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. 2012;7(9):e44151.
doi: 10.1371/journal.pone.0044151. Epub 2012 Sep 6.

Molecular and cellular mechanisms of cigarette smoke-induced myocardial injury: prevention by vitamin C

Archita Das  1 , Neekkan Dey, Arunava Ghosh, Shovanendu Das, Dhruba J Chattopadhyay, Indu B Chatterjee
Affiliations

Molecular and cellular mechanisms of cigarette smoke-induced myocardial injury: prevention by vitamin C

Archita Das et al. PLoS One. 2012.

Abstract

Background: 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.

Methodology/principal findings: 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.

Conclusions/significance: 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.

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Conflict of interest statement

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.

Figures

Figure 1
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.
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.
Figure 3
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.
Figure 4
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.
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.
Figure 6
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.
Figure 7
Figure 7. Mechanisms of CS-induced myocardial injury and its prevention by vitamin C.
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