1. 1 In Vivo Detection of Cell Death in the Area at Risk in Acute Myocardial Infarction 報告者 : 實習醫師 唐之航 指導者 : 住院醫師 謝宏仁 J Nucl Med. 2003 Mar; 44(3): 391-6 J Nucl Med. 2003 Mar; 44(3): 391-6

2. 2 Apoptosis / Programmed Cell Death Apoptosis, a Greek word that describes the process of leaves falling from trees or petals falling from flowers Coined by Kerr and colleagues in 1972 to describe a form of cell death exhibiting a distinct set of morphological features Cell death that is a normal part of the life of a multicellular organism

3. 3 Characteristics of Apoptosis Chromatin condensation Fragmentation of DNA into oligonucleosomal fragments Membrane blebbing and “boiling” (zeiosis) Loss of membrane asymmetry leading to the exposure of phosphatidylserine (PS) Induced by stress or death signals (Fas ligand) Activate caspase pathway

4. 4 Measurements of Apoptosis Annexin V assay: externalization of phosphatidylserine (PS) Electron microscopy: apoptotic cells Immunohistochemistry: detection of caspase cleavage in tissue sections TUNEL [terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick- end labeling] assay: detection of DNA fragmentation

5. 5 PS Externalization and Annexin A5 Imaging Anionic phospholipids such as PS and phosphatidyl ethanolamine are actively recruited to the inner leaflet of the lipid bilayer by “translocase”. “Floppase” actively pumps cationic phospholipids such as phosphatidyl choline and sphingomyelin to the outer leafle. During apoptosis, translocase and floppase enzymes are deactivated and the asymmetric pattern of distribution is lost. The random bidirectional distribution is further exaggerated by activation of “scramblase” enzyme. PS is then expressed on the cell membraneand detected by annexin A5.

6. 6 Externalization of PS During Apoptosis Blankenberg et al., Nucl Med Comm 21 (3), 241-50 ↑Cationic PC, sphigomyelin Anionic PS, P ethanolamine↓

7. 7 Van Engeland et al., Cytometry 31 (1), 1-9 Van Engeland et al., Cytometry 31 (1), 1-9

8. 8 ApoptosisNecrosis Patterns of death Single cells Groups of neighboring cells Cell size Shrinkage Fragmentation Swelling Plasma membrane Preserved continuity Blebbed Phosphatidylserine on surface Smoothing Early lysis Nuclei Chromatin: Clumps & Fragmented Membrane disruption Organelle shape Contracted "Apoptotic bodies“ Swelling Disruption

9. 9 ApoptosisNecrosis Mitochondria Increased membrane permeability Contents released into cytoplasm Cytochrome c; Apaf1 Structure relatively preserved Swelling Disordered structure DNA degradation Fragmented Internucleosomal cleavage Free 3' ends Laddering on electrophoresis DNA appears in cytoplasm Diffuse & Random Cell degradation Phagocytosis No inflammation Inflammation Macrophage invasion

10. 10 Introduction Previous study showed the presence of programmed cell death (PCD) in the hearts of animals and humans after acute myocardial infarction (AMI) by detecting PS with annexin A5 on necropsy. 99m Tc-4,5-bis(thioacetamido)pentanoyl-annexin A5 (BTAP-anxA5) (Apomate, Theseus Imaging Corp., Cambridge, MA) was used to monitor the cell death in human hearts noninvasively and reconstruction of oblique images as commonly done in cardiac perfusion studies.

11. 11 Study Aim To demonstrate the area at risk in the acute phase of MI using 99m Tc-sestamibi (MIBI) To localize uptake of 99m Tc-annexin A5 in vivo in the area at risk using 99m Tc-BTAP-anxA5 To study To study 99m Tc-annexin A5 activities in the subacute phase of AMI To determined the size of 99m Tc-MIBI defects in acute and subacute phases of AMI

12. 12 Materials and Methods Nine patients (6 male, 3 female; mean age, 61 y; range, 42–77 y) with primary AMI who presented within 6 h of the onset of symptoms were included in this study. All patients underwent percutaneous transluminal coronary angioplasty (PTCA) of the infarct-related vessels (primary PTCA: 4; rescue PTCA: 5). In 2 patients, the 99m Tc-annexin A5 study was repeated in the subacute phase.

13. 13 Thimister et al., J Nucl Med. 44 (3), 391-6 Time Line of the Study Protocol

14. 14 Patient Characteristics Thimister et al., J Nucl Med. 44 (3), 391-6

15. 15 Results-1 All patients showed decreased uptake of 99m Tc-MIBI in an area corresponding to the location of the infarct as diagnosed on electrocardiography but an increased uptake of 99m Tc-annexin A5 was seen indicating the presence of cardiomyocytes with externalized PS in the infarct area. Best images were obtained 15 h after the infusion, showing 99m Tc-annexin A5 activity at the infarct site and little 99m Tc-MIBI rest activity (“MIBI ghost”). SPECT reconstruction was difficult in the acquisitions 20 h after the injection because of advanced decay of 99m Tc-MIBI and 99m Tc-annexin A5 activity.

16. 16 Thimister et al., J Nucl Med. 44 (3), 391-6

17. 17 Results-2 99m Tc-MIBI imaging 5–19 d after onset of the MI (n=7) still showed a defect, although much smaller, corresponding to the area of increased 99m Tc-annexin A5 uptake and no sign of ischemia. Quantification on 99m Tc-MIBI SPECT pictures: all patients on whom 99m Tc-MIBI in both the acute and the subacute phase was performed (n=6) showed decreased 99m Tc-MIBI defects on the subacute phase images when compared with the acute phase of 12% on average.

18. 18 Thimister et al., J Nucl Med. 44 (3), 391-6

19. 19 Results-3 99m Tc-annexin A5 uptake was reduced on the 4- d images when compared with the initial images. After 8 d, no 99m Tc-annexin A5 activity was present at the infarct site. 201 Tl scintigraphy performed 1 mo after the acute MI showed complete normalization of the affected area (i.e., reversible lesions). There was no 99m Tc-annexin A5 activity present in the myocardium of the patient studied 3 mo after the MI onset.

20. 20 Thimister et al., J Nucl Med. 44 (3), 391-6

21. 21 Discussion-1 Apoptosis in AMI may cause Apoptosis in AMI may cause cardiomyocyte dysfunction and irreversible cell loss which can lead to the decline in left ventricular contraction. Better understanding of the cellular apoptotic pathway may lead to the development of new therapeutic drugs that might limit this cardiomyocyte loss. Animal studies have shown that increased 99m Tc- annexin A5 uptake in transplanted hearts of rats correlated well with the in vitro detection of apoptosis using annexin A5 immunofluorescence microscopy. Animal studies also indicated that ischemia of the heart, followed by reperfusion, induces reperfusion injury with a substantial loss of cardiomyocytes through apoptosis.

22. 22 Discussion-2 Combining 99m Tc-MIBI and 99m Tc-annexin A5 images could well define the area at risk on the MIBI pictures. Because of the storage of 99m Tc-MIBI in the mitochondrion of cardiomyocytes (and lack of redistribution), imaging could be postponed until after revascularization was achieved, which is advantageous to the use of 201 Tl-chloride for the detection of cardiovascular perfusion defects. A MIBI image of the heart could still be seen vaguely (“ 99m Tc-MIBI ghost-image”) in the unaffected myocardial area on the 99m Tc-annexin A5 scintigraphy. This technique allowed better SPECT reconstruction and much better localization of the 99m Tc-annexin A5 activity in the heart.

23. 23 Discussion-3 Necrosis features were also reported in the Necrosis features were also reported in the annexin A5–positive cardiomyocytes More necrosis than apoptosis? The decreased 99m Tc-MIBI defect in the subacute phase, when compared with the acute phase of the MI, strongly suggests that at least part of the myocardial 99m Tc-annexin A5 activity as present in the acute phase represent potentially reversible myocardial cell damage rather than necrosis. Cardiac biopsy is more straightforward but not ethically acceptable.

24. 24 Narula et al., J Nucl Med. 44 (3), 397-9

25. 25 Discussion-4 Decreased 99m Tc-annexin A5 activity in a patient who was repeatedly studied 3 and 8 d after the MI onset. Because no activity remained after 8 d, regeneration of myocardial cells might have taken place, in which case PS expression is no longer present. This would imply either that the ischemic part of the MI has been restored to viable tissue or that cells have been removed as necrotic material.

26. 26 Discussion-5 The main clinical utility of this in vivo detection of cell death with 99m Tc-annexin A5 scintigraphy would be the evaluation of new therapeutic strategies that intervene in myocardial cell death, namely, cell death inhibitors. The application of this methodology may be extended to any situation of progressive myocardial dysfunction. Performing 99m Tc-annexin A5 scintigraphy will soon be possible to provide more information about the kinetics of the cell death process in different clinical forms of myocardial disease.

27. 27 Conclusion Apoptosis can be detected in vivo at the infarct area. The area at risk can be well defined with this protocol. The decrease in 99m Tc-MIBI defect size in the subacute phase of the MI further suggests that in parts of the area at risk, reversible myocardial damage rather than necrosis is present in cardiomyocytes.

28. 28 Future Studies Can ischemia be subsumed within the spectrum of apoptosis? Could annexin-positive myocytes constitute an indication for intervention? What is the temporal window for treating PS- expressing ischemic cells? Is it possible to develop a model of annexin uptake vis-a`-vis the extent of troponin leakage as an index of reversibility? Can inhibitors of apoptosis abrogate ischemic injury beyond reperfusion?

29. 29 Prospects If annexin positivity identifies a significant fraction of salvageable myocytes, we can expect a major shift in the management of acute coronary syndromes, somewhat similar to the one that began 2 decades ago with the advent of thrombolytic therapy.

30. 30 Thank You for Your Attention!