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Globally, tobacco smoke exposure remains a significant risk for the development of acute myocardial infarction (MI) irrespective of ethic or gender disparities. Exposure to tobacco smoke increases inflammation, oxidative stress1, 2, 3, 4, 5, 6, 7, 8, 9, 10 and decreases antioxidant expression.11, 12, 13, 14, 15 Direct and indirect studies have demonstrated that oxidative stress from reactive oxygen species (ROS) generated through mitochondria, xanthine oxidase, and the NADPH oxidase system16, 17, 18, 19, 20, 21 is one of the important factors in the pathogenesis of myocardial ischemia. Decreased infarct size in superoxide dismutase (SOD)-producing transgenic mice suggests direct evidence for the role of involvement of ROS in MI.22, 23 In limited investigations, the use of antioxidants such as mercaptopropionyl glycine, edaravone and probucol have demonstrated varied effects on remodeling in animal models of MI,24, 25, 26, 27, 28, 29, 30, 31, 32 with improvement in collagen content and histological changes. In this study, we sought to delineate the role of chronic tobacco smoke leading up to an acute MI by focusing on oxidative stress coupled with inflammation in the genesis of events leading to cardiac dysfunction and histological extent of damage. To further develop therapeutic targets, we tested the efficacy of N-acetyl cysteine (NAC), an antioxidant and anti-inflammatory agent in ameliorating the extent of myocardial damage and also investigated if facilitated repair could be augmented in a rat model of left anterior descending artery ligation-induced MI.

MATERIALS AND METHODS

Exposure of Rats to Cigarette Smoke

This study was conducted according to the Guidelines for Animal Experiments of the NIH. Sprague–Dawley (SD) rats were exposed to cigarette smoke in a specially constructed smoke chamber.1 The rats were restrained in the chambers and ventilated by the smoking machine. In this machine, smoke is sucked from lit cigarettes by syringes and pumped around the chamber. Rats that head to the smog compartment are additively inhaling the smoke from the tubing triggered from automatic controlled smoking generator. The smoking machine is set to inhale and exhale at intervals mimicking human smoking, with each cigarette lasting 8–10 min. Animals were under constant supervision to ensure that the animals were not distressed. The University of Maryland, Baltimore Animal Care and Use Committee approved all procedures. 3R4F research grade cigarettes with a regular amount of nicotine were used in this study (KTRDC Tobacco Biotechnology Group, University of Kentucky, Lexington, KY, USA). Animals were exposed to cigarette smoke 5 days per week for a total of 6 weeks with four cigarettes given intermittently throughout the day. Control rats were also kept in smoking chambers for four times a day, but without cigarette smoke equivalent to the time, the rats termed as tobacco naïve were exposed to cigarette smoke. The efficacy of cigarette smoke exposure was assessed by quantification of circulating levels of cotinine, the metabolite of nicotine using a kit from Calbiotech (Spring Valley, CA, USA).

The Rat Model of MI

Ligation of left descending artery

Model of MI in rats was created as described by us.33 Before the induction of anesthesia, animals were examined to identify any pre-existing conditions that may complicate surgical outcome. Physical examination included visual inspection and measurement of heart rate, respiratory rate, body temperature and weight. MI was performed in SD rats based on sterilized microsurgery under general anesthesia on the water-circulating heating pad, which maintains the warm body temperature. First, the rats were atropinenized by the administration of atropine, intracerebrally (0.04 mg/kg), 15 min in advance of anesthesia. Then, the rats were anesthetized with ketamine/xylazine (100 mg/kg and 10 mg/kg) cocktail injected by peritoneum and followed by non-invasive ventilation (Harvard 683 rodent ventilator) with pure oxygen, and fixed on the surgical table covered by a sterile surgical drape. All instruments were autoclaved before first surgery and were sterilized with a hot bead sterilizer (Fine Science Tools 250 Hot Bead Sterilizer) for subsequent surgeries. The animal's left thorax was incised and the heart was exposed. LAD was confirmed and ligated with 7-0 polypropylene suture at 3 mm underneath the left atrium appendage. No sooner tighten ligation was made, and then infarction size and position were confirmed by visible blanching of the infarct area simultaneously and the elevated S–T segment in ECG to confirm the infarction. The chest was then closed. For sham group, the procedure is the same as above only with the intact LAD artery

Study design

MI in rats was created by ligation of left anterior descending artery and rats without ligation were used as sham; tobacco-naïve sham (n=3), tobacco-exposed sham (n=3), tobacco-naïve MI (n=3) and tobacco-exposed MI (n=4), tobacco-naïve MI plus NAC (n=5) and tobacco-exposed MI plus NAC (n=5) rats were used. Rats were exposed with four cigarettes per day for 6 weeks. Cardiac function was monitored by echocardiography, histological changes by Masson's trichrome staining (MTS) and molecular changes of pro-inflammatory cytokines, tissue repair molecules and markers of oxidative stress was studied by real-time PCR. All the studies were performed in animals killed after 6 weeks.

Treatment strategy

MI rats were treated with NAC (10 mg/kg per day), administered intraperitoneally, beginning the first day of surgery till 6 weeks. The treated animals (n=5, each MI group with or without tobacco exposure) were killed on week 6 after performing detailed echocardiography. Gross pathological analysis of the heart was performed; heart tissue, spleen and serum was saved at −80 °C till analysis. All the experiments were performed blindly based on the ID of each animal.

Echocardiography analysis of sham and MI rats with and without exposure to cigarette smoke exposure

We performed echocardiogram-based morphological and functional assessment of all animals. After 30 days of cigarette smoke exposure, surgery was performed (sham or ligation of LAD). At 1 week after the surgery, transthoracic echocardiography (770 VisualSonics Ultrasound System, 17.5 MHz linear array transducer) was implemented under anesthesia induced by 5% isoflurane and maintained by 2% isoflurane in O2 with activated charcoal absorption filters (VaporGuard). Short- and long-axis 2D M-mode and Doppler data were collected. All static and dynamic images were recorded. The Doppler analyze the LV input and output blood flow spectrum. In addition, all the animals were also scanned randomly 24 h in advance of MI procedure to obtain baseline imaging data. The image of the left ventricle cavity was obtained by positioning the cursor in the M-mode at the papillary muscles, below the mitral valve plane. The heart structures were measured in at least five consecutive heart cycles. Measurement parameters included LV end-diastolic and end-systolic diameter (LVEDD and LVESD) in short- and long axis, respectively, end-diastolic LV anterior wall thickness, posterior wall thickness and lateral wall thickness (only in short axis). Fractional shortening (FS) was calculated as FS (%)=(100)·((LVEDD−LVESD)/LVEDD). Each measurement was repeated three times for statistical analysis to avoid intraobserver errors.

Detection of mRNA by real-time PCR

We performed real-time quantitative RT-PCR using a Bio-Rad iCycler system (Bio-Rad, Hercules, CA, USA) as described earlier.1 RNAs were isolated from cardiac tissues using a kit from Promega (Madison, WI, USA) and reverse-transcribed into cDNAs by using a cDNA synthesis kit from Invitrogen (Carlsbad, CA, USA). The primer sequences for each gene are shown in Table 1. The mRNA level of each sample for each gene was normalized to that of the β-actin mRNA. Relative mRNA level was presented as 2((Ct/β-actin−Ct/gene of interest)).

Table 1 Primer sequences of genes for real-time PCR
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Quantification of Circulating Levels of Cytokines

The Bio-Plex rat cytokine assay was used for simultaneous quantitation of pro-inflammatory cytokines: IL-1α, IL-1β, IL-2, IL-6, IFN-γ and TNF-α according to the recommended procedure. In brief, the premixed standards were reconstituted in 0.5 ml of sample diluent, generating a stock concentration of 10 000 pg/ml for each cytokine as described.1 The standard stock was serially diluted in the same diluent to generate 8 points for the standard curve. Serum samples from rats were diluted 1:10. The assay was performed in a 96-well filtration plate supplied with the assay kit. Premixed beads (50 μl) coated with target capture antibodies were transferred to each well of the filter plate and washed twice with Bio-Plex wash buffer. Premixed standards or samples (50 μl) were added to each well containing washed beads. The plate was shaken for 30 s, and then incubated at room temperature for 30 min with low-speed shaking. After incubation and washing, premixed detection antibodies (50 μl) were added to each well. The incubation was terminated after shaking for 10 min at room temperature. After washing three times, the beads were resuspended in 125 μl of Bio-Plex assay buffer. Beads were read on the Bio-Plex suspension array system, and the data were analyzed using the Bio-Plex Manager™ software with 5PL curve fitting.

Tissue processing, assessment of infarction and fibrosis

Upon removal, part of hearts were immediately suspended in formalin-containing storage vials, a part of cardiac tissue was rapidly frozen in liquid nitrogen and then at −80°C until processing. Two 5-μm sections of the hearts, one near the base and one at mid-ventricle, were stained with hematoxylin and eosin and MTS to facilitate determination of the degree of infarction, myocardial fibrosis and collagen deposition. The rats were killed immediately after 6 weeks of postoperative echocardiography. Hearts were excised with heparin (30 μg/kg, intraperitoneally), weighed and the entire heart from base to apex was cut into 1-mm-thick slices along the short axial plane. The parts of middle capillary muscle were kept in 10% formalin. For histological staining, the slices were further sectioned into 4-μm slides to examine infarct size and its percentile by MTS. Briefly, heart sections were deparaffinized and treated with a gradient (100–70%) of alcohol, washed in distilled water and stained with Weigert's iron hematoxylin solution for 10 min and slides were washed. For the cytoplasm and muscle, the sections were then stained in Biebrich scarlet-acid fuchsin solution for 10–15 min, again washed with distilled water and treated with phosphomolybdic-phosphotungstic acid solution for 10–15 min, at this step, and for collagen staining, slides were transferred to aniline blue solution for 5–10 min and rinsed with water and 1% acetic acid. Slides were dehydrated with 95% alcohol and mounted with resinous mounting medium. The infarct area was measured on short axial section from the section of slice corresponding to the position of middle papillary muscle of LV. Infarct size was calculated using computer-based planimetry software (ImageJ, NIH). Infarction area percentage was defined as the infarcted area divided by whole LV muscular area.

Statistical analysis

Student's t-test was used to assess differences between the group means for FS, LVES, LVED dimensions, gene expression and cytokine levels. The analysis was performed using a statistical software program (GraphPad, San Diego, CA, USA). Results are expressed as the mean±s.e.m. and two-tailed significance determined at the level of P<0.05.

RESULTS

Circulating Levels of Cotinine

The levels of cotinine, expressed as mean±s.e.m. (ng/ml), were 77.2±8.4 and 0.83±0.13 in serum samples from SD rats exposed to cigarette smoke and air, respectively.

Effect of Chronic Tobacco Smoke Exposure on Extent of MI

Cardiac function by echocardiography

Typical M-mode images of parasternal short- and long-axis view at the middle of the papillary muscle level were as follows: in the sham group without exposure to cigarette smoke (Figure 1a) and when exposed to chronic cigarette smoke (Figure 1b). Figure 1a shows normal wall motion in contrast to the hypokinetic motion in Figure 1b. The wall motion in hearts with infarction as shown in Figures 1c and d demonstrates decreased ventricular function. (The Figures also show how the LVEDD and LVESD were measured). A significant difference in calculated FS (%) was seen in sham rats with and without chronic tobacco exposure, and those with MI and chronic tobacco exposure demonstrated the most decrease in ventricular function. Interestingly, the FS (%) was similar in sham with chronic tobacco smoke exposure (Figure 1b) and MI rats without exposure to cigarette smoke (Figure 1c). Comparison of FS (%) in tobacco-naïve sham (n=3), tobacco-naïve MI (n=3), tobacco-exposed sham (n=3) and tobacco-exposed MI (n=4) rats is shown in Figure 2. A significantly decreased FS was observed in tobacco-exposed sham (35.1±1.3%; P<0.001), tobacco-exposed MI (15.4±0.2%; P<0.001) and tobacco-naïve MI (27.4±2.3%; P<0.001) when compared to tobacco-naïve (55.1±3.5%) rats.

Figure 1
figure 1

Typical M-mode images of parasternal short-axis view at the middle of the papillary muscle level: (a) representative tobacco-naïve sham group; (b) representative tobacco-exposed sham group; (c) representative tobacco-naïve myocardial infarction (MI) group; and (d) representative tobacco-exposed MI group. Calculated fractional shortening (%) is also shown for each animal.

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Figure 2
figure 2

Effect of tobacco exposure on cardiac function: fractional shortening (%) in tobacco-naïve sham (n=3) and tobacco-exposed sham (n=3), and tobacco-naïve myocardial infarction (MI) (n=3) and tobacco-exposed MI (n=4) rats, expressed as mean±s.e.m. Statistical analysis is represented as P-values for each group.

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Histological analysis of myocardium

Micrographs of Masson's trichrome-stained myocardial section are shown in Figure 3a. Short-axis MTS (magnification: × 10) images from rats: sham (panel A) tobacco-naïve MI without (panel B) and with NAC treatment (panel C) and MI rats with chronic cigarette smoke without (panel D) and with (panel E) NAC treatment are shown. The tissue with blue staining is the infarcted area. The scar comprises dense collagen that signifies an infarct at the peri-infarction zone, and the collagen inter-digitates with viable myofibers.

Figure 3
figure 3figure 3

(a) Comparison of histology between the tobacco-naïve myocardial infarction (MI), tobacco-exposed MI and tobacco-exposed rats treated with N-acetyl cysteine: short-axis Masson's trichrome staining (MTS; magnification: × 10) images; the tissue with blue staining is the infarcted area, which is shown for a representative rat from each group. (b) Comparison of histology at high power of magnification between the tobacco-naïve MI, tobacco-exposed MI and tobacco-exposed rats treated with N-acetyl cysteine: short-axis MTS (magnification: × 60) images; the tissue with blue staining is the infarcted area, which is shown for a representative rat from each group; panel A, representative tobacco-naïve sham group; panel B, representative tobacco-exposed sham group; panel C, representative tobacco-naïve MI group; and panel D, representative tobacco-exposed MI group. (c) Mean±s.e.m. of percent infarct size for each group is also shown. *P=0.004, **P=0.0008 and ***P=0.0001.

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Histopathological analysis at × 60 is shown in Figure 3b. There was no significant fibrosis in tobacco-naïve sham, and development of fibrosis in tobacco-exposed sham was seen (blue stain in panel A). The photomicrographs of Masson's trichrome-stained heart sections in the infarct and peri-infarct regions from the tobacco-naïve MI rats group show comparatively less fibrosis (blue stain panel B) in the infarct and peri-infarct zone compared with tobacco-exposed MI (panel D), and marked attenuation of fibrosis in NAC-treated rats tobacco-naïve MI (panel C) and tobacco-exposed MI (panel E).

Quantification of infarct size is also shown in Figure 3c. Infract size in sham-operated rats was 0.6±0.04 and significantly increased infarct size in cigarette smoke-exposed rats with MI was observed compared to tobacco-naïve rats (45.7±2.1 vs 37.6±0.56; P=0.004). NAC treatment of tobacco-exposed rats with MI resulted in a significant improvement in myocardial infarct size (45.7±2.1 vs 25.4±2.5; P=0.0008). Similarly, NAC treatment resulted in a significant decrease in infarct size (37.6±0.56 vs 16.8±0.86. P=0.0001) in tobacco-naïve rats. The results from each group of animals studied for weeks 1–6 are shown in Table 2.

Table 2 Sequential changes of fractional shortening in different groups studieda
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Effect of the antioxidant NAC on tobacco smoke-related cardiac structure and function

Post-MI, rats were treated with NAC (10 mg/kg per day intraperitoneally) till 6 weeks. Echocardiography analysis was performed each week. A representative echocardiogram without and with NAC treatment is shown in Figures 4a and b. Compared to the first week, the FS (%) (15.4±0.2) increased significantly (P<0.001) at 6 weeks (Figure 4b). A significantly increased LVESD compared to tobacco-naïve (3.45±0.33) rats was observed in tobacco-exposed sham (4.92±0.41; P<0.05), tobacco-exposed MI (8.24±0.3; P<0.01) and tobacco-naïve MI (6.1±0.49; P<0.01) rats. Treatment with NAC resulted in a significant decrease in LVESD (6.76±0.48; P<0.02) compared to untreated group (8.24±0.3). However, we did not observe any differences for LVEDD with or without either tobacco exposure or NAC treatment.

Figure 4
figure 4

(a) Comparison of echocardiography analysis of tobacco-exposed myocardial infarction (MI) rats with and without N-acetyl cysteine: typical M-mode images of parasternal short-axis view at the middle of the papillary muscle level, which are shown for representative tobacco-exposed MI rat without and with N-acetyl cysteine. Individual fractional shortening (%) for each rat is shown. (b) Mean±s.e.m. of fraction shortening (%) for tobacco-exposed MI rats and tobacco-exposed MI rats with N-acetyl cysteine is also shown.

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mRNA expression of inflammation, tissue repair and oxidative stress pathways and modulation by NAC

We demonstrate evidence of modulation of mRNA expression of inflammation, oxidative stress, tissue repair and antioxidant genes with and without NAC (Figures 5a, the mRNA expression was significantly higher in tobacco-exposed MI rats compared to tobacco-naïve MI rats for IL-6 (P=0.001) and TNF-α (P=0.001), IFN-γ (P=0.001) and p22phox (P=0.001)). Conversely, expression of SOD was significantly lower in tobacco-exposed MI rats compared to tobacco-naïve MI (P=0.001). However, there was no detectable thioredoxin (TXN) mRNA in either tobacco-exposed or -naïve MI rats. The expression of nuclear factor-E2-related factor 2 (Nrf2) mRNA increased significantly in tobacco-exposed MI compared to tobacco-naïve MI (P=0.001) rats; Kelch ECH-associating protein 1 (Keap-1) followed a similar trend (P=0.05) (Figure 5a). Similar results were obtained with mRNA expression of MMP-2 (P=0.01), stromal-derived factor-1 (SDF-1) (P=0.01), tumor growth factor (TGF)-β (P=0.001) and poly-(ADP-ribose) polymerase (PARP) that occurs in response to DNA damage (P=0.001) (Figure 5b). The mRNA expression for IL-6 (P=0.001), TNF-α (P=0.001), IFN-γ (P=0.001), p22phox (P=0.001), Nrf2 (P=0.001) and Keap-1 (P=0.001) in NAC-treated rats decreased significantly compared to tobacco-exposed MI rats (Figure 5b). Similar decrease in mRNA expression for MMP-2 (P=0.001), SDF-1 (P=0.001), TGF-β (P=0.001) and PARP (P=0.001) was observed in NAC-treated rats. In contrast, mRNA expression for antioxidants SOD (P=0.001) and TXN (P=0.0001) increased significantly in response to NAC.

Figure 5
figure 5

Effect of N-acetyl cysteine on tobacco-induced intracardiac mRNA expression. (a) Pro-inflammatory cytokines: the mRNA expression of interleukin (IL)-6, tumor necrosis factor (TNF)-α and interferon (IFN)-γ in cardiac tissues from tobacco-naïve sham (n=3), tobacco-exposed sham (n=3), tobacco-naïve myocardial infarction (MI) (n=3) and tobacco-exposed MI rats (n=4) was studied by real-time polymerase chain reaction (PCR). The results are presented as normalized fold expression (mean±s.e.m., n=3) using β-actin as a reference gene. *P=0.001 for TNF-α, IL-6 and IFN-γ mRNA (tobacco-naïve vs tobacco-exposed rats with MI); **P=0.001 for TNF-α, IL-6 and IFN-γ mRNA (tobacco-exposed MI vs and tobacco-exposed MI rats vs N-acetyl cysteine-treated tobacco-exposed MI rats). Oxidative stress molecules: *P=0.001, increased p22phox and decreased superoxide dismutase (SOD) mRNA; **P<0.001, decreased thioredoxin (TXN) mRNA (tobacco-naïve vs and tobacco-exposed rats with MI); *P=0.001, increased SOD mRNA, P=0.0001, increased TXN mRNA (tobacco-exposed MI vs and tobacco-exposed MI rats vs N-acetyl cysteine-treated tobacco-exposed MI rats), p22phox mRNA was not detectable. $P=0.05 for Kelch ECH-associating protein 1 (Keap-1) mRNA (tobacco-naïve vs and tobacco-exposed rats with MI); **P=0.001 for nuclear factor-E2-related factor 2 (Nrf2) mRNA (tobacco-naïve vs and tobacco-exposed rats with MI); **P=0.001 for Nrf2-2 and Keap-1 mRNA (tobacco-exposed MI vs and tobacco-exposed MI rats vs N-acetyl cysteine-treated tobacco-exposed MI rats). (b) Tissue repair molecules: $P=0.05 for MMP-2 mRNA (tobacco-naïve vs tobacco-exposed rats with MI); #P=0.01 for stromal-derived factor-1 (SDF-1) mRNA (tobacco-naïve vs and tobacco-exposed rats with MI); **P=0.001 for MMP-2 and SDF-1 mRNA (tobacco-exposed MI vs tobacco-exposed MI rats vs N-acetyl cysteine-treated tobacco-exposed MI rats); *P=0.001, increased tumor growth factor (TGF)-β and poly-(ADP-ribose) polymerase (PARP) mRNA (tobacco-naïve vs tobacco-exposed MI rats); **P=0.001, decreased TGF-β and PARP mRNA (tobacco-exposed MI vs and tobacco-exposed MI rats vs N-acetyl cysteine-treated tobacco-exposed MI rats).

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Effect of NAC on circulating glutathione and pro-inflammatory cytokine levels

Glutathione levels: NAC is known to rapidly metabolize to intracellular glutathione, which acts as an antioxidant in the body. Therefore, we quantified serum levels of glutathione. There was a significant decrease in serum levels of glutathione in tobacco-exposed sham compared to tobacco-naïve sham rats (11.0±0.37 vs 8.9±0.7; P=0.04), and tobacco-exposed MI vs tobacco-naïve MI rats (8.0±0.2 vs 7.0±0.12; P=0.009). However, treatment of tobacco-exposed MI rats with NAC resulted in significantly increased levels of glutathione (7±0.12 vs 10±0.18; P=<0.001), the levels being almost identical to the tobacco-naïve sham rats (Figure 6).

Figure 6
figure 6

Effect of N-acetyl cysteine on glutathione levels. We quantified serum levels of glutathione using a luciferase activity based kit from Promega in serum samples from tobacco-exposed sham, tobacco-naïve sham, tobacco-exposed myocardial infarction (MI) rats and tobacco-naïve MI rats. *P=0.04, tobacco exposed sham vs tobacco-naïve sham; **P=0.009, tobacco-exposed MI vs tobacco-naïve MI; ***P⩽0.001, tobacco-exposed MI vs tobacco-exposed MI+N-acetyl cysteine.

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Pro-inflammatory cytokine levels: We compared the circulating levels of cytokines IL-1α, IL-1β, IL-2, IL-6, IFN-γ and TNF-α in the sera obtained from sham-operated and MI rats with and without chronic exposure to tobacco (Figure 7). The circulating levels of IL-1α were significantly higher in tobacco-exposed sham compared to tobacco-naïve sham (963±75 vs 243±62; P=0.001) and in tobacco-exposed MI compared to tobacco-naïve MI rats (1271±143 vs 849±88; P=0.05). The circulating levels of IL-1β were significantly higher in tobacco-exposed sham compared to tobacco-naïve sham rats (2862±28 vs 466±16; P=0.001) and in tobacco-exposed MI compared to tobacco-naïve MI rats (3014±326 vs 1916±110; P=0.01); IL-2 levels were significantly higher in tobacco-exposed sham compared to tobacco-naïve sham rats (1278±8 vs 24±28; P=0.001) and in tobacco-exposed MI compared to tobacco-naïve MI rats (1766±266 vs 799±266; P=0.05). The circulating levels of IL-6 were significantly higher in tobacco-exposed sham compared to tobacco-naïve sham rats (28 177±2415 vs 464±83; P=0.001) and in tobacco-exposed MI compared to tobacco-naïve MI rats (29 192±3273 vs 13 082±1664; P=0.001) and IFN-γ levels were also significantly higher in tobacco-exposed sham compared to tobacco-naïve sham (4023±616 vs 2438±343; P=0.05) and in tobacco-exposed MI compared to tobacco-naïve MI rats (7753±626 vs 3634±379; P=0.01). Similarly, the levels of TNF-α were significantly higher in tobacco-exposed sham compared to tobacco-Naïve sham (1600±214 vs 379±83; P=0.05) and in tobacco-exposed MI compared to tobacco-naïve MI rats (2494±143 vs 888±234; P=0.001). The serum levels in NAC-treated rats compared to tobacco-exposed MI decreased significantly for IL-1α (1271±143 vs 369±72; P=0.0005), IL-1β (3014±326 vs 676±56; P=0.0001), IL-2 (1766±266 vs 1380±224; P=0.01), IL-6 (29 192±3273 vs 10 515±1241; P=0.0006), IFN-γ (7753±626 vs 1334±60; P=0.00001) and TNF-α (2494±182 vs 511±120; P=0.001).

Figure 7
figure 7

Effect of N-acetyl cysteine on tobacco-induced circulating levels of pro-inflammatory cytokines. We quantified circulating levels of pro-inflammatory cytokines using a BioPlex rat cytokine assay kit (Bio-Rad) in the serum obtained from tobacco-exposed sham, tobacco-naïve sham, tobacco-exposed myocardial infarction (MI) and tobacco-naïve MI rats. The results are expressed as mean±s.e.m. (pg/ml). A significant decreased circulating level of cytokines in N-acetyl cysteine-treated rats is shown.

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DISCUSSION

Our results are supportive of the concept that chronic exposure to tobacco results in the depression of cardiac function, which worsens even further in the setting of an acute MI. These sentinel findings confirm the damaging effect of chronic tobacco exposure in determining post-infarction ventricular remodeling. The model used in our investigation was distinctive. We first exposed rats to conditions that simulate the condition of chronic habitual smoking. The validity of this model has been published previously.1 We then investigated the outcomes of these rats with respect to ventricular function and structural damage in the setting an acute MI and with abrupt cessation of smoking exposure (as is typically noted in the human condition). Thereafter, we tested whether the extent of damage could be ameliorated by using an easily available therapy in the form of dietary supplement NAC, which specifically targeted those molecular pathways that are aberrant in the setting of tobacco-related damage. We find that NAC can not only abrogate the inflammatory and pro-oxidant pathways upregulated in this model, but also results in improved ventricular remodeling, reduced myocardial damage, and thus facilitates myocardial repair in acute MI.

Previous studies, using an acute cigarette inhalation model, have demonstrated changes in diastolic function.34, 35, 36 We extend these findings in a model of chronic tobacco exposure, which indicate the presence of systolic function impairment and that arbiters of this response include molecular pathways of increased inflammation and oxidative stress. It is known that there is no detectable mRNA expression of pro-inflammatory cytokines in normal myocardial tissue,37 but once cardiac injury sets in (such as with an MI), the intracardiac expression of IL-6, TNF-α and IFN-γ mRNA expression possibly mediate the events of remodeling in the myocardium as reported earlier.38, 39, 40, 41, 42 An increased expression of these cytokines may result in enhanced production of tissue repair molecules. Our results support this assumption, as myocardial mRNA expression of MMP-2, SDF-1 and TGF-β was significantly higher in chronic tobacco-exposed rats compared with tobacco-naïve rats with MI. It is likely that the increased expression of these molecules in tobacco-exposed rats not only signifies their role in altered remodeling in the myocardium, but also the direct effect of tobacco exposure on the expression of these tissue repair participants. Similarly, tobacco exposure also resulted in a significantly higher mRNA expression of PARP, a marker of DNA damage and indicator of apoptosis,43, 44 suggesting that the more extensive MI in tobacco-exposed rats is also accompanied by an increase in cardiomyocyte apoptosis.

We also show a marked increase in oxidative stress in rats with MI previously exposed to chronic tobacco smoke compared to tobacco-naïve rats. This was demonstrated by increased expression of the NADPH oxidase component p22phox. The integral membrane protein p22phox is an indispensable component of the superoxide-generating phagocyte NADPH oxidase, which is a central molecule in the production of superoxides.45, 46, 47, 48, 49 Conversely, the expression of antioxidants SOD and TXN decreased significantly in infarcted myocardium of rats exposed to tobacco smoke compared to tobacco naïve.

We also explored the expression of the Keap 1-Nrf2--antioxidant response element signaling pathway that is known to regulate several protective mechanisms, including the expression of antioxidant and anti-inflammatory events.50, 51, 52 It is also known that the adaptive response induced by 4-hydroxynonenal, one of the important determinants of oxidative stress, is positively mediated by gene expression of cyto-protective protein (Nrf2/Keap-1) pathway.53 Our results suggest the critical role of Nrf2/Keap1 pathway in the stimulation of oxidative stress by chronic tobacco exposure because cigarette smoke exposure resulted in the activation of Nrf2 mRNA expression that counteract the immense oxidative stress that accompanies this trigger. These results are supported by a recent study,54 which demonstrated that Nrf2 is activated in the small airway epithelium of healthy smokers. Other in vitro studies on different cells types have also suggested the increased Nrf2 expression in response to cigarette smoke extracts,55 indicating the protective role of Nrf2 in cigarette smoke-induced health disorders. A significant inhibition of Nrf2 mRNA expression in NAC-treated rats supports this assumption, as in the absence of oxidative stress, Nrf2 expression was not activated. Alternatively, in the presence of cigarette smoke, Nrf2 may stay in the cytoplasm, and in the presence of NAC, it travels to the nucleus and ensue the transcription of antioxidants and inhibits inflammation.

The positive effects of the treatment with NAC that reverses the sequel of acute MI suggest that it targets both vascular inflammation and oxidative stress leading to the consequent facilitation of tissue repair. Although our intent was to see if NAC could counter the deleterious effects of inflammatory and oxidative stress pathways in chronic tobacco-exposed situations, we cannot ignore the fact that this may be an equally viable strategy for non-smokers with MI.

One of the weaknesses of this study is that the gene and protein expression reported are based on a single time point of 6 weeks; however, a time course of the effect of NAC on cardiac function could provide better results with regard to the efficacy of NAC on MI. Also, these results are obtained during continued smoking till 6 weeks; the studies in animals post-cessation of smoking may also provide interesting data on the effect of smoking on cardiac dysfunction. The numbers for each group are between 3 and 5 due to very laborious effort during exposure to cigarette smoke as well as the analysis of cardiac dysfunction using echocardiography each week for all the animals.

In conclusion, we demonstrate the efficacy of antioxidant glutathione precursor NAC to limit the extent of MI and facilitate tissue repair and preservation of ventricular function in the setting of chronic tobacco exposure. Such a simple therapy was based on a dietary supplement.