Mechanisms of cardiac arrhythmias and sudden death in transgenic rabbits with long QT syndrome.

Long QT syndrome (LQTS) is a heritable disease associated with ECG QT interval prolongation, ventricular tachycardia, and sudden cardiac death in young patients. Among genotyped individuals, mutations in genes encoding repolarizing K+ channels (LQT1:KCNQ1; LQT2:KCNH2) are present in approximately 90% of affected individuals. Expression of pore mutants of the human genes KCNQ1 (KvLQT1-Y315S) and KCNH2 (HERG-G628S) in the rabbit heart produced transgenic rabbits with a long QT phenotype. Prolongations of QT intervals and action potential durations were due to the elimination of IKs and IKr currents in cardiomyocytes. LQT2 rabbits showed a high incidence of spontaneous sudden cardiac death (>50% at 1 year) due to polymorphic ventricular tachycardia. Optical mapping revealed increased spatial dispersion of repolarization underlying the arrhythmias. Both transgenes caused downregulation of the remaining complementary IKr and IKs without affecting the steady state levels of the native polypeptides. Thus, the elimination of 1 repolarizing current was associated with downregulation of the reciprocal repolarizing current rather than with the compensatory upregulation observed previously in LQTS mouse models. This suggests that mutant KvLQT1 and HERG interacted with the reciprocal wild-type alpha subunits of rabbit ERG and KvLQT1, respectively. These results have implications for understanding the nature and heterogeneity of cardiac arrhythmias and sudden cardiac death.

99mTc-annexin V imaging for in vivo detection of atherosclerotic lesions in porcine coronary arteries.

UNLABELLED: We used a model of porcine coronary atherosclerosis characterized by smooth muscle cell apoptosis to test the hypothesis that apoptosis of cells in the vascular wall of coronary arteries can be detected on SPECT images using a technetium-labeled radiotracer that targets apoptosis. METHODS: Eleven juvenile male swine received a high-fat diet combined with injury to 22 coronary vessels. After 51 +/- 9 d (mean +/- SD), the animals underwent coronary angiography, were injected with 403.3 +/- 48.1 MBq of 99mTc-annexin V, underwent SPECT, and were sacrificed. The coronary arteries underwent autoradiography and well counting, and immunostaining was performed for alpha-actin, caspase, and macrophages. RESULTS: Atherosclerotic lesions were predominantly of American Heart Association class II. Thirteen of the 22 injured vessels showed focal uptake of 99mTc-annexin V in vivo (scan positive), and 9 injured vessels and all control vessels showed no focal uptake (scan negative). The count ratios of the injured vessels to the control vessels were 2.38 +/- 0.61 for scan-positive vessels and 1.27 +/- 0.23 for scan-negative vessels (P < 0.001). The percentages of injected dose for the scan-positive and scan-negative vessels were 1.73 +/- 0.83 x 10(-3) and 0.68 +/- 0.20 x 10(-3), respectively (P < 0.001). Immunohistopathologic examination found that the cells undergoing apoptosis were smooth muscle cells. The apoptotic index (caspase-positive cells to total cells) was 63% +/- 7% for scan-positive vessels and 16% +/- 10% for scan-negative vessels (P < 0.001). Both the count ratio of injured vessels to control vessels and the percentage injected dose correlated significantly with death rate by regression analysis. CONCLUSION: Annexin is a noninvasive method to identify plaque apoptosis in the coronary vessels.

Smooth muscle cell proliferation index correlates with 111In-labeled antibody Z2D3 uptake in a transplant vasculopathy swine model.

UNLABELLED: Transplant vasculopathy is a major cause of morbidity and mortality in heart transplantation. The proliferation of coronary vascular smooth muscle cells is a hallmark of transplant vasculopathy. The goal of this study was to detect coronary vascular smooth muscle cell proliferation in a swine model by imaging regions of uptake of a monoclonal antibody (Z2D3) labeled with 111In. METHODS: Coronary-to-right carotid artery transplantation was performed in 10 Yucatan minipigs with coronary arteries from farm pigs as donors. In 5 of these experiments, the right carotid artery was also grafted to the left carotid artery as a homograft. In 1 farm pig, the left and right carotid arteries were switched. After 44 +/- 22 days (mean +/- SE), animals were injected with 5-bromo-2-deoxyuridine (BrDU) and 111In-Z2D3 F(ab')2. Approximately 24 h later, the pigs underwent planar and SPECT imaging. After the imaging session, the pigs were sacrificed and the vessels were removed. Ex vivo autoradiography of all grafts was performed. Next, the tissues were immersion fixed, paraffin embedded, sectioned, and stained for histologic or immunohistologic examination. Quantitative morphometry was performed. A smooth muscle cell proliferation index, calculated as (BrDU- and actin-stained cells/actin-stained cells) x 100, was correlated with in vivo and ex vivo radiotracer uptake. RESULTS: Patency or neovascularization was demonstrated in 10 of 10 allografts and 5 of 6 homografts. Ten of the scans were positive for focal tracer uptake in the neck in the area corresponding to the graft site, and 6 were negative. Actin- and BrDU-stained cells were seen in the media of allografts and in the recanalized lumen of occluded homografts. A smooth muscle cell proliferation index of 30 was used as a cutoff for scan positivity, on the basis of previous work. Analysis by the chi2 test indicated significant concordance (P < 0.01). Ex vivo vessel count ratios were significantly correlated with the smooth muscle cell proliferation index (r2 = 0.528, P < 0.01). CONCLUSION: The use of monoclonal antibody Z2D3 tagged with 111In allows the detection of proliferating smooth muscle cells and correlates with the intensity of cell proliferation. This diagnostic method could allow early noninvasive detection of transplant vasculopathy.

Effect of endomyocardial laser channels on regional innervation shown with (125)I-MIBG and autoradiography.

UNLABELLED: The aim of this study was to map regional innervation against regional flow early after laser channel placement using autoradiography in a porcine model. METHODS: Four juvenile male swine underwent left ventricular mapping using a catheter-based mapping system and laser treatment with 20-30 channels to the mid and distal anterior wall of the left ventricle. Three days later animals were injected with 37 MBq (125)I-metaiodobenzylguanidine (MIBG) followed in 3 h with 1,110 MBq (99m)Tc-sestamibi; 1 h later the animals were killed. Hearts were removed, perfusion fixed, and sliced into 1-cm slices. The slices best showing laser holes were selected, and circumferential sections were taken for autoradiography and hematoxylin-eosin staining. Phosphor screens were exposed for (99m)Tc and (125)I, and images were processed. The MIBG image was subtracted from the methoxyisobutylisonitrile (MIBI) image and vice versa, and color tables were applied to the difference images and overlaid on the perfusion images. Quantitative analysis of the light image data was also performed. RESULTS: Thirty-three sections from the last 3 experiments were analyzed. Acoustic damage from 30 laser channels was identified from the hematoxylin-eosin sections. Reduced MIBG relative to regional flow was seen in surrounding tissue corresponding to only 1 channel. There was no statistically significant difference in light units expressed as (MIBG - MIBI)/maximal MIBG value between laser channels and unmarked myocardial map regions. The regions identified from the color table on the map as low MIBG relative to MIBI were significantly lower than remaining laser channels and remaining myocardium. Mean light units for the regions with high MIBG relative to MIBI were significantly higher than the remaining laser channels and remaining myocardium. CONCLUSION: Using a high-resolution technique correlated with microscopic pathology in an animal model, there is negligible regional denervation 3 d after placement of endomyocardial laser channels.

Radiolabeled arginine-glycine-aspartic acid peptides to image angiogenesis in swine model of hibernating myocardium.

OBJECTIVES: Our aim was to image angiogenesis produced by endomyocardial injection of phVEGF165 in a swine model of hibernating myocardium using [123I]Gluco-arginine-glycine-aspartic acid (RGD) targeting the alphavbeta3 integrins. BACKGROUND: A noninvasive test to monitor the efficacy of therapy inducing angiogenesis is needed. The interaction between extracellular matrix and endothelial cells in sprouting capillaries is effected primarily by alphavbeta3 integrins that bind through RGD motifs. METHODS: At 21 +/- 4 days, after left circumflex coronary artery ameroid constrictor placement, 8 swine received endomyocardial injection of 1.2 mg phVEGF165 divided into 6 sites and 6 swine received saline (S) using nonfluoroscopic 3-dimensional endocardial mapping system (Noga)-guided delivery. After 20 +/- 6 days, 13 animals were injected with 6.4 +/- 1.7 mCi [123I]Gluco-RGD, 1 VEGF (vascular endothelial growth factor)-injected animal with I-123-labeled peptide control, and all animals with 2.5 +/- 0.4 mCi of Tl-201 and underwent single-photon emission computed tomography imaging. Blood flow and echocardiographic measurements were made at both time points and tissue analyzed for fibrosis and capillary density by lectin staining. RESULTS: Hibernating myocardium in the ameroid constrictor territory at time of injections was documented by reduced wall thickening compared with remote. Ratio of myocardial blood flow in left circumflex coronary artery/left anterior descending coronary artery territories increased by 15 +/- 11% in the VEGF animals and fell 13 +/- 12% in S-injected (p < 0.01). There was a small increase in wall thickening in constrictor territory after VEGF (8 +/- 17%) while in S-injected animals wall thickening fell by 23 +/- 31% (p = 0.01 vs. VEGF). Lectin staining as percent positive tissue staining for ameroid territory was higher in VEGF-injected compared with S-injected animals (2.5 +/- 1.5% vs. 0.87 +/- 0.52%, p = 0.01). Focal uptake of [123I]Gluco-RGD corresponding to Tl-201 defects was seen in VEGF-injected but not in S-injected animals. [123I]Gluco-RGD uptake in the ameroid territory as percent injected dose correlated with lectin staining (R2 = 0.80, p = 0.002). CONCLUSIONS: These data suggest that single-photon emission computed tomography imaging of radiolabeled RGD peptides may be a useful noninvasive method to monitor therapy that induces angiogenesis in the heart.