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First published online 13 February 2007
doi: 10.1242/jcs.03393

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Survivin mediates the anti-apoptotic effect of -opioid receptor stimulation in cardiomyocytes

Ling-Ling Yao^1,*, Yong-Gang Wang^2,*, Wen-Jie Cai¹, Tai Yao¹ and Yi-Chun Zhu^1,

¹ Department of Physiology and Pathophysiology, Fudan University Shanghai Medical College, 138 Yi Xue Yuan Road, Shanghai 200032, China
² Department of Neurobiology, Second Military Medical University, Shanghai 200433, China

View larger version (23K): [in this window] [in a new window]   Fig. 1. Schematic illustration of the treatment protocol. DEPV, serum and glucose deprivation; MOR, morphine; NAL, naloxone; GNTI, guanidinyl-naltrindole; NAT, natrindole; DADLE, [D-Ala2, D-Leu5]-enkephalin acetate salt; PD98059, 2'-amino-3'-methoxyflavone; U0126, 1,4-diamino-2,3-dicyano-1,4-bis(2-aminophenylthio)-butadiene; LY294002, 2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one; GLY, glybenclamide.

View larger version (15K): [in this window] [in a new window]   Fig. 2. Ultrastructure of DEPV-induced cardiomyocytes apoptosis. (A) Scanning electron micrographs of control cardiomyocytes (a) and those cultured under DEPV conditions (b-f). The DEPV-treated cardiomyocytes were characterized by detachment of the cell, which became spherical (b) as well as formation of many small blebs (arrowhead) on the cell surface (c-e) or apoptotic bodies (e,f; arrow). Bars, 10 µm. (B) Transmission electron micrographs of control cardiomyocytes (a) and those cultured under DEPV conditions (b-e). (a) The normal nucleus (NN), mitochondria (Mt), myofibrils (Mf) and cross striations (arrows) in a control cardiomyocyte. (b) The apoptotic nucleus (AN) with bounding condensed chromatin, shriveled cytoplasm, mitochondria (Mt), myofibrils (Mf) and a lot of vacuoles (arrowheads) in an apoptotic cardiomyocyte. (c) Apoptotic bodies (arrows) with condensed chromatin. (d) A phagocytosed apoptotic body (arrow). (e) Condensed chromatin in the round, oncotic nucleus (ON), disrupted organelles and droplets (L) in an oncotic cardiomyocyte. Bars, 2 µm.

View larger version (110K): [in this window] [in a new window]   Fig. 3. Effects of opioid receptor stimulation on DEPV-induced cardiomyocyte apoptosis. (A) Representative electrophoretic analysis of nucleosomal DNA fragmentation from six experiments, showing the effect of morphine or DADLE on DEPV-induced cardiomyocyte apoptosis in the presence or absence of the nonselective-, -, or -opioid receptor antagonist. (B) Representative Hoechst 33258 staining from six experiments, showing morphine-induced protection against DEPV-induced cardiomyocyte apoptosis. a, control; b, DEPV; c, DEPV + MOR; d, DEPV + MOR + NAL. Bar, 20 µm. (C) The effect of morphine or DADLE on DEPV-induced cardiomyocyte apoptosis in presence or absence of the nonselective-, -, or -opioid receptor antagonist. Bar chart shows the percentage of apoptotic cells, as assessed by TUNEL staining, in each group (n=6 in each group). Values are the mean ± s.d. *P<0.05 among the indicated groups. DEPV, serum and glucose deprivation; DADLE, [D-Ala2, D-Leu5]-enkephalin acetate salt; NAT, natrindole; MOR, morphine; NAL, naloxone; GNTI, guanidinyl-naltrindole.

View larger version (30K): [in this window] [in a new window]   Fig. 4. (A) ROS production increased in cardiomyocytes cultured under DEPV conditions in a time-dependent manner reaching a peak at 6 hours. DADLE (10 µmol/l) pretreatment inhibited this increase in ROS production at 4, 6 and 8 hours. Values are the mean ± s.d. *P<0.05 vs control, #P<0.05 vs DEPV. (B) Cardiomyocyte mitochondrial damage was assessed by examining mitochondrial membrane potential, which was determined by the ratio of red and green signals in the micrographs. DEPV-induced depolarization of the mitochondria appeared as a decrease in the red signals with a concomitant increase in the green signals (upper-panel). DADLE (10 µmol/l) inhibited the destruction of mitochondrial membrane potential at 45, 60, 90 and 120 minutes. These effects were blunted by 1 µmol/l glybenclamide (GLY; lower-panel). Values are the mean ± s.d. *P<0.05 vs control; #P<0.05 vs DEPV; P<0.05 vs DEPV + DADLE. DEPV, serum and glucose deprivation; DADLE, [D-Ala2, D-Leu5]-enkephalin acetate salt.

View larger version (35K): [in this window] [in a new window]   Fig. 5. Time-dependent action of DADLE on ERK phosphorylation, expression of survivin and Bcl-2, as well as Bax translocation from the cytosolic to the mitochondrial fraction in cardiomyocytes cultured under DEPV conditions. (A) Representative blots showing time course of p-ERK, ERK, survivin, Bcl-2 and the cytosolic and mitochondrial fractions of Bax in cardiomyocytes cultured under DEPV conditions with or without pretreatment with DADLE (A). (B-F) Quantification by densitometry of the bands of pERK (B), survivin (C), Bcl-2 (D), the mitochondrial fractions and the cytosolic fractions of Bax (E) (n=3 in each group). Values are the mean ± s.d. *P<0.05 between the indicated groups. DEPV, serum and glucose deprivation; DADLE, [D-Ala2, D-Leu5]-enkephalin acetate salt.

View larger version (46K): [in this window] [in a new window]   Fig. 6. Role of DADLE-induced ERK1/2 phosphorylation in Bax translocation, Bcl-2 expression, survivin expression and cardiomyocyte apoptosis. (A) DADLE pretreatment induced a significant increase in ERK1/2 phosphorylation at 6 hours. This increase was prevented by either natrindole, or the ERK inhibitors, PD98059 and U0126, in cardiomyocytes cultured in DEPV conditions. (B) Translocation of Bax from cytosol into the mitochondria was assessed by comparing the cytosolic and mitochondrial Bax levels measured by western blot analysis at 6 hours. An increase in mitochondrial Bax with a concomitant decrease in cytosolic Bax indicates increased translocation of the protein into mitochondria. DEPV increased translocation of Bax from cytosol into the mitochondria. This translocation was prevented by DADLE. The effect of DADLE was abolished by natrindole, PD98059, or U0126. (C) DADLE-induced increase in Bcl-2 protein levels was prevented by either natrindole, PD98059, or U0126 at 6 hours. (D) Survivin level was significantly decreased in the cardiomyocytes cultured under DEPV conditions as assessed by western blot analysis at 6 hours. Values are mean ± s.d. (*P<0.05, n=6 in each group). (E) The effect of DADLE in protecting the cardiomyocytes against apoptosis as assessed by TUNEL staining was prevented by PD98059 or U0126 (a, control; b, DEPV; c, DEPV+DADLE; d, DEPV+DADLE+PD98059). Values are mean ± s.d. (*P<0.05, n=6 in each group). DEPV, serum and glucose deprivation; DADLE, [D-Ala2, D-Leu5]-enkephalin acetate salt; NAT, natrindole; PD98059, 2'-amino-3'-methoxyflavone; U0126, 1,4-diamino-2, 3-dicyano-1, 4-bis (2-aminophenylthio)-butadiene.

View larger version (28K): [in this window] [in a new window]   Fig. 7. Survivin RNAi abrogates DADLE-induced increase in survivin expression in cardiomyocytes. (A) Survivin expression, identified by immunofluorescent staining, was significantly decreased by transfection of survivin siRNA in cultured cardiomyocytes (red signals for survivin). Successful transfection was confirmed by the green signals of GFP labeling conjugated with the siRNAs. Micrographs are representative of four independent experiments. (B) 48 hours after transfection, mRNA transcripts of survivin as measured by real-time RT-PCR were significantly reduced by survivin RNAi in cardiomyocytes cultured under control condition, as well as in cardiomyocytes cultured under DEPV conditions with or without DADLE treatment. (C) Representative blots and quantification of survivin levels by densitometry. DADLE-induced increase in survivin levels was abrogated by transfection of survivin RNAi 72 hours after transfection. Transfection of survivin siRNA and GAPDH siRNA specifically knocked down the expression of their respective target genes without affecting each other's targets. Values are the mean ± s.d., n=6. *P<0.05. DADLE, [D-Ala2, D-Leu5]-enkephalin acetate salt; DEPV, serum and glucose deprivation; RNAi, RNA interference; siRNA, small interfering RNA.

View larger version (34K): [in this window] [in a new window]   Fig. 8. Survivin RNAi blunts DADLE-induced protection of cardiomyocytes against apoptosis. (A) Representative micrographs and (B) percentage of apoptotic cells as assessed by TUNEL staining. In cardiomyocytes cultured under DEPV conditions, survivin RNAi increased apoptosis in the absence or presence of DADLE. However, survivin RNAi did not change the number of apoptotic cardiomyocytes cultured under control condition. (C) Survivin siRNA blunted the effect of DADLE in reducing apoptosis of cardiomyocytes cultured under DEPV conditions. There was a survivin RNAi-induced increase in the number of apoptotic cardiomyocytes cultured under DEPV conditions as compared with that of the non-transfected or the GAPDH RNAi group in either the absence or the presence of DADLE (A,B). However, survivin RNAi did not change the number of apoptotic cardiomyocytes cultured under control condition. DADLE-induced reduction in the cardiomyocytes transfected with survivin siRNA was significantly less than that in the non-transfected cardiomyocytes or those transfected with GAPDH siRNA (C). Values are the mean ± s.d. *P<0.05. n=6 in each group. DEPV, serum and glucose deprivation; DADLE, [D-Ala2, D-Leu5]-enkephalin acetate salt; RNAi, RNA interference; siRNA, small interfering RNA; TUNEL, TdT-mediated dUTP nick end labeling.

View larger version (20K): [in this window] [in a new window]   Fig. 9. Survivin overexpression protected DEPV-induced apoptosis but not ROS production and mitochondrial membrane depolarization. (A) Western blots analysis showing high levels of the GFP-survivin fusion protein in the cardiomyocytes transfected with GFP-survivin vectors but not in the cells transfected with GFP control vector. (B,C) Representative micrographs (B) and quantification of apoptotic cardiomyocytes expressed as the ratio of the TUNEL positive cells to GFP positive cells (C). (D,E) Survivin overexpression had no effect on ROS production (D) and mitochondrial membrane potential depolarization (E). (F) Glybenclamide was not effective in preventing DADLE-induced increase in survivin expression. Values are the mean ± s.d. *P<0.05. n=6 in each group. DEPV, serum and glucose deprivation; DADLE, [D-Ala2, D-Leu5]-enkephalin acetate salt; GFP, green fluoresce protein; TUNEL, TdT-mediated dUTP nick end labeling; ROS, reactive oxygen species.

View larger version (36K): [in this window] [in a new window]   Fig. 10. Schematic illustration of the intracellular signaling mechanisms of the anti-apoptotic effect of DADLE shown in the present study.

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