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Expression of Constitutively Active Phosphatidylinositol 3-Kinase Inhibits Activation of Caspase 3 and Apoptosis of Cardiac Muscle Cells*

Open AccessPublished:December 22, 2000DOI:https://doi.org/10.1074/jbc.M004108200
      Apoptosis of cardiac muscle cells contributes to the development of cardiomyopathy. Recent studies showed that insulin-like growth factor I (IGF-I) inhibits apoptosis of cardiac muscle cells and improves myocardial function in experimental heart failure. This study was carried out to elucidate the role of phosphatidylinositol 3-kinase (PI 3-kinase) in the anti-apoptotic actions of IGF-I in cardiomyocytes and to explore whether expression of constitutively active PI 3-kinase can inhibit apoptosis in cardiomyocytes. Apoptosis of primary cardiomyocytes was induced by doxorubicin treatment and serum withdrawal. Transduction of cardiomyocytes with constitutively active PI 3-kinase specifically lead to serine phosphorylation of Akt, whereas phosphorylation of IGF-I receptor, IRS1/2 and p44/42 mitogen-activated protein kinase were not increased. In the cardiomyocytes transduced with constitutively active PI 3-kinase, activation of the pro-apoptotic caspase 3 was attenuated and fragmentation of DNA was reduced. Preincubating cells with PI 3-kinase inhibitor LY294002 was associated with loss of anti-apoptotic actions of IGF-I and PI 3-kinase. Neither IGF-I nor constitutively active PI 3-kinase lead to serine phosphorylation of Bad, suggesting that the anti-apoptotic effects of PI 3-kinase are not mediated through Bad phosphorylation in cardiac muscle cells. To determine whether activation of caspase 3 is sufficient to induce apoptosis in cardiomyocytes, an engineered TAT-caspase 3 protein was introduced to cardiomyocytes. Significant reduction of cell viability occurred in the cardiomyocytes transduced with active caspase 3, indicating that activation of caspase 3 is sufficient to cause cardiomyocyte death. These findings indicate the existence of an IGF-I receptor-PI 3-kinase-caspase 3 pathway in cardiomyocytes that plays an important role in the anti-apoptotic actions of IGF-I in heart. Moreover, these data suggest that modulation of PI 3-kinase activities may represent a potential therapeutic strategy to counteract the occurrence of apoptosis in cardiomyopathy.
      IGF-I
      insulin-like growth factor I
      PI 3-kinase
      phosphatidylinositol 3-kinase
      Akt/PKB
      protein kinase B
      MEK
      MAP kinase kinase
      FBS
      fetal bovine serum
      MAP
      mitogen-activated protein
      DMEM
      Dulbecco's modified Eagle's medium
      PAGE
      polyacrylamide gel electrophoresis
      PBS
      phosphate-buffered saline
      IRS
      insulin receptor substrate
      Apoptosis of cardiac muscle cells have been observed during fetal development of cardiac tissue, in various models of myocardial injuries, and during the development of heart failure (
      • MacLellan W.R.
      • Schneider M.D.
      ,
      • Williams R.S.
      ,
      • James T.N.
      ,
      • Cheng W.
      • Li B.
      • Kajstura J.
      • Li P.
      • Wolin M.
      • Sonnenblick E.H.
      • Hintze T.H.
      • Olivetti G.
      • Anversa P.
      ,
      • Delpy E.
      • Hatem S.N.
      • Andrieu N.
      • de Vaumas C.
      • Henaff M.
      • Rucker-Martin C.
      • Jaffrezou J.P.
      • Laurent G.
      • Levade T.
      • Mercadier J.J.
      ,
      • Saraste A.
      • Pulkki K.
      • Kallajoki M.
      • Heikkila P.
      • Laine P.
      • Mattila S.
      • Nieminent M.S.
      • Parvinen M.
      • Voipio-Pulkki L.-M.
      ,
      • Von Harsdorf R.
      • Li P.F.
      • Dietz R.
      ,
      • Yue T.-L.
      • Wang C.
      • Romanic A.M.
      • Kikly K.
      • Keller P.
      • DeWolf Jr., W.E.
      • Hart T.K.
      • Thomas H.C.
      • Storer B.
      • Gu J.-L.
      • Wang X.
      • Feuerstein G.Z.
      ,
      • Zhu W.
      • Zou Y.
      • Aikawa R.
      • Harada K.
      • Kudoh S.
      • Uozumi H.
      • Hayashi D.
      • Gu Y.
      • Yamazaki T.
      • Nagai R.
      • Yazaki Y.
      • Komuro I.
      ). Because myocytes rarely proliferate in adult cardiac muscles, loss of cardiac muscle cells leads to a permanent loss of cardiac functional units. Thus myocardial apoptosis may contribute to or aggravate the development of myocardial dysfunction in various cardiac diseases. The occurrence of cardiac apoptosis can be suppressed with insulin-like growth factor I (IGF-I)1(
      • Buerke M.
      • Murohara T.
      • Skurk C.
      • Nuss C.
      • Tomaselli K.
      • Lefer A.M.
      ,
      • Foncea R.
      • Anderson M.
      • Ketterman A.
      • Blakesley V.
      • Sapag-Hagar M.
      • Sugden P.H.
      • LeRoith D.
      • Lavandero S.
      ,
      • Li Q.
      • Li B.
      • Wang X.
      • Leri A.
      • Jana K.P.
      • Liu Y.
      • Kajstura J.
      • Baserga R.
      • Anversa P.
      ,
      • Wang L.
      • Ma W.
      • Markovich R.
      • Chen J.-W.
      • Wang P.H.
      ,
      • Wang L.
      • Ma W.
      • Markovich R.
      • Lee W.-L.
      • Wang P.H.
      ,
      • Lee W.-L.
      • Chen J.-W.
      • Ting C.-T.
      • Ishiwata T.
      • Lin S.-J.
      • Korc M.
      • Wang P.H.
      ). IGF-I is a growth factor that activates multiple intracellular signaling pathways. How these pathways modulate apoptosis signaling in cardiomyocytes is not yet clear. Recent studies have shown that in various types of cells the anti-apoptotic effects of IGF-I disappeared when activation of PI 3-kinase was inhibited. Thus it appears that the anti-apoptotic effects of IGF-I require activation of PI 3-kinase. PI 3-kinase is composed of an 85-kDa (p85) regulatory subunit and a 110-kDa catalytic subunit (p110). Activation of PI 3-kinase leads to activation of downstream signaling molecules such as Akt/PKB. How PI 3-kinase/Akt signaling modulates apoptosis signaling is not completely understood. Datta et al. (
      • Datta S.R.
      • Dudek H.
      • Tao X.
      • Masters S.
      • Fu H.
      • Gotoh Y.
      • Greenberg M.E.
      ) have observed that activation of Akt lead to serine phosphorylation of Bad, a pro-apoptotic protein of Bcl-2 family, and in turn suppressed apoptosis of transformed cells. However, whether activation of PI 3-kinase can lead to phosphorylation of Bad in cardiac muscle cells is not known. Our laboratory (
      • Wang L.
      • Ma W.
      • Markovich R.
      • Chen J.-W.
      • Wang P.H.
      ,
      • Wang L.
      • Ma W.
      • Markovich R.
      • Lee W.-L.
      • Wang P.H.
      ) has observed that doxorubicin increased the activity of caspase 3 and induced apoptosis of cardiac muscle cells, whereas IGF-I suppressed activation of caspase 3 and apoptosis of cardiomyocytes. Caspase 3 is a key component of apoptotic signaling that mediates both mitochondria-dependent and -independent apoptotic signaling. Although the anti-apoptotic effects of IGF-I may be in part mediated by PI 3-kinase, it is unclear whether activation of PI 3-kinase pathway alone suppresses activation of caspase 3 in cardiac muscle or in other tissues. The aim of this study was to investigate whether activation of PI 3-kinase can lead to suppression of caspase 3 activation and in turn inhibit the occurrence of apoptosis in cardiac muscle cells. The results show that transduction of a constitutively active PI 3-kinase into cardiomyocytes suppressed activation of caspase 3 and attenuated apoptosis of cardiomyocytes. Moreover, transduction of active caspase 3 into cardiomyocytes was associated with decreased cardiomyocyte viability. Since activation of PI 3-kinase was not associated with serine phosphorylation of Bad, we concluded that in cardiac muscle cells, PI 3-kinase/Akt signaling inhibited caspase cascades through a mechanism independent of Bad phosphorylation. These results thus provide a novel link between PI 3-kinase pathway and suppression of caspase 3 activation in a tissue where apoptosis plays a significant pathophysiological role.

      DISCUSSION

      The findings of this study delineated the relationship of PI 3-kinase, caspase 3 activation, and apoptosis in cardiac muscle cells. Apoptosis of cardiac muscle cells was recently recognized as a new paradigm that occurs in human and experimental models of heart failure. This emerging concept of cardiomyocyte apoptosis has important implications on cardiac function because loss of cardiomyocytes could be a fundamental part of the myocardial process that initiates or aggravates heart failure. For example, administration of doxorubicin is associated with heart failure and reduced number of cardiac muscle cells per functional units (
      • Olson R.D.
      • Mushlin P.S.
      ), and in vitro incubation of cardiomyocytes with doxorubicin lead to activation of caspase 3 and occurrence of apoptosis (
      • Wang L.
      • Ma W.
      • Markovich R.
      • Chen J.-W.
      • Wang P.H.
      ,
      • Wang L.
      • Ma W.
      • Markovich R.
      • Lee W.-L.
      • Wang P.H.
      ). IGF-I can attenuate apoptosis of cardiac muscle cells induced by doxorubicin and apoptosis of cardiac muscle cells in various experimental models of cardiomyopathy (
      • Buerke M.
      • Murohara T.
      • Skurk C.
      • Nuss C.
      • Tomaselli K.
      • Lefer A.M.
      ,
      • Foncea R.
      • Anderson M.
      • Ketterman A.
      • Blakesley V.
      • Sapag-Hagar M.
      • Sugden P.H.
      • LeRoith D.
      • Lavandero S.
      ,
      • Wang L.
      • Ma W.
      • Markovich R.
      • Chen J.-W.
      • Wang P.H.
      ,
      • Wang L.
      • Ma W.
      • Markovich R.
      • Lee W.-L.
      • Wang P.H.
      ,
      • Lee W.-L.
      • Chen J.-W.
      • Ting C.-T.
      • Ishiwata T.
      • Lin S.-J.
      • Korc M.
      • Wang P.H.
      ,
      • Imai Y.
      • Clemmons D.R.
      ,
      • Matsui T.
      • Li L.
      • Monte F.D.
      • Fukui Y.
      • Franke T.F.
      • Hajjar R.J.
      • Rosenzweig A.
      ). Although considerable progress has been made in understanding the signaling pathways of IGF-I, relatively little is known about how IGF-I signaling modulates apoptotic signaling. The results of this study indicate that activation of caspase 3 was sufficient to cause cardiomyocyte death and that activation of PI 3-kinase signaling can suppress activation of caspase 3 and hence inhibit the occurrence of apoptosis.
      There is ample evidence suggesting that activation of PI 3-kinase may lead to suppression of caspase 3 and DNA fragmentation in a variety of cells. Most of the evidence derived from studies using pharmacological inhibitors of PI 3-kinase (LY294002 and wortmannin), inhibition of PI 3-kinase with these compounds lead to cell apoptosis or loss of anti-apoptotic effects of growth factors (
      • Imai Y.
      • Clemmons D.R.
      ,
      • Matsui T.
      • Li L.
      • Monte F.D.
      • Fukui Y.
      • Franke T.F.
      • Hajjar R.J.
      • Rosenzweig A.
      ,
      • Parrizas M.
      • Saltiel A.
      • LeRoith D.
      ,
      • Crowder R.J.
      • Freeman R.S.
      ,
      • Kulik G.
      • Weber M.
      ,
      • Ryu B.R.
      • Ko H.W.
      • Jou I.
      • Noh J.S.
      • Gwag B.J.
      ). Several other studies used dominant negative constructs of PI 3-kinase to inhibit PI 3-kinase signaling and further confirmed that PI 3-kinase is essential in maintaining cell survival (
      • Buerke M.
      • Murohara T.
      • Skurk C.
      • Nuss C.
      • Tomaselli K.
      • Lefer A.M.
      ,
      • Crowder R.J.
      • Freeman R.S.
      ). However, from these studies it is not clear whether activation of PI 3-kinase alone is sufficient to protect the cells from apoptosis induction and whether PI 3-kinase activation suppresses caspase activation. A downstream target of PI 3-kinase that may mediate the anti-apoptotic actions of PI 3-kinase is Akt. Akt pathways may modulate sequential steps of apoptosis signaling. Activation of Akt may lead to phosphorylation of Bad (
      • Datta S.R.
      • Dudek H.
      • Tao X.
      • Masters S.
      • Fu H.
      • Gotoh Y.
      • Greenberg M.E.
      ), induction of Bcl-2 family of proteins (
      • Matsuzaki H.
      • Tamatani M.
      • Mitsuda N.
      • Namikawa K.
      • Kiyama H.
      • Miyake S.
      • Tohyama M.
      ), inhibition of cytochrome crelease from mitochondria (
      • Kennedy S.G.
      • Kandel E.S.
      • Cross T.K.
      • Hay N.
      ), and phosphorylation and inactivation of caspase 9 (
      • Fujita E.
      • Jinbo A.
      • Matuzaki H.
      • Konishi H.
      • Kikkawa U.
      • Momoi T.
      ). Some of these observations appear to be dependent upon the experimental system used. For example, Akt phosphorylation of caspase 9 was reported in human neoplastic cells, but murine caspase 9 lacks the phosphorylation motif and cannot be phosphorylated by Akt (
      • Fujita E.
      • Jinbo A.
      • Matuzaki H.
      • Konishi H.
      • Kikkawa U.
      • Momoi T.
      ). Moreover, we were unable to find phosphorylation of Bad by IGF-I or by activation of PI 3-kinase in cardiomyocytes. Thus it appears survival signaling may employ different mechanisms to antagonize apoptosis in different cells. These signaling steps can be variably dependent on the type of tissue, the type of species, the state of cell transformation or differentiation, and the nature of apoptosis inducers. The lack of Bad phosphorylation in IGF-I-stimulated and in Ad5-p110*-tranduced cardiomyocytes suggests that PI 3-kinase/Akt signaling may utilize survival pathways independent of Bad.
      When cardiomyocytes were incubated with the PI 3-kinase inhibitor LY294002, the protective effects of IGF-I and Ad5-p110* were reversed and caspase 3 activities increased to a level higher than the caspase 3 activities induced by doxorubicin alone. LY294002 is a very effective inhibitor of PI 3-kinase signaling as phosphorylation of Akt in LY294002-treated cells was suppressed to a level lower than the basal phosphorylation of Akt in control cardiomyocytes. Adding LY294002 alone to culture medium for a prolonged period of time lead to induction of moderate cardiomyocyte apoptosis (data not shown). It is possible that LY294002 completely inhibited the basal activities of PI 3-kinase and thus lead to a greater induction of apoptosis in the cardiomyocytes treated with both doxorubicin and LY294002. Together with our finding that activation of PI 3-kinase inhibited induction of apoptosis in cardiomyocytes, these observations indicate PI 3-kinase is essential for cardiomyocyte viability.
      Caspases are stored as proenzymes and can be cleaved into active fragments upon apoptosis induction. The process of cleavage is mediated by proteolysis and generates two subunits (10 and 20 kDa), these two subunits form heterodimers that eventually produce the enzymatic activities of caspases (
      • Fernandes-Alnemri T.
      • Litwack G.
      • Alnemri E.S.
      ). Caspase 3 plays a pivotal role in execution of apoptosis; ES cells deficient in caspase 3 were resistant to apoptosis induction (
      • Colussi P.A.
      • Kumar S.
      ). In human cardiomyopathy, apoptosis of cardiac muscle cells is associated with release of cytochromec and activation of caspase 3 (
      • Narula J.
      • Pandey P.
      • Arbustini E.
      • Haider N.
      • Narula N.
      • Kolodgie F.D.
      • Dal Bello B.
      • Semigran M.J.
      • Bielsa-Masdeu A.
      • Dec G.W.
      • Israels S.
      • Ballester M.
      • Virmani R.
      • Saxena S.
      • Kharbanda S.
      ). Activation of myocardial caspase 3 was also observed in experimental models of post-transplant cardiac rejection (
      • Koglin J.
      • Granville D.J.
      • Glysing-Jensen T.
      • Mudgett J.S.
      • Carthy C.M.
      • McManus B.M.
      • Russell M.E.
      ), which are accompanied by inflammatory reactions and myocardial apoptosis. These studies suggest that caspase 3 represents a potential therapeutic target for suppression of cardiomyocyte apoptosis.
      Caspase 3 activation may lead to propagation of apoptosis signaling and structural disintegration of cells. Intracellular targets of caspases cascades include ICAD, gelsolin, PAK2, PKC, lamins, NuMa, actin, catenin, FAK, PARP, and several transcription factors (
      • Rosen A.
      • Casciola-Rosen L.
      ). The exact roles of these molecules in the execution of apoptosis are not entirely clear at present. However, studies thus far suggest that these molecules represent different components of apoptosis machinery that ultimately terminate survival signaling, dissolve cell integrity, promote cell packaging, and facilitate phagocytosis of cell remnants. Using peptide inhibitor of caspases, Bialik et al. (
      • Bialik S.
      • Cryns V.L.
      • Drincic A.
      • Miyata S.
      • Wollowick A.L.
      • Srinivasan A.
      • Kitsis R.N.
      ) also have shown that cardiomyocytes apoptosis induced by serum and glucose withdrawal can be attenuated. Our data show that activation of caspase 3 alone was sufficient to cause cell death in cardiac muscle, suggesting preservation of myocardial viability can be achieved with inhibition of caspase 3. Since PI 3-kinase has additional beneficial effects on cell growth and metabolism, gene transfer of active PI 3-kinase to cardiomyocytes may have potential therapeutic implications on the preservation of cardiac muscle in cardiac diseases.
      Parrizas et al. (
      • Parrizas M.
      • Saltiel A.R.
      • LeRoith D.
      ) have reported that inhibition of PI 3-kinase or MEK signaling with chemical inhibitors lead to attenuation of the anti-apoptotic actions of IGF-I in cardiomyocytes. In IGF-I signaling pathways, p85 PI 3-kinase subunit interacts with phosphorylated IRS 1/IRS 2 and then activates p110 catalytic subunit. There are conflicting data on whether activation of PI 3-kinase lead to activation of Ras and MAP kinase signaling. Activation of PI 3-kinase resulted in activation of Raf-1/MEK pathways in some experimental systems, whereas other studies failed to demonstrate activation of MAP kinase signaling by PI 3-kinase (
      • Egawa K.
      • Sharma P.M.
      • Nakashima N.
      • Huang Y.
      • Huver E.
      • Boss G.R.
      • Olefsky J.M.
      ). Activation of MEK/ERK signaling was not observed in the cardiomyocytes transduced with active PI 3-kinase. These findings support the notion that in cardiac muscle cells MEK/ERK signaling is not downstream from PI 3-kinase, and suggest that the anti-apoptotic effects of PI 3-kinase activation in cardiomyocytes does not involve activation of MEK/ERK pathway.

      Acknowlegements

      We thank Dr. Steven Dowdy for providing TAT fusion protein vectors and Dr. Robert J. Smith for providing anti-IGF-I receptor antibodies. We are grateful to Jeffrey Hsu for excellent technical assistance in preparing primary cultures of cardiomyocytes.

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