Author + information
- Received August 23, 2017
- Revision received November 28, 2017
- Accepted November 29, 2017
- Published online December 3, 2018.
- Hideki Kawai, MD, PhDa,∗,
- Farhan Chaudhry, MSa,∗,
- Aditya Shekhara,
- Artiom Petrov, PhDa,∗∗ (, )
- Takehiro Nakahara, MD, PhDa,
- Takashi Tanimoto, MD, PhDa,
- Dongbin Kim, MDa,
- Jiqiu Chen, MDa,
- Djamel Lebeche, PhDa,
- Francis G. Blankenberg, PhDb,
- Koon Y. Pak, PhDc,
- Frank D. Kolodgie, PhDd,
- Renu Virmani, MDd,
- Partho Sengupta, MDa,
- Navneet Narula, MDe,
- Roger J. Hajjar, MDa,
- Harry W. Strauss, MDa and
- Jagat Narula, MD, PhDa,∗ ()
- aIcahn School of Medicine at Mount Sinai, New York, New York
- bLucile Salter Packard Children’s Hospital, Stanford, California
- cMolecular Targeting Technologies, Inc., West Chester, Pennsylvania
- dCardiovascular Pathology Institute, Inc., Gaithersburg, Maryland
- eWeill-Cornell Medical College, New York, New York
- ↵∗Address for correspondence:
Dr. Jagat Narula, Mount Sinai Heart, One Gustave L. Levy Place, Guggenheim Pavilion 1-West, 1190 Fifth Avenue, New York, New York 10029.
- ↵∗∗Dr. Artiom Petrov, Mount Sinai Heart, One Gustave L. Levy Place, Guggenheim Pavilion 1-West, 1190 Fifth Avenue, New York, New York 10029.
Objectives The purpose of this study was to evaluate the feasibility of imaging apoptosis in experimental ischemia-reperfusion model by technetium-99m (99mTc)-labeled Duramycin, and compare it to an established tracer, 99mTc-labeled Annexin-V, which has a relative disadvantage of high radiation burden to nontarget organs.
Background During apoptosis, the cell membrane phospholipids-phosphatidylserine (PS) and phosphatidylethanolamine (PE) are exposed and can be targeted by Annexin-V and Duramycin, respectively, for in vivo imaging. Identification of a reversible cell death process should permit therapeutic intervention to help reduce myocyte loss and left ventricle dysfunction.
Methods In a 40-min left coronary artery ischemia-reperfusion model in 17 rabbits, 7 mCi of 99mTc-labeled Duramycin (n = 10), 99mTc-linear Duramycin (a negative tracer control; n = 3), or 99mTc-Annexin-V (a positive tracer-control; n = 4) were intravenously administered 30 min after reperfusion. Of the 10 Duramycin group animals, 4 animals were treated with an antiapoptotic agent, minocycline at the time of reperfusion. In vivo and ex vivo micro–single-photon emission computed tomography (μSPECT) and micro-computed tomography (μCT) imaging was performed 3 h after reperfusion, followed by quantitative assessment of tracer uptake and pathological characterization. Fluorescent Duramycin and Annexin-V were injected in 4 rats to visualize colocalization in infarct areas in a 40-min left coronary artery occlusion and 30-min reperfusion model.
Results Intense uptake of Duramycin and Annexin-V was observed in the apical (infarcted) areas. The percent injected dose per gram uptake of Duramycin in apical region (0.751 ± 0.262%) was significantly higher than remote area in same animals (0.045 ± 0.029%; p < 0.01). Duramycin uptake was insignificantly lower than Annexin-V uptake (1.23 ± 0.304%; p > 0.01) but demonstrated substantially lower radiation burden to kidneys (0.358 ± 0.210% vs. 1.58 ± 0.316%, respectively; p < 0.001). Fluorescence studies with Duramycin and Annexin V showed colocalization in infarct areas. Minocycline treatment substantially resolved Duramycin uptake (0.354% ± 0.0624%; p < 0.01).
Conclusions Duramycin is similarly effective in imaging apoptotic cell death as Annexin-V with lower nontarget organ radiation. Clinical feasibility of apoptosis imaging with a PE-seeking tracer should be tested.
Apoptosis is central to acute myocardial injury and contributes to subsequent process of adverse left ventricle remodeling (1,2). Tissue loss during myocardial infarction is usually initiated by apoptosis; it is accelerated in severely afflicted cells during reperfusion (3) and may culminate into myocardial necrosis. There is evidence that inhibition of apoptosis reduces infarct size and could help preserve myocyte mass and attenuate remodeling and heart failure (4–7). An ability to noninvasively detect and track apoptosis with in vivo imaging may allow systematic assessment of targeted therapeutic interventions (8).
Molecular imaging of apoptosis has traditionally targeted loss of phospholipid asymmetry of the dying cell membrane (9–11). Phosphatidylserine (PS) and phosphatidylethanolamine (PE), which normally reside within the inner layer of the sarcolemma are exteriorized to the outer cell membrane layer during apoptosis. Exteriorized PS and PE are considered reliable signatures of cellular apoptosis and constitute attractive targets for noninvasive imaging. Annexin-V, a naturally occurring protein that binds to PS with high affinity, radiolabeled with technetium-99m (99mTc) has successfully allowed clinical imaging of apoptosis. 99mTc-Annexin-V is a large molecule (approximately 32 kDa) with significant positive charge and has limitations. It inflicts significant nontarget organ radiation burden, especially on the kidneys and liver; the latter interferes with the specificity of cardiac imaging (9–11).
Another constituent of the inner cell membrane layer, PE, is also exteriorized during apoptosis similar to PS. It is more abundant (20% to 25% vs. 3% to 15% of PS) in the cell membrane in health and after injury (12,13) and therefore is an attractive target for noninvasive imaging. Duramycin, a small, 19-amino-acid antibiotic molecule with neutral charge, binds avidly to PE and should result in less nontarget radiation than Annexin-V (14–17). Not only do feasibility and validation studies need to be conducted with respect to imaging of 99mTc-Duramycin for ischemia and reperfusion injury, radiolabeled Duramycin must also be tested for noninferiority and for any potential benefits compared to the norm of in vivo apoptosis imaging. We tested 99mTc-labeled Duramycin in an ischemia-reperfusion model in rabbits, comparing its efficacy for the detection of myocardial damage with 99mTc-labeled Annexin-V and concurrent radiation burden to the kidneys. A parallel study using Cy5.5-labeled Duramycin and Alexa Fluor 488-labeled Annexin-V was also undertaken in a rat ischemia-reperfusion model to determine if PS- and PE-seeking probes colocalized in the same regions of injury. Finally, the efficacy of Duramycin to detect longitudinal apoptotic modulation during therapeutic interventions was also evaluated with minocycline, a known antiapoptotic agent with cardioprotective effects (18–20).
Preparation of Duramycin, linear Duramycin, and Annexin-V radiotracers
Radiolabeling of targeting agents
HYNIC-Duramycin and HYNIC-linear Duramycin kits were provided by Molecular Targeting Technologies, Inc. (MTTI, West Chester, Pennsylvania). Briefly, Duramycin was covalently modified with succinimidyl 6-hydrazinonicotinate acetone hydrazone (S-HYNIC) at the distal end from the PE-binding pocket. 99mTc-labeled linear Duramycin (ARQAAAFGPFAFVADGNAR), which has the same sequence as Duramycin except that lysine amino acids at positions 2 and 19 are substituted with arginine and the thioether-linked amino acids are replaced by alanine, was used as a negative tracer control (BAChem, Torrance, California). Linear Duramycin was modified with S-HYNIC in a similar fashion to Duramycin. The molecular weights of the HYNIC-Duramycin (expected MW = 2,188.4 g/mole; actual MW = 2,188.0 g/mole) and HYNIC-linear Duramycin (expected MW = 2,112.0 g/mole, actual MW = 2,112.0 g/mole) were confirmed using mass spectroscopy. For radiolabeling, ∼30 mCi of 99mTc-pertechnetate in 0.5 ml of saline was added to the vials, and excess vial pressure was vented. The vials were heated at 80°C in lead-lined heating blocks for 30 min. The radiolabel incorporation was assessed using high-performance liquid chromatography (HPLC) analysis.
99mTc-Annexin-V was used as the apoptosis imaging-positive control agent. Human Annexin-V was produced by expression in Escherichia coli and labeled with technetium-99m as described before like the labeling of Duramycin (5).
Fluorescent labeling of Duramycin and Annexin-V
The molecular weight of Cy5.5-Duramycin (MTTI) was confirmed using mass spectroscopy (expected MW = 2,637.8 g/mole, actual MW = 2,638.0 g/mole). Two-hundred nmol of Cy5.5-Duramycin was mixed with 800 μl of 1% dimethyl sulfoxide water prior to injection. Alexa Fluor-488 Annexin-V (Thermofisher Scientific, Waltham, Massachusetts) was obtained in a solution of 25 mM HEPES, 140 mM NaCl, 1 mM EDTA, pH 7.4, and 0.1% bovine serum albumin in a kit format; 50 μl of the kit was mixed with 500 μl of saline prior to injection.
All procedures followed the recommendations of the Guide for the Care and Use of Laboratory Animals (Department of Health and Human Services publication no. NIH 78–23, 1996), and all experiments were approved by the Icahn School of Medicine at Mount Sinai Institutional Animal Care and Use Committee.
Acute experimental MI
Acute experimental MI was induced in anesthetized New Zealand white male rabbits (n = 17; weight: 2.5 to 3.0 kg) by occluding left coronary artery (LCA). The rabbits were anesthetized by using a mixture of ketamine and xylazine (0.5 to 0.6 mg/kg administered subcutaneously; 10:1 vol/vol mixture of 100 mg/ml each).
Following intubation, anesthesia was maintained with 2.0% to 5.0% isoflurane inhalation; ventilation was maintained with a volume-cycled rodent respirator (Harvard Apparatus, Holliston, Massachusetts). The heart was exposed through parasternal thoracotomy, and the pericardium was removed. LCA was identified, and a monofilament suture was placed around the vessel and threaded through a polyethylene tube to create a snare; LCA was occluded by tightening the snare. Prior to reperfusion, minocycline hydrochloride (PiChemicals, Shanghai, China) was administered intravenously (30 mg/kg dose; n = 4), or saline placebo was administered intravenously similarly (n = 13); 4 randomly allocated animals received minocycline treatment. The snare was removed after 40-min occlusion to allow reperfusion. Lead II or III of the electrocardiogram was continuously monitored during the experiments.
At 30 min after reperfusion, ∼7 mCi of 99mTc-Duramycin (n = 6), 99mTc-linear Duramycin (n = 3), or 99mTc-Annexin-V (n = 4) was administered intravenously to placebo-treated rabbits; each placebo-treated rabbit was randomly allocated to a group. All 4 minocycline-treated rabbits (n = 4) received radiolabeled Duramycin.
In vivo imaging of the thorax was performed at 3 h after radiotracer administration using a dual-head micro–single-photon emission computed tomography (μSPECT) gamma camera combined with micro-computed tomography (μCT) (X-Spect, Trifoil Imaging, Inc., Chatsworth, California). A μCT scan was acquired using an x-ray tube operating at 80 kVp and 0.8 mA. Immediately after μCT imaging, μSPECT images of the heart were acquired in a 64 × 64 matrix at 32 steps with 60 s per step on the 140 keV photo peak of 99mTc with 15% windows, using a low-energy high-resolution parallel tube collimator.
The μSPECT images and μCT studies were fused, allowing scintigraphic and anatomic views of the tomographic scans in all 3 spatial axes: transverse, sagittal, and coronal.
Ex vivo imaging
After in vivo imaging, the LCA was reoccluded at the original site of the snare. Evans blue dye was infused from the ascending aorta to visualize the area at risk (AAR). The rabbits were euthanized using pentobarbital (150 mg/kg) administered intravenously. The heart was removed, rinsed in saline, and imaged with an ex vivo 900-s static scan at centerline 140 keV with a 20% window.
After ex vivo imaging, the hearts were cut into 4 short-axis slices followed by further sectioning into 29 to 31 fragments. Each piece was weighed and gamma counted for calculation of percent injected dose per gram (%ID/g). Tissue samples of the main organs were also gamma counted. This protocol is summarized in Figure 1A.
Histopathological and immunohistochemical characterization
Processing and staining of the myocardial tissues were done at Weill-Cornell Medicine and Cardiovascular Pathology Institute, Inc. Samples of rabbit myocardium were dehydrated in a graded series of alcohols and xylenes. After they were embedded in paraffin, histologic sections were prepared on a rotary microtome at 4 to 6 μm, mounted on charged slides, and stained by routine hematoxylin and eosin staining for light microscopy review. Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining was performed (at the Cardiovascular Pathology Institute, Inc.) according to the manufacturer’s prescription (TACS in-situ apoptosis detection kit, Trevigen, Gaithersburg, Maryland). Histologic sections underwent mild digestion in proteinase K (catalog ENZ-33801, Enzo, Farmingdale, New York). Normal nuclei were counterstained with hematoxylin solution (catalog S3302, Dako, Carpinteria, California) in blue, whereas apoptotic nuclei were visualized using Nova Red chromogen (catalog SK-4800, Vector, Burlingame, California) in red. Tissue from involuting rat mammary glands served as positive controls.
TUNEL-stained minocycline-treated heart sections (n = 12) and untreated heart sections (n = 6) from randomly selected animals were captured using a light microscope. Images of foci with the highest TUNEL-positive nuclei were acquired. Sixty frames were acquired from the minocycline-treated group, and 29 from the placebo group. TUNEL-positive cells were counted and then divided by the total number of cells to get the TUNEL-positive ratio. The TUNEL-positive ratio was averaged for each animal.
Fluorescence experiments in rats
Acute MI in rats
Male Sprague-Dawley rats (n = 4) were anesthetized with pentobarbital (60 mg/kg) by intraperitoneal injection and placed on a ventilator. The chest was accessed from the left side through the fourth intercostal space and the pericardium was incised. LCA was identified and encircled with a 7-0 silk suture. Successful ligation was verified by visual inspection of the left ventricle apical area for myocardial blanching. The snare was removed 40 min later to induce reperfusion.
Alexa Fluor-488 Annexin-V and Cy5.5-Duramycin were administrated intravenously through the tail vein after 30 min of reperfusion (approximately 50 nm each). Both rats were injected with both fluorescent materials at the same time. These acute MI rats were sacrificed 1 h after fluorescent tracer administration. The hearts were removed and frozen in optimal cutting temperature compound at −80°C. For localization of the fluorescent Duramycin and Annexin-V, 7-μm thick frozen sections were prepared, mounted in 4,6-diamino-2-phenylindole-containing medium (DAPI, Vector Laboratories) and examined by fluorescence microscopy. This protocol is summarized in Figure 1B.
Prior publications of 99mTc-Annexin-V, 99mTc-Duramycin imaging, and minocycline treatment for apoptosis were reviewed to assess predictable effect for power calculations (10,17,21). It was assumed that %ID/g of 99mTc-Duramycin in an untreated MI rabbit heart would be like that of 99mTc-Annexin-V uptake from the previous studies of approximately 0.65 ± 0.10%ID/g. Whereas the %ID/g 99mTc-Duramycin in a minocycline-treated MI rabbit model would be like that of 99mTc-Annexin-V of approximately 0.38 ± 0.07%ID/g (21). The power calculation took into consideration these assumptions to generate a sample size which met a power over 80%. The choice of imaging agent and placebo versus minocycline prescription were delivered just before perfusion (by A.P., H.K., or F.C.). A Shapiro-Wilk test was performed to test for normality. Data distribution was considered not normal if p value was <0.05. A Levene test was performed for groups with normally distributed %ID/g or TUNEL-positive ratio to test for homogeneity of variance. Equal variances were assumed if p value was >0.05. The mean %ID/g or TUNEL-positive ratio of 2 normally distributed independent groups with equal variances were compared with an independent 1-tailed Student’s t-test. The mean %ID/g of 2 normally distributed paired groups with equal variances were compared with a paired 1-tailed Student’s t-test. Means were considered significantly different if p value was <0.01. If the distribution of the mean values were not normally distributed, then the median %ID/g of similarly distributed independent groups were tested by Mann-Whitney U test. Medians of the 1-tailed p values and the test statistic from the Mann-Whiney U test were stated and found to be significant at p value of <0.05. Values were expressed as mean ± SD or specified otherwise. All calculations were performed using SPSS software version 24 (IBM, Armonk, New York).
Duramycin imaging and uptake in placebo-treated acute MI model
In vivo μSPECT-μCT imaging at 3 h and ex vivo images of the heart revealed intense uptake of radiolabeled Duramycin or Annexin-V in the apical (infarcted) region (Figure 2A). In contrast, no focal uptake was observed in animals injected with linear Duramycin. The radiochemical purity for labeled Duramycin, linear Duramycin, and Annexin-V were >90%. The organ uptake for the untreated Duramycin and Annexin-V animals is shown in Table 1 and Figures 3A and 3B. Most notably, at the same threshold, there was much less uptake of radiolabeled Duramycin than that of Annexin-V in the kidney cortex visually using in vivo μSPECT (Figure 3A). Also, quantitatively there is significantly lower average uptake in the cortex of the kidneys for Duramycin (0.358 ± 0.210%ID/g) compared to that of Annexin-V (1.58 ± 0.316%ID/g; p < 0.001) (Figure 3B).
Gamma counting confirmed these findings (Figure 2B): %ID/g uptakes were found to be normally distributed for the 99mTc-Duramycin, the 99mTc-Annexin, and the minocycline-treated groups as assessed by the Shapiro-Wilk test (p > 0.05). However, %ID/g uptake for the 99mTc-linear Duramycin group was not normally distributed (p = 0.013). The %ID/g uptake of Duramycin and Annexin-V in the apical area (corresponding to the AAR) were both significantly higher than in the remote area for each untreated group; 0.751 ± 0.262%ID/g in the infarcted area versus 0.045 ± 0.029%ID/g in the remote area for Duramycin (p < 0.01 via paired Student’s t-test), and 1.23 ± 0.304% ID/g in the infarcted area versus 0.058 ± 0.009%ID/g in the remote area for Annexin-V (p < 0.01 via paired Student’s t-test). A Mann-Whitney U test was used to determine the differences in %ID/g uptake between Duramycin and linear Duramycin. The distributions of the %ID/g uptake were similar between the groups by visual inspection. There was minimal uptake of linear Duramycin with a median uptake of 0.0775%ID/g which was significantly less than that of Duramycin uptake of 0.727%ID/g (U = 0; p = 0.024). Although radiolabeled Annexin-V uptake was slightly higher than the Duramycin uptake in the infarcted region, the difference between the 2 radiotracers was statistically not significant (p > 0.01 via independent Student’s t-test).
Duramycin uptake in minocycline-treated acute MI Model
%ID/g uptake of radiolabeled Duramycin in the infarct area of minocycline-treated animals (0.354 ± 0.0624%ID/g) was significantly lower than 0.751 ± 0.262%ID/g from the placebo-treated rabbits (p < 0.01). There was no statistically significant difference in 99mTc-Duramycin uptake between the remote areas in the untreated and treated animal groups (Figure 2B).
Histologic and immunohistochemical characterization
Coagulative necrosis and contraction band necrosis were seen in the infarcted regions by light microscopy with the presence of TUNEL-positive evidence of myocyte apoptosis in both treated and untreated animals. Only a few neutrophils were seen in the AAR; the inflammatory changes are not expected at this early stage. TUNEL-verified apoptosis was substantially lower visually in the minocycline-treated infarcts. The average TUNEL-positive ratio for the minocycline group (0.118 ± 0.068) was significantly less than that of the untreated animals (0.447 ± 0.110; p < 0.0001), indicating a 74% reduction in nuclear changes with minocycline treatment (Figures 4A and 4B).
Fluorescence uptake in rat ischemia-perfusion model
In the infarcted area, the red Cy5.5-Duramycin and the green Alexa Fluor-488 Annexin-V showed intense uptake; there was minimal uptake of both tracers in the remote region. The overlap of the Cy5.5, Alexa Fluor-488, and DAPI, showed co-localization of the tracer in the infarcted region. (Figure 5)
We demonstrated, using an ischemia-reperfusion model to mimic the clinical scenario, that noninvasive imaging with 99mTc-Duramycin, which avidly targets exteriorized PE on apoptotic cell surfaces, can detect and track myocardial apoptosis as effectively as 99mTc-Annexin-V directed at PS. Tracer uptake and distribution in the infarcted region delineated by 99mTc-Duramycin was similar to that delineated by 99mTc-Annexin-V. Second, 99mTc-Duramycin uptake was specific since 99mTc-linear Duramycin did not show visual uptake in the infarcted region. Moreover, very little 99mTc-Duramycin was found in the remote myocardium suggesting high specificity for region with increased apoptotic activity. Third, 99mTc-Duramycin had good dynamic range for detecting apoptosis as it could track diminishing levels of apoptosis following antiapoptotic therapy. Fourth, additional studies in rats showed that both tracers colocalized in the infarcted regions. Fifth, 99mTc-Duramycin revealed a significantly lower radiation burden to the nontarget organs, especially the kidney, thus overcoming an important clinical limitation of the 99mTc-Annexin-V imaging.
Detecting apoptosis noninvasively by targeting PE and PS expression
Aminophospholipids such as PE and PS, normally residents of the inner layer of the sarcolemma, are rapidly exteriorized during apoptosis and are thus attractive targets for noninvasive imaging (12,13). Annexin-V, a positively-charged, naturally occurring protein with anticoagulant activity, has high calcium dependent affinity to negatively charged PS on cell surface of apoptotic cells (22). 99mTc-Annexin-V has been used to image apoptosis in various cardiac diseases and in various cancers before and after therapy (23–27). It has also been safely tested in humans (24–27). However, adoption of Annexin-V into clinical practice has been slow due to some inherent limitations, and there has been no significant clinical advance for the last decade. This positively charged tracer results in significant off target radiation, especially to the kidney. Nonetheless, Annexin-V imaging is the molecular imaging strategy with availability of the most data, including in humans.
Similar to PS, PE is abundantly expressed; 20% to 25% of the inner-phospholipid bilayer is PE compared to under 10% for PS. Therefore, PE is a signal-rich target. Apoptotic cells with exteriorized PE can be detected in ischemic animal models with radiolabeled Duramycin as seen in this study. Duramycin is a clinically safe antibiotic (28). We have previously shown that 99mTc-Duramycin had significantly greater uptake in atherosclerotic lesions (29) than 99mTc-Annexin-V, whereas there is significantly less radiation burden to the kidneys. In this study, however, although 99mTc-Duramycin was advantageous in terms of radiation burden, its uptake was not greater than 99mTc-Annexin-V for in vivo characterization of apoptosis. The reason for nonsuperior uptake of Duramycin despite much higher apoptotic cell surface PE expression remains unclear. The avidity of Annexin-V binding to PS is highly dependent on intracellular calcium. Ischemia-reperfusion is usually associated with substantial calcium overload and might enhance Annexin-V uptake and outweigh the difference in cell surface dominance of PE versus PS in this model (30–32).
Monitoring apoptosis for defining therapeutic efficacy
Apoptosis is a common occurrence in many cardiac diseases. In addition to extensive apoptosis during the acute event, slow ongoing apoptotic cell loss continues to occur over years and contributes to ventricular remodeling and progression of heart failure (1,2). Modulating apoptosis after myocardial infarction should be beneficial in near term to prevent myocyte loss (4,6,33) as also in long term to attenuate remodeling and inexorable decline in function in post-infarct patients (5). There is the need to be able to noninvasively monitor myocyte apoptosis. Such imaging might detect early damage, stratify patients for advanced therapies and might allow serial monitoring of damage and improvement with therapeutic intervention. Apoptosis imaging has been successfully used to noninvasively understand the effects of passivating therapies (34). We observed that 99mTc-Duramycin imaging could not only detect apoptosis, but had a sufficient diagnostic range to detect the effects of minocycline in attenuating myocyte apoptosis. Duramycin uptake correlated with the degree of apoptosis on histology.
Radiation burden in nontarget organs during apoptosis imaging
Any noninvasive therapy, particularly one that will be used for serial monitoring will need to have minimal radiation burden. 99mTc-Duramycin is a small 19 amino acid peptide and is rapidly cleared from the kidneys; having a neutral charge it is likely to have less off target radiation than 99mTc-Annexin-V. The latter tracer shows higher radiation burden on the renal cortex, which could adversely influence likelihood of its widespread acceptance in clinical practice. It is not clear why 99mTc-Duramycin showed so much less radiation burden in our studies even accounting for differences in size of the molecule. Size, although it contributes partially, may not be the main explanation given that some molecules larger than Annexin-V pass through the glomerulus easily. It is likely that Annexin-V, a positively charged protein, could potentially bind to the strongly negatively charged apical membrane of epithelial cells in the lumen of the proximal and/or distal convoluted tubule or in the negatively charged glomerular basement membrane of the nephron in the cortical region of the kidney (35,36). Nevertheless, with the renewed emphasis on reducing radiation burden, 99mTc-Duramycin is a potentially attractive alternative to 99mTc-Annexin-V for molecular imaging of apoptosis (37). In addition, lower uptake in nontarget abdominal region decreases potential interference during cardiovascular imaging and might improve signal-to-noise ratio (38).
A limitation of this study was that the linear Duramycin was only available for administration in 3 animals, and so %ID/g uptake distribution of the group was not normal according to Shapiro-Wilk test. However, visually the linear Duramycin revealed negligible uptake compared to Duramycin and Annexin in Figure 2A.
The present study demonstrates the feasibility of 99mTc-Duramycin to detect acute myocardial injury in a rabbit ischemia-reperfusion model, and the results are comparable to those obtained with the established apoptosis imaging tracer, 99mTc-Annexin-V. A substantially reduced radiation burden to the kidney, suggests that it should be theoretically safer to use 99mTc-Duramycin. In addition, because it is an antibiotic molecule that has been safely used in humans, 99mTc-Duramycin may allow for rapid translation to apoptosis imaging in clinical practice.
COMPETENCY IN MEDICAL KNOWLEDGE: This experimental study addresses the clinical need for a specific molecular imaging agent to noninvasively identify apoptosis without high radiation burden. We demonstrated in a rabbit ischemia-reperfusion model that 99mTc-Duramycin that binds to phosphatidylethanolamine exposed on the surface of apoptotic cells with high-affinity, can be imaged in vivo. Monitoring the process of apoptosis also allows for evaluation of therapeutic intervention targeted at restricting myocellular loss. 99mTc-Duramycin imaging results in low radiation burden to nontarget organs such as the kidney.
TRANSLATIONAL OUTLOOK: 99mTc-Duramycin is an attractive molecular imaging agent for noninvasive imaging of apoptosis in myocardial ischemia. The study suggests that the administration of an antiapoptotic agent before reperfusion may help restrict loss of myocardial mass. Theoretically, 99mTc-Duramycin may also be used to assess apoptotic damage in other diseases such as cancer.
↵∗ Drs. Kawai and Chaudhry contributed equally to this work.
Dr. Pak is an employee of and owns stock in of Molecular Targeting Technologies. Dr. Virmani as an employee of CVPath Institute; and has a relationship with 480 Biomedical, Abbott Vascular, ART, BioSensors International, Biotronik, Boston Scientific, Celonova, Claret Medical, Cook Medical, Cordis, Edwards Lifescience, Medtronic, MicroPort, MicroVention, Celonova, OrbusNeich, ReCore, SINO Medical Technology, Spectranetics, Surmodics, Terumo Corporation, W.L. Gore, and Xeltis; and has received honoraria from 480 Biomedical, Abbott Vascular, Boston Scientific, Cook Medical, Lutonix, Medtronic, Terumo Corporation, and W.L. Gore. All other authors have reported that they have no relationships relevant to the contents of this paper to disclose. Thomas Schindler, MD, served as the Guest Editor for this paper.
- Abbreviations and Acronyms
- Received August 23, 2017.
- Revision received November 28, 2017.
- Accepted November 29, 2017.
- 2018 American College of Cardiology Foundation
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