Heider SH. Qassam MSc. PH. & TH.

1. Heider SH. QassamMSc. PH. & TH. MK-886 and DITPA Attenuate Global Myocardial Ischemia Reperfusion Injury after Heterotopic Heart Transplantation in Male Rats

2. Introduction

3. Heart transplantation • It is a widely accepted therapy for most patients under 65 years of age with advanced heart failure who remain symptomatic with the expectation of high intermediate term mortality, despite optimal heart failure medications.

4. Heterotopic Heart Transplantation • The phrase `heterotopic' describes placing the heart in an ectopic position without removing the native heart. The heterotopic technique has been used in experimental transplantation. • It can be categorized into`working' and `nonworking' models. • It has several advantages , including technical simplicity, better accessibility for biopsies, and survival of the recipient even in case of graft rejection.

5. Transplantation–Induced (I/R) Injury • Organ transplantation is a unique situation where grafts are successively subjected to global cold ischemia, warm ischemia, and blood reperfusion. • These events are believed to impair graft function. • The pathophysiology of (I/R) injury shows several characteristics of inflammatory responses including activation of complement, endothelial cells, infiltration of neutrophils, the release of oxygen–derived free radicals, and cytokines.

6. Pathogenesis of Myocardial (I/R) Injury • Ion accumulation • Intracellular calcium overload. • Increased intracellular sodium. • Decrease in pH with rapid normalization upon reperfusion.

7. Dissipation of mitochondrial membrane potential • MPTP • Free radical formation/ROS • Generation from xanthine oxidase. • Release of reactive mitochondrial intermediates. • Neutrophil infiltration.

8. NO metabolism • Loss of NO-vasodilation. • Accumulation of reactive peroxynitrite. • Apoptosis • Endothelial dysfunction • Cytokine and chemokine signaling. • Expression of cellular adhesion markers. • Impaired vasodilation.

9. Immune activation • Innate immunity (e.g., complement activation, expression of Toll-like receptors) • Neutrophil accumulation • Cell-mediated damage (macrophage and T cell)

10. Proinflammatory Mediators inMyocardial (I/R) Injury • Tumour necrosis factor alpha • It is a pleiotropic cytokine that has many proinflammatory actions with negative inotropic effects. • It is produced by a host in response to inflammation, tissue injury and has recently been recognized as a myocardial depressant substance. • TNF-α is released from macrophages, monocytes and mast cells within minutes in response to myocardial I/R injury.

11. Interleukine-1 Beta • IL-1β is a proinflammatory cytokine produced by activated macrophages and monocytes. It functions in the generation of systemic and local responses to infection, injury, and is the primary cause of inflammation. • It has functions similar to TNF-α such as induction of chemokine production and expression of adhesion molecules by endothelial cells. • IL-1β plays a crucial role in initiating inflammatory responses and in recruitment and activation neutrophils to inflammation sites.

12. Intercellular Adhesion Molecule (ICAM-1) • It is a member of the immunoglobulin superfamily and is critical for the firm arrest and transmigration of leukocytes out of blood vessels and into tissues. • ICAM-1 plays an important role in the early stages of cardiac damage following ischemia–reperfusion injury. In this process, ICAM-1-mediated leukocyte adhesion and subsequent infiltration into the infarct area could be responsible for myocyte damage via released free radicals.

13. Cardiac Troponin I • Troponin I is a contractile protein " Part of the thin filament regulatory complex that confers calcium sensitivity to the ATP activity of the striated muscle actin-myosin complex". • cTnI was found exclusively in cardiac muscle and thus making it a specific marker for myocardial injury.

14. MK-886 • It is a highly potent inhibitor of leukotriene formation. • This compound inhibits leukotriene biosynthesis indirectly by a mechanism through the binding of a membrane bound (FLAP), thereby inhibiting the translocation and activation of 5-lipoxygenase which is catalyses the conversion of arachidonic acid to (LTA4).

15. It was found to be a potent and specific inhibitor of both LTB4 and LTC4 synthesis in human phagocytes and moderately potent PPARα antagonist. • It was also found to prevents postischemic leukotriene accumulation after I/R .

16. DITPA • It is a TH analog with positive inotropic activity but minimal effect on heart rate or metabolic rate compared with thyroid hormone itself. • DITPA can promote angiogenesis by interacting with membrane-bound integrin αVβ3 and activating the MAPK cascade. • DITPA attenuates the acute inflammatory response and reduces myocardial infarct size following myocardial ischemia .

17. DITPA enhances endothelial NO-mediated vasorelaxation. • DITPA prevent abnormalsarcoplasmic reticulum calcium transport and abnormal contractile function associated with MI.

18. Aim of the Study • this study was undertaken to assess the possible protective effect of MK-886 and DITPA against global myocardial I/R injury after heart transplantation via interfering with inflammatory pathways.

19. Materials and Methods

20. Animals and Study Design The rats were randomized into six groups as follow • Sham group:rats underwent the same anesthetic and surgical procedures but with no HHT . • Control group:rats underwent 30 min of global myocardial ischemia followed by 60 min of reperfusion via HHT.

21. Control vehicle (1) group: Donors rats received vehicle of MK-886 30 min before HHT, and the same dose was repeated for recipients upon reperfusion. • Control vehicle (2) group:Donors and recipients ratspretreated with DITPA vehicle for 7days before HHT. • MK-886 treated group: Donors rats received MK-886 (0.6 mg/kg) i.p. 30 min before HHT , and the same dose was repeated for recipients upon reperfusion. • DITPA treated group:Donors and recipients ratspretreated with DITPA(3.75 mg/kg) s.c. for 7days before HHT.

22. Methods Overview Isolate donor heart Prepare recipient animals Heart transplantation • Bilateral thoractomy was made, and interior chest wall was opend. • All the vessels, except the aorta and pulmonary artery, were ligated • The heart removed & stored in cold lactated ringer solution. • The right CA was exposed and then occluded with clamp. • Distal portion of CA was ligated • CA was then incised and passed through cuff. • REJV was prepared the in same way. • The donor heart was placed in the right neck of the recipient. • The arterial cuff was inserted into donor aorta and fixed with a ligature. • IV cuff was inserted into the donor PA and fixed with ligature.

23. Isolated donor heart Heart transplantation Operation Prepare recipient rats

24. Preparation of Samples • Blood Sampling for measurement of plasma cTnI • At the end of experiment, about 2 ml of blood was collected from the heart and placed in a tube containing Na2EDTA and used for determination plasma cTnI. • Tissue Sampling for Histopathology • The apical part of the heart was fixed in 10% formalin to be used for histopathological evaluation

25. Tissue preparation for TNF-α, IL-1β and ICAM-1 measurement • 10% homogenates of heart tissue were prepared in PBS that contained 1% Triton X-100 and a protease inhibitor cocktail. • The homogenate was centrifuged at 2,500 g for 20 min at 4°C. The supernatant was collected for determination of TNF-α, IL-1β and ICAM-1.

26. Histopathological Evaluation of Rat Heart • The scoring system for histological findings was divided into 4 scores: • Score 0, no damage. • Score 1, interstitial edema and focal necrosis. • Score 2, diffuse myocardial cell swelling and necrosis. • Score 3, necrosis with the presence of contraction bands, neutrophil infiltration and the capillaries were compressed. • Score 4, widespread necrosis with the presence of contraction bands, neutrophil infiltration, capillaries compressing and hemorrhage.

27. Statistical Analysis • ANOVA testwas used for the multiple comparison among all groups followed by post-hoc tests using LSD method. • Mann-Whitney and Krusakl-Wallis tests were used to assess the statistical significance of histopathological parameters. • Pearson correlation coefficientwas used to assess the associations between two quantitative variable. • Spearman correlationcoefficient was used to assess the associations between two quantitative variable when one of them was non-normally distributed.

28. Results

29. This result is consistent with Gurevitchet al. (1996) were the first to demonstrate a significant release of TNF-α in the rat coronary effluent at 1 min after reperfusion. * †,* Ѱ,* Figure (6): The mean of cardiac TNF-α level (pg/ml) in the six experimental groups at the end of the experiment. * vs. sham group, ѱ vs. control vehicle (1) group, †vs. control vehicle (2) group. The data expressed as mean ±SEM (p<0.05)

30. This result is consistent with Herskowitz et al . (1995) demonstrated induced myocardial gene expression of IL-1β after permanent LAD occlusion and temporary LAD occlusion followed by reperfusion. * †,* Ѱ,* Figure (7): The mean of cardiac IL-1β level (pg/ml) in the six experimental groups at the end of the experiment. *vs. sham group, ѱvs. control vehicle (1) group, †vs. control vehicle (2) group. The data expressed as mean ±SEM (p<0.05)

31. This result is in agreement with Kukielkaet al. (1993)) were reported that ICAM-1 gene expression increased in reperfused myocardium after 1 hr coronary occlusion and 1 hr reperfusion * Ѱ,* †,* Figure (8): The mean of cardiac ICAM-1 level (pg/ml) in the six experimental groups at the end of the experiment *vs. sham group,ѱ vs. control vehicle (1) group,†vs. control vehicle (2) group. The data expressed as mean ±SEM (p<0.05)

32. This result is consistent with Bertinchantet al. (1999) ) showed that cTnI released in 1 min during 60 min of reperfusion after 20 min, 30 min, 40 min or 60 min of global ischemiaby using Langendorff-perfused rat hearts model. * †,* Ѱ,* Figure (9): The mean of plasma (cTnI) level (ng/ml) in the six experimental groups at the end of the experiment *vs. sham group, ѱvs. control vehicle (1) group, †vs. control vehicle (2) group. The data expressed as mean ±SEM (p<0.05)

33. This result is cosistent with zingarelli et al. (2002) showed that a marked disruption of the myocardial structure in I/R injury was characterized by appearance of extensive necrosis and contraction bands. * †,* Ѱ,* Figure (11): Error bar chart shows the difference in mean ±SEM values of total severity scores in the six experimental groups. *vs. sham group, ѱvs. control vehicle (1) group, †vs. control vehicle (2) group. The data expressed as mean ±SEM (p<0.05)

34. Figure (12 ): Photomicrograph of cardiac section of normal rats shows the normal architecture. The section stained with Haematoxylin and Eosin (X 40).

35. Figure (13): Photomicrograph of cardiac section showed interstitial edema and focal necrosis. The section stained with Haematoxylin and Eosin (X 40).

36. Figure (14): Photomicrograph of cardiac section showed neutrophil infiltration (arrows). The section stained with Haematoxylin and Eosin (X 40).

37. Figure (15): Photomicrograph of cardiac section showed capillary compressing. The section stained with Haematoxylin and Eosin (X 40).

38. Figure (16 ): Photomicrograph of cardiac section showed contraction bands. The section stained with Haematoxylin and Eosin (X 40).

39. Figure (17): Photomicrograph of cardiac section showed hemorrhage. The section stained with Haematoxylin and Eosin (X 40).

40. Figure (18 ): Photomicrograph of cardiac section in MK-886 treated group. The section stained with Haematoxylin and Eosin (X 40). Figure (19): Photomicrograph of cardiac section in DITPA treated group. The section stained with Haematoxylin and Eosin (X 40).

41. Conclusions • The findings of this study suggest the followings: • Both MK-886 and DITPA ameliorate myocardial injury associated with HT by reducing inflammatory mediators and adhesion molecule. This may give an evidence for their protective effect. • Both MK-886 and DITPA reduce cTnIassociated with MI/R injury induced by HT which is a specific marker for cardiac injury. • This study supports the hypothesis that inflammatory pathways are involved in global MI/R injury induced by HT.

42. Recommendations we recommend the following: • These findings have put MK-886 and DITPA as a future therapeutic agents in situations including global MI/R injury following heart transplantation through their interfering with inflammatory pathways. • Measuring of cardiac myeloperoxidase activity to estimate tissue PMNs accumulation in inflamed hearts. • Measuring of infarct size to evaluate the cytoprotection of drugs used in this study and this is done by (TTC) staining. • Measuring of LTB4 and LTC4 in the heart to further support their role in the pathogenesis of MI/R injury.

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