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• W2093056834 abstract "Expression of the wild type α subunit of Gq (GqWT) in cardiomyocytes induces hypertrophy, whereas a constitutively active Gαq subunit (GqQ209L) induces apoptosis. Akt phosphorylation increases with GqWT expression but is markedly attenuated in cardiomyocytes expressing GqQ209L or in those expressing GqWT and treated with agonist. A membrane-targeted Akt rescues GqQ209L-expressing cardiomyocytes from apoptotic cell death. In contrast, leukemia inhibitory factor fails to activate Akt or promote cell survival in these cells. Association of Akt and PDK-1 with the membrane is also diminished in GqQ209L-expressing cardiomyocytes. Phosphatidylinositol 3,4,5-trisphosphate (PIP3), the primary regulator of Akt, increases significantly in GqWT-expressing cells but not in cardiomyocytes expressing GqQ209L. Levels of phosphatidylinositol 4,5-bisphosphate (PIP2), the immediate precursor of PIP3, are also markedly lower in GqQ209L-expressing compared to control cells. Expression of a GqQ209L mutant that has diminished capacity to activate phospholipase C does not decrease PIP2 or Akt or induce apoptosis. In transgenic mice with cardiac Gαq overexpression, heart failure and increased cardiomyocyte apoptosis develop during the peripartal period. Akt phosphorylation and PIP2 levels decrease concomitantly. Our findings suggest that an Akt-mediated cell survival pathway is compromised by the diminished availability of PIP2 elicited by pathological levels of Gq activity. Expression of the wild type α subunit of Gq (GqWT) in cardiomyocytes induces hypertrophy, whereas a constitutively active Gαq subunit (GqQ209L) induces apoptosis. Akt phosphorylation increases with GqWT expression but is markedly attenuated in cardiomyocytes expressing GqQ209L or in those expressing GqWT and treated with agonist. A membrane-targeted Akt rescues GqQ209L-expressing cardiomyocytes from apoptotic cell death. In contrast, leukemia inhibitory factor fails to activate Akt or promote cell survival in these cells. Association of Akt and PDK-1 with the membrane is also diminished in GqQ209L-expressing cardiomyocytes. Phosphatidylinositol 3,4,5-trisphosphate (PIP3), the primary regulator of Akt, increases significantly in GqWT-expressing cells but not in cardiomyocytes expressing GqQ209L. Levels of phosphatidylinositol 4,5-bisphosphate (PIP2), the immediate precursor of PIP3, are also markedly lower in GqQ209L-expressing compared to control cells. Expression of a GqQ209L mutant that has diminished capacity to activate phospholipase C does not decrease PIP2 or Akt or induce apoptosis. In transgenic mice with cardiac Gαq overexpression, heart failure and increased cardiomyocyte apoptosis develop during the peripartal period. Akt phosphorylation and PIP2 levels decrease concomitantly. Our findings suggest that an Akt-mediated cell survival pathway is compromised by the diminished availability of PIP2 elicited by pathological levels of Gq activity. Gαq signaling plays a key role in the hypertrophic growth of cardiomyocytes. Hormones that act through receptors coupled to the heterotrimeric protein Gq (e.g. norepinephrine, PGF2α, 1The abbreviations used are: PGF2α, prostaglandin F2α; PLC, phospholipase C; NRVMs, neonatal rat ventricular myocytes; PI3K, phosphoinositide 3-kinase; PDK-1, phosphoinositide-dependent kinase-1; PIP3, phosphoinositide 3,4,5-trisphosphate; PIP2, phosphoinositide 4,5-bisphosphate; CT-1, cardiotrophin-1; LIF, leukemia inhibitory factor; ITS, insulin/transferrin/selenium; myr-Akt, myristoylated Akt; PP, protein phosphatase; HPLC, high pressure liquid chromatography. endothelin, and angiotensin II) induce cardiomyocyte hypertrophy in vitro (1Clerk A. Sugden P.H. Am. J. Cardiol. 1999; 83: H64-H69Abstract Full Text Full Text PDF PubMed Scopus (103) Google Scholar). Further evidence for a physiological role of Gαq activation is that blocking Gq signaling in vivo prevents pressure-overload hypertrophy (2Akhter S.A. Luttrell L.M. Rockman H.A. Iaccarino G. Lefkowitz R.J. Koch W.J. Science. 1998; 280: 574-577Crossref PubMed Scopus (394) Google Scholar, 3Wettschureck N. Rutten H. Zywietz A. Gehring D. Wilkie T.M. Chen J. Chien K.R. Offermanns S. Nat. Med. 2001; 7: 1236-1240Crossref PubMed Scopus (317) Google Scholar). Hypertrophy can transition to heart failure under conditions of enhanced Gαq signaling induced by overexpression of Gq-coupled receptors, of phospholipase C-β (PLC), of constitutively active Gαq, or of wild type Gαq in combination with stress (4Milano C.A. Dolber P.C. Rockman H.A. Bond R.A. Venable M.E. Allen L.F. Lefkowitz R.J. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 10109-10113Crossref PubMed Scopus (332) Google Scholar, 5Akhter S.A. Milano C.A. Shotwell K.F. Cho M.-C. Rockman H.A. Lefkowitz R.J. Koch W.J. J. Biol. Chem. 1997; 272: 21253-21259Abstract Full Text Full Text PDF PubMed Scopus (141) Google Scholar, 6Engelhardt S. Hein L. Wiesmann F. Lohse M.J. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 7059-7064Crossref PubMed Scopus (658) Google Scholar, 7Hein L. Stevens M.E. Barsh G.S. Pratt R.E. Kobilka B.K. Dzau V.J. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 6391-6396Crossref PubMed Scopus (194) Google Scholar, 8Adams J.W. Sakata Y. Davis M.G. Sah V.P. Wang Y. Liggett S.B. Chien K.R. Brown J.H. Dorn G.W. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 10140-10145Crossref PubMed Scopus (472) Google Scholar, 9Sakata Y. Hoit B.D. Liggett S.B. Walsh R.A. Dorn G.W. Circulation. 1997; 97: 1488-1495Crossref Scopus (177) Google Scholar, 10Mende U. Kagen A. Cohen A. Aramburu J. Schoen F.J. Neer E.J. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 13893-13898Crossref PubMed Scopus (226) Google Scholar, 11Wakasaki H. Koya D. Schoen F.J. Jirousek M.R. Ways D.K. Hoit B.D. Walsh R.A. King G.L. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 9320-9325Crossref PubMed Scopus (353) Google Scholar). Thus studies in isolated cardiomyocytes and in transgenic animals have led to the conclusion that although Gαq signaling is critical for cardiac hypertrophy, prolonged or marked enhancement of Gq activity is deleterious to the heart. Our laboratory has characterized a model of in vitro cardiomyocyte hypertrophy and apoptosis by expressing the α subunit of Gq (8Adams J.W. Sakata Y. Davis M.G. Sah V.P. Wang Y. Liggett S.B. Chien K.R. Brown J.H. Dorn G.W. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 10140-10145Crossref PubMed Scopus (472) Google Scholar). When the wild type α subunit (GqWT) is expressed by adenoviral infection, neonatal rat ventricular myocytes (NRVMs) undergo hypertrophy (8Adams J.W. Sakata Y. Davis M.G. Sah V.P. Wang Y. Liggett S.B. Chien K.R. Brown J.H. Dorn G.W. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 10140-10145Crossref PubMed Scopus (472) Google Scholar). In contrast, when the constitutively active α subunit (GqQ209L) is expressed, or GqWT-expressing cardiomyocytes are treated with Gq-coupled receptor agonists, the NRVMs undergo apoptosis (8Adams J.W. Sakata Y. Davis M.G. Sah V.P. Wang Y. Liggett S.B. Chien K.R. Brown J.H. Dorn G.W. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 10140-10145Crossref PubMed Scopus (472) Google Scholar, 12Adams J.W. Pagel A.L. Means C.K. Oksenberg D. Armstrong R.C. Brown J.H. Circ. Res. 2000; 87: 1180-1187Crossref PubMed Scopus (99) Google Scholar). Varying the amount of Gαq signaling in myocytes therefore results in distinct cellular phenotypes. Although modest levels of signaling induce hypertrophy, at higher levels survival is diminished and apoptosis ensues. This provides a paradigm for elucidating the molecular mechanisms by which Gαq signaling can transition from stimulation of hypertrophic growth to the development of apoptosis. We demonstrated previously (12Adams J.W. Pagel A.L. Means C.K. Oksenberg D. Armstrong R.C. Brown J.H. Circ. Res. 2000; 87: 1180-1187Crossref PubMed Scopus (99) Google Scholar) that expression of GqQ209L leads to apoptosis through mitochondrial permeability (PT) pore opening, cytochrome c release, and caspase activation. Sustained Gαq signaling, beyond the level that causes hypertrophy, may also compromise normal survival pathways. A well documented cell survival pathway is the PI3K/phosphoinositide-dependent kinase-1/Akt (PI3K/PDK-1/Akt) pathway (13Krasilnikov M.A. Biochemistry. 2000; 65: 59-67PubMed Google Scholar). PI3K generates phosphatidylinositol 3,4,5-trisphosphate (PIP3) from phosphatidylinositol 4,5-bisphosphate (PIP2) to stimulate the membrane colocalization of PDK-1 and Akt (14Matsui T. Li L. del Monte F. Fukui Y. Franke T.F. Hajjar R.J. Rosenzweig A. Circulation. 1999; 100: 2373-2379Crossref PubMed Scopus (338) Google Scholar, 15Fujio Y. Nguyen T. Wencker D. Kitsis R.N. Walsh K. Circulation. 2000; 101: 660-667Crossref PubMed Scopus (737) Google Scholar, 16Rameh L.E. Cantley L.C. J. Biol. Chem. 1999; 274: 8347-8350Abstract Full Text Full Text PDF PubMed Scopus (852) Google Scholar). Akt/protein kinase B is phosphorylated by PDK-1 at Thr-308 and subsequently autophosphorylates at Ser-473 (17Toker A. Newton A.C. J. Biol. Chem. 2000; 275: 8271-8274Abstract Full Text Full Text PDF PubMed Scopus (422) Google Scholar). In cardiomyocytes, Akt has been shown to be activated by agonists that stimulate tyrosine kinase receptors (e.g. insulin and insulin-like growth factor (15Fujio Y. Nguyen T. Wencker D. Kitsis R.N. Walsh K. Circulation. 2000; 101: 660-667Crossref PubMed Scopus (737) Google Scholar, 18Pham F.H. Sugden P.H. Clerk A. Circ. Res. 2000; 86: 1252-1258Crossref PubMed Scopus (114) Google Scholar)) and by cytokines (e.g. cardiotrophin-1 (CT-1) and leukemia inhibitory factor (LIF) (19Kuwahara K. Saito Y. Harada M. Ishikawa M. Ogawa E. Miyamoto Y. Hamanaka I. Kamitani S. Kajiyama N. Takahashi N. Nakagawa O. Masuda I. Nakao K. Circulation. 1999; 100: 1116-1124Crossref PubMed Scopus (106) Google Scholar, 20Hirota H. Chen J. Betz U.A.K. Rajewsky K. Gu Y. Ross Jr., J. Muller W. Chien K.R. Cell. 1999; 97: 189-198Abstract Full Text Full Text PDF PubMed Scopus (588) Google Scholar, 21Oh H. Fujio Y. Kunisada K. Hirota H. Matsui H. Kishimoto T. Yamauchi K.-T. J. Biol. Chem. 1998; 273: 9703-9710Abstract Full Text Full Text PDF PubMed Scopus (188) Google Scholar)). Akt has been reported to protect cardiomyocytes from apoptosis induced by serum deprivation or hypoxia (14Matsui T. Li L. del Monte F. Fukui Y. Franke T.F. Hajjar R.J. Rosenzweig A. Circulation. 1999; 100: 2373-2379Crossref PubMed Scopus (338) Google Scholar, 15Fujio Y. Nguyen T. Wencker D. Kitsis R.N. Walsh K. Circulation. 2000; 101: 660-667Crossref PubMed Scopus (737) Google Scholar). Furthermore, in vivo gene transfer of activated Akt protects against cell death and the development of cardiomyopathies caused by ischemia-reperfusion and doxorubicin treatment (22Matsui T. Tao J. del Monte F. Lee K.H. Li L. Picard M. Force T.L. Franke T.F. Hajjar R.J. Rosenzweig A. Circulation. 2001; 104: 330-335Crossref PubMed Scopus (597) Google Scholar, 23Miao W. Luo Z. Kitsis R.N. Walsh K. J. Mol. Cell. Cardiol. 2000; 32: 2397-2402Abstract Full Text PDF PubMed Scopus (124) Google Scholar, 24Taniyama Y. Walsh K. J. Mol. Cell. Cardiol. 2002; 34: 1241-1247Abstract Full Text PDF PubMed Google Scholar). The critical role of Akt in cardioprotection underscores the need to examine the regulation of PI3K, PIP3 formation, and PDK-1 activity and determine how these control Akt activation in cardiomyocytes. We report here that increasing Gq signaling to pathological levels results in a loss of the protective PI3K/Akt signaling pathway. This occurs at a time point that precedes the appearance of apoptotic markers such as cytochrome c release and nuclear fragmentation. This contrasts directly with the enhanced Akt phosphorylation seen in cardiomyocytes that undergo hypertrophy in response to expression of GqWT. Our investigation into the pathways leading to the down-regulation of Akt activation in cardiomyocytes expressing GqQ209L revealed an unexpected decrease in cellular levels of PIP2, the substrate for both PLC and PI3K, which appeared to be related to the Gq-mediated increase in PLC activity. Importantly, in vivo studies also revealed decreased Akt phosphorylation and PIP2 levels in a Gq-expressing model of heart failure. We suggest that PIP3 generation and Akt activation are influenced by the availability of PIP2 and that this serves as a nodal point in determining the balance between hypertrophic and apoptotic pathways. Cardiomyocyte Culture and Adenoviral Infection—NRVMs were prepared and infected with adenoviruses as described previously (8Adams J.W. Sakata Y. Davis M.G. Sah V.P. Wang Y. Liggett S.B. Chien K.R. Brown J.H. Dorn G.W. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 10140-10145Crossref PubMed Scopus (472) Google Scholar). Briefly, cardiomyocytes were plated at a density of 1.5 × 106 cells per 6-cm dish or 3 × 105 per well of a 6-well plate in serum-containing media overnight. The cells were then washed, and the medium was replaced with serum-free medium supplemented with insulin/transferrin/selenium (ITS). Cells were infected with adenoviruses at 200-500 viral particles/cell for 16-18 h or as indicated. Cells were subsequently washed and maintained in serum-free medium with supplements. The p110WT and p110caax expressing adenoviruses were kindly provided by Dr. Jerry Olefsky (University of California, San Diego) (25Egawa K. Sharma P.M. Nakashima N. Huang Y. Huver E. Boss G.R. Olefsky J.M. J. Biol. Chem. 1999; 274: 14306-14314Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar). The virus encoding myristoylated Akt was obtained from Dr. Kenneth Walsh (Tufts University) (26Fujio Y. Guo K. Mano T. Mitsuuchi Y. Testa J.R. Walsh K. Mol. Cell. Biol. 1999; 19: 5073-5082Crossref PubMed Scopus (188) Google Scholar). The GqWT and GqQ209L adenoviruses have been characterized previously, and the GqQ209LDNE adenovirus was subcloned into adenovirus from a plasmid obtained from Dr. John Exton (Vanderbilt University), by using our method published previously (8Adams J.W. Sakata Y. Davis M.G. Sah V.P. Wang Y. Liggett S.B. Chien K.R. Brown J.H. Dorn G.W. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 10140-10145Crossref PubMed Scopus (472) Google Scholar). Apoptosis Measurements—Oligonucleosomal DNA fragmentation was analyzed by DNA laddering as described previously (12Adams J.W. Pagel A.L. Means C.K. Oksenberg D. Armstrong R.C. Brown J.H. Circ. Res. 2000; 87: 1180-1187Crossref PubMed Scopus (99) Google Scholar). Nuclear condensation was visualized using fluorescence microscopy after staining with Hoechst 33342 as described previously (12Adams J.W. Pagel A.L. Means C.K. Oksenberg D. Armstrong R.C. Brown J.H. Circ. Res. 2000; 87: 1180-1187Crossref PubMed Scopus (99) Google Scholar). Western Blot Analysis—Cells were harvested in lysis buffer, and Bradford analysis was performed. Equal micrograms of protein (10-30 μg) were loaded onto SDS-PAGE, run, transferred to an Immobilon membrane, and the resulting blot probed using the following antibodies. The Ser-473 and Thr-308 phosphospecific antibodies for Akt, as well as the Pan-Akt antibody, were purchased from Cell Signaling Technologies. Anti-PTEN and anti-p85 were purchased from Upstate Biotechnology, Inc. Anti-SHIP-2 was purchased from Santa Cruz Biotechnology. The antibody against the cardiac sodium/calcium exchanger was purchased from Swant. p110α (Transduction Laboratories), p110β (Santa Cruz Biotechnology), and p110γ (Santa Cruz Biotechnology) antibodies were kind gifts from Dr. Stephanie Watts (Michigan State University). The phospho-PDK-1 antibody (Cell Signaling) was a kind gift from Dr. Alexandra Newton's laboratory (University of California, San Diego). Measurement of [3H]PIP 3 —NRVMs plated at 4 × 106 cells on 10-cm plates were labeled with 30 μCi/ml myo-[3H]inositol for 36 h, and then infected with control, GqWT, or GqQ209L adenoviruses overnight, and harvested 24 h later. Cells were extracted with 5% trichloroacetic acid, 5 mm EDTA, 5 mm phytic acid; the lysates were sonicated for 10 s, and lipid-containing material was pelleted by centrifugation. [3H]Inositol-labeled phospholipids in the trichloroacetic acid pellet were extracted with chloroform/methanol/hydrochloric acid (200:100:1, v/v) and subsequently deacylated using methylamine/methanol/butanol (42:47:9) for 45 min at 50 °C as described previously (27Woodcock E.A. Suss M.B. Anderson K.E. Circ. Res. 1995; 76: 252-260Crossref PubMed Google Scholar), treated with proteinase K (28Woodcock E.A. Mol. Cell. Biochem. 1997; 172: 121-127Crossref PubMed Scopus (20) Google Scholar), and then separated by anion-exchange HPLC and quantified using an on-line β-counter (28Woodcock E.A. Mol. Cell. Biochem. 1997; 172: 121-127Crossref PubMed Scopus (20) Google Scholar). Measurement of PIP 2 —To measure [3H]PIP2, cells (1.5 × 106 on 6-cm dishes) were incubated with 5 μCi/ml myo-[3H]inositol for 36 h, prior to infection with the indicated adenoviruses. [3H]Inositol phospholipids were extracted and deacylated as described above. The final aqueous phase containing the deacylated lipids was loaded onto Dowex-1 (formate) columns. The columns were washed with 400 mm ammonium formate, 100 mm formic acid to remove [3H]glycerophosphoinositol (deacylated phosphatidylinositol) and [3H]glycerophosphoinositol(4)monophosphate. [3H]Glycerophosphoinositol(4,5)bisphosphate was eluted with 10 ml of 1 m ammonium formate, 100 mm formic acid. Samples were counted in a β-counter. For the mass measurement of PIP2, unlabeled lipids were extracted and deacylated as described above except 5 mm ATP replaced phytic acid in the extraction buffer. Deacylated lipids were deglycerated by treating with periodate (50 mm) for 30 min with the reaction being terminated by adding 10% ethylene glycol followed by 0.3% dimethylhydrazine (27Woodcock E.A. Suss M.B. Anderson K.E. Circ. Res. 1995; 76: 252-260Crossref PubMed Google Scholar). The resulting Ins(1,4,5)P3 was assayed by competitive binding assay (BioTrak IP3 Assay protocol, Amersham Biosciences), as described by the supplier. Cell Fractionation Studies—Fractionation was performed as described (29Sonnenburg E.D. Gao T. Newton A.C. J. Biol. Chem. 2001; 276: 45289-45297Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar). Briefly, NRVMs were harvested in detergent-free lysis buffer (20 mm HEPES, pH 7.5, 2 mm EDTA, 2 mm EGTA, 1 mm dithiothreitol, 300 μm phenylmethylsulfonyl fluoride, 40 μg/ml leupeptin, 1 mm sodium vanadate (NaVO3)), frozen at -80 °C for 5 min, and then sonicated for 2 min in a bath sonicator. The resulting cytoplasmic fraction was cleared by ultracentrifugation at 100,000 × g for 30 min. After removing the supernatant (cytosolic fraction), the pellet was resuspended in lysis buffer (50 mm Tris, 5 mm MgCl2, 1 mm EGTA, 0.5% Nonidet P-40, 2.5% glycerol, 50 mm NaF, 1 mm NaVO3, 300 μm phenylmethylsulfonyl fluoride, 10 μg/ml aprotinin, 40 μg/ml leupeptin), sonicated for 2 min, and centrifuged again at 100,00 × g for 30 min. The resulting supernatant was analyzed as the membrane fraction, and the pellet as the insoluble fraction. G q Transgenic Mouse Studies—Female Gq transgenic mice (kindly provided by Dr. G. W. Dorn II, University of Cincinnati Medical Center) and nontransgenic littermate control mice were mated, and 5 days after parturition, hearts were excised and snap-frozen in liquid nitrogen. Age-matched virgin mice were used as controls. Procedures were performed in accordance with the Guide for the Care and Use of Laboratory Animals and approved by the Institutional Animal Care and Use Committee. The frozen hearts were powdered and homogenized in lysis buffer, and 30 μg of protein was loaded onto SDS-PAGE for Western blot analysis as described above. For PIP2 mass analysis, 10 mg of powder was homogenized in trichloroacetic acid; then the lipids were deacylated and deglycerated before being subjected to the same mass assay as described above. Statistical Analysis—All results are reported as mean ± S.E. Comparisons of two groups only were accomplished using unpaired Student's t test. Experiments with more than two groups were compared by one-way analysis of variance followed by the Tukey post-hoc test for comparison between groups. Phospho-Akt Levels Are Diminished in G q Q209L-expressing NRVMs—Expression of a constitutively active mutant of Gαq (GqQ209L) induces apoptosis in NRVMs, whereas expression of the GqWT protein induces a sustained hypertrophy with no evidence of cell death (8Adams J.W. Sakata Y. Davis M.G. Sah V.P. Wang Y. Liggett S.B. Chien K.R. Brown J.H. Dorn G.W. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 10140-10145Crossref PubMed Scopus (472) Google Scholar). We hypothesized that this might reflect differential activation of protective pathways such as those initiated by phosphorylation of Akt. NRVMs were infected with viruses expressing GqWT, GqQ209L, or control virus backbone for 16 h followed by washing out of the viruses. Cells were harvested immediately after removal of the virus or after a further 8 or 24 h. All of these time points precede the development of apoptosis (36-48 h after overnight infection) (12Adams J.W. Pagel A.L. Means C.K. Oksenberg D. Armstrong R.C. Brown J.H. Circ. Res. 2000; 87: 1180-1187Crossref PubMed Scopus (99) Google Scholar) and exhibited similar results. As shown in Fig. 1, at 8 h after virus removal, GqWT expression caused a 3-fold increase in Akt phosphorylation (Fig 1, A and B) relative to control. Treating GqWT-expressing myocytes with PGF2α to further enhance Gq activity abolished this increase in Akt phosphorylation. Enhancing Gq activity even further, via expression of the constitutively active GqQ209L, resulted in a marked (45%) decrease in Akt phosphorylation, relative to control. Total Akt levels did not differ in GqWT or GqQ209L-expressing NRVMs versus control (Fig. 1A). Akt Can Prevent G q Q209L-induced Apoptosis—The observation that levels of phospho-Akt were markedly decreased in GqQ209L-expressing cells suggested that the loss of this survival pathway could be a critical factor in the apoptotic response to GqQ209L. If this is so, then direct activation of Akt should prevent GqQ209L-induced apoptosis. To examine this possibility, NRVMs were coinfected with GqQ209L along with adenovirus expressing a constitutively activated Akt construct in which the c-Src myristoylation sequence was fused to the N terminus of Akt (myr-Akt). This membrane-targeted form of Akt has been reported to prevent serum deprivation-induced apoptosis in cultured cardiomyocytes and to reduce apoptosis caused by ischemia-reperfusion in vivo (15Fujio Y. Nguyen T. Wencker D. Kitsis R.N. Walsh K. Circulation. 2000; 101: 660-667Crossref PubMed Scopus (737) Google Scholar) or hypoxia in vitro (14Matsui T. Li L. del Monte F. Fukui Y. Franke T.F. Hajjar R.J. Rosenzweig A. Circulation. 1999; 100: 2373-2379Crossref PubMed Scopus (338) Google Scholar). Several independent indices of apoptosis were examined 36-48 h after infection to establish whether myr-Akt could protect the myocytes from GqQ209L-induced apoptosis. The characteristic laddering pattern resulting from oligonucleosomal DNA fragmentation is prominent in GqQ209L-expressing cells undergoing apoptosis (8Adams J.W. Sakata Y. Davis M.G. Sah V.P. Wang Y. Liggett S.B. Chien K.R. Brown J.H. Dorn G.W. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 10140-10145Crossref PubMed Scopus (472) Google Scholar), and this was reduced in cells in which myr-Akt was expressed along with GqQ209L (Fig. 2A). In addition, whereas disorganization of myofilaments is evident in cells expressing GqQ209L (12Adams J.W. Pagel A.L. Means C.K. Oksenberg D. Armstrong R.C. Brown J.H. Circ. Res. 2000; 87: 1180-1187Crossref PubMed Scopus (99) Google Scholar), the expression of myr-Akt along with GqQ209L preserved myofilament organization (Fig. 2B). Finally, Hoechst staining was used to quantitate the number of cells with fragmented nuclei following infection with GqQ209L, myrAkt, or both viruses together. Expression of myr-Akt reduced the number of fragmented nuclei in GqQ209L-infected cells by ∼60% (Fig. 2C). The effect of enhancing Akt activity supports the notion that decreases in Akt activity play a central role in the development of GqQ209L-induced apoptosis. LIF Does Not Prevent Apoptosis or Activate Akt in G q Q209L-expressing NRVMs—Cytokines that act via the gp130 receptor, including LIF and cardiotrophin 1 (CT-1), induce PI3K-dependent Akt phosphorylation (21Oh H. Fujio Y. Kunisada K. Hirota H. Matsui H. Kishimoto T. Yamauchi K.-T. J. Biol. Chem. 1998; 273: 9703-9710Abstract Full Text Full Text PDF PubMed Scopus (188) Google Scholar, 30Kuwahara K. Saito Y. Kishimoto I. Miyamoto Y. Harada M. Ogawa E. Hamanaka I. Kajiyama N. Takahashi N. Izumi T. Kawakami R. Nakao K. J. Mol. Cell. Cardiol. 2000; 32: 1385-1394Abstract Full Text PDF PubMed Scopus (93) Google Scholar) and can prevent apoptosis induced by serum deprivation in NRVMs (31Sheng Z. Knowlton K. Chen J. Hoshijima M. Brown J.H. Chien K.R. J. Biol. Chem. 1997; 272: 5783-5791Abstract Full Text Full Text PDF PubMed Scopus (350) Google Scholar). When NRVMs were placed in serum-free medium lacking supplementation with ITS, apoptosis ensued. LIF was able to prevent the development of apoptosis as assessed by DNA laddering (Fig. 3A, 5th and 6th lanes). In contrast, LIF failed to protect against GqQ209L-induced DNA laddering (Fig. 3A, 2nd to 4th lanes). Because LIF may not be sufficiently stable to signal for the periods needed for protection from cell death, we added additional LIF to the GqQ209L-expressing cells, throughout the course of GqQ209L expression (data not shown). Even under these conditions, LIF failed to rescue the GqQ209L phenotype. In addition, whereas acute treatment with LIF markedly increased Akt phosphorylation in control NRVMs, it failed to do so in GqQ209L-expressing myocytes (Fig. 3B). The lack of Akt activation by LIF would appear to be responsible for the inability of this cytokine to prevent GqQ209L-expressing NRVMs from undergoing apoptosis. Phosphatase Inhibition Does Not Restore Akt Phosphorylation in G q Q209L-expressing NRVMs—We considered the possibility that GqQ209L expression might diminish Akt phosphorylation by activating phosphatases, such as PP2A, that can dephosphorylate Akt (32Schmidt M. Evellin S. Weernink P.A. von Dorp F. Rehmann H. Lomasney J.W. Jakobs K.H. Nat. Cell Biol. 2001; 3: 1020-1024Crossref PubMed Scopus (282) Google Scholar). Calyculin, a phosphatase inhibitor with specificity for PP1 and PP2A, was used to test this possibility. In both control-infected and GqQ209L-expressing NRVMs pretreated for 30 min with calyculin, basal Akt phosphorylation was enhanced, demonstrating some endogenous control by phosphatases PP1 and PP2A. However, LIF was unable to increase Akt phosphorylation in GqQ209L-expressing cells even when these phosphatases were inhibited (Fig. 3C). This argues against enhanced dephosphorylation of Akt as the mechanism by which GqQ209L expression limits Akt activation and cell survival. G q Q209L-expressing Cells Do Not Exhibit an Increase in PIP 3 —Because it was not possible to stimulate Akt phosphorylation using the cytokine LIF, and a calyculin-sensitive phosphatase did not appear to be involved, we turned our focus upstream of Akt phosphorylation. Activation of Akt is often used as a surrogate for changes in PIP3 levels because Akt phosphorylation by PDK-1 is PIP3-dependent. We therefore asked whether there was diminished PIP3 production in GqQ209L-expressing cells. To our knowledge, there are no published reports of direct PIP3 measurements in cardiomyocytes, and PIP3 levels at rest are thought to be extremely low. To establish the feasibility of measuring [3H]PIP3 in NRVMs, we labeled cells (4 × 106 per plate) with 30 μCi/ml of myo-[3H]inositol for 36 h and used an adenoviral vector to express a membrane-targeted, and thus constitutively active, PI3K catalytic subunit (p110caax). The wild type p110 subunit of PI3K (p110WT), which does not target to membranes, served as a control. [3H]Inositol phospholipids were extracted and quantified using HPLC as described under “Experimental Procedures.” Chromatographic analysis of extracts from cells expressing p110caax revealed a peak at the expected elution time of PIP3. The corresponding peak in p110WT-expressing cells was substantially smaller, confirming the identity of the peak as PIP3 (Fig. 4). We then examined [3H]PIP3 levels in cells infected with control, GqWT, and GqQ209L-expressing adenoviruses. HPLC analysis revealed significant levels of [3H]PIP3 in GqWT-expressing cells (Fig. 5). In contrast, PIP3 was barely detectable in cells expressing GqQ209L.Fig. 5Reduced [3H]PIP3 content in GqQ209L-versus GqWT-expressing NRVMs. NRVMs were labeled with 30 μCi/ml myo-[3H]inositol for 36 h prior to infection with control, GqWT-, or GqQ209L-expressing adenovirus overnight. 24 h later, 3H-labeled phospholipids were extracted and quantified by HPLC, as described under “Experimental Procedures.” Representative HPLC traces showing PIP2 and PIP3 peaks are shown for each group. Values shown are mean ± S.E. in counts/min of [3H]PIP3 per 10-cm plate of cells. *, p < 0.05 compared with control or GqQ209L.View Large Image Figure ViewerDownload Hi-res image Download (PPT) PIP 3 -dependent Membrane Translocation Is Not Stimulated in G q Q209L-expressing Cardiomyocytes—Akt phosphorylation, and thus activation, occurs when PDK-1 and Akt are colocalized at the membrane following PIP3 generation (33Toker A. Newton A.C. Cell. 2000; 103: 185-188Abstract Full Text Full Text PDF PubMed Scopus (367) Google Scholar). The localization of these enzymes was therefore examined by fractionation and immunoblotting of cells to determine whether alterations in PIP3 formation led to d" @default.
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• W2093056834 title "Akt-mediated Cardiomyocyte Survival Pathways Are Compromised by Gαq-induced Phosphoinositide 4,5-Bisphosphate Depletion" @default.
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