Adeno-sh-beta-catenin abolishes ischemic preconditioning-mediated cardioprotection by downregulation of its target genes VEGF, Bcl-2, and survivin in ischemic rat myocardium

Abstract

beta-Catenin, the downstream target of glycogen synthase kinase-3beta (GSK-3beta), plays a vital role in ischemic preconditioning (IP)-mediated cardioprotection. In the present study, we investigated the mechanism of IP-mediated cardioprotection through suppression of beta-catenin expression by intramyocardial injection of adeno-sh-RNA against beta-catenin (BCT) (4 x 10(8) pfu). Adeno-LacZ (LZ) was used as control. The rats were randomized into (a) LZ + ischemia-reperfusion (IR); (b) LZIPIR; (c) BCTIR; and (d) BCTIPIR. Isolated hearts from each group were subjected to 30 min of I followed by 2 h of R. Both IPIR group hearts were subjected to IP (5 min I + 10 min R; four cycles) before IR. Significant reduction in left ventricular functional recovery (78 vs. 88 mm Hg), dp/dt(max) (1,802 vs. 2,189 mm Hg/sec), and aortic flow (4 vs. 9 ml/min) was observed in BCTIPIR compared with LZIPIR at 120 min of reperfusion. Increased infarct size (42 vs. 24%) and apoptotic cardiomyocytes (122 vs. 58 counts/60 HPF) were observed in BCTIPIR compared with LZIPIR. Realtime PCR and Western blot analysis showed significant downregulation in mRNA and protein expression of VEGF, Bcl-2, and survivin in BCTIPIR compared with LZIPIR. These findings indicated for the first time that silencing beta-catenin abolished IP-mediated cardioprotection, probably through inhibition of VEGF-Bcl-2 and survivin.

FIG. 1.

Effect of Ad-sh-β-cat (BCT) on cardiac functional parameters at baseline (BL) and after 30 min of ischemia at 30, 60, 90, and 120 min of reperfusion. The results in (A) represent LVDP. (B) dp/dt_max. (C) Aortic flow. Results are shown in six animals per group. *p < 0.05 represents comparison with LZIR. ^†p < 0.05 represents comparison with LZIPIR. Blank bar, LZIR; wide upward diagonal bar, LZIPIR; black bar, BCTIR; grey bars, BCTIPIR group.

FIG. 2.

Effect of Ad-sh-β-cat (BCT) on infarct size. Graph represents the percentage (%) of infarct size between the comparative groups after 30 min of ischemia and 2 h of reperfusion. Results are shown in six animals per group. *p < 0.05 represents comparison with LZIR. ^†p < 0.05 represents comparison with LZIPIR.

FIG. 3.

(A) Effect of Ad-sh-β-cat (BCT) on cardiomyocyte apoptosis. Graph represents the number of cardiomyocyte apoptosis between the comparative groups after 30 min of ischemia and 2 h of reperfusion. Results are shown in six animals per group.*p < 0.05 represents comparison with LZIR; ^†p < 0.05 represents comparison with LZIPIR. (B) Representative pictures of cardiomyocyte apoptosis. Apoptotic cells identified with anti-α sarcomeric actin labeling colocalizes with those identified with Tunel labeling. Rat heart sections were labeled with immunofluorescence and visualized by using confocal microscopy. Cross section of the fluorescently labeled vasculature demonstrates the presence of (I, IV, VII, and X, red fluorescence) and (II, V, VIII, and XI, green fluorescence). III, VI, IX, and XII show the superimposed pictures representing the apoptotic cardiomyocytes. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article at www.liebertonline.com/ars).

FIG. 3.

(A) Effect of Ad-sh-β-cat (BCT) on cardiomyocyte apoptosis. Graph represents the number of cardiomyocyte apoptosis between the comparative groups after 30 min of ischemia and 2 h of reperfusion. Results are shown in six animals per group.*p < 0.05 represents comparison with LZIR; ^†p < 0.05 represents comparison with LZIPIR. (B) Representative pictures of cardiomyocyte apoptosis. Apoptotic cells identified with anti-α sarcomeric actin labeling colocalizes with those identified with Tunel labeling. Rat heart sections were labeled with immunofluorescence and visualized by using confocal microscopy. Cross section of the fluorescently labeled vasculature demonstrates the presence of (I, IV, VII, and X, red fluorescence) and (II, V, VIII, and XI, green fluorescence). III, VI, IX, and XII show the superimposed pictures representing the apoptotic cardiomyocytes. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article at www.liebertonline.com/ars).

FIG. 4.

Representative pictures of beta-catenin by using DAB staining. Green arrows, The β-catenin expression in the cytosol; yellow arrows, the β-catenin translocation into the nucleus. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article at www.liebertonline.com/ars).

FIG. 5.

Representative bar graphs showing the various mRNA expression. (A) VEGF. (B) Bcl-2. (C) Survivin. *p < 0.05 represents comparison with LZIR. ^†p < 0.05 represents comparison with LZIPIR. ^#p < 0.05 represents comparison with BCTIR (n = 6).

FIG. 6.

(A) Representative Western blot showing the expression of β-catenin in the cytosolic fraction. GAPDH was used as the loading control. Bar graph represents the quantitative comparison between the groups. (B) Representative Western blot showing the expression of β-catenin in the nuclear fraction. Histone H3 was used as the loading control. Bar graph represents the quantitative comparison between the groups. *p < 0.05 represents comparison with LZIR. ^†p < 0.05 represents comparison with LZIPIR. ^#p < 0.05 represents comparison with BCTIR (n = 6).

FIG. 6.

(A) Representative Western blot showing the expression of β-catenin in the cytosolic fraction. GAPDH was used as the loading control. Bar graph represents the quantitative comparison between the groups. (B) Representative Western blot showing the expression of β-catenin in the nuclear fraction. Histone H3 was used as the loading control. Bar graph represents the quantitative comparison between the groups. *p < 0.05 represents comparison with LZIR. ^†p < 0.05 represents comparison with LZIPIR. ^#p < 0.05 represents comparison with BCTIR (n = 6).

FIG. 7.

Representative Western blot showing the expression of VEGF, Bcl-2, and survivin in the cytosolic fraction. GAPDH was used as the loading control. Bar graph represents the quantitative comparison between the groups for VEGF, Bcl-2, and survivin. *p < 0.05 represents comparison with LZIR. ^†p < 0.05 represents comparison with LZIPIR. ^#p < 0.05 represents comparison with BCTIR (n = 6).