Cardiovascular disease (CVD) remains one of the major killers in modern society. One strong risk factor of CVD is cigarette smoking that causes myocardial injury and leads to the genesis of pathological cardiovascular events. However, the exact toxic component(s) of cigarette smoke (CS) and its molecular and cellular mechanisms for causing myocardial injury leading to heart damage and its prevention are largely unknown.
Using a guinea pig model, here we show that chronic exposure to CS produces myocardial injury that is prevented by vitamin C. Male guinea pigs were fed either vitamin C-deficient (0.5 mg/day) or vitamin C-sufficient (15 mg/day) diet and subjected to CS exposure from 5 Kentucky Research cigarettes (3R4F)/day (6 days/week) in a smoke chamber up to 8 weeks. Pair-fed sham controls were subjected to air exposure instead of CS exposure under similar conditions. Myocardial injury was produced in CS-exposed marginal vitamin C-deficient guinea pigs as evidenced by release of cardiac Troponin-T and I in the serum, oxidative stress, inflammation, apoptosis, thrombosis and collagen deposition in the myocardium. Treatment of rat cardiomyocyte cells (H9c2) in vitro and guinea pigs in vivo with p-benzoquinone (p-BQ) in amounts derived from CS revealed that p-BQ was a major factor responsible for CS-induced myocardial damage. A moderately large dose of vitamin C (15 mg/day) prevented CS/p-BQ-induced myocardial injury. Population based studies indicated that plasma vitamin C levels of smokers without disease were significantly lower (p = 0,0000) than that of non-smokers. Vitamin C levels of CS-related cardiovascular patients were further lower (p = 0.0000) than that of smokers without disease.
The results indicate that dietary supplementation of vitamin C may be a novel and simple therapy for the prevention of pathological cardiovascular events in habitual smokers.
Citation: Das A, Dey N, Ghosh A, Das S, Chattopadhyay DJ, et al. (2012) Molecular and Cellular Mechanisms of Cigarette Smoke-Induced Myocardial Injury: Prevention by Vitamin C. PLoS ONE 7(9): e44151. doi:10.1371/journal.pone.0044151
Editor: Partha Mukhopadhyay, National Institutes of Health, United States of America
Received: March 25, 2012; Accepted: July 30, 2012; Published: September 6, 2012
Copyright: © Das et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This research was supported by Juthika Research Foundation of Calcutta University, Department of Biotechnology, Government of West Bengal and Council of Scientific and Industrial Research, Government of India. Indu B. Chatterjee is INSA (Indian National Science Academy) Honorary Scientist. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: SD is an In-House Physician in VLCC Health Care. This does not alter the authors' adherence to all the PLoS ONE policies on sharing data and materials.
Cardiovascular disease (CVD) including coronary heart disease and myocardial infarction (MI) remains one of the major killers in modern society. CVD is believed to account for about 12 million deaths annually , . Epidemiologic studies have established worldwide that besides lung cancer and emphysema, cigarette smoking is a strong risk factor of CVD –. Numerous studies indicate that pathogenesis of most of the cigarette smoke (CS)-induced degenerative diseases is associated with oxidative damage . The existing evidence also support the view that oxidative stress may play a crucial role in cardiac and vascular abnormalities in different types of CVD .
While the deleterious effects of cigarette smoke (CS) on cardiovascular morbidity and mortality are well established, the molecular mechanisms and temporal progression of CS-induced pathophysiological disorders are not clear , , . Cigarette smoke (CS) is a complex mixture of about 4000 compounds including free radicals and long-lived radicals , . It has been indicated that free radical-mediated oxidative stress may play a prominent role in causing myocardial injury and damage of vascular endothelium that leads to the genesis of pathological cardiovascular events , –. However, the exact toxic component(s) of CS and its mechanism of action for causing myocardial injury leading to heart damage remain poorly understood.
Earlier we had shown that p-benzoquinone (p-BQ), derived from p-benzosemiquinone (p-BSQ) of CS, produces oxidative damage that is accompanied by inflammation and apoptosis leading to emphysema . p-BQ is also responsible for CS-induced myelodysplastic syndromes (MDS) . p-BSQ is present in substantial amounts (100–200 µg/cigarette) in smoke from all commercial cigarettes examined as well as Kentucky Research cigarettes , . In the smoker’s lungs, p-BSQ is converted to p-benzoquinone (p-BQ) apparently through disproportionation and oxidation by transition metal containing proteins , . We have shown that p-BQ formed in the lungs goes to the blood and alters the structure and function of serum albumin . p-BQ is a redox cycling agent that produces reactive oxygen species (ROS) leading to oxidative damage . We considered that p-BQ carried by the blood to the heart might cause oxidative damage leading to myocardial injury and cardiomyocyte death. Adult cardiomyocytes are terminally differentiated cells, and once destroyed, are rarely replaced. Until recently, the mode of cell death involved was not clear and was attributed to necrosis. However, it is now believed that apoptosis also plays an important role .
The available evidence supports the importance of antioxidant mechanisms in cardioprotection . Vitamin C is a strong antioxidant that reduces p-BQ and thereby inactivates it. Smoking reduces the plasma vitamin C level , , , . This may be at least in part due to consumption of vitamin C by p-BQ. Epidemiological studies have shown that low-plasma vitamin C is a risk factor for coronary heart disease and its acute clinical manifestations . Earlier we had reported that a moderately large dose of vitamin C prevents CS-induced oxidative damage, inflammation and apoptosis by reducing and thereby inactivating p-BQ , . Some studies show the protective effect of vitamin C on cardiomyocyte damage and heart failure , . We therefore considered that the nutritional status of vitamin C might be a critical determining factor for causing CS-induced myocardial injury.
However, investigations on the effects of CS exposure in vitamin C-deficient subjects leading to myocardial injury in population-base studies are difficult to achieve. Animal models have proven to be valuable tools for the investigation of CS-induced diseases , . In some laboratories, rat and mouse models have been used for the investigation of CS-induced CVD. Rats and mice synthesize vitamin C that would reduce the toxicity of CS , . We considered that as used for the studies on CS-induced emphysema  and MDS , marginal vitamin C-deficient guinea pig would be a better animal model to study the pathophysiology and molecular mechanisms of CS-induced myocardial injury. The guinea pig, like human, is incapable of synthesizing vitamin C and is dependent on the dietary source of the vitamin , . Thus it is possible to manipulate the nutritional status of vitamin C in the guinea pig through dietary means. Moreover, the guinea pig has anatomical and CS-induced pathophysiological similarities to human .
In this paper we show that chronic exposure of CS to marginal vitamin C-deficient guinea pigs produces myocardial injury that is accompanied by oxidative stress, inflammation, thrombosis, apoptosis and collagen deposition. We further show that p-BQ is largely responsible for CS-induced myocardial damage. A moderately large dose of vitamin C prevents CS/p-BQ-induced myocardial injury.
Biochemistry and Histopathology of CS-induced Myocardial Injury and its Prevention by Vitamin C
The most preferred biomarker for myocardial injury is cardiac Troponin (T and I), which has nearly absolute myocardial tissue specificity, as well as high sensitivity . Here we show that the levels of cardiac Troponin T and I are markedly elevated in the serum of marginal vitamin C-deficient guinea pigs exposed to CS for 6–8 weeks (Figure1A). Serum Troponin T and I are absent in CS-exposed vitamin C-sufficient group (Figure 1A). It is known that CVD, particularly myocardial ischemia, is usually accompanied by reduction of blood and oxygen delivery due to thrombus formation and thereby obstruction of circulation to one or more regions of the heart . Histopathology using H&E shows that in contrast to air-exposed guinea pigs, blood vessel of CS-exposed marginal vitamin C-deficient guinea pig is filled with thrombus that almost blocks the lumen at the end of 8 weeks (Figure 1B, I, II, III, IV, V). Thrombus formation is negligible in CS-exposed vitamin C-sufficient group (Figure 1B, VI). In contrast to normal striated cardiac mussels (Figure 1B, VII) or that observed after 2 weeks of exposure (Figure 1B, VIII), CS exposure after 4 weeks causes loss of striation and the cytoplasm becomes eosinophilic at the end of 6 weeks (Figure 1B, IX, X). After exposure of 8 weeks, there is formation of fibrous tissues (Figure 1B, XI). Fibrous tissue formation has been confirmed by deposition of collagen, as evidenced by marked blue colour taken up after Masson’s trichrome staining of the left ventricular part of the heart of the guinea pig (Figure 1B, XVI and XVII). No fibrous tissue formation is observed in the air–exposed or 2–4 weeks of CS-exposed myocardium (Figure 1B, XIII, XIV, XV). Loss of striation, fibrous tissue formation and collagen deposition are very low in CS-exposed vitamin C-sufficient guinea pigs (Figure 1B, XII and XVIII). Figure 1C shows quantitative evaluation of the progression of thrombus formation and fibrous tissue deposition. Collagen deposition is supported by the fact that synthesis of collagen (type I) is markedly increased in myocardial tissue of marginal vitamin C-deficient guinea pigs after exposure to CS for 6–8 weeks (Figure 1D). Collagen (type I) synthesis indicates enhancement of myocardial fibrosis . No such collagen synthesis takes place in CS-exposed vitamin C-sufficient guinea pigs (Figure 1D).
Figure 1. Progression and detection of myocardial injury in CS-exposed guinea pigs and prevention by vitamin C.
(Panel A) Immunoblots of cardiac Troponin T and I in serum. Bottom panel indicates ponceau S staining of the membrane as the loading control. (Panel B) Hematoxylin and Eosin (H&E) and Masson’s trichrome stain showing histological changes in cardiomyocytes as well as blood vessels; → indicate thrombus formation and fibrous tissue (FT) deposition (magnification 400X). (Panel C) Bar graphs depicting progression of thrombus formation (% area within blood vessel by H&E staining) and fibrous tissue deposition (% area in left ventricle by Masson’s trichrome staining). (Panel D) Detection of collagen type I. (Panel E) Lipid profile in serum. Bar over the respective columns represents mean ± SEM (n = 6), * indicates significant (p<0.05) increase from AIR-exposed sham control, ** indicates significant (p<0.05) decrease in vitamin C-sufficient group. Vit C means vitamin C.doi:10.1371/journal.pone.0044151.g001
It is reported that smokers have significantly higher serum cholesterol, triglyceride, and low-density lipoprotein levels  that appear to be strong and independent predictors of future risk of CVD . Figure 1E shows that in contrast to air-exposed sham controls or CS-exposed vitamin C-sufficient guinea pigs, the levels of serum total cholesterol, LDL cholesterol and triglyceride are gradually and significantly increased (p<0.05) in CS-exposed marginal vitamin C-deficient guinea pigs.
Vitamin C Status in CS-related Cardiovascular Patients
Earlier we had shown that CS exposure resulted in lowering of plasma vitamin C levels in guinea pigs . This is because CS consumes vitamin C . A total of 24 newly diagnosed smoke-related cardiovascular patients, 25 non-smokers and 25 smokers without disease were used in this study. Here we show that as observed in the CS-exposed guinea pigs, plasma vitamin C levels of human smokers without disease (0.18±0.01 mg/dL) are significantly lower (p = 0.0000) than that of non-smokers (0.50±0.16 mg/dL) (Table I). Similar observations were also made in other laboratories , . The plasma vitamin C levels of CS-related cardiovascular patients (0.15±0.01 mg/dL) are further lower (p = 0.0000) than that of smokers without disease (Table I).
Table 1. Plasma vitamin C levels in non-smokers, smokers without disease and CS-related cardiovascular patients.doi:10.1371/journal.pone.0044151.t001
CS-induced Oxidative Stress in the Myocardium
We had previously shown that after exposure to CS, p-BSQ of CS is oxidized to p-BQ in the lungs that gets into the blood stream , . p-BQ is a strong arylating agent  and here we show that p-BQ carried in the blood goes to the heart and forms p-BQ-protein adducts (Figure 2A). Adduct formation is absent in the air-exposed sham controls or negligible in CS-exposed vitamin C-sufficient group (Figure 2A). Besides being an arylating agent, p-BQ is a strong redox cycling agent that produces ROS and oxidative damage , . It is known that formations of protein carbonyl and 8-oxo-2′-deoxyguanosine (8-oxodG) are hallmarks of oxidative damage. We show that after exposure of CS to the marginal vitamin C-deficient guinea pigs for 2 weeks, both protein carbonyls (Figure 2B) and 8-oxodG (Figure 2C and 2D) are produced in the myocardium that increased with increased exposure to CS up to 8 weeks. It is reported that oxidative stress induces structural damage of cardiac myocytes and plays a key role in the pathophysiology of heart failure , . Oxidative damage is almost prevented when the CS-exposed guinea pigs are fed 15 mg vitamin C/day (Figure 2).
Figure 2. Identification of p-BQ-protein adducts and detection of oxidative stress in myocardium of CS-exposed guinea pigs and prevention by vitamin C.
(Panel A) p-BQ protein adducts in the myocardial tissue. (Panel B) Protein carbonyl formation by Oxyblot™. (Panel C) DNA oxidation; upper row: red fluorescence indicates formation of 8-oxo-2′-deoxyguanosine (8-oxodG); lower row: stained with 6-diamidino-2-phenylindole (DAPI); (magnification 200X). (Panel D) Quantitative evaluation of 8-oxodG; Bars over the respective columns represent means ± SEM (n = 6) * indicates significant (p<0.05) increase from AIR exposed sham control. Vit C means vitamin C.doi:10.1371/journal.pone.0044151.g002
Oxidative damage has the potential to trigger inflammatory cytokines, such as tumor necrosis factor-alpha (TNF-α) and interleukin IL-1β , . TNF-α and IL-1β activate NF-kB. We demonstrate that oxidative damage is the initial event, which is followed by inflammation. While oxidative damage starts after 2 weeks of CS exposure to marginal vitamin C-deficient guinea pigs, up regulation of both TNF- α and NF-kB takes place after 4 weeks of smoke exposure that persists up to 8 weeks (Figure 3A). We further show that IL-1β is significantly increased (p<0.05) in the serum of guinea pigs, which is apparently released from the injured myocardium (Figure 3B). Inflammation is known to be accompanied by infiltration of neutrophils. Histopathology shows marked neutrophil adhesion to the endothelium of a blood vessel after 4 weeks of CS exposure to marginal vitamin C-deficient guinea pigs. After 6–8 weeks of CS exposure, neutrophil infiltration is observed in the myocardium (Figure 3C). No neutrophil infiltration is observed either in sham control or CS-exposed vitamin C-sufficient group (Figure 3C).
Figure 3. Detection of inflammatory response, nutrophil infiltration in the injured myocardium, activation of metallo matrix proteinases (MMPs) in CS-exposed guinea pigs and prevention by vitamin C.
(Panel A) Immunoblots of TNF-α and NF-kB in myocardial tissue lysate lysate. (Panel B) ELISA test indicating IL-1β (pg/ml) level in serum. (Panel C) Hematoxylin and Eosin (H&E) stain showing neutrophil infiltration in the injured myocardium; → indicates neutrophils within injured myocardium (magnification 200X) (Panel D) Immonoblots of MMP-9, MMP-12 in myocardial tissue lysate. Vit C means vitamin C.doi:10.1371/journal.pone.0044151.g003
Marginal neutrophils exert potent cytotoxic effects through the release of poteolytic enzymes like matrix metalloproteinases (MMPs) , . Figure 3D shows that both MMP-9 and MMP-12 are up regulated in the myocardium of marginal vitamin C-deficient guinea pigs after exposure to CS for 4–8 weeks. No such up regulation is noticed in the sham controls or CS-exposed guinea pigs fed 15 mg vitamin C/day (Figure 3D).
CS-induced Apoptosis in Injured Myocardium
It is now believed that besides necrosis apoptosis plays an important role in CVD . Over the past few years, there have been several reports of the occurrence of cardiomyocyte apoptosis during myocardial injury , . According to the earlier reports oxidative stress can trigger either the ‘intrinsic’ (mitochondrial) or extrinsic (extra mitochondrial) apoptotic pathways via multiple mechanisms . It has been suggested that cytochrome c-mediated apoptosis is important in cardiomyocyte death . Figure 4A indicates that after exposure of marginal vitamin C-deficient guinea pigs to CS for 4 weeks cytochrome c is almost absent in the mitochondria that is accompanied by its marked presence in the cytosol. Figure 4B shows quantitative estimation of both fractions depicting release of cytochrome c in the cytosolic fraction under ‘intrinsic’ apoptotic condition. Apoptosis of the cardiomyocytes after CS exposure is further confirmed by activation (phosphorylation) of p53, increase in the ratio of Bax/Bcl-2 and activation of caspase 3, as revealed by the formation of cleaved caspase 3 (Figure 4C). Extra mitochondrial apoptosis is evidenced by the up regulation of TNF- α (Figure. 3A, row 1) and activation of caspase 8 (Figure 4D). It is reported that TNF-α plays a role in the progression of CVD . Neither mitochondrial nor extra mitochondrial apoptosis has been observed in CS-exposed vitamin C-sufficient guinea pigs (Figure 4C and 4D).
Figure 4. Assessment of apoptosis in the myocardium of CS-exposed guinea pigs and prevention by vitamin C.
(Panel A) Release of cytochrome c from mitochondria to cytosol. Bottom panel indicates cropped SDS-PAGE with coomassie stain as the loading control. (Panel B) Quantitative estimation of cytochrome c of both fractions; * significantly increased (p<0.05) in cytosolic fraction with respect to AIR exposed sham controls; ** significantly decreased (p<0.05) in mitochondrial fraction with respect to AIR exposed sham controls. (Panel C) Overexpression of pp53 (phospho-p53), Bax, and formation of cleaved caspase 3 (CC3). (Panel D) Formation of cleaved caspase 8 (CC8). (Panel E) TUNEL positive cells in the myocardium (green fluorescence); respective lower rows stained with, 6-diamidino-2-phenylindole (DAPI) (magnification 200X). (Panel F) Quantitative evaluation of TUNEL positive cells; bars over the columns indicate means ± SEM (n = 6); * significantly increased (p<0.05) with respect to AIR exposed sham controls. Vit C means vitamin C.doi:10.1371/journal.pone.0044151.g004
DNA fragmentation is also a criterion of early apoptosis. TUNEL assay shows that compared to sham controls, there is progressive increase in the DNA fragmentation after 4–6 weeks of CS exposure to the marginal vitamin C-deficient guinea pigs (Figure 4E). Figure 4F shows quantitative evaluation of TUNEL positive cells. DNA fragmentation is almost absent in CS-exposed vitamin C-sufficient group.
p-Benzoquinone (p-BQ) Induces Oxidative Damage, Inflammation and Apoptosis Leading to Myocardial Injury in Guinea Pigs in vivo
The calculated amount of total p-BQ available from CS exposure/guinea pig/day was 25 µg (see Materials and Methods). When CS exposure was replaced by p-BQ treatment (25 µg/animal/day), significant release of cardiac Troponin T and I occurred in the serum of marginal vitamin C-deficient guinea pigs (Figure 5A). This was accompanied by myocardial injury and fibrosis, as evidenced by collagen deposition (Figure 5B). Figure 5C depicts quantitative estimation of myocardial injury and deposition of collagen fibres. p-BQ treatment also resulted in up regulation of MMP-9 and MMP-12 as well as increased synthesis of collagen type 1 (Figure 5D). Furthermore, p-BQ produced proteins adducts (Figure 5E) and oxidative damage, as evidenced by the formation of protein carbonyls and 8-oxodG (Figures 5F and 5G). Similar to that observed by CS exposure, p-BQ treatment caused increase in the inflammatory markers TNF-α and NF-kB (Figure 5H). p-BQ also produced apoptosis as revealed by the overexpression phospho-p53 (pp53), increase in the Bax/Bcl-2 ratio and formation of cleaved caspase 3 (Figure 5I). All the aforesaid pathophysiological events were not produced in the vitamin C-sufficient group (Figure 5).
Figure 5. Detection of myocardial injury in p-BQ-treated guinea pigs and prevention by vitamin C.
(Panel A) Cardiac troponin T and I in the serum. Bottom panel represents ponceau S staining of the membrane as the loading control. (Panel B) Histology showing myocardial injury and deposition of collagen fibres (magnification 200X). (Panel C) Quantitative analyses of histological images of panel B. (Panel D) Immunoblots of collagen type1, MMP-9 and MMP-12. (Panel E) p-BQ protein adducts in the myocardial tissue. (Panel F) Protein oxidation as evidenced by Oxyblot™. (Panel G) DNA oxidation as evidenced by the formation of 8-oxo-2′-deoxyguanosine (8-oxodG) (red fluorescence); lower row: stained with 6-diamidino-2-phenylindole (DAPI); (magnification 200X). (Panel H) Immunoblots of TNF-α and NF-kB. (Panel I) Over expression of phospho-p-53 (pp53) and Bax and formation of cleaved caspase 3 (CC3). Vit C means vitamin C.doi:10.1371/journal.pone.0044151.g005
p-BQ is Responsible for Aqueous Extract of Cigarette Smoke (AECS)-induced Oxidative Stress and Apoptosis in Rat Cardiomyocytes (H9c2)
AECS (50 µl/mL)-induced ROS generation, formation of protein carbonyl and protein adducts in rat cardiomyocytes were not only prevented by antibody to p-BQ but also mimicked by p-BQ in amounts (2.5 µg/ml) present in AECS (Figure 6A, 6B and 6C). Also, AECS-mediated apoptosis, as evidenced by Annexin V/PI staining, phosphorylation of p53 as well as activation of caspases 3 and 8, were prevented by antibody to p-BQ and mimicked by p-BQ (Figure 6D, 6E and 6F). The results indicate that p-BQ is responsible for AECS-mediated oxidative stress, formation of protein adduct and apoptosis in vitro. Like that observed in vivo, all the aforesaid events were also prevented by addition of vitamin C (40 µg/ml) to the incubation medium.
Figure 6. AECS-induced pathophysiological disorders are mimicked by p-BQ in rat cardiomyocytes (H9c2).
(Panel A) ROS generation. (Panel B) protein oxidation by Oxyblot™. (Panel C) Formation of p-BQ-protein adducts. (Panel D) Aoptosis as evidenced by Annexin V/PI staining. (Panel E) Activation of p53 and caspase3. (Panel F) Activation of caspase 8.doi:10.1371/journal.pone.0044151.g006
In this paper using a guinea pig model, we have addressed the fundamental questions that remain poorly answered regarding the initiation, molecular mechanisms and temporal progression of CS-induced myocardial injury and its prevention. Myocardial injury has been evidenced by histology indicating thrombosis, fibrosis of the myocardium and collagen deposition in the left ventricular part of the heart of guinea pigs. Histology is supported by lipid profile that shows increase in triglyceride and LDL cholesterol. Also, release of cardiac Troponin-T and I in serum confirm the onset of myocardial injury .
Existing evidence indicate a strong association between CS, oxidative stress and CVD . Here we show both in vivo as well as in vitro that oxidative stress is the initial event and p-BQ derived from CS is a major factor responsible for CS-induced oxidative damage of proteins and DNA in the myocardium. We have also demonstrated that oxidative damage is followed by inflammation and apoptosis that ultimately leads to myocardial injury. All the pathophysiological events are prevented by oral administration of vitamin C (15 mg/animal/day).
We show that inflammation is accompanied by thrombus formation. Thrombosis results in ischemia. In the ischemic region and surrounding myocardium, inflammatory cytokines, such as TNF-α, IL-1ß are triggered as a host reaction. TNF-α and IL-1ß are inducers of NF-kB, a family of transcription factors that plays a major role in the regulation of diverse genes involved in the inflammatory response , . Here we show that TNF- α and NF-kB are activated in the myocardium after 4 weeks and persisted up to 8 weeks of the experimental condition of CS exposure. After 6 weeks of CS exposure, IL-1ß, apparently secreted by the injured myocardium, is released in the serum. Previous studies suggest that inflammation is a critical factor in the initiation and progression of CVD .
A hall mark of acute inflammation is recruitment of leukocytes including neutrophils to the site of injury. After 4 weeks of CS exposure, neutrophil transmigration occurs into the injured myocardium that continues up to 8 weeks. Activated neutrophils and macrophages release proteolytic enzymes, including collagenolytic matrix metalloproteinases (MMPs), including MMP-9 and MMP-12 , . Our results indicate that both MMP-9 and MMP-12 are released in the myocardium after 4 weeks of CS exposure. The release of MMPs is accompanied by an increase in collagen type 1 synthesis and its deposition in the myocardium.
Although cardiomyocyte death may occur via necrosis or apoptosis, apoptosis is now emerging as major contributor . The apoptotic signals in mammals may be transduced by “intrinsic” and/or “extrinsic” pathways. The intrinsic pathway is primarily activated in myocytes by ischemic hypoxia and oxidative stress that incite increased permeability of mitochondrial membranes, release of cytochrome c from the mitochondria to the cytosol, and activation of caspase 3, a key effector protein in the execution of downstream event in apoptosis. Extrinsic apoptotic pathway involves transduction of a signal from membrane receptors belonging to the tumor receptor factor such as Fas or TNF receptor 1. Stimulation of death receptors results in activation of caspase 8, which in turn activates caspase 3. We have shown in this paper that exposure of CS to marginal vitamin C–deficient guinea pigs induces both “intrinsic” and “extrinsic” pathways of apoptosis in a time-dependent manner. Besides activation of caspase 8 and caspase 3, CS exposure also resulted in DNA fragmentation, activation of p53 and increase in the Bax/Bcl-2 ratio. Alteration of balance between Bax and Bcl-2 by p-BQ treatment was reported in human lung carcinoma cells (A549) in vitro . We have further shown that oral administration of vitamin C (15 mg/day) to the guinea pigs almost completely prevents CS-induced apoptosis. We consider that vitamin C prevents CS-induced myocardial injury by preventing oxidative stress and thereby inflammation and apoptosis.
Previously we had shown that p-BQ, a redox cycling agent produced from p-BSQ of CS, is a major factor that produces ROS, oxidative damage and apoptosis , , . Here we show both in the guinea pig in vivo and rat cardiomyocytes (H9c2) ex vivo that p-BQ is a causative factor responsible for producing CS-induced ROS, oxidative damage, inflammation and apoptosis leading to myocardial injury. Vitamin C is a known scavenger of free radicals that prevents oxidative damage, lipid peroxidation and myocardial injury , . Vitamin C has also other beneficial effects in smokers. It is known that CS-induced leukocyte aggregation and adhesion to endothelium may lead to thrombosis. It is reported that vitamin C almost entirely prevents CS-induced leukocyte adhesion to micro- and macrovascular endothelium and leukocyte-platelet aggregate formation . This might be one of the reasons how sufficient vitamin C prevents thrombosis in CS-induced guinea pigs.
We have shown in this paper that compared to non-smokers (0.50±0.16 mg/dL), plasma vitamin C levels of smokers without disease (0.18±0.01 mg/dL) are very low (p = 0.0000). This has also been reported before , . The plasma vitamin C levels of CS-related cardiovascular patients (0.15±0.01 mg/dL) are further lower (p = 0.0000) than that of smokers without disease. This may be an effect of CVD. Vitamin C (Éo ±0.08V) is strongly oxidized and thereby consumed by p-BQ (Éo ±0.71V). We consider that a major part of the decline of plasma vitamin C in smokers is due to consumption of vitamin C by p-BQ. The inverse association between smoking and plasma vitamin C levels occurs independently of dietary intake , .
In a recent report, the effects of N-acetyl cysteine (NAC), an antioxidant and anti-inflammatory agent, was studied in a rat model of MI created by ligation of the left anterior descending artery followed by chronic exposure of CS . NAC, administered intraperitoneally, produced significant decrease in oxidative damage, inflammation and circulating levels of cytokines resulting in marked amelioration of the extent of myocardial damage. Previously we examined the efficacy of oral administration of NAC in the treatment of CS-induced emphysema in our guinea pig model. Whereas vitamin C protected CS-induced emphysema almost completely, NAC prevented the extent of lung damage to about 33% . However once CS-induced damage is done, vitamin C cannot ameliorate the injury.
Figure 7. Mechanisms of CS-induced myocardial injury and its prevention by vitamin C.doi:10.1371/journal.pone.0044151.g007
In conclusion we state that p-BQ, derived from CS, is a major risk factor responsible for causing CS-induced oxidative damage, which is followed by inflammation and apoptosis, ultimately leading to myocardial injury (Figure 7). The injury is accompanied by vascular thrombosis, collagen deposition in the left ventricular part of the heart and release of Troponin T and I in the serum. All these events indicate occurrence of MI. However, this needs to be confirmed by doing ECG and echocardiogram. We have shown that dietary supplementation of vitamin C prevents CS-induced myocardial injury apparently by reducing and thereby inactivating p-BQ. In population-based studies, we have observed that plasma vitamin C levels of habitual smokers are very low, which may make them susceptible to CVD. We consider that intake of a moderately large dose of vitamin C may protect the smokers from CS-induced myocardial injury.
CS →p – BSQ →p – BQ
Oxidative damage → Inflammation → Apoptosis → Myocardial Injury
Materials and Methods
All methods were approved by the Institutional Animal Ethics Committee, Permit No. 797/CPCSEA, University of Calcutta. All efforts were made to minimize suffering of the animals. The collection of human blood and subsequent experiments with plasma were approved by the Institutional Bioethics Committee for animal and human research studies, University of Calcutta, permission No. 1096, following the Code of Ethics of the World Medical Association (Declaration of Helsinki) for experiments involving humans.
Exposure of Guinea Pigs to CS
Male short hair inbred albino guinea pigs weighing 400–500 g were fed vitamin C-free diet for 7 days to minimize the vitamin C level of tissues ,  (see Materials and Methods S1). After 7 days, the guinea pigs were subjected to cigarette smoke exposure from 5 Kentucky research cigarettes 3R4F/day @ 2 puffs/cigarette/animal in a smoke chamber, as described before , , . (see Materials and Methods S1). Pair-fed sham controls were subjected to air exposure instead of CS under similar conditions. The guinea pigs were divided into the following weight-matched experimental groups (n = 6/group). (i) exposed to CS and supplemented with 0.5 mg vitamin C/animal/day (CS-exposed marginal vitamin C-deficient group); (ii) exposed to CS and supplemented with 15 mg vitamin C/animal/day (CS-exposed vitamin C-sufficient group); (iii) exposed to air and fed 0.5 mg vitamin C/animal/day (sham control); exposed to air and fed 15 mg vitamin C/animal/day. At the end of 8 weeks of CS exposure, the guinea pigs were euthanized under deep anesthesia using i.p. injection of ketamine hydrochloride (100 mg/kg body weight) and blood, heart and other tissues were collected for further experiments.
Treatment of Guinea Pigs with p-BQ
After feeding vitamin C-free diet for 7 days, as mentioned above under “Exposure of guinea pigs to cigarette smoke”, the guinea pigs (n = 4) were given intramuscular injection (lumber muscle) of 25 µg p-BQ/animal/day in 100 µl of Milli Q water for 8 weeks. The p-BQ was purchased from HImedia (RM-489) and freshly crystallized from n-hexane before use. The amount of p-BQ injected (25 µg/day/guinea pig, i.m.) was to mimic the calculated amount of total p-BQ available per day/guinea pig inhaled from 5 Kentucky research cigarettes in the smoke chamber (see Materials and Methods S1). The sham controls received injection of only water. After 8 weeks, the guinea pigs were euthanized under deep anesthesia using i.p. injection of ketamine hydrochloride (100 mg/kg body weight) and blood, heart and other tissues were collected for further experiments.
Preparation of Aqueous Extract of CS (AECS)
Measurement of p-BQ in AECS
Collection of Blood from Human Volunteers
Written consents were obtained from all the volunteers prior to collection of blood. Venous blood (≈2 ml) was collected and plasma separated immediately. For estimation of vitamin C, dithiothreitol (DTT) (1 mmol/L final concentration) was added to the plasma to prevent loss of vitamin C by autooxidation.
Measurement of Vitamin C in Plasma
Histology for Assessing Myocardial Damage and Fibrosis
The left ventricular part of the heart was surgically removed and fixed in 10% neutral buffered formalin for 24 hours, embedded in paraffin and subjected to microtomy (4 µm) routinely. A part of the cardiac tissue was stored at −80°C until processing. The deparaffinized sections were stained with i) conventional hematoxylin-eosin (H&E) (Sigma) to determine the degree of cardiomyocytes damage, neutrophil infiltration and myocardial fibrosis ii) collagen content assessment by Masson’s Trichrome stain. Images were captured with a bright field microscope (Dewinter) equipped with high resolution digital Digieye 330/210 camera and quantified using Dewinter Biowizard 4.1 software.
Lipid Profile in Serum of CS-exposed Guinea Pigs
Blood samples were collected from CS-exposed guinea pigs by cardiac puncture and serum separated. Total cholesterol, HDL, LDL, VLDL, and triglyceride were measured in serum in a fully automated analyzer, Model: XL-300 (Eeba Mannheim).
Myocardial Marker Analysis
The serum of CS-exposed guinea pigs was used immediately for the detection of cardiac troponin-T and troponin-I by immunoblots using cardiac anti-troponin-T and I antibody (abcam, UK.).
Frozen heart tissues were homogenized, first by pestle and then glass homogenizer in cold lysis buffer (50 mmol/L Tris–HCl; 1 mmol/L DTT, 1mmol/L EDTA (in place of 100 AMo-phenanthroline), 0.2% Nonidet P-40, pH 7.4) supplemented with protease inhibitor cocktail (Sigma, USA), phosphatase inhibitor cocktail (Sigma, USA) . Rat cardiomyocytes (H9c2) were also extracted in the same lysis buffer. Samples were spun at 19,000 g at 4°C for 15 minutes and the supernatants collected.
Collagen Type 1 Synthesis
Synthesis of collagen (type 1) was measured by immunoblot using antibody to collagen type 1 (Santa Cruz biotechnology, Inc., California, USA).
Identification of p-BQ-protein Adduct
Tissue/cell lysates were immunoblotted using antibody against p-BQ (rabbit, polyclonal; Abexome Biosciences, Bangalore, India) .
Detection of Oxidative Damage in Myocardial Tissue
Protein oxidation was measured by the formation of protein carbonyls by immunoblot using antibody to 2, 4-dinitropheny-hydrazine (DNPH) as per Oxyblot™ protein oxidation detection kit (Intergen, NY) . DNA oxidation was detected by immunofluorescence using antibody to 8-oxodG as per manufacturer’s (Abcam, UK) instruction .
Inflammation and Quantification of Cytokine Level
Cytokine (IL-1β) was analyzed using a sandwich ELISA kit (Human IL-1β ELISA Kit II, BD optEIA™). Other inflammatory markers like TNF-α, NF-kB, MMP-9 and MMP-12 were measured by immunoblot using respective antibodies (TNF-α and NF-kB: Cell Signaling Technology, USA; MMP-9 and MMP-12: Santa Cruz biotechnology, Inc., California, USA).
Assessment of Apoptosis
Thirty µg protein/lane were separated on 10% SDS-PAGE, blotted on PVDF membrane, and probed with respective antibodies against p53, phospho-p53, Bax, Bcl-2, caspase 3, cleaved caspase 3, caspase 8, cleaved caspase 8, α-tubulin (Cell Signaling Technology, USA). Preparation of mitochondrial extract and immunoblot of cytochrome c (Cell Signaling Technology, USA) were carried out as described previously . Quantitative estimation of cytochrome c was made by densitometric scanning using Image Quant TL software. Apoptosis was also measured by the terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay using “In situ cell death detection kit, fluorescein” (Roche, USA) according to the manufacturer’s instruction . Two fields per section of 6 independent sections in each group were evaluated.
Rat cardiomyocyte (H9c2) cells (source: National Centre for Cell Science, India) were grown to 50–60% confluence in Dulbecco’s Modified Eagle Medium (DMEM) containing 10% fetal calf serum ((GIBCO)-BRL, USA), 100 units/ml penicillin and 100 µg/ml streptomycin.
Detection of Reactive Oxygen Species (ROS) and Protein Oxidation in H9c2 Cells
ROS was measured by flow cytometry (FACS Caliber, Becton Dickinson, USA) using dichlorodihydrofluorescein diacetate (H2DCFDA) (Sigma-Aldrich) as per manufacturer’s instruction. Data were acquired and analyzed using the CELL Quest Programme. Protein oxidation was measured by the formation of protein carbonyls as described previously , .
Assessment of Apoptosis in H9c2 Cells by FACS
Cultured cells (3×106), after treatment with/without AECS (50 µL/mL) for 1hour  were washed with PBS solution (Hyclone, Thermo Scientific) and incubated in fresh culture medium for 12 hours. Apoptosis was assessed by Annexin V and propidium iodide (PI) staining according to manufacturer’s protocol (Becton Dickinson), and analyzed using the FACS Calibur-Cell Quest software (Becton Dickinson) as described earlier . A total of 10,000 events were acquired and dual parameter dot plot of FL-2 (X-axis; PI-fluorescence, log scale) vs. FL-1 (Y-axis; Annexin V-FITC-fluorescence, log scale) was recorded. Immunoblots were also done to confirm apoptosis.
All values are expressed as mean ± SEM except in human patients where the values are given as mean ± SD. Statistical significance was carried out using a two factor ANOVA, with factors being CS and vitamin C, or one way ANOVA as needed. The p-values were calculated using appropriate F-tests. Difference with p-values <0.05 was considered significant.
We gratefully acknowledge Dr. Prakash Mandal, M.D. and Prof. Gourisankar Ghosh of UCSD, USA for critically reading the manuscript before submission. We thank Dr. Arun Bandopadhya, IICB, India for kindly providing the H9c2 cell line.
Conceived and designed the experiments: IBC. Performed the experiments: AD ND AG SD DJC. Analyzed the data: AD ND IBC. Wrote the paper: IBC AD.
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