Exploring the role of dimethylarginine dimethylaminohydrolase-mediated reduction in tissue asymmetrical dimethylarginine levels in cardio-protective mechanism of ischaemic postconditioning in rats

Document Type: Original Article


1 Department of Pharmaceutical Sciences and Drug Research, Punjabi University, Patiala

2 Department of Pharmacology, Khalsa College of Pharmacy, Amritsar



Objective(s): Reperfusion of ischaemic myocardium results in reduced nitric oxide (NO) biosynthesis by endothelial nitric oxide synthase (eNOS) leading to endothelial dysfunction and subsequent tissue damage. Impaired NO biosynthesis may be partly due to increased levels of asymmetrical dimethylarginine (ADMA), an endogenous inhibitor of eNOS. As dimethylarginine dimethylaminohydrolase (DDAH) is a key enzyme responsible for degradation of ADMA, the present study was designed to explore the role of DDAH/ADMA/NO pathway in cardio-protective mechanism of ischaemic postconditioning.
Materials and Methods: Isolated rat hearts were subjected to myocardial ischaemia for 30 min followed by reperfusion for 2 hours in control group. Myocardial injury was assessed by measurement of infarct size, left ventricular developed pressure (LVDP), lactate dehydrogenase (LDH) and creatine kinase (CK) enzymes in coronary effluents. The reperfused hearts were homogenised and tissue concentration of nitrite, ADMA level and DDAH enzyme activity was determined.
Results: A significant increase in infarct size, LDH, CK release in coronary effluents and ADMA level in myocardial tissue was observed in control group. The increase in tissue ADMA coincided with reductions of NO tissue concentrations and DDAH activity. Ischaemic postconditioning significantly attenuated ischaemia-reperfusion induced myocardial injury manifested in the terms of decreased infarct size, LDH, CK, tissue ADMA along with increase in NO levels and DDAH enzyme activity. Pretreatment with L-Homocysteine (300 µM), a competitive inhibitor of DDAH, and L-NG-nitroarginine methyl ester (L-NAME; 100 µM), an inhibitor of eNOS, completely abolished ischaemic postconditioning-induced myocardial protection.
Conclusion: Enhancing DDAH activity by postconditioning may be a novel target to reduce ADMA level and increase NO bioavailability to prevent myocardial ischaemia-reperfusion injury.


1. Benjamin EJ, Virani SS, Callaway CW, Chang AR, Cheng S, Chiuve SE et al. Heart Disease and Stroke Statistics 2018 update: a report from the American Heart Association. Circulation 2018; 137 (e67–e492).
2. Kristensen SD, Laut KG, Fajadet J, Kaifoszova Z, Kala P, Mario CD et al. Reperfusion therapy for ST elevation acute myocardial infarction 2010/2011: current status in 37 ESC countries. Eur Heart J 2014; 35:1957-1970.
3. Yellon DM and Hausenloy DJ.  Myocardial reperfusion injury. N Engl J Med 2007; 357: 1121−1135.
4. Murry CE, Jennings RB, Reimer KA. Preconditioning with ischemia: a delay of lethal cell injury in ischemic myocardium. Circulation 1986; 74:1124-36.
5. Laskey WK and Beach Dana. Frequency and clinical significance of ischemic preconditioning during percutaneous coronary intervention.J Am Coll Cardiol 2003; 42: 998–1003.
6. Cung TT, Morel O, Cayla G, Rioufol G, Garcia-Dorado D, Angoulvant, Moulin F et al. Cyclosporine before PCI in patients with acute myocardial infarction. N Engl J Med 2015; 373:1021-1031.
7. Zhao ZQ, Corvera JS, Halkos ME, Kerendi F, Wang NP, Guyton RA, Vinten- Johansen J. Inhibition of myocardial injury by ischemic postconditioning during reperfusion: comparison with ischemic preconditioning. Am J Physiol Heart Circ Physiol 2003; 285:579-588.
8. Sorensson P, Saleh N, Bouvier F, Bohm F, Settergren M, Caidahl K, Tornvall P et al. Effect of postconditioning on infarct size in patients with ST elevation myocardial infarction. Heart 2010; 96:1710–1715.
9. Ross AM, Gibbons RJ, Stone GW, Kloner RA, Alexander RW. A randomized, double-blinded, placebo-controlled multicenter trial of adenosine as an adjunct to reperfusion in the treatment of acute myocardial infarction (AMISTAD-II). J Am Coll Cardiol 2005; 45:1775-1780.
10. Garcia-Dorado D, Otaegui I, Rodrı´guez Palomares JF, Evangelista E, Pineda V, Ruiz Salmero´n R et al. Primary results of the PROMISE trial: myocardial protection with intracoronary adenosine given before reperfusion in patients with STEMI. Eur Heart J 2014; 34:669.
11. Woo JS, Kim W, Ha SJ, Kim JB, Kim SJ, Kim WS, Seon HJ, Kim KS. Cardioprotective effects of exenatide in patients with ST-segment-elevation myocardial infarction undergoing primary percutaneous coronary intervention: results of exenatide myocardial protection in revascularization study. Arterioscler Thromb Vasc Biol 2013; 33: 2252–2260.
12. Koyama T, Munakata M, Akima T, et al. Impact of postconditioning with lactate enriched blood on in-hospital outcomes in patients with with ST-segment elevation myocardial infarction. Int J Cardiol 2016; 220:146–148.
13. Tsao PS, and Lefer AM. Time course and mechanism of endothelial dysfunction in isolated ischemic and hypoxic perfused rat hearts. Am J Physiol 1996; 259: H1660-H1666.
14. Stühlinger MC, Conci E, Haubner BJ, Stocker E-M, Schwaighofer J, Cooke JP, Tsao PS, et al. Asymmetric Dimethyl L-Arginine (ADMA) is a critical regulator of myocardial reperfusion injury. Cardiovas Res 2007; 75: 417–425.
15. Tsikas D, Bo¨ger RH, Sandmann J, et al. Endogenous nitric oxide synthase inhibitors areresponsible for the L-arginine paradox. FEBS Lett 2000; 478:1-3.
16. Bae SW, Stuhlinger MC, Yoo HS, Yu KH, Park HK, Choi BY, et al. Plasma asymmetric dimethylarginine concentrations in newly diagnosed patients with acute myocardial infarction or unstable angina pectoris during two weeks of medical treatment. Am J Cardiol 2005; 95:729–733.
17. Gray GA, Patrizio M, Sherry L, Miller LA, Malak M, Alison F. Wallace AF, Leiper JM, Vallance P. Immunolocalisation and activity of DDAH I and II in the heart and modification post-myocardial infarction. Acta histochemica 2010; 112: 413-423.
18. Schlesinger S, Sonntag SR, Lieb W, Maa R. Asymmetric and symmetric dimethylarginine as risk markers for total mortality and cardiovascular outcomes: a systematic reviewand meta-analysis of prospective studies. PLoS ONE2016; 11: e0165811.
19. Hsu C-P, Zhao J-F,  Lin S-J, Shyue S-K, Guo B-C, MS; Lu T, Lee T-S. Asymmetric dimethylarginine limits the efficacy of simvastatin activating endothelial nitric oxide synthase. J Am Heart Assoc 2016; 5:e003327.
20. Blokhin IO, Stevens JW, Murry DJ, Galagudza MM, Shlyakhto EV, Leiper JM, Lentz SR Deficiency of dimethylarginine dimethylaminotransferase-1 exacerbates myocardial ischemia-reperfusion injury in mice. Circulation 2009; 120:S784.
21. He H, Li X, Wang H, Zhang W, Jiang H, Wang S, Yuan L, Liu Y, Liu X. Effects of salvianolic acid A on plasma and tissue dimethylarginine levels in a rat model of myocardial infarction. J Cardiovasc Pharmacol 2013; 61: 482-488.
22. Stühlinger MC, Tsao PS, Her J-H, Kimoto M, Balint RF, Cooke JP. Homocysteine impairs the nitric oxide synthase pathway role of asymmetric dimethylarginine. Circulation 2001; 104: 2569-2575.
23. Hong L and Fast W. Inhibition of human dimethylarginine dimethylaminohydrolase-1 by S-Nitroso-L-homocysteine and hydrogen peroxide analysis, quantification, and implications for hyperhomocysteinemia. J Biol Chem 2007; 282:34684-34692.
24. Langendorff O. Investigations on the surviving mammalian heart. Pflugers Arch fur die Gesante Physiologie des Menschen und der Tiere 1895; 61: 291–332
25. Fishbein M C, Meerbaum S, Rit J. Early phase acute myocardial infarct size quantification, validation of triphenyltetrazolium chloride tissue enzyme staining technique. Am Heart J 1981; 101: 593-600.
26. King J. A routine method for the estimation of lactic dehydrogenase activity. J Med Lab Tech1959; 16: 265–272.
27. Huges BP. A method for the estimation of serum creatine kinase and its use in comparing creatine kinase and aldolase activity in normal and pathological sera. Clinica Chimica Acta 1962; 7 Suppl 5: 597-603.
28. Green LC, Wagner DA, Godowsky J, Skipper PL, Wishnok JS, Tannenbaum SR. Analysis of nitrate, nitrite and [15N] nitrate in biological fluids. Anal. Biochem1982; 126:131-138.
29. Jeddi S, Zaman J, Ghasemi A. Effects of ischemic postconditioning on the hemodynamic  parameters and heart nitric oxide levels of hypothyroid rats. Arq Bras Cardiol 2015; 104:136-143.
30. Trocha M,  Merwid-L A,  Sozañski T, Chlebda-Sieragowska E, Szuba A, Dziêgiel P et al. Influence of ezetimibe on ADMA-DDAH-NO pathway in rat liver subjected to partial ischemia followed by global reperfusion. Pharmacological Reports 2013; 65: 122-133.
31. Bell RM, Mocanu MM, Yellon DM. Retrograde heart perfusion: the Langendorff technique of isolated heart perfusion. J Mol Cell Cardiol 2011; 50:940-950.
32. Sigg DC and Iaizzo PA. In vivo versus In vitro comparison of swine cardiac performance: induction of cardiodepression with halothane. Eur J Pharmacol 2006; 543:97–107.
33. Sutherland FJ, Hearse DJ. The isolated blood and perfusion fluid perfused heart. Pharmacol Res 2000;  41:613–627
34. Hill AJ, Coles JA Jr, Sigg DC, Laske TG, Iaizzo PA. Images of the human coronary sinus ostium obtained from isolated working hearts. Ann Thorac Surg 2003; 76:2108.
35. Banka N, Anand IS, Sharma PL, Wahi PL. Bull. P.G.I. 1981; 15: 147-150.
36. Domanski MJ, Mahaffey K, Hasselblad V. et al. Association of myocardial enzyme elevation and survival following coronary artery bypass graft surgery. J Am Med Ass 2011; 305: 585–591.
37. Jaggi AS, Singh M, Sharma A, Singh D, Singh N. Cardioprotective effects of mast cell modulators in ischemia-reperfusion-induced injury in rats. Methods Find Exp Clin Pharmacol 2007; 29: 593-600.
38. Kaur K, Singh M, Singh N, Jaggi AS. Possible mechanism of rottlerin induced modulation of ischemia reperfusion injury in isolated rat hearts. Biol Pharm Bull 2008; 31:1745-1748.
39. Zhao ZQ, Corvera JS, Halkos ME, Kerendi F, Wang NP, Guyton RA, Vinten- Johansen J Inhibition of myocardial injury by ischemic postconditioning during reperfusion: comparison with ischemic preconditioning. Am J Physiol Heart Circ Physiol 2003; 285:579-588.
40. Cardounel AJ, Cui H, Samouilov A, Johnson W, Kearn P, Tsai AL, Berka V, Zweier JL Evidence for the pathophysiological role of endogenous methylarginines in regulation of endothelial NO production and vascular function. J Biol Chem 2007; 282: 879-887.
41. Leiper J, Murray-Rust J, McDonald N, Vallance P.  S-nitrosylation of dimethylarginine dimethylaminohydrolase regulates enzyme activity: further interactions between nitric oxide synthase and dimethylarginine dimethylaminohydrolase. Proc Natl Acad Sci 2002; USA 99, 13527-13532.
42. Lin KY, Ito A, Asagami T, Tsao PS, Adimoolam S, Kimoto M, Tsuji H et al. Impaired nitric oxide synthase pathway in diabetes mellitus: role of asymmetric dimethylarginine and dimethylarginine dimethylaminohydrolase. Circulation 2002; 106:987-992.
43. Xiong Y, Fu YF, Fu SH, Zhou HH Elevated levels of the serum endogenous inhibitor of nitric oxide synthase and metabolic control in rats with streptozotocin-induced diabetes. J Cardiovasc Pharmacol 2003; 42:191-196.
44. Stuhlinger MC, Tsao PS, Her JH, Kimoto M, Balint RF, Cooke JP. Homocysteine impairs the nitric oxide synthase pathway: role of asymmetric dimethylarginine. Circulation. 2001; 104: 2569-2575.
45. Trocha M, Merwid-Ląd A, Szuba A, Sozański T, Magdalan J, Szeląg A, Kopacz A et al. Effect of Quercetin-5’-sulfonic Acid Sodium Salt on SOD Activity and ADMA/DDAH Pathway in Extracorporeal Liver Perfusion in Rats. Adv Clin Exp Med. 2012; 21: 423-431.
46. Shenoy V, Veena Mehendale V, Prabhu K,  Shetty R, and  Rao P. Correlation of Serum Homocysteine Levels with the Severity of Coronary Artery Disease. Indian J Clin Biochem. 2014; 29: 339-344.
47. Mahalle N, Kulkarni MV, Garg M K, Naik S S. Vitamin B12 deficiency and hyperhomocysteinemia as correlates of cardiovascular risk factors in Indian subjects with coronary artery disease. J Cardiol. 2013; 61: 289-294.
48. Patel JD, Chakrabarti C. Homocysteine, folate and vitamin B12 status in patients with coronary artery disease in young age. Int J Clin Biochem and Res. 2017; 4:129-135
49. Melhem A, Desai A and Hofmann MA. Case report: Acute myocardial infarction and pulmonary embolism in a young man with pernicious anemia-induced severe hyperhomocysteinemia. Thromb J. 2009, 7:5.
50. Jiang X, Yang F, Tan H, et al. Hyperhomocystinemia impairs endothelial function and eNOS activity via PKC activation. Arterioscler Thromb Vasc Biol. 2005; 25: 2515-2521.