Chronic Recurrent Myocardial Ischemic Injury Is Significantly Attenuated by Pre-Emptive Adeno-Associated Virus Heme Oxygenase-1 Gene Delivery
Alok S. Pachori, PhD*,
Luis G. Melo, PhD ,
Lunan Zhang, MD*,
Scott D. Solomon, MD and
Victor J. Dzau, MD*,*
* Department of Medicine, Duke University Medical Center, Durham, North Carolina
Department of Medicine, Brigham and Women’s Hospital, Harvard Medical School, Boston, Massachusetts
Department of Physiology, Queen’s University, Kingston, Ontario, Canada
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Figure 1 Schematic representation of repetitive ischemia and reperfusion (I/R) protocol and adeno-associated virus (AAV)-2-mediated intramyocardial gene transfer. (A) Illustration of the experimental protocol for repetitive ischemia. AAV-HO-1 was administered five weeks in advance of the injury, followed by left anterior descending coronary artery (LAD) occlusion for 15 min and release performed daily for five days. Twelve days after the last ischemic episode, animals were subjected to echocardiography followed by tissue harvest and analyses. (B) Schematic diagram of the sites of injection (indicated by filled circles). (C) Representative electrocardiogram (ECG) at basal showing normal tracing and showing an increase in ST-segment following LAD ligation. (D) Semi-quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) detection of HO-1 transcript in left ventricle five weeks after gene transfer. (E) Western blot analysis of distribution of HO-1 protein in injected (ischemic) and non-injected (remote) areas of the left ventricle.
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Figure 2 Effect of heme-oxygenase (HO)-1 gene transfer and repeated I/R on ventricular function. (A) Echocardiographic analyses of rat myocardium 12 days after the last ischemic episode demonstrates significant improvement in ejection fraction in HO-1-treated animals as compared to lacZ-treated controls. (* vs. sham; ** vs. lac Z. p < 0.05, n = 5 to 6/group). (B) Representative picture of M-Mode echocardiogram showing significant increase in left ventricle cavity and decrease in wall thickness in lacZ-treated animals when compared to HO-1-treated group. Other abbreviations as in Figure 1.
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Figure 3 Effect of HO-1 gene transfer on ventricular remodeling and fibrosis after repeated I/R. (A) Masson Trichrome staining shows significant anterior wall thinning and collagen deposition two weeks after the last ischemic episode in lacZ-treated animals in contrast to HO-1–treated rats with decreased collagen deposition. Quantification of the fibrotic area as a percentage of total left ventricular area shows significant attenuation of fibrosis in the HO-1–treated animals as compared to saline or lacZ-treated controls. (n = 4/group, * vs. saline and lacZ, p < 0.05) (B) Representative alpha-smooth muscle actin antibody staining demonstrates positive staining both in vessel walls as well as in the myocardium in lacZ-treated animals indicating higher phenotypic conversion to cardiac fibroblasts as compared to HO-1–treated animals, which demonstrated positive staining only in the vessel walls. Abbreviations as in Figure 1.
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Figure 4 Expression of markers of apoptosis after repeated I/R. (A) Representative Western blots of two samples from each group (except in sham) from myocardial samples obtained 12 h after the last episode of intermittent I/R. (B) Bcl-2 and Bcl-xl were highly induced in lacZ-treated animals, while phospho-bad was elevated in the HO-1–treated animals. There was no change in levels of bax and alpha-sacrcomeric actin levels. HO-1–injected animal without I/R injury was used as a negative control. Abbreviations as in Figure 1.
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Figure 5 Effect of HO-1 gene transfer on myocardial apoptosis after repeated I/R. Representative micrograph demonstrating apoptosis as determined by terminal deoxynucleotidyltransferase dUTP nick end-labeling (TUNEL) staining in heart sections after five days of repeated I/R. There were significantly fewer numbers of TUNEL-positive cells in HO-1–treated animals in contrast to lacZ-treated controls (0.31 ± 0.045 vs. 0.59 ± 0.035, p < 0.005, n = 5 to 6/group). Apoptotic index was determined as described in Methods. Other abbreviations as in Figure 1.
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Figure 6 In situ detection of reactive oxygen species in rat myocardium after repeated I/R. Representative fluorescent photomicrographs of confocal microscopic sections of myocardium labeled with the oxidative dye dihydroethidium (DHE). Red fluorescence is generated when dihydroethidium DHE is oxidized to EtBr by O2 –. At identical laser settings, fluorescence in the HO-1–treated animals is markedly decreased compared with lacZ-treated controls and is similar to the sham controls.
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