Cyclic GMP-dependent Protein Kinase Iα Attenuates Necrosis and Apoptosis Following Ischemia/Reoxygenation in Adult Cardiomyocyte
2006; Elsevier BV; Volume: 281; Issue: 50 Linguagem: Inglês
10.1074/jbc.m606142200
ISSN1083-351X
AutoresAnindita Das, Albert Smolenski, Suzanne M. Lohmann, Rakesh C. Kukreja,
Tópico(s)Cardiac electrophysiology and arrhythmias
ResumoCyclic GMP-dependent protein kinases protein kinase G (PKG) Iα and PKGIβ are major mediators of cGMP signaling in the cardiovascular system. PKGIα is present in the heart, although its role in protection against ischemia/reperfusion injury is not known. We investigated the direct effect of PKGIα against necrosis and apoptosis following simulated ischemia (SI) and reoxygenation (RO) in cardiomyocytes. Adult rat cardiomyocytes were infected with adenoviral vectors containing hPKGIα or catalytically inactive mutant hPKGIαK390A. After 24 h, the cells were subjected to 90 min of SI and 2 h RO for necrosis (trypan blue exclusion and lactate dehydrogenase release) or 18 h RO for apoptosis studies. To evaluate the role of KATP channels, subgroups of cells were treated with 5-hydroxydecanoate (100 μm), HMR1098 (30 μm), or glibenclamide (50 μm), the respective blockers of mitochondrial, sarcolemmal, or both types of KATP channels prior to SI. The necrosis observed in 33.7 ± 1.6% of total myocytes in the SI-RO control group was reduced to 18.6 ± 0.8% by PKGIα (mean ± S.E., n = 7, p < 0.001). The apoptosis observed in 17.9 ± 1.3% of total myocytes in the SI-RO control group was reduced to 6.0 ± 0.6% by PKGIα (mean ± S.E., n = 7, p < 0.001). In addition, PKGIα inhibited the activation of caspase-3 after SI-RO in myocytes. Myocytes infected with the inactive PKGIαK390A mutant showed no protection. PKGIα enhanced phosphorylation of Akt, ERK1/2, and JNK, increased Bcl-2, inducible nitric-oxide synthase, endothelial nitric-oxide synthase, and decreased Bax expression. 5-Hydroxydecanoate and glibenclamide abolished PKGIα-mediated protection against necrosis and apoptosis. However, HMR1098, had no effect. A scavenger of reactive oxygen species, as well as inhibitors of phosphatidylinositol 3-kinase, ERK, JNK1, and NOS, also blocked PKGIα-mediated protection against necrosis and apoptosis. These results show that opening of mitochondrial KATP channels and generation of reactive oxygen species, in association with phosphorylation of Akt, ERK, and JNK, and increased expression of NOS and Bcl-2, play an essential role in the protective effect of PKGIα. Cyclic GMP-dependent protein kinases protein kinase G (PKG) Iα and PKGIβ are major mediators of cGMP signaling in the cardiovascular system. PKGIα is present in the heart, although its role in protection against ischemia/reperfusion injury is not known. We investigated the direct effect of PKGIα against necrosis and apoptosis following simulated ischemia (SI) and reoxygenation (RO) in cardiomyocytes. Adult rat cardiomyocytes were infected with adenoviral vectors containing hPKGIα or catalytically inactive mutant hPKGIαK390A. After 24 h, the cells were subjected to 90 min of SI and 2 h RO for necrosis (trypan blue exclusion and lactate dehydrogenase release) or 18 h RO for apoptosis studies. To evaluate the role of KATP channels, subgroups of cells were treated with 5-hydroxydecanoate (100 μm), HMR1098 (30 μm), or glibenclamide (50 μm), the respective blockers of mitochondrial, sarcolemmal, or both types of KATP channels prior to SI. The necrosis observed in 33.7 ± 1.6% of total myocytes in the SI-RO control group was reduced to 18.6 ± 0.8% by PKGIα (mean ± S.E., n = 7, p < 0.001). The apoptosis observed in 17.9 ± 1.3% of total myocytes in the SI-RO control group was reduced to 6.0 ± 0.6% by PKGIα (mean ± S.E., n = 7, p < 0.001). In addition, PKGIα inhibited the activation of caspase-3 after SI-RO in myocytes. Myocytes infected with the inactive PKGIαK390A mutant showed no protection. PKGIα enhanced phosphorylation of Akt, ERK1/2, and JNK, increased Bcl-2, inducible nitric-oxide synthase, endothelial nitric-oxide synthase, and decreased Bax expression. 5-Hydroxydecanoate and glibenclamide abolished PKGIα-mediated protection against necrosis and apoptosis. However, HMR1098, had no effect. A scavenger of reactive oxygen species, as well as inhibitors of phosphatidylinositol 3-kinase, ERK, JNK1, and NOS, also blocked PKGIα-mediated protection against necrosis and apoptosis. These results show that opening of mitochondrial KATP channels and generation of reactive oxygen species, in association with phosphorylation of Akt, ERK, and JNK, and increased expression of NOS and Bcl-2, play an essential role in the protective effect of PKGIα. Apoptotic cell death in cardiac myocytes is well recognized to be responsible for myocardial infarction following ischemia/reperfusion injury (1Fliss H. Gattinger D. Circ. Res. 1996; 79: 949-956Crossref PubMed Scopus (773) Google Scholar), hypertrophy (2Badorff C. Ruetten H. Mueller S. Stahmer M. Gehring D. Jung F. Ihling C. Zeiher A.M. Dimmeler S. J. Clin. Investig. 2002; 109: 373-381Crossref PubMed Scopus (145) Google Scholar), and development of heart failure (3Narula J. Haider N. Virmani R. DiSalvo T.G. 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In recent years, there has been considerable interest in the role of the NO-cGMP-protein kinase G (PKG) 2The abbreviations used are: PKG, protein kinase G; SI, simulated ischemia; RO, reoxygenation; 5-HD, 5-hydroxydecanoate; Glib, glibenclamide; ERK, extracellular signal-regulated kinase; JNK, c-Jun N-terminal kinase; NOS, nitric-oxide synthase; eNOS, endothelial NOS; iNOS, inducible NOS; nNOS, neuronal NOS; ROS, reactive oxygen species; PI, phosphatidylinositol; MAPK, mitogen-activated protein kinase; MEKK, MAPK/ERK kinase kinase; MPG, N-(2 mercaptopropionyl)glycine; l-NAME, l-nitro-amino-methyl-ester; TUNEL, terminal deoxynucleotidyl transferase-mediated nick end label. 2The abbreviations used are: PKG, protein kinase G; SI, simulated ischemia; RO, reoxygenation; 5-HD, 5-hydroxydecanoate; Glib, glibenclamide; ERK, extracellular signal-regulated kinase; JNK, c-Jun N-terminal kinase; NOS, nitric-oxide synthase; eNOS, endothelial NOS; iNOS, inducible NOS; nNOS, neuronal NOS; ROS, reactive oxygen species; PI, phosphatidylinositol; MAPK, mitogen-activated protein kinase; MEKK, MAPK/ERK kinase kinase; MPG, N-(2 mercaptopropionyl)glycine; l-NAME, l-nitro-amino-methyl-ester; TUNEL, terminal deoxynucleotidyl transferase-mediated nick end label. pathway in protection of the heart against ischemia/reperfusion injury (8Han J. Kim N. Kim E. Ho W.E. Earm Y.E. J. Biol. Chem. 2001; 276: 22140-22147Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar). Inhibition of cGMP-specific phosphodiesterase 5A with sildenafil citrate (Viagra) induced protective effects against ischemia/reperfusion injury in the intact heart and adult cardiomyocytes (9Ockaili R. Salloum F. Hawkins J. Kukreja R.C. Am. J. Physiol. 2002; 283: H1263-H1269Crossref PubMed Scopus (257) Google Scholar, 13Das A. Xi L. Kukreja K.C. J. Biol. Chem. 2005; 280: 12944-12955Abstract Full Text Full Text PDF PubMed Scopus (306) Google Scholar). Conceptually, sildenafil inhibits the enzymatic hydrolysis of cGMP, which in turn maintains the tissue accumulation of cGMP, leading to downstream protective mechanisms involving PKG activation and opening of mitochondrial ATP-sensitive potassium (mitoKATP) channels (6Kukreja R.C. Salloum F. Das A. Ockaili R. Yin C. Bremer Y.A. Fisher P.W. Wittkamp M. Hawkins J. Chou E. Kukreja A.K. Wang X. Marwaha V.R. Xi L. Vasc. Pharmacol. 2005; 42: 219-232Crossref PubMed Scopus (178) Google Scholar). It has been shown that sildenafil induces preconditioning through NO generated from endothelial and/or inducible nitric-oxide synthase (eNOS/iNOS) and via activation of protein kinase C and opening of the mitoKATP channels (9Ockaili R. Salloum F. Hawkins J. Kukreja R.C. Am. J. Physiol. 2002; 283: H1263-H1269Crossref PubMed Scopus (257) Google Scholar, 10Salloum F. Yin C. Xi L. Kukreja R.C. Circ. Res. 2003; 92: 595-597Crossref PubMed Scopus (209) Google Scholar, 12Das A. Ockaili R. salloum F. Kukreja R.C. Am. J. Physiol. 2004; 286: H1455-H1460Crossref PubMed Scopus (77) Google Scholar). Phosphodiesterase 5A inhibition also attenuated cell death resulting from necrosis and apoptosis by increasing the Bcl-2/Bax ratio through NO signaling (13Das A. Xi L. Kukreja K.C. J. Biol. Chem. 2005; 280: 12944-12955Abstract Full Text Full Text PDF PubMed Scopus (306) Google Scholar).A recent study also showed that phosphodiesterase 5A inhibition by sildenafil generates a potent anti-hypertrophic effect by enhancing PKGI activity, without increasing total cGMP in mice (14Takimoto E. Champion H.C. Li M. Belardi D. Ren S. Ridriguez E.R. Bedja D. Gabrielson K.L. Wang Y. Kass D.A. Nat. Med. 2005; 11: 214-222Crossref PubMed Scopus (734) Google Scholar). Adenoviral gene transfer of PKGIβ selectively enhanced anti-hypertrophic effects of NO, without increasing apoptosis (15Wollert K.C. Fiedler B. Gambaryan S. Smolenski A. Heineke J. Butt E. Trautwein C. Lohmann S.M. Drexler H. Hypertension. 2002; 39: 87-92Crossref PubMed Scopus (116) Google Scholar). Qin et al. (16Qin Q. Yang X.M. Cui L. Critz S.D. Cohen M.V. Browner N.C. Lincoln T.M. Downey J.M. Am. J. Physiol. 2004; 287: H712-H718Crossref PubMed Scopus (92) Google Scholar) showed that exogenous NO triggers preconditioning effect by stimulation of guanylyl cyclase to make cGMP, activation of PKG, opening of mitoKATP channels, and production of reactive oxygen species (ROS). Peptide blockers of PKG inhibited the ROS generation by pharmacological preconditioning agents such as acetylcholine and bradykinin in cardiomyocytes (17Krieg T. Philipp S. Cui L. Dostmann W.R. Downey J.M. Cohen M.V. Am. J. Physiol. 2005; 288: H1976-H1981Crossref PubMed Scopus (33) Google Scholar). Nevertheless, the direct role of PKGIα and the downstream signaling pathways that lead to protection of cardiomyocytes against apoptosis after ischemia/reoxygenation injury remain to be elucidated.PKGI has a number of effects that may be relevant to its regulation of apoptosis. PKGI can modulate gene expression in a variety of cell types by either stimulating or inhibiting extracellular signal-regulated kinase (ERK1/2)/mitogen-activated protein kinase (MAPK) (18Hofmann F. Ammendola A. Schlossmann J. J. Cell Sci. 2000; 113: 1671-1676Crossref PubMed Google Scholar, 19Pilz R.B. Casteel D.E. Circ. Res. 2003; 93: 1034-1046Crossref PubMed Scopus (242) Google Scholar). PKGI has been shown to suppress cell proliferation by inhibiting the Ras/MAPK pathway in baby hamster kidney cells transfected with PKGIβ (20Suhasini M. Li H. Lohmann S.M. Boss G.R. Pilz R.B. Mol. Cell Biol. 1998; 18: 6983-6994Crossref PubMed Scopus (99) Google Scholar). PKGI also induced the expression of MAPK phosphatase-1, which reverses activation of the MAPK pathway (20Suhasini M. Li H. Lohmann S.M. Boss G.R. Pilz R.B. Mol. Cell Biol. 1998; 18: 6983-6994Crossref PubMed Scopus (99) Google Scholar). PKGIα induced ERK1/2 by activating MAPK/ERK kinase in smooth muscle cells (21Komalavilas P. Shah P.K. Jo H. Lincoln T.M. J. Biol. Chem. 1999; 274: 34301-34309Abstract Full Text Full Text PDF PubMed Scopus (124) Google Scholar) but activated p38 MAPK in fibroblasts (22Browning D.D. McShane M.P. Marty C. Ye R.D. J. Biol. Chem. 2000; 275: 2811-2816Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar). In human colon cancer cells, PKG activated the MEKK1-SEK1-JNK1 pathway, by directly phosphorylating and activating MEKK1 (23Soh J.W. Mao Y. Liu L. Thompson W.J. Pamukcu R. Weinstein I.B. J. Biol. Chem. 2001; 276: 16406-16410Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar).Although PKG effects on cell survival/proliferation pathways have been observed in various cells types, the mechanism of PKG attenuation of ischemia/reoxygenation injury in the heart is not clear. The present study was designed to examine whether the expression of PKGIα protects cardiomyocytes from necrosis and apoptosis following simulated ischemia and reoxygenation. We also examined whether opening of mitoKATP channel, ROS generation and activation of multiple signaling pathways including Akt, NOS, and MAPKs are essential to PKG-mediated cellular protection. Our findings define mechanisms by which PKGIα inhibits necrosis and apoptosis signaling pathways to protect cardiomyocytes from ischemia/reperfusion injury.EXPERIMENTAL PROCEDURESIsolation of Ventricular Cardiomyocytes and Adenoviral Infection— Adult male Wistar rats (300 g) were purchased from Harlan Sprague-Dawley Inc. (Indianapolis, IN). The animal experimental protocol was approved by the Institutional Animal Care and Use Committee of Virginia Common-wealth University. Ventricular cardiomyocytes were isolated using an enzymatic technique as previously reported (13Das A. Xi L. Kukreja K.C. J. Biol. Chem. 2005; 280: 12944-12955Abstract Full Text Full Text PDF PubMed Scopus (306) Google Scholar). The freshly isolated cardiomyocytes were plated with Medium 199 containing 2 mm l-carnitine, 5 mm creatine, 5 mm taurine, 5 mm glucose, 0.1 μm insulin, and 1% penicillin-streptomycin. After 1 h of plating, the myocytes were infected with adenoviral vectors containing hPKGIα (Ad.PKGIα) (24Begum N. Sandu O.A. Ito M. Lohmann S.M. Smolenski A. J. Biol. Chem. 2002; 277: 6214-6222Abstract Full Text Full Text PDF PubMed Scopus (140) Google Scholar) or catalytically inactive hPKGIαK390A (25Smolenski A. Poller W. Walter U. Lohmann S.M. J. Biol. Chem. 2000; 275: 25723-25732Abstract Full Text Full Text PDF PubMed Scopus (117) Google Scholar) in serum-free growth medium for 24 h. The cells were routinely infected with the viruses at a concentration of 1 × 103 particles/cell.Simulated Ischemia/Reoxygenation Protocol—After 24 h of adenoviral infection, the cells were subjected to simulated ischemia (SI) for 90 min by replacing the cell medium with an "ischemia buffer" that contained 118 mm NaCl, 24 mm NaHCO3, 1.0 mm NaH2PO4, 2.5 mm CaCl2-2H2O, 1.2 mm MgCl2, 20 mm sodium lactate, 16 mm KCl, 10 mm 2-deoxyglucose (pH adjusted to 6.2) as reported previously (13Das A. Xi L. Kukreja K.C. J. Biol. Chem. 2005; 280: 12944-12955Abstract Full Text Full Text PDF PubMed Scopus (306) Google Scholar). The cells were incubated at 37 °C in tri-gas incubator adjusting 1-2% O2 and 5% CO2 during the entire SI period. RO was accomplished by replacing the ischemic buffer with normal cell medium under normoxic conditions. Cell necrosis and apoptosis were assessed after 2 or 18 h of reoxygenation, respectively.Evaluation of Cell Viability and Apoptosis—Trypan blue exclusion assay and lactate dehydrogenase release into the medium were used to assess cell necrosis (13Das A. Xi L. Kukreja K.C. J. Biol. Chem. 2005; 280: 12944-12955Abstract Full Text Full Text PDF PubMed Scopus (306) Google Scholar). Cardiomyocyte apoptosis was analyzed by TUNEL staining as reported previously (13Das A. Xi L. Kukreja K.C. J. Biol. Chem. 2005; 280: 12944-12955Abstract Full Text Full Text PDF PubMed Scopus (306) Google Scholar).Detection of Activated Caspase 3—Activated caspase was detected using the CaspaTagTM Caspase 3,7 in situ assay kit (Chemicon, Temecula, CA) according to the manufacturer's instructions (13Das A. Xi L. Kukreja K.C. J. Biol. Chem. 2005; 280: 12944-12955Abstract Full Text Full Text PDF PubMed Scopus (306) Google Scholar).Inhibitor Studies—Each experiment was started with a change of medium in the wells. To evaluate the involvement of KATP channels in PKG1α-mediated protection, Ad.P-KGIα-infected cells were treated for 30 min before SI-RO with 5-hydroxydecanoate (5-HD, 100 μm), HMR1098 (30 μm), or glibenclamide (Glib, 50 μm), the respective blockers of mitochondrial, sarcolemmal, or sarcolemmal/mitoKATP channels. To test the effect of a ROS scavenger on PKG-induced protection, a subgroup of cells were treated with N-(2 mercaptopropionyl)glycine (MPG, 1 mm) for 30 min prior to SI-RO. Other subgroups of cells were treated similarly with the NOS inhibitor l-nitro-amino-methyl-ester (l-NAME, 100 μm), PI 3-kinase inhibitors wortmannin (100 nm) and LY-294002 (10 μm), the ERK inhibitor PD-98059 (25 μm), and the JNK inhibitor SP600125 (10 μm).Western Blot Analysis—Western blots were performed as described previously (13Das A. Xi L. Kukreja K.C. J. Biol. Chem. 2005; 280: 12944-12955Abstract Full Text Full Text PDF PubMed Scopus (306) Google Scholar). The blots were incubated with rabbit polyclonal primary antibody or mouse monoclonal antibody at a dilution of 1:1000 for each of the respective proteins, i.e. PKGIα, nNOS, iNOS, eNOS, Bcl-2, Bax, pAkt, Akt, pERK, ERK1, pp38, p38, pJNK, JNK, and actin (all purchased from Santa Cruz) for 2 h. The membranes were then incubated with anti-rabbit or anti-mouse horseradish peroxidase-conjugated secondary antibody (1:2000 dilution; Amersham Biosciences) for 1 h. The blots were developed using a chemiluminescent system, and the bands were scanned and quantified by densitometric analysis.Data Analysis and Statistics—The data are presented as the means ± S.E. The difference between the groups was analyzed respectively with an unpaired t test or one-way analysis of variance followed by Student-Newman-Keul post-hoc test. p < 0.001 was considered to be statistically significant.RESULTSPKGIα Overexpression Inhibits Necrosis and Apoptosis—Fig. 1A shows a typical preparation of our isolated adult rat cardiomyocytes. At least 90% of the cardiomyocytes had rod-shaped morphology. After SI (90 min)-RO (2 h), the percentage of necrotic, trypan blue-positive cardiomyocytes increased to 33.7 ± 1.6 as compared with non-SI-RO controls (2.0 ± 0.2) (n = 7; p < 0.001; Fig. 1, A, B, and E). PKGIα expression in cardiomyocytes exposed to SI-RO reduced trypan blue-positive cardiomyocytes (n = 7; p < 0.001 versus SI-RO alone; Fig. 1, C and E). The overexpression of catalytically inactive PKGIαK390A failed to protect cells (Fig. 1, D and E). Similarly, PKGIα overexpression attenuated the release of lactate dehydrogenase as compared with SI-RO alone, whereas cardiomyocytes overexpressing PKGIαK390A did not (Fig. 1F).Apoptosis was not detectable after 90 min of SI and 2 h of RO. With an extended RO period of 18 h, TUNEL-positive cells increased as compared with the control group (p < 0.001 versus control, n = 7; Fig. 2, A and G). PKGIα overexpression reduced the TUNEL-positive cells (n = 7; p < 0.001 versus SI-RO group; Fig. 2, B and G), whereas PKGIαK390A failed to do so (Fig. 2, C and G). Representative staining of total nuclei with 4′,6-diamidino-2-phenylindole is shown for SI-RO control (Fig. 2D) and cells overexpressing PKGIα (Fig. 2E) or PKGIαK390A (Fig. 2F). Likewise, the red fluorescence of active caspase-positive cells increased in cardiomyocytes following SI and 18 h of RO (Fig. 2I) as compared with the control group (Fig. 2H), however clearly reduced by PKGIα overexpression (Fig. 2J) but not by PKGIαK390A (Fig. 2K). Representative panels (Fig. 2, L-O) show that 4′,6-diamidino-2-phenylindole staining of total nuclei was more or less similar in three groups, suggesting that the observed changes in caspase-3 staining represented true differences in apoptosis, not the number of nuclei. Quantitative measurements showed that the active caspase-positive cardiomyocytes are significantly higher in SI-RO-treated control (25. 6 ± 0.6%) and PKGIαK390A cardiomyocytes (26.3 ± 0.7%) as compared with the cells overexpressing PKG1α (7.2 ± 0.5%) (p < 0.001, n = 7; Fig. 2P). Nonischemic group showed only 1.3 ± 0.1% (n = 7) of the caspase-positive cardiomyocytes.FIGURE 2PKGIα inhibits apoptosis in cardiomyocytes. Apoptotic nuclei were observed using TUNEL assay after 90 min of SI and 18 h of RO in cardiomyocytes with no adenoviral transfection (A and D), with overexpression of PKGIα (B and E) or catalytically inactive PKGIαK390A (C and F). A-C, TUNEL-positive myocyte nuclei (stained in green fluorescent color); D-F, total nuclei (4′,6-diamidino-2-phenylindole staining). Note that PKGIα overexpression protects cardiomyocytes from apoptotic cell death following SI-RO in comparison with control SI-RO-treated cardiomyocytes. G, bar diagram shows quantitative data of TUNEL-positive cells from seven independent experiments. *, p < 0.001 versus SI-RO; Δ, p < 0.001 versus control without SI-RO. H-P, caspase 3 activity was detected by using CaspaTag reagent and a fluorescence microscope. The red fluorescent signal is a direct measure of activated caspase 3 in the cell (red, left panels) and nuclei stained by Hoechst (blue, right panels). H and L, control cardiomyocytes; I and M, cardiomyocytes subjected to 90 min of SI and 18 h of RO; J and N, PKGIα-overexpressing cardiomyocytes exposed to SI-RO; K and O, cardiomyocytes expressing catalytically inactive PKGIαK390A. Note that PKGIα-overexpressing cardiomyocytes show a negligible amount of red fluorescence signal as compared with the control SI-RO or PKGIαK390A expressing cardiomyocytes. P, bar diagram showing quantitative data of active caspase-positive cells from seven independent experiments *, p < 0.001 versus SI-RO; Δ, p < 0.001 versus control without SI-RO.View Large Image Figure ViewerDownload Hi-res image Download (PPT)FIGURE 2PKGIα inhibits apoptosis in cardiomyocytes. Apoptotic nuclei were observed using TUNEL assay after 90 min of SI and 18 h of RO in cardiomyocytes with no adenoviral transfection (A and D), with overexpression of PKGIα (B and E) or catalytically inactive PKGIαK390A (C and F). A-C, TUNEL-positive myocyte nuclei (stained in green fluorescent color); D-F, total nuclei (4′,6-diamidino-2-phenylindole staining). Note that PKGIα overexpression protects cardiomyocytes from apoptotic cell death following SI-RO in comparison with control SI-RO-treated cardiomyocytes. G, bar diagram shows quantitative data of TUNEL-positive cells from seven independent experiments. *, p < 0.001 versus SI-RO; Δ, p < 0.001 versus control without SI-RO. H-P, caspase 3 activity was detected by using CaspaTag reagent and a fluorescence microscope. The red fluorescent signal is a direct measure of activated caspase 3 in the cell (red, left panels) and nuclei stained by Hoechst (blue, right panels). H and L, control cardiomyocytes; I and M, cardiomyocytes subjected to 90 min of SI and 18 h of RO; J and N, PKGIα-overexpressing cardiomyocytes exposed to SI-RO; K and O, cardiomyocytes expressing catalytically inactive PKGIαK390A. Note that PKGIα-overexpressing cardiomyocytes show a negligible amount of red fluorescence signal as compared with the control SI-RO or PKGIαK390A expressing cardiomyocytes. P, bar diagram showing quantitative data of active caspase-positive cells from seven independent experiments *, p < 0.001 versus SI-RO; Δ, p < 0.001 versus control without SI-RO.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Effect of KATP Channel Blockers and a ROS Scavenger on PKGIα-induced Protection—Both Glib and 5-HD abolished PKGIα protection of cardiomyocytes exposed to SI-RO (p < 0.001 versus PKGIα; n = 3; Fig. 3A). Similarly, Glib and 5-HD blocked PKGIα protection against apoptosis, as demonstrated by an increase in TUNEL-positive cells (p < 0.001 versus PKGIα; n = 3; Fig. 3B). HMR1098 failed to block the protective effect of PKGIα against necrosis (Fig. 3A) as well as apoptosis (Fig. 3B) (p > 0.05 versus PKGIα; n = 3), ruling out a role of sarcolemmal KATP channels in PKGIα-induced protection. Glib, 5-HD, and HMR1098 had no effect on necrotic or apoptotic cell death after SI-RO in control or PKGIαK390A-expressing cardiomyocytes, thereby ruling out any potential PKG-independent effects of these agents. MPG abolished protective effects of PKGIα, i.e. increased the percentage of trypan blue-positive cells (Fig. 3A) and TUNEL-positive nuclei (Fig. 3B) after SI-RO, suggesting a role for ROS in PKGI-dependent cardiomyocyte protection.FIGURE 3Effect of KATP channel blockers and ROS scavenger on PKGIα-induced cardiomyocyte protection. 24 h after infection with PKGIα, cardiomyocytes were treated with 5-HD (100μm), HMR1098 (HMR,30μm), or Glib (50 μm) for 30 min before SI-RO. Another subgroup of cells was treated with MPG (1 mm) for 30 min before SI-RO. A, cell necrosis as determined by trypan blue-positive cells after 90 min of SI and 2 h of RO; B, apoptotic nuclei identified using TUNEL assay after 90 min of SI and 18 h of RO. Note that the protection against necrosis and apoptosis by PKGIα was abolished by 5-HD, Glib, and MPG.View Large Image Figure ViewerDownload Hi-res image Download (PPT)PKGIα Increases Phosphorylation of Akt—Because phosphorylation of Akt at Ser473 is required for its full activation, we used an antibody that specifically recognized Akt phosphorylated at Ser473. PKGIα overexpression increased the phosphorylation of Akt as compared with control (without infection) and PKGIαK390A in cardiomyocytes (Fig. 4, A and B). No further increases in Akt phosphorylation were observed when cardiomyocytes were subjected to SI and 15 or 30 min of reoxygenation following transfection with PKGIα (data not shown). Wortmannin and LY-294002 increased the percentage of trypan blue-positive cardiomyocytes from 18.1 ± 0.3 (PKGIα overexpression group) to 33.5 ± 0.6 and 34.7 ± 0.9, respectively (n = 3; p < 0.001; Fig. 4C). The number of TUNEL-positive nuclei was also increased from 6.8 ± 0.8 (in PKGIα overexpression group) to 23.5 ± 0.2 and 23.8 ± 1.0 by wortmannin and LY-294002, respectively (n = 3; p < 0.001; Fig. 4D). Wortmannin and LY-294002 had no effect on necrosis or apoptosis in control or PKGIαK390A-overexpressing cardiomyocytes exposed to SI-RO.FIGURE 4Role of Akt phosphorylation in PKGIα-induced cardiomyocyte protection. A, representative Western blots showing expression of phosphorylated Akt and total Akt after 24 h of infection with PKGIα or PKGIαK390A. B, bar diagram showing average ratio of p-Akt to total Akt. PKGIα expression increased significantly compared with control (without infection) and PKGIαK390A expression. Treatment with wortmannin (Wort, 100 nm) or LY-294002 (LY, 10 μm) before SI-RO abolished PKG-dependent protection. C, cell necrosis is determined by trypan blue-positive cells after 90 min of SI and 2 h of RO. D, apoptotic nuclei identified using TUNEL assay after 90 min of SI and 18 h of RO. Note that wortmannin and LY-294002 abolished PKGIα-induced protection against necrosis and apoptosis. Wortmannin and LY-294002 had no effect on control or PKGIαK390A-overexpressing cardiomyocytes exposed to SI-RO.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Effect of PKGIα on Expression of NOS, Bcl-2, and Bax—Adenoviral transfection with PKGIα and PKGIαK390A augmented the expression of PKGIα protein after 24 h (Fig. 5A). PKGIα overexpression clearly increased iNOS, eNOS, and Bcl-2 expression as compared with the control cells or cells infected with PKGIαK390A (Fig. 5, A and B). However, Bax was significantly decreased with overexpression of PKGIα as compared with control and PKGIαK390A-overexpressing cells (Fig. 5, A and B). No increase in nNOS was observed after PKGIα or PKGIαK390A overexpression as compared with control cells (Fig. 5, A and B). Similar results were obtained when cardiomyocytes were subjected to SI and 15 or 30 min of reoxygenation following transfection with PKGIα (data not shown).FIGURE 5Effect of PKGIα on expression of NOS isoforms, Bax, and Bcl-2. A, representative Western blots show expression levels of PKGIα, nNOS, iNOS, eNOS, Bax, Bcl-2, and β-actin 24 h after infection with PKGIα or PKGIαK390A. B, bar diagrams showing density of bands after normalization to β-actin in the same samples. Note that iNOS, eNOS, and Bcl-2 proteins were significantly increased, but Bax was decreased by PKGIα overexpression.View Large Image Figure ViewerDownload Hi-res image Download (PPT)To demonstrate the
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