Experimental study of myocardial apoptosis with 99Tcm-FM2 imaging

Objective: To evaluate the value of 99Tcm-C2A domain of synaptotagmin I-glutathione-S-transferase fusion protein (FM2) for the non-invasive assessment of myocardial apoptosis using SPECT. Methods: Recombinant C2A domain of synaptotagmin I was overexpressed in E. Coli, and purified as glutathione-S-transferase, FM2 was produced through this process. FM2 was thiolated with 2-iminothiolane(2-IT) and labeled with 99Tcm. Radiochemical purity was determined with thin layer chromatography. To ensure that the binding activity of 99Tcm-FM2 and Ca2+-dependent phosphatidylserine (PS) was retained, 99Tcm-FM2 was tested by using lyses blood cell membranes in both presence and absence of Ca2+. The radiotracer was tested with ischemia-reperfusion models in 6 pigs. Briefly, occlusion of the left descending coronary artery or circumflex branch or right coronary artery of adult pigs was induced by balloon angioplasty for 20-30 min, then followed by reperfusion. 99Tcm-FM2 was intravenously injected and SPECT images were acquired at 3 h post-injection. Results: 99Tcm-FM2 had good radiochemical purity (>95%); and the binding activity of 99Tcm-FM2 and Ca2+ -dependent PS was well preserved. There was focal uptake of radioactivity at the experimental infarct areas, with target/background (T/B) ratio being 3.36±0.74 in pigs at 3 h post-injection of the radiotracer using SPECT, while T/B ratio was 11.68±4.02 in direct counting of the infarct tissue and normal myocardium. Myocardial apoptosis in infarct areas was confirmed by flow cytometry and electron microscopy. Conclusions: 99Tcm-FM2 binds to apoptotic cells by recognizing exposed PS as a molecular marker. It should be a potential radiotracer for imaging of myocardial apoptosis. (authors)