Rat Model of Cardiotoxic Drug-Induced Cardiomyopathy.

Cardiotoxicity from cancer drugs remains a clinical problem. To find reliable markers of cardiotoxicity, animal models were proposed and potential new diagnostic markers have been actively investigated using these models. Here we describe our protocols, using male Sprague-Dawley rats, for inducing cardiomyopathy by single injection of high-dose doxorubicin (5-10 mg/kg) or multiple injections (2-4 times) of low-dose doxorubicin (2.5 mg/kg) with combined single injection of trastuzumab (10 mg/kg). The cardiotoxicity is evaluated by imaging modalities (echocardiography and nuclear imaging), serum troponin levels, and histopathological analyses.

Cardioprotective Effects of HSP72 Administration on Ischemia-Reperfusion Injury.

BACKGROUND: Although early reperfusion is the most desirable intervention after ischemic myocardial insult, it may add to damage through oxidative stress. OBJECTIVES: This study investigated the cardioprotective effects of a single intravenous dose of heat shock protein-72 (HSP72) coupled to a single-chain variable fragment (Fv) of monoclonal antibody 3E10 (3E10Fv) in a rabbit ischemia-reperfusion model. The Fv facilitates rapid transport of HSP72 into cells, even with intact membranes. METHODS: A left coronary artery occlusion (40 min) reperfusion (3 h) model was used in 31 rabbits. Of these, 12 rabbits received the fusion protein (Fv-HSP72) intravenously. The remaining 19 control rabbits received a molar equivalent of 3E10Fv alone (n = 6), HSP72 alone (n = 6), or phosphate-buffered saline (n = 7). Serial echocardiographic examinations were performed to assess left ventricular function before and after reperfusion. Micro-single-photon emission computed tomography imaging of 99mTc-labeled annexin-V was performed with micro-computed tomography scanning to characterize apoptotic damage in vivo, followed by gamma counting of the excised myocardial specimens to quantify cell death. Histopathological characterization of the myocardial tissue and sequential cardiac troponin I measurements were also undertaken. RESULTS: Myocardial annexin-V uptake was 43% lower in the area at risk (p = 0.0003) in Fv-HSP72-treated rabbits compared with control animals receiving HSP72 or 3E10Fv alone. During reperfusion, troponin I release was 42% lower and the echocardiographic left ventricular ejection fraction 27% higher in the Fv-HSP72-treated group compared with control animals. Histopathological analyses confirmed penetration of 3E10Fv-containing molecules into cardiomyocytes in vivo, and treatment with Fv-HSP72 showed fewer apoptotic nuclei compared with control rabbits. CONCLUSIONS: Single-dose administration of Fv-HSP72 fusion protein at the time of reperfusion reduced myocardial apoptosis by almost one-half and improved left ventricular functional recovery after myocardial ischemia-reperfusion injury in rabbits. It might have potential to serve as an adjunct to early reperfusion in the management of myocardial infarction.

2-deoxy-2-[18F]fluoro-D-mannose positron emission tomography imaging in atherosclerosis.

Progressive inflammation in atherosclerotic plaques is associated with increasing risk of plaque rupture. Molecular imaging of activated macrophages with 2-deoxy-2-[(18)F]fluoro-D-glucose ([(18)F]FDG) has been proposed for identification of patients at higher risk for acute vascular events. Because mannose is an isomer of glucose that is taken up by macrophages through glucose transporters and because mannose receptors are expressed on a subset of the macrophage population in high-risk plaques, we applied (18)F-labeled mannose (2-deoxy-2-[(18)F]fluoro-D-mannose, [(18)F]FDM) for targeting of plaque inflammation. Here, we describe comparable uptake of [(18)F]FDM and [(18)F]FDG in atherosclerotic lesions in a rabbit model; [(18)F]FDM uptake was proportional to the plaque macrophage population. Our FDM competition studies in cultured cells with 2-deoxy-2-[(14)C]carbon-D-glucose ([(14)C]2DG) support at least 35% higher [(18)F]FDM uptake by macrophages in cell experiments. We also demonstrate that FDM restricts binding of anti-mannose receptor antibody to macrophages by approximately 35% and that mannose receptor targeting may provide an additional avenue for imaging of plaque inflammation.

Annexin A5 uptake in ischemic myocardium: demonstration of reversible phosphatidylserine externalization and feasibility of radionuclide imaging.

UNLABELLED: 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 gamma-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.

Small-animal SPECT and SPECT/CT: application in cardiovascular research.

Preclinical cardiovascular research using noninvasive radionuclide and hybrid imaging systems has been extensively developed in recent years. Single photon emission computed tomography (SPECT) is based on the molecular tracer principle and is an established tool in noninvasive imaging. SPECT uses gamma cameras and collimators to form projection data that are used to estimate (dynamic) 3-D tracer distributions in vivo. Recent developments in multipinhole collimation and advanced image reconstruction have led to sub-millimetre and sub-half-millimetre resolution SPECT in rats and mice, respectively. In this article we review applications of microSPECT in cardiovascular research in which information about the function and pathology of the myocardium, vessels and neurons is obtained. We give examples on how diagnostic tracers, new therapeutic interventions, pre- and postcardiovascular event prognosis, and functional and pathophysiological heart conditions can be explored by microSPECT, using small-animal models of cardiovascular disease.

Use of an oxygen-carrying blood substitute to improve intravascular optical coherence tomography imaging.

Optical coherence tomography (OCT) is a catheter-based imaging technology with powerful resolution capable of identifying vulnerable plaques and guiding coronary intervention. However, a significant limitation of intravascular OCT imaging is its attenuation by blood. We propose that the use of an oxygen-carrying blood substitute could potentially optimize OCT image quality. Surgical isolation of the descending thoracic aorta of six rabbits is performed, followed by intravascular OCT imaging of the abdominal aorta. Perfluorodecalin (PFD) is oxygenated using a bubble-through technique with 100% oxygen. OCT imaging is performed and compared using three different flushing modalities: PFD; saline; and blood. OCT imaging of the rabbit abdominal aorta is successful in all of the subjects. In each of the six studied subjects, flushing with PFD consistently provides dramatically better imaging of the vessel wall tissue structures. OCT image quality is highly dependent on the ability of the flushing modality to remove blood from the imaging field. From this proof-of-concept study, we demonstrate that endovascular flushing with an oxygen-carrying blood substitute (PFD) is optically superior to saline flushing for intravascular imaging.

Noninvasive imaging of angiotensin receptors after myocardial infarction.

OBJECTIVES: The purpose of this study was to evaluate the feasibility of noninvasive imaging of angiotensin II (AT) receptor upregulation in a mouse model of post-myocardial infarction (MI) heart failure (HF). BACKGROUND: Circulating AT levels do not reflect the status of upregulation of renin-angiotensin axis in the myocardium, which plays a central role in ventricular remodeling and evolution of HF after MI. Appropriately labeled AT or AT receptor blocking agents should be able to specifically target AT receptors by molecular imaging techniques. METHODS: AT receptor imaging was performed in 29 mice at various time points after permanent coronary artery ligation or in controls using a fluoresceinated angiotensin peptide analog (APA) and radiolabeled losartan. The APA was used in 19 animals for intravital fluorescence microscopy on a beating mouse heart. Tc-99m losartan was used for in vivo radionuclide imaging and quantitative assessment of AT receptor expression in 10 mice. After imaging, hearts were harvested for pathological characterization using confocal and 2-photon microscopy. RESULTS: No or little APA uptake was observed in control animals or within infarct regions on days 0 and 1. Distinct uptake occurred in the infarct area at 1 to 12 weeks after MI; the uptake was at maximum at 3 weeks and reduced markedly at 12 weeks after MI. Ultrasonographic examination demonstrated left ventricular remodeling, and pathologic characterization revealed localization of the APA tracer with collagen-producing myofibroblasts. Tc-99m losartan uptake in the infarct region (0.524 +/- 0.212% injected dose/g) increased 2.4-fold as compared to uptake in the control animals (0.215 +/- 0.129%; p < 0.05). CONCLUSIONS: The present study demonstrates the feasibility of in vivo molecular imaging of AT receptors in the remodeling myocardium. Noninvasive imaging studies aimed at AT receptor expression could play a role in identification of subjects likely to develop heart failure. In addition, such a strategy could allow for optimization of anti-angiotensin therapy in patients after MI.