Annexin A5 Uptake in Ischemic Myocardium: Demonstration of Reversible Phosphatidylserine Externalization and Feasibility of Radionuclide Imaging

Abstract

Ischemic insult to the myocardium is associated with cardiomyocyte apoptosis. Because apoptotic cell death is characterized by phosphatidylserine externalization on cell membrane and annexin-A5 (AA5) avidly binds to phosphatidylserine, we hypothesized that radiolabeled AA5 should be able to identify the regions of myocardial ischemia. Methods: Models of brief myocardial ischemia by the occlusion of the coronary artery for 10 min (I-10) and reperfusion for 180 min (R-180) for the detection of phosphatidylserine exteriorization using 99m Tc-labeled AA5 and g-imaging were produced in rabbits. 99m Tc-AA5 uptake after brief ischemia was compared with an I-40/R-180 infarct model. Histologic characterization of both myocardial necrosis and apoptosis was performed in ischemia and infarct models. Phosphatidylserine exteriorization was also studied in a mouse model, and the dynamics and kinetics of phosphatidylserine exposure were assessed using unlabeled recombinant AA5 and AA5 labeled with biotin, Oregon Green, or Alexa 568. Appropriate controls were established. Results: Phosphatidylserine exposure after ischemia in the rabbit heart could be detected by radionuclide imaging with 99m Tc-AA5. Pathologic characterization of the explanted rabbit hearts did not show apoptosis or necrosis. Homogenization and ultracentrifugation of the ischemic myocardial tissue from rabbit hearts recovered two thirds of the radiolabeled AA5 from the cytoplasmic compartment. Murine experiments demonstrated that the cardiomyocytes expressed phosphatidylserine on their cell surface after an ischemic insult of 5 min. Phosphatidylserine exposure occurred continuously for at least 6 h after solitary ischemic insult. AA5 targeted the exposed phosphatidylserine on cardiomyocytes; AA5 was internalized into cytoplasmic vesicles within 10-30 min. Twenty-four hours after ischemia, cardiomyocytes with internalized AA5 had restored phosphatidylserine asymmetry of the sarcolemma, and no detectable phosphatidylserine remained on the cell surface. The preadministration of a pan-caspase inhibitor, zVAD-fmk, prevented phosphatidylserine exposure after ischemia. Conclusions: After a single episode of ischemia, cardiomyocytes express phosphatidylserine, which is amenable to targeting by AA5, for at least 6 h. Phosphatidylserine exposure is transient and internalized in cytoplasmic vesicles after AA5 binding, indicating the reversibility of the apoptotic process.

Cell surface-expressed phosphatidylserine as therapeutic target to enhance phagocytosis of apoptotic cells

“…We showed previously that murine cardiomyocytes exposed to ischemic/reperfusion stress externalized and internalized PS continuously during a period of more than 60 min. 22 RGD-anxA5-fluorescein and RGT-anxA5-alexa568 stained the same cardiomyocytes in the area at risk if administered intravenously ( Figure 4). No uptake of RGD-anxA5 and RGT-anxA5 was observed in control mouse hearts (data not shown) and RGD-M1234 failed to Figure 4 (a-c) Ex vivo images of sections of a mouse heart that was exposed to 30 min ischemia and 24 h of reperfusion.…”

The Extent of Coronary Atherosclerosis Is Associated With Increasing Circulating Levels of High Sensitive Cardiac Troponin T

“…7 It is unlikely that only minutes of ischemia would have resulted in myocardial cell death in the patients exhibiting significant ischemia. Recent work 25 of our group (unpublished data, 2009) demonstrated that 5-minute episodes of cardiac ischemia in a mouse model, followed by reperfusion, results in exposure of phosphatidylserine to the surface of cardiomyocytes and activation of caspase-3, both of which are indicative of apoptosis. 26 However, no demonstration of cardiomyocyte cell death was made.…”

Cardiac Biomarkers and Noninvasive Predictors of Atherosclerosis in Chronic Peritoneal Dialysis Patients

“…Traditionally it was thought that the release of cTnT is equivalent to myocardial necrosis; however, some recent animal studies have suggested that short episodes of ischemia may result in the release of cTnT, without demonstration of cell death [30]. These studies also suggested that elevated hs-cTnT levels observed in patients with coronary artery disease may be the result of activation of caspase-3 within cardiac myocytes and the resulting cleavage and release of cTnT, but they do not necessarily implicate myocardial cell death [31,32,33]. Atherosclerosis-related decreased coronary perfusion may cause the activation of caspase-3 and release of cTnT into the circulation.…”