Noninvasive molecular imaging of apoptosis in a mouse model of anthracycline-induced cardiotoxicity

Abstract

Background: Anthracycline-induced cardiotoxicity and myocardial dysfunction may be associated with apoptosis. Caspase 3 catalyzes a terminal step in apoptosis, and its expression may serve as a marker of cardiomyocyte apoptosis. We synthesized 18F-CP18, a caspase-3 substrate and evaluated cardiac 18F-CP18 uptake in a mouse model of anthracycline cardiotoxicity.

Methods and results: For 12 weeks, mice were injected with doxorubicin, 3 mg/kg/week, or vehicle (control). Left ventricular fractional shortening was quantified by echocardiography. CP18 uptake after intravenous injection of 250 μCi of 18F-CP18, 24 hours post-doxorubicin treatment was quantified by microPET, autoradiography, and gamma counting. Apoptosis was assessed by enzymatic assay of myocardial caspase 3 and TUNEL staining of tissue sections. Compared with controls, at 6 and 12 weeks of doxorubicin treatment, fractional shortening was reduced (20.7%±2.5% versus 31%±3.5%, P=0.010; and 20.3%±3.1% versus 32.4%±2.1%, P=0.011). Doxorubicin treatment was associated with increased 18F-CP18 uptake in %ID/g by gamma counting from 0.36±0.01 (week 1) to 0.78±0.01 (week 12), P=0.003. A similar increase in 18F-CP18 uptake was observed by microPET (0.41±0.04 versus 0.73±0.1, P=0.014) and autoradiography (1.1±0.3 versus 2.8±0.2 P=0.001). Caspase 3 enzymatic activity and apoptosis by TUNEL staining were also increased after 12 weeks of doxorubicin compared with weeks 1 and 3. CP18 uptake in controls was relatively unchanged at weeks 1, 3, and 12.

Conclusions: In a mouse model of cardiotoxicity, doxorubicin treatment is associated with increased myocardial caspase 3 expression and an increase in CP18 uptake. 18F-CP18 may be useful for detection of anthracycline-induced myocardial apoptosis.

Figure 1

The upper panel represents the chemical structure of 18F-CP18 (amino acid sequence: DEVD) and the lower panel represents the chemical structure of 18F-CP32 (amino acid sequence: EDDV).

Figure 2

A, Myocardial uptake of 18F-CP18 was quantified by gamma counting of the heart explanted from doxorubicin-treated (Doxo) mice and mice treated with vehicle (control). Uptake of both myocardium measured in %ID/g. Data points represent median and interquartile range of myocardial uptake from 5 mice each at weeks 1, 3, 6, and 12 of treatment expressed as %ID/g. B, Enzymatic activity of caspase 3 was measured in homogenized samples of myocardium in control and Doxo mice (n=5), and data points represent median and interquartile range of myocardial caspase activity in 5 mice each at weeks 1, 3, 6, and 12 of treatment expressed in nmol/min/mg of tissue.

Figure 3

A, 18F-CP18 uptake by the myocardium was quantified in regions of interest (ROI) in the ex vivo microPET images obtained 1 h post injection of 200 μCi of 18F-CP18 and expressed as a ratio of %ID/g of myocardium. Individual data points represent median and interquartile range of myocardial CP18 uptake activity in 5 mice each at weeks 1, 3, 6, and 12 of treatment. B, In vivo microPET imaging was performed in doxorubicin-treated (Doxo) mice at week 1, 6, and 12 of treatment using an INVEON Multimodality scanner. a and b, In vivo microPET images from Doxo mice for 1 week and 6 weeks, respectively. c–e, Imaging performed at 12 weeks of doxorubicin treatment; the CT angiogram (c), microPET showing prominent myocardial uptake of 18F-CP18 at week 12 of treatment with doxorubicin (d), and coregsitration of microPET with CT angiogram (e).

Figure 4

A, Heart explanted from doxorubicin-treated (Doxo) mice and mice treated with vehicle (control) was fixed and 10 μm sections of myocardium were placed on an imaging plate and exposed overnight at −80°C for autoradiography. Doxorubicin treatment for 3, 6, and 12 weeks was associated with increase in 18F-CP18 uptake of myocardial tissue sections compared with minimal 18F-CP18 uptake seen in control. Regions in red represent foci with high uptake, yellow and green represent intermediate uptake, and blue represents background. Each slide contains myocardial sections from the base, mid, and distal segments of the heart from 1 individual animal. B, 18F-CP18 uptake was quantified by measuring count densities using an FLA-7000 scanner. An image analysis software was used to quantify signal intensity of 18F-CP18 retained in the myocardium and expressed as PSL/mm². Data points represent median and interquartile range of 18F-CP18 uptake in 3 to 4 myocardial tissue sections from 3 mice each at weeks 1, 3, 6, and 12 of treatment.

Figure 5

TUNEL staining of tissue sections were performed using the in situ cell death detection kit-TMR red. TUNEL-positive apoptotic nuclei and DAPI-stained nuclei were visualized at ×200 magnification. Representative sections obtained from mice at weeks 1, 3, 6, and 12 after doxorubicin treatment and a contrasting section from a control mouse treated with vehicle are shown.

Figure 6

Comparison of CP18 uptake by autoradiography with apoptosis by TUNEL staining. 18F-CP18 uptake quantified by measuring count densities and expressed as PSL/mm² was compared with the % of TUNEL-positive apoptotic nuclei among DAPI-stained nuclei per high power field. The association between CP18 uptake and TUNEL staining of adjacent myocardial 5 μm sections from 4 individual mice treated with doxorubicin for 1 week, 3 weeks, 6 weeks, and 12 weeks, respectively, was given by a Spearman’s rank correlation coefficient.