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ORIGINAL ARTICLE
Year : 2013  |  Volume : 3  |  Issue : 2  |  Page : 84-87  

Effect of sildenafil-induced nitric oxide on the histomorphology of cardiomyocytes in male rats


1 Department of Anatomy, Kasturba Medical College, Manipal University, Mangalore, Karnataka, India
2 Department of Anatomy, Gulf Medical University, Ajman, United Arab Emirates
3 Department of Pathology, Kasturba Medical College, Manipal University, Mangalore, Karnataka, India
4 Department of Physiology, Kasturba Medical College International Center, Manipal University, Manipal, Karnataka, India

Date of Submission25-Oct-2012
Date of Acceptance12-Mar-2013
Date of Web Publication27-Aug-2013

Correspondence Address:
Rajalakshmi Rai
Department of Anatomy, Kasturba Medical College, Bejai, Mangalore 576 104, Karnataka
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/2229-516X.117056

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   Abstract 

Introduction: Although sildenafil citrate, one of the selective phosphodiesterase-5 (PDE5) inhibitors, is considered the best treatment for erectile dysfunction, studies have shown that it has also a beneficial effect on a variety of cardiovascular conditions. In spite of reports of a significant protective effect of sildenafil against necrosis in intact hearts, there are also contradictory reports regarding its beneficial effect on the heart. Since there are not enough reports regarding the histomorphological changes in the cardiomyocytes after exposure to sildenafil citrate, the present study was conducted to observe the same along with other biochemical parameters. Materials and Methods: Adult male albino rats of Wistar strain were used in the present study. The animals were divided into a control group and two experimental groups containing six rats each. The animals were treated with a solution of sildenafil citrate dissolved in distilled water. Histomorphological changes were observed by light microscopy and the levels of nitric oxide (NO) and PDE in the heart were measured by spectrophotometry. Results: It was observed that animals treated with sildenafil citrate showed a highly significant increase in NO and a decrease in PDE level, but the histological architecture of the cardiomyocytes did not show much change other than a slightly elongated and swollen nucleus. Conclusions: This study shows that sildenafil citrate at low dosage is well tolerated by cardiac muscle cells, but as dosage increases, it may become detrimental through its NO and PDE activity.

Keywords: Cardiomyocytes, erectile dysfunction, nitric oxide, phosphodiesterase-5 inhibitor, sildenafil


How to cite this article:
Prabhu LV, Rai R, Ranade AV, Kini H, Krishnamurthy A, Bernhardt KL. Effect of sildenafil-induced nitric oxide on the histomorphology of cardiomyocytes in male rats. Int J App Basic Med Res 2013;3:84-7

How to cite this URL:
Prabhu LV, Rai R, Ranade AV, Kini H, Krishnamurthy A, Bernhardt KL. Effect of sildenafil-induced nitric oxide on the histomorphology of cardiomyocytes in male rats. Int J App Basic Med Res [serial online] 2013 [cited 2020 Aug 4];3:84-7. Available from: http://www.ijabmr.org/text.asp?2013/3/2/84/117056


   Introduction Top


Among all phosphodiesterase-5 (PDE5) inhibitors sildenafil citrate is considered one of the prize-winning because of its efficacy [1],[2],[3],[4] and pleiotropic effects. [5] Also in recent years, there have been several studies on sildenafil for its therapeutic applications in diseases other than erectile dysfunction (ED). [6],[7],[8],[9],[10],[11],[12] Kumar et al., [13] stated that patients with heart failure and preserved ejection fraction might derive particular benefit from these drugs. However, unfortunately, as the availability of sildenafil has become easier, it is being used and misused by many. [14] Reports in letters to the Editor of the British Medical Journal state that sildenafil is used as a recreational drug in younger generations. [15] On account of the increasing incidences of sudden cardiac deathsamong ED patients treated with sildenafil citrate, it becomes essential to understand how this drug affects the heart. Therefore the present study was conducted to find out the histological changes in the cardiomyocytes and correlate it with the biochemical parameters.


   Materials and Methods Top


Adult (14-16 weeks old) male albino rats of Wistar strain were used in the present study. The animals were divided into two groups. Group-I (control group (n = 6)) rats were intraperitoneally injected distilled water for the duration of the experiment on alternate days. Group-1a and1b (experimental groups (n = 6 in each group)) rats were intraperitoneally injected 10 mg/kg body weight (bw) and 8 mg/kg bw of sildenafil citrate, respectively, for 30 days, on every alternate day at a regular time interval. The dosing solution was prepared by grinding Viagra tablets into powder and dissolving in distilled water (concentration: 3.5 mg/ml of water). [16],[17] On the last day of the treatment, rats were anaesthetized with pentobarbital sodium (45 mg/kg bw) 1 h after injecting sildenafil citrate. The experimental protocol was approved by the Institutional Animal Ethics Committee of Manipal University (Vide No.: 245/2005). The heart was removed from the killed rats and part of the left ventricle was used for histopathological analysis and the other part for biochemical investigations.

The part of the tissue for histological analysis was fixed in 10% formalin for 18 h. Following fixation, the specimens were dehydrated, embedded and then sectioned to 5 μm thickness. For histological examinations, sections were stained with Ehrlich hematoxylin and eosin. [18] Multiple slides were examined for each group: At least three slides from different areas of the tissue were examined. Each tissue was examined with a standard light microscope.

Tissue for biochemical estimation was homogenated in 10 ml of phosphate-buffered saline (pH 7.4). The homogenate was tested for nitric oxide (NO) and PDE. NO was estimated by Griess reagent method [19] and PDEwas measured by spectrophotometry. [20],[21]

Data analysis

Results are expressed as means ± standard deviation (SD). Kruskal - Wallis test was performed to measure any differences between the mean values of the different groups. If a difference was found, groups were compared using Wilcoxon signed rank sum test. Probability value (P-value) less than 0.05 was considered significant.


   Results Top


Results of biochemical analysis

NO level

Animals in Group-Ia showed a highly significant (P < 0.05) increase and those in Group-Ib showed a significant (P < 0.01) increase in the level of NO in the heart when compared with their control group (Group-I) [Table 1].
Table 1: NO level in heart in the control and experimental groups


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PDE level

There was a highly significant decrease (P < 0.01) in the level of PDE in the heart of animals in Group-Ia in comparison with the control group, whereas changes in PDE level in Group-Ib animals were not significant when compared with those in the control group animals [Table 1].

Results of histological analysis of heart

Histological evaluation of heart sections from animals of Group-Ia showed a few morphological changes in their microstructure. The nucleus of the muscle fibers in this group was slightly swollen and elongated. In Group-Ib animals the cardiac muscles did not show any morphological changes in their microstructure when compared with the control group animals.


   Discussion Top


Although studies of intact hearts have demonstrated a significant protective effect of sildenafil against necrosis (infarction), there is absolutely no information in the literature regarding the histological changes in cardiomyocytes. After an early case report suggesting that PDE5A inhibitors might increase the risk of heart attack, [22] several studies attemptedto define the cardiac effects of this class of drugs. Results of direct analysis of cardiac effects, which have been obtained in vitro, remain limited and conflicting. It is said that PDE1 is abundant in ventricular myocytes and sildenafil shows far less affinity for other PDE isozymes, including PDE1. [23] There has been a dominant view that PDE5 is not present in the myocardium. [23],[24] Cheitlin et al., [24] stated that PDE5 is not present in cardiomyocytes, instead PDE3 is found and sildenafil is comparatively less potent on this isoenzyme. [24] The present study shows that changes in the level of total PDE were statistically significant at a dosage of 10 mg/kg bw when compared with the 8-mg/kg bw and the control groups. This suggests that as the dosage of sildenafil increases it becomes more potent on PDE in heart, indicating the presence of PDE5. This is in agreement with the results of a few previous studies, which showed that PDE5 is found in specific compartments within myocytes (specifically at z-bands). [17],[25] Cremers et al., [26] found no effect of 10 μM sildenafil (a relatively high dose) on isoprenaline-stimulated function in human papillary muscle strips. PDE5 inhibition has also been shown to suppress and reverse pressure over-load-induced ventricular hypertrophy, [27] attenuate apoptosis [17] and reduce post-ischemic dysfunction [28] in mice, which appear to be critically coupled to the NO synthase. In the present study, at 8 mg/kg bw cardiomyocytes did not show any histological changes, indicating the negligible effect of sildenafil citrate on the heart at a low dosage. However, as dosage increased (10 mg/kg bw) the nucleus of the cardiac muscle became swollen and elongated. Tracqui et al., [29] have described the death of a 56-year-old man associated from an overdose of sildenafil citrate. Histological examination of the ventricular myocardium revealed some areas of patchy fibrosis and moderate hypertrophy of the myocytes.

Fisher et al., [28] hypothesized that the vasodilatory action of sildenafil could potentially release endogenous mediators of preconditioning such as adenosine or bradykinin from endothelial cells, triggering the phosphorylation of NO synthase (NOS) and subsequent release of NO. If the increased production of NO is well in balance with a moderate increase in oxygen radicals then NO will exert beneficial effects. [30] In the present study, although there was significantly high (P < 0.05) level of NO when compared with the control group at a high dosage (10mg/kg bw), histological differences were fewerthan the control group animals [Figure 1]. Therefore it can be concluded that sildenafil citrate at a low dosage does not appear to influence cardiomyocytes. However, NO-induced changes may become detrimental to the cardiac tissue as the dosage of the sildenafil increases.
Figure 1: Photomicrographs of cardiac muscle tissue. Magnification: (H and E, ×40). Control: Animals treated with distilled water. (a) Group-Ia (10 mg/kg bw sildenafil citrate). (b) Group-Ib (8 mg/kg bw sildenafil citrate); N: Nucleus of cardiomyocytes; F: Fibrocytes

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   References Top

1.Wright PJ. Comparison of phosphodiesterase type 5 (PDE5) inhibitors. Int J Clin Pract 2006;60:967-75.  Back to cited text no. 1
    
2.Cirino G, Fusco F, Imbimbo C, Mirone V. Pharmacology of erectile dysfunction in man. Pharmacol Ther 2006;111:400-23.  Back to cited text no. 2
    
3.Burnett AL. Phosphodiesterase 5 mechanisms and therapeutic applications. Am J Cardiol 2005;96:29-31M.  Back to cited text no. 3
    
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13.Kumar P, Francis GS, Tang WH. Phosphodiesterase 5 inhibition in heart failure: Mechanisms and clinical implications. Nat Rev Cardiol 2009;6:349-55.  Back to cited text no. 13
    
14.Enomoto M, Sakaguchi H, Ominami M, Iwai S, Morikawa H, Tamori A, et al. Sildenafil-induced severe cholestatic hepatotoxicity. Am J Gastroenterol 2009;104:254-5.  Back to cited text no. 14
    
15.Aldridge J, Measham F. Sildenafil (Viagra) is used as a recreational drug in England. BMJ 1999;318:669.  Back to cited text no. 15
    
16.Behn D, Potter MJ. Sildenafil-mediated reduction in retinal function in heterozygous mice lacking the gamma-subunit of phosphodiesterase. Invest Ophthalmol Vis Sci 2001;42:523-7.  Back to cited text no. 16
    
17.Das A, Xi L, Kukreja RC. Phosphodiesterase-5 inhibitor sildenafil preconditions adult cardiac myocytes against necrosis and apoptosis. Essential role of NO signaling. J Biol Chem 2005;280:12944-5.  Back to cited text no. 17
    
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19.Ding AH, Nathan CF, Stuehr DJ. Release of reactive nitrogen intermediates and reactive oxygen intermediates from mouse peritoneal macrophages: Comparison of activating cytokines and evidences for independent production. J Immunol 1988;141:2407-12.  Back to cited text no. 19
    
20.Razzell WE, Khorana HG. Studies on polynucleotides. IV. Enzymic degradation. The stepwise action of venom phosphodiesterase on deoxyribo-oligonucleotides. J Biol Chem 1959;234:2114.  Back to cited text no. 20
    
21.Mamillapalli R, Haimovitz R, Ohad M, Shinitzky M. Enhancement and inhibition of snake venom phosphodiesterase activity by lysophospholipids. FEBS Lett 1998;436:256-8.  Back to cited text no. 21
    
22.Feenstra J, van Drie-Pierik RJ, Lacle CF, Stricker BH. Acute myocardial infarction associated with sildenafil. Lancet 1998;352:957-8.  Back to cited text no. 22
    
23.Wallis RM, Corbin JD, Francis SH, Ellis P. Tissue distribution of phosphodiesterase families and the effects of sildenafil on tissue cyclic nucleotides, platelet function, and the contractile responses of trabeculaecarneae and aortic rings in vitro. Am J Cardiol 1999;83:3-12C.  Back to cited text no. 23
    
24.Cheitlin MD, Hutter AM, Brindis RG, Ganz P, Kaul S, Russell RO. Use of sildenafil (Viagra) in patients with cardiovascular disease. Circulation 1999;99:168-77.  Back to cited text no. 24
    
25.Takimoto E, Champion HC, Belardi D, Moslehi J, Mongillo M, Mergia E, et al. cGMP catabolism by phosphodiesterase 5A regulates cardiac adrenergic stimulation by NOS3-dependent mechanism. Circ Res 2005a; 96:100-9.  Back to cited text no. 25
    
26.Cremers B, Scheler M, Maack C, Wendler O, Schafers HJ, Sudkamp M, et al. Effects of sildenafil (Viagra) on human myocardial contractility, in vitro arrhythmias, and tension of internal mammaria arteries and saphenous veins. J Cardiovasc Pharmacol 2003;41:734-43.  Back to cited text no. 26
    
27.Takimoto E, Champion HC, Li M, Belardi D, Ren S, Rodriguez ER, et al. Chronic inhibition of cGMP phosphodiesterase 5A prevents and reverses cardiac hypertrophy. Nat Med 2005;11:214-22.  Back to cited text no. 27
    
28.Fisher PW, Salloum F, Das A, Hyder H, Kukreja RC. Phosphodiesterase-5 inhibition with sildenafil attenuates cardiomyocyte apoptosis and left ventricular dysfunction in a chronic model of doxorubicin cardiotoxicity. Circulation 2005;111:1601-10.  Back to cited text no. 28
    
29.Tracqui A, Miras A, Tabib A, Raul JS, Ludes B, Malicier D. Fatal overdosage with sildenafil citrate (Viagra): First report and review of the literature. Hum Exp Toxicol 2002;21:623-9.  Back to cited text no. 29
    
30.Berges A, Van Nassauw L, Bosmans J, Timmermans JP, Vrints C. Role of nitric oxide and oxidative stress in ischaemic myocardial injury and preconditioning. Acta Cardiol 2003;58:119-32.  Back to cited text no. 30
    


    Figures

  [Figure 1]
 
 
    Tables

  [Table 1]


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