell growth, the remodelling process may help to maintain or restore cardiac function. Chronically, however, biochemical, genetic, and structural alterations occur, resulting in progressive ventricular dysfunction [2-4]. In agreement with this concept, our study showed that in rats exposed to cigarette smoke, the remodelling process was accompanied by a significant decrease in systolic function, assessed by fractional shortening and ejection fraction. Our study showed no alteration in diastolic function, at least in the variables assessed through transmitral flow.

Cigarette smoking increases the risk of coronary artery disease, cerebrovascular disease and peripheral vascular disease. It is known that chronic smoking causes endothelial dysfunction, increased oxidation of LDL-cholesterol, reduction of HDL-cholesterol blood levels, and increased blood levels of adhesion molecules and fibrinogen, joint factors which may lead to platelet aggregation and eventually to vascular spasm [11,17,18]. However, the mechanisms involved in the morphological and functional cardiac alterations induced by ETS are still incompletely understood.

The extracellular matrix, which is composed of structural elements such as collagen, and other proteins like fibronectin, proteoglycans and matricellular proteins, plays a critical role in the maintenance of ventricular geometry and function. An important component of the extracellular matrix is a family of extracellular matrix degrading enzymes, the MMPs [19]. Recently, a clear relationship between MMPs and remodelling has been demonstrated in animal models. In a paced LV-failure animal model, a time-dependent increase in myocardial MMP levels accompanying the progression of LV dilation and dysfunction has been demonstrated [20]. The same phenomenon has been demonstrated after myocardial infarction [5,19,21]. In spontaneously hypertensive rats, myocardial MMP levels were shown to increase during the transition from compensated LV hypertrophy to decompensated heart failure [22]. In addition, MMP inhibition has been shown to attenuate ventricular remodelling due to different types of injury, such as hypertension and myocardial infarction [22,23].

Therefore, there are now several pieces of evidence to indicate that MMP activation contributes to remodelling secondary to different stimuli.

The noteworthy finding in the present study was that, contrary to our original hypothesis, we found that MMP-2 and MMP-9 were not involved in the ventricular remodelling induced by tobacco smoke exposure. In fact, our study did not detect any differences in MMP-2 and MMP-9 expression between those animals exposed to tobacco smoke and the control animals, although cardiac remodelling was observed in the animals exposed to tobacco smoke. This finding is surprising because MMP-2 and MMP-9 are increased in different models of cardiac injury, such as myocardial infarction, pressure and volume overload, and heart failure induced by pacing [5-7,19-22]. In addition, although fibrosis is considered to be a hallmark modulator of myocardial function, this study did not show collagen accumulation. However, a number of factors may contribute to myocardial dysfunction in the remodelled heart. These include changes in the contractile proteins, alteration in the β-adrenergic pathway, alterations in cytoskeletal proteins, abnormalities in calcium-handling proteins, increased apoptosis, modifications in chamber geometry, metabolic remodelling, and gap junction alterations [24,25]. Therefore, at this point, our data suggest that other mechanisms not related to MMP-2 and MMP-9 activation or fibrosis might be involved in ventricular dysfunction. Importantly, we cannot comment on the participation of other MMPs in ventricular remodelling induced by tobacco smoke exposure.

In conclusion, based on our findings, we assume that MMP-2 and MMP-9 do not participate in the remodelling process induced by tobacco smoke exposure in rats.


   References
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
 References
 

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