Effects of CXCR4 gene transfer on cardiac function after ischemia-reperfusion injury

Abstract

Acute coronary occlusion is the leading cause of death in the Western world. There is an unmet need for the development of treatments to limit the extent of myocardial infarction (MI) during the acute phase of occlusion. Recently, investigators have focused on the use of a chemokine, CXCL12, the only identified ligand for CXCR4, as a new therapeutic modality to recruit stem cells to individuals suffering from MI. Here, we examined the effects of overexpression of CXCR4 by gene transfer on MI. Adenoviruses carrying the CXCR4 gene were injected into the rat heart one week before ligation of the left anterior descending coronary artery followed by 24 hours reperfusion. Cardiac function was assessed by echocardiography couple with 2,3,5-Triphenyltetrazolium chloride staining to measure MI size. In comparison with control groups, rats receiving Ad-CXCR4 displayed an increase in infarct area (13.5% +/- 4.1%) and decreased fractional shortening (38% +/- 5%). Histological analysis revealed a significant increase in CXCL12 and tumor necrosis factor-alpha expression in ischemic area of CXCR4 overexpressed hearts. CXCR4 overexpression was associated with increased influx of inflammatory cells and enhanced cardiomyocyte apoptosis in the infarcted heart. These data suggest that in our model overexpressing CXCR4 appears to enhance ischemia/reperfusion injury possibly due to enhanced recruitment of inflammatory cells, increased tumor necrosis factor-alpha production, and activation of cell death/apoptotic pathways.

Figure 1

CXCR4-overexpressed rats exhibit larger infarcts after ischemia-reperfusion in vivo. One week after CXCR4 gene transfer, myocardial infarction was induced by LAD ligation (30 minutes followed by 24 hours reperfusion) in rats in vivo. A: Histological analysis of infarct size was performed by TTC staining, white = infarct area; red = risk area; blue = normal area. B–D: Infarct size as a percentage of risk area (B) and evaluation of risk area and infarct size: quantitative assessment (mean ± SD) of risk area as a percentage of LV (C) for control groups (β-gal and control) and the CXCR4-overexpressed group. Although risk areas are similar for all groups, infarct sizes as a percentage of risk area are statistically different and are larger in CXCR4 group. Infarct data: infarct/ischemic area = infarct area/(infarct area + risk area) × 100. P > 0.05 in risk area, *P < 0.05 in infarct and ischemic area, compared with β-gal and control, **P < 0.05 compared with β-gal control. Mean ± SD.

Figure 2

CXCR4-overexpressed rats exhibit worsening cardiac function after ischemia-reperfusion in vivo. A: Echocardiography was performed on rats’ hearts post 30 minutes ischemia and 24 hours reperfusion. Short axis. Left, blank control without IR; Middle, β-gal gene transfer with IR; Right, CXCR4 gene transfer with IR. B: Percentage of fractional shortening (%) was calculated: *P < 0.05 compared with blank, **P < 0.05 compared with β-gal and control. Mean ± SD. C: Echocardiography was performed on rats’ hearts before Ad-CXCR4 gene transfer and one week after injection in an absence of any ischemic injury.

Figure 3

CXCR4 protein expression on myocardium after one week gene transfer and ischemia 30 minutes followed by 24 hours reperfusion. A: X-gal staining for β-gal expression, blue color is positive, (B) H-E staining, (C–D) immunostaining (DAB) for CXCR4 protein expression. C: CXCR4 group. D: β- gal control. E: CXCR4-overexpressed rats exhibit increased CXCR4 protein expression in ischemic myocardium after IR in vivo. Protein lysates were prepared from rats’ hearts one week after CXCR4 gene transfer followed by 30 minutes LAD ligation and 24 hours reperfusion. The ischemic and remote tissues were separated, lysed, and analyzed by Western blot. CXCR4 Mr; 40 to 47 kDa. Representative gel of three independent experiments is shown. Densitometric analysis of data from three different experiments is shown. *P < 0.05.

Figure 4

CXCL12 is upregulated in ischemic myocardium of CXCR4-overexpressed rat. A: CXCL12 protein expression was assessed in hearts injected with either CXCR4 or β-gal and/or control (saline-injected; with IR) by immunofluorescence staining. Frozen sections were fixed and stained with anti-CXCL12 (green) and anti–α actinin (red). Primary Abs visualized with FITC or Texas Red conjugate. Nuclei were stained with DAPI. Images are taken with confocal microscopy. B: Protein lysates were prepared from rats’ hearts one week after CXCR4 gene transfer followed by 30 minutes LAD ligation and 24 hours reperfusion. The ischemic and remote tissues were lysed and analyzed by Western blot. Representative gel of three independent experiments is shown. C: CXCL12 mRNA levels were determined by quantitative real-time PCR (QRT-PCR) using a QuantiTect SYBR Green RT-PCR Kit and using specific primers for CXCL12 and 18S. Primers were designed to generate short amplification products. Densitometric analysis of data from three different experiments is shown. *P < 0.05.

Figure 5

CXCR4-overexpressed rats exhibit increased leukocyte counts in ischemic myocardium after IR in vivo. A: Frozen sections from hearts either injected with Ad-CXCR4, or β-gal and/or control (saline injected) hearts (with IR) stained with hematoxylin and eosin, and examined by light microscopy at ×10 and ×40 objectives for the presence of inflammatory cells. B: The number of inflammatory cells was quantified per mm². Inflammatory infiltrate is significantly increased in CXCR4-overexpressed hearts (*P < 0.05). C and D: The number of monocytes and macrophages were also assessed by DAB staining using cell specific antibody (VP-M640). Number of monocyte/macrophages were counted in high-power field (HPF; ×40), and average cell count was graphed (n = 3).

Figure 6

CXCR4-overexpressed rats exhibit significant increase in cell death/apoptosis after ischemia-reperfusion in vivo. A: Increased cardiomyocyte susceptibility to ischemic injury was examined by assessing apoptosis using TUNEL assay. A double-staining technique was used (ie, TUNEL staining by using an In Situ Cell Death Detection Kit [Roche, USA] for apoptotic cell nuclei and DAPI staining for all cell nuclei. Representative images are shown. B: Apoptotic index was determined (ie, number of TUNEL-positive myocytes/total number of myocytes stained with anti-α-actinin × 100) from a total of 40 fields per heart. Assays were performed in a blinded manner. C: Increased cardiomyocyte susceptibility to ischemic injury was examined by assessing apoptosis using Western blot analysis of caspase 3 activation. Densitometric analysis of data from three different experiments is shown. *P < 0.05. D and E: The expression of inflammatory cytokine TNF-α was assessed in those hearts using immunofluorscence and QRT-PCR assay using a QuantiTect SYBR Green RT-PCR Kit. Specific primers for TNF-α and 18S were used. Primers were designed to generate short amplification products. Densitometric analysis of data from three different experiments is shown. *P < 0.05.