99mTc-labeled C2A domain of synaptotagmin I as a target-specific molecular probe for noninvasive imaging of acute myocardial infarction.
The exposure of phosphatidylserine (PtdS) is a common molecular marker for both apoptosis and necrosis and enables the simultaneous detection of these distinct modes of cell death. Our aim was to develop a radiotracer based on the PtdS-binding activity of the C2A domain of synaptotagmin I and assess 99mTc-C2A-GST (GST is glutathione S-transferase) using a reperfused acute myocardial infarction (AMI) rat model.


METHODS
The binding of C2A-GST toward apoptosis and necrosis was validated in vitro. After labeling with 99mTc via 2-iminothiolane thiolation, radiochemical purity and radiostability were tested. Pharmacokinetics and biodistribution were studied in healthy rats. The uptake of 99mTc-C2A-GST within the area at risk was quantified by direct gamma-counting, whereas nonspecific accumulation was estimated using inactivated 99mTc-C2A-GST. In vivo planar imaging of AMI in rats was performed on a gamma-camera using a parallel-hole collimator. Radioactivity uptake was investigated by region-of-interest analysis, and postmortem tetrazolium staining versus autoradiography.


RESULTS
Fluorescently labeled and radiolabeled C2A-GST bound both apoptotic and necrotic cells. 99mTc-C2A-GST had a radiochemical purity of >98% and remained stable. After intravenous injection, the uptake in the liver and kidneys was significant. For 99mTc-C2A-GST, radioactivity uptake in the area at risk reached between 2.40 and 2.63 %ID/g (%ID/g is percentage injected dose per gram) within 30 min and remained plateaued for at least 3 h. In comparison, with the inactivated tracer the radioactivity reached 1.06 +/- 0.49 %ID/g at 30 min, followed by washout to 0.52 +/- 0.23 %ID/g. In 7 of 7 rats, the infarct was clearly identifiable as focal uptake in planar images. At 3 h after injection, the infarct-to-lung ratios were 2.48 +/- 0.27, 1.29 +/- 0.09, and 1.46 +/- 0.04 for acute-infarct rats with (99m)Tc-C2A-GST, sham-operated rats with (99m)Tc-C2A-GST, and acute-infarct rats with 99mTc-C2A-GST-NHS (NHS is N-hydroxy succinimide), respectively. The distribution of radioactivity was confirmed by autoradiography and histology.


CONCLUSIONS
The C2A domain of synaptotagmin I labeled with fluorochromes or a radioisotope binds to both apoptotic and necrotic cells. Ex vivo and in vivo data indicate that, because of elevated vascular permeability, both specific binding and passive leakage contribute to the accumulation of the radiotracer in the area at risk. However, the latter component alone is insufficient to achieve detectable target-to-background ratios with in vivo planar imaging.
Zhao
Ming
M
Department of Biophysics, Medical College of Wisconsin, Milwaukee, Wisconsin 53226, USA. mzhao@mcw.edu
Zhu
Xiaoguang
X
Ji
Shundong
S
Zhou
Jundong
J
Ozker
Kutlan S
KS
Fang
Wei
W
Molthen
Robert C
RC
Hellman
Robert S
RS
D000818:Animals; D017209:Apoptosis; D016650:Fluorescein-5-isothiocyanate / Q000494:pharmacology; D005982:Glutathione Transferase / Q000378:metabolism; D006801:Humans; D019169:Jurkat Cells; D008297:Male; D009203:Myocardial Infarction / Q000175:diagnosis* / Q000473:pathology*; D009336:Necrosis; D017434:Protein Structure, Tertiary; D051381:Rats; D017207:Rats, Sprague-Dawley; D015427:Reperfusion Injury; D050863:Synaptotagmin I / Q000737:chemistry*; D013667:Technetium
D050863:Synaptotagmin I; D013667:Technetium; D005982:Glutathione Transferase; D016650:Fluorescein-5-isothiocyanate