臺灣博碩士論文加值系統

心肌梗塞引發缺血與缺氧是造成心肌細胞凋亡的主要原因，而惟獨缺氧亦足以誘發心肌細胞的凋亡。然而在缺氧心臟中，自噬作用調控心肌凋亡亦扮演重要的角色。於先前的文獻指出，缺氧誘導因子HIF-1α與BH3-only bcl-2家族蛋白BNIP3在缺氧下可能引起適應或是傷害的機轉。同時缺氧也能夠誘導類胰島素生長因子結合蛋白IGFBP-3，進而箝制IGF1R/PI3K/Akt心肌抗凋亡的途徑。因此本篇論文希望研究心肌細胞受到極度缺氧下所引發的分子機轉以及缺氧蛋白HIF-1α、BNIP3與IGFBP-3之間調控的相關性。研究中，我們採用了老鼠心臟衍生的H9c2肌原母細胞以及新生鼠初代培養的心室細胞，將其長時間培養於含氧量正常（21% O2）或缺氧（1% O2）的環境。結果顯示，過度缺氧首先造成HIF-1α表現增加並活化下游基因BNIP3與IGFBP-3，進一步引發自噬作用與凋亡的途徑。此外IGF1R/PI3K/Akt訊息途徑受到HIF-1α下游IGFBP-3的負調控而有顯著性的減弱，進而增強缺氧誘導的凋亡現象。另一方面，當自噬作用受到3MA或ATG5或是Beclin-1 siRNA的抑制均能顯著的降低缺氧引發的心肌細胞凋亡。同時結果也指出在缺氧情形下，自噬作用的活化明顯地受到FoxO3a與下游BNIP3所調控；更重要地，當以siRNA阻斷FoxO3a或BNIP3蛋白表現時，缺氧引發的自噬作用及粒線體依賴型細胞凋亡均有顯著的抑制。綜合上述結果發現，自噬作用主要透過FoxO3a影響下游BNIP3訊息路徑的調控，對於缺氧誘導的粒線體凋亡扮演一個關鍵的調節者。然而，長期缺氧心肌細胞，藉HIF-1α活化正調控BNIP3並藉誘發IGFBP-3來箝制IGF1R/PI3K/Akt存活路徑，造成粒線體依賴型細胞凋亡。同時透過FoxO3a影響下游BNIP3進而誘發Atg5-LC3-II依賴型自噬作用，更進一步促進心肌細胞凋亡作用。相信藉由HIF-1α及FoxO3a之阻斷，應可防止極度缺氧所造成之心肌細胞的傷害。

Myocardial infarction (MI) is the common cause of cardiomyocyte death. Even hypoxia alone is sufficient to induce apoptosis of cardiomyocytes. In hearts, autophagy play important roles in hypoxia-mediated cardioprotection or myocardial injury effects. To date, the hypoxia-inducible factor-1α (HIF-1α) transcriptional factor and the BH-3 only protein, Bcl-2 adenovirus E1B 19 kDa interacting protein 3 (BNIP3), are known to play fundamental roles in adaptive or death process in response to hypoxia. In addition, hypoxia induces insulin-like growth factor binding protein 3 (IGFBP-3) to block the IGF1R/PI3K/Akt survival pathway. Therefore, we aim to investigate the molecular mechanisms and the correlation of HIF-1α, BNIP3 and IGFBP-3 in hypoxia-induced cardiomyocytes injuries. In the present study, heart-derived H9c2 cells and neonatal rat ventricular myocytes (NRVMs) were incubated in normoxic (21% oxygen) or hypoxic (1% oxygen) conditions for up to 48 h. Results showed that hypoxia primarily highly increased HIF-1α expression, then activated downstream genes such as BNIP3 and IGFBP-3, and further triggered mitochondria-dependent apoptotic pathways. Moreover, IGF1R/PI3K/Akt signaling obviously attenuated by up-regulated expression of HIF-1α-dependent IGFBP-3 expression to enhance hypoxia-induced cell apoptosis. In addition, suppression of autophagy with 3-methyladenine (3MA) or siRNA of ATG5 or Beclin-1 significantly decreased the myocardial apoptosis under hypoxic conditions. The data also showed that the activation of autophagy during hypoxia was obviously induced by Forkhead box O3 (FoxO3a)-dependent BNIP3 expression. Importantly, knockdown of FoxO3a or BNIP3 significantly abrogated hypoxia-induced autophagy and mitochondria-dependent apoptosis effects. Taken together, our present results confirmed that autophagy is a pivotal regulator for hypoxia-induced cardiomyocyte apoptosis modulated by FoxO3a-dependent BNIP3 expression. Moreover, prolonged-hypoxia induced HIF-1α not only stimulated BNIP3 expression but also enhanced IGFBP-3 activation to inhibit IGF1R/PI3K/Akt survival pathway and mediate mitochondria-dependent cardiomyocyte apoptosis. We believe that HIF-1α and FoxO3a blockage are sufficient to annul the change of excessive hypoxia of hearts.

學位考試委員審定書 I
誌謝 II
Contents IV

Part I:
Chinese abstract 2
English abstract 4
Abbreviation 6
Introduction 9
1. Hypoxia in the heart diseases 9
2. Hypoxia regulation of cell-death pathways 9
3. Hypoxia-inducible factor-1α (HIF-1α) 10
4. Insulin-like growth factor binding protein 3 (IGFBP-3) 11
5. Bcl-2 adenovirus E1B 19 kDa interacting protein 3 (BNIP3) 13
6. Autophagy (self-eating) 15
7. Role of autophagy in heart: a double-edged sword 17
8. Mechanism of autophagy during hypoxia 17
Aims 19
Materials and Methods 20
1. Human cardiovascular tissue microarray 20
2. Immunohistochemistry 20
3. Cell culture 21
4. Western blot 21
5. Nuclear extraction 22
6. Immunofluorescence 22
7. Semi quantitative RT-PCR 23
8. Gene overexpression through transient transfection 24
9. Gene silencing through siRNA 24
10. Annexin V-FITC/PI staining 25
11. Transient-transfected GFP-LC3 and autophagy detection 25
12. DAPI staining and TUNEL assay 26
13. Co-immunoprecipitation (Co-IP) 26
14. Primary cardiomyocyte culture 27
15. Statistical analysis 27
Results 28
Discussion 35
References 39
Figures 48

Part II:
Chinese abstract 65
English abstract 67
Abbreviation 69
Introduction 71
1. The gender difference in heart disease 71
2. Function of the sex steroid hormone: estrogen 72
3. Nuclear proteins of steroid hormone: estrogen receptors (ERs) 73
4. The role of ERα and ERβ in cardioprotection 74
Aims 76
Materials and Methods 77
1. Construct Tet-On ERβ gene expression system 77
2. Cell culture 78
3. ERβ overexpression through transient transfection 78
4. Western blot 79
5. Primary cardiomyocyte culture 80
6. TUNEL assay and DAPI staining 80
7. Statistical analysis 80
Results 81
Discussion 83
References 86
Figures 90

Part I:
Arany, Z., Huang, L.E., Eckner, R., Bhattacharya, S., Jiang, C., Goldberg, M.A., Bunn, H.F., Livingston, D.M., 1996. An essential role for p300/cbp in the cellular response to hypoxia. Proc Natl Acad Sci U S A 93 (23), 12969-73.
Bakker, W.J., Harris, I.S., Mak, T.W., 2007. Foxo3a is activated in response to hypoxic stress and inhibits hif1-induced apoptosis via regulation of cited2. Mol Cell 28 (6), 941-53.
Baxter, R.C., 2000. Insulin-like growth factor (igf)-binding proteins: Interactions with igfs and intrinsic bioactivities. Am J Physiol Endocrinol Metab 278 (6), E967-76.
Bayes-Genis, A., Conover, C.A., Schwartz, R.S., 2000. The insulin-like growth factor axis: A review of atherosclerosis and restenosis. Circ Res 86 (2), 125-30.
Bialik, S., Geenen, D.L., Sasson, I.E., Cheng, R., Horner, J.W., Evans, S.M., Lord, E.M., Koch, C.J., Kitsis, R.N., 1997. Myocyte apoptosis during acute myocardial infarction in the mouse localizes to hypoxic regions but occurs independently of p53. J Clin Invest 100 (6), 1363-72.
Brunet, A., Bonni, A., Zigmond, M.J., Lin, M.Z., Juo, P., Hu, L.S., Anderson, M.J., Arden, K.C., Blenis, J., Greenberg, M.E., 1999. Akt promotes cell survival by phosphorylating and inhibiting a forkhead transcription factor. Cell 96 (6), 857-68.
Bursch, W., 2001. The autophagosomal-lysosomal compartment in programmed cell death. Cell Death Differ 8 (6), 569-81.
Burton, T.R., Gibson, S.B., 2009. The role of bcl-2 family member bnip3 in cell death and disease: Nipping at the heels of cell death. Cell Death Differ 16 (4), 515-23.
Butt, A.J., Firth, S.M., Baxter, R.C., 1999. The igf axis and programmed cell death. Immunol Cell Biol 77 (3), 256-62.
Carrero, P., Okamoto, K., Coumailleau, P., O''brien, S., Tanaka, H., Poellinger, L., 2000. Redox-regulated recruitment of the transcriptional coactivators creb-binding protein and src-1 to hypoxia-inducible factor 1alpha. Mol Cell Biol 20 (1), 402-15.
Chen, G., Cizeau, J., Vande Velde, C., Park, J.H., Bozek, G., Bolton, J., Shi, L., Dubik, D., Greenberg, A., 1999. Nix and nip3 form a subfamily of pro-apoptotic mitochondrial proteins. J Biol Chem 274 (1), 7-10.
Cheng, W., Kajstura, J., Nitahara, J.A., Li, B., Reiss, K., Liu, Y., Clark, W.A., Krajewski, S., Reed, J.C., Olivetti, G., Anversa, P., 1996. Programmed myocyte cell death affects the viable myocardium after infarction in rats. Exp Cell Res 226 (2), 316-27.
Chinnadurai, G., Vijayalingam, S., Gibson, S.B., 2008. Bnip3 subfamily bh3-only proteins: Mitochondrial stress sensors in normal and pathological functions. Oncogene 27 Suppl 1, S114-27.
Cizeau, J., Ray, R., Chen, G., Gietz, R.D., Greenberg, A.H., 2000. The c. Elegans orthologue cebnip3 interacts with ced-9 and ced-3 but kills through a bh3- and caspase-independent mechanism. Oncogene 19 (48), 5453-63.
Clemmons, D.R., 1998. Role of insulin-like growth factor binding proteins in controlling igf actions. Mol Cell Endocrinol 140 (1-2), 19-24.
Crow, M.T., 2002. Hypoxia, bnip3 proteins, and the mitochondrial death pathway in cardiomyocytes. Circ Res 91 (3), 183-5.
Daido, S., Kanzawa, T., Yamamoto, A., Takeuchi, H., Kondo, Y., Kondo, S., 2004. Pivotal role of the cell death factor bnip3 in ceramide-induced autophagic cell death in malignant glioma cells. Cancer Res 64 (12), 4286-93.
Dammrich, J., Pfeifer, U., 1983. Cardiac hypertrophy in rats after supravalvular aortic constriction. Ii. Inhibition of cellular autophagy in hypertrophying cardiomyocytes. Virchows Arch B Cell Pathol Incl Mol Pathol 43 (3), 287-307.
Delafontaine, P., Song, Y.H., Li, Y., 2004. Expression, regulation, and function of igf-1, igf-1r, and igf-1 binding proteins in blood vessels. Arterioscler Thromb Vasc Biol 24 (3), 435-44.
Dice, J.F., 2007. Chaperone-mediated autophagy. Autophagy 3 (4), 295-9.
Diwan, A., Matkovich, S.J., Yuan, Q., Zhao, W., Yatani, A., Brown, J.H., Molkentin, J.D., Kranias, E.G., Dorn, G.W., 2nd, 2009. Endoplasmic reticulum-mitochondria crosstalk in nix-mediated murine cell death. J Clin Invest 119 (1), 203-12.
Dorn, G.W., 2nd, Kirshenbaum, L.A., 2008. Cardiac reanimation: Targeting cardiomyocyte death by bnip3 and nix/bnip3l. Oncogene 27 Suppl 1, S158-67.
Ebert, B.L., Bunn, H.F., 1998. Regulation of transcription by hypoxia requires a multiprotein complex that includes hypoxia-inducible factor 1, an adjacent transcription factor, and p300/creb binding protein. Mol Cell Biol 18 (7), 4089-96.
Ema, M., Hirota, K., Mimura, J., Abe, H., Yodoi, J., Sogawa, K., Poellinger, L., Fujii-Kuriyama, Y., 1999. Molecular mechanisms of transcription activation by hlf and hif1alpha in response to hypoxia: Their stabilization and redox signal-induced interaction with cbp/p300. EMBO J 18 (7), 1905-14.
Giaccia, A.J., Simon, M.C., Johnson, R., 2004. The biology of hypoxia: The role of oxygen sensing in development, normal function, and disease. Genes Dev 18 (18), 2183-94.
Graham, R.M., Frazier, D.P., Thompson, J.W., Haliko, S., Li, H., Wasserlauf, B.J., Spiga, M.G., Bishopric, N.H., Webster, K.A., 2004. A unique pathway of cardiac myocyte death caused by hypoxia-acidosis. J Exp Biol 207 (Pt 18), 3189-200.
Halterman, M.W., Federoff, H.J., 1999. Hif-1alpha and p53 promote hypoxia-induced delayed neuronal death in models of cns ischemia. Exp Neurol 159 (1), 65-72.
Hamacher-Brady, A., Brady, N.R., Gottlieb, R.A., 2006. Enhancing macroautophagy protects against ischemia/reperfusion injury in cardiac myocytes. J Biol Chem 281 (40), 29776-87.
Harris, A.L., 2002. Hypoxia--a key regulatory factor in tumour growth. Nat Rev Cancer 2 (1), 38-47.
Hein, S., Arnon, E., Kostin, S., Schonburg, M., Elsasser, A., Polyakova, V., Bauer, E.P., Klovekorn, W.P., Schaper, J., 2003. Progression from compensated hypertrophy to failure in the pressure-overloaded human heart: Structural deterioration and compensatory mechanisms. Circulation 107 (7), 984-91.
Henson, M., Damm, D., Lam, A., Garrard, L.J., White, T., Abraham, J.A., Schreiner, G.F., Stanton, L.W., Joly, A.H., 2000. Insulin-like growth factor-binding protein-3 induces fetalization in neonatal rat cardiomyocytes. DNA Cell Biol 19 (12), 757-63.
Hsieh, Y.C., Athar, M., Chaudry, I.H., 2009. When apoptosis meets autophagy: Deciding cell fate after trauma and sepsis. Trends Mol Med 15 (3), 129-38.
Hwang, J.M., Weng, Y.J., Lin, J.A., Bau, D.T., Ko, F.Y., Tsai, F.J., Tsai, C.H., Wu, C.H., Lin, P.C., Huang, C.Y., Kuo, W.W., 2008. Hypoxia-induced compensatory effect as related to shh and hif-1alpha in ischemia embryo rat heart. Mol Cell Biochem 311 (1-2), 179-87.
Itoh, G., Tamura, J., Suzuki, M., Suzuki, Y., Ikeda, H., Koike, M., Nomura, M., Jie, T., Ito, K., 1995. DNA fragmentation of human infarcted myocardial cells demonstrated by the nick end labeling method and DNA agarose gel electrophoresis. Am J Pathol 146 (6), 1325-31.
Kallio, P.J., Okamoto, K., O''brien, S., Carrero, P., Makino, Y., Tanaka, H., Poellinger, L., 1998. Signal transduction in hypoxic cells: Inducible nuclear translocation and recruitment of the cbp/p300 coactivator by the hypoxia-inducible factor-1alpha. EMBO J 17 (22), 6573-86.
Kanzawa, T., Zhang, L., Xiao, L., Germano, I.M., Kondo, Y., Kondo, S., 2005. Arsenic trioxide induces autophagic cell death in malignant glioma cells by upregulation of mitochondrial cell death protein bnip3. Oncogene 24 (6), 980-91.
Ke, Q., Costa, M., 2006. Hypoxia-inducible factor-1 (hif-1). Mol Pharmacol 70 (5), 1469-80.
Kim, J.Y., Ahn, H.J., Ryu, J.H., Suk, K., Park, J.H., 2004. Bh3-only protein noxa is a mediator of hypoxic cell death induced by hypoxia-inducible factor 1alpha. J Exp Med 199 (1), 113-24.
Klionsky, D.J., 2005. The molecular machinery of autophagy: Unanswered questions. J Cell Sci 118 (Pt 1), 7-18.
Klionsky, D.J., Emr, S.D., 2000. Autophagy as a regulated pathway of cellular degradation. Science 290 (5497), 1717-21.
Knaapen, M.W., Davies, M.J., De Bie, M., Haven, A.J., Martinet, W., Kockx, M.M., 2001. Apoptotic versus autophagic cell death in heart failure. Cardiovasc Res 51 (2), 304-12.
Kong, D.K., Georgescu, S.P., Cano, C., Aronovitz, M.J., Iovanna, J.L., Patten, R.D., Kyriakis, J.M., Goruppi, S., 2010. Deficiency of the transcriptional regulator p8 results in increased autophagy and apoptosis, and causes impaired heart function. Mol Biol Cell 21 (8), 1335-49.
Kothari, S., Cizeau, J., Mcmillan-Ward, E., Israels, S.J., Bailes, M., Ens, K., Kirshenbaum, L.A., Gibson, S.B., 2003. Bnip3 plays a role in hypoxic cell death in human epithelial cells that is inhibited by growth factors egf and igf. Oncogene 22 (30), 4734-44.
Kunz, J.B., Schwarz, H., Mayer, A., 2004. Determination of four sequential stages during microautophagy in vitro. J Biol Chem 279 (11), 9987-96.
Lee, S.D., Kuo, W.W., Lin, J.A., Chu, Y.F., Wang, C.K., Yeh, Y.L., Wang, S.G., Liu, J.Y., Chang, M.H., Huang, C.Y., 2007. Effects of long-term intermittent hypoxia on mitochondrial and fas death receptor dependent apoptotic pathways in rat hearts. Int J Cardiol 116 (3), 348-56.
Lee, S.D., Kuo, W.W., Wu, C.H., Lin, Y.M., Lin, J.A., Lu, M.C., Yang, A.L., Liu, J.Y., Wang, S.G., Liu, C.J., Chen, L.M., Huang, C.Y., 2006. Effects of short- and long-term hypobaric hypoxia on bcl2 family in rat heart. Int J Cardiol 108 (3), 376-84.
Lee, S.H., Wolf, P.L., Escudero, R., Deutsch, R., Jamieson, S.W., Thistlethwaite, P.A., 2000. Early expression of angiogenesis factors in acute myocardial ischemia and infarction. N Engl J Med 342 (9), 626-33.
Levine, B., Klionsky, D.J., 2004. Development by self-digestion: Molecular mechanisms and biological functions of autophagy. Dev Cell 6 (4), 463-77.
Levine, B., Kroemer, G., 2008. Autophagy in the pathogenesis of disease. Cell 132 (1), 27-42.
Maiuri, M.C., Zalckvar, E., Kimchi, A., Kroemer, G., 2007. Self-eating and self-killing: Crosstalk between autophagy and apoptosis. Nat Rev Mol Cell Biol 8 (9), 741-52.
Mammucari, C., Milan, G., Romanello, V., Masiero, E., Rudolf, R., Del Piccolo, P., Burden, S.J., Di Lisi, R., Sandri, C., Zhao, J., Goldberg, A.L., Schiaffino, S., Sandri, M., 2007. Foxo3 controls autophagy in skeletal muscle in vivo. Cell Metab 6 (6), 458-71.
Mammucari, C., Schiaffino, S., Sandri, M., 2008. Downstream of akt: Foxo3 and mtor in the regulation of autophagy in skeletal muscle. Autophagy 4 (4), 524-6.
Martinet, W., Knaapen, M.W., Kockx, M.M., De Meyer, G.R., 2007. Autophagy in cardiovascular disease. Trends Mol Med 13 (11), 482-91.
Masson, N., Willam, C., Maxwell, P.H., Pugh, C.W., Ratcliffe, P.J., 2001. Independent function of two destruction domains in hypoxia-inducible factor-alpha chains activated by prolyl hydroxylation. EMBO J 20 (18), 5197-206.
Matsui, Y., Takagi, H., Qu, X., Abdellatif, M., Sakoda, H., Asano, T., Levine, B., Sadoshima, J., 2007. Distinct roles of autophagy in the heart during ischemia and reperfusion: Roles of amp-activated protein kinase and beclin 1 in mediating autophagy. Circ Res 100 (6), 914-22.
May, D., Gilon, D., Djonov, V., Itin, A., Lazarus, A., Gordon, O., Rosenberger, C., Keshet, E., 2008. Transgenic system for conditional induction and rescue of chronic myocardial hibernation provides insights into genomic programs of hibernation. Proc Natl Acad Sci U S A 105 (1), 282-7.
Mizushima, N., Levine, B., Cuervo, A.M., Klionsky, D.J., 2008. Autophagy fights disease through cellular self-digestion. Nature 451 (7182), 1069-75.
Mohan, S., Baylink, D.J., 2002. Igf-binding proteins are multifunctional and act via igf-dependent and -independent mechanisms. J Endocrinol 175 (1), 19-31.
Nakai, A., Yamaguchi, O., Takeda, T., Higuchi, Y., Hikoso, S., Taniike, M., Omiya, S., Mizote, I., Matsumura, Y., Asahi, M., Nishida, K., Hori, M., Mizushima, N., Otsu, K., 2007. The role of autophagy in cardiomyocytes in the basal state and in response to hemodynamic stress. Nat Med 13 (5), 619-24.
Nakayama, H., Chen, X., Baines, C.P., Klevitsky, R., Zhang, X., Zhang, H., Jaleel, N., Chua, B.H., Hewett, T.E., Robbins, J., Houser, S.R., Molkentin, J.D., 2007. Ca2+- and mitochondrial-dependent cardiomyocyte necrosis as a primary mediator of heart failure. J Clin Invest 117 (9), 2431-44.
Narula, J., Kolodgie, F.D., Virmani, R., 2000. Apoptosis and cardiomyopathy. Curr Opin Cardiol 15 (3), 183-8.
Nishida, K., Kyoi, S., Yamaguchi, O., Sadoshima, J., Otsu, K., 2009. The role of autophagy in the heart. Cell Death Differ 16 (1), 31-8.
Palojoki, E., Saraste, A., Eriksson, A., Pulkki, K., Kallajoki, M., Voipio-Pulkki, L.M., Tikkanen, I., 2001. Cardiomyocyte apoptosis and ventricular remodeling after myocardial infarction in rats. Am J Physiol Heart Circ Physiol 280 (6), H2726-31.
Pfeffer, M.A., Braunwald, E., 1990. Ventricular remodeling after myocardial infarction. Experimental observations and clinical implications. Circulation 81 (4), 1161-72.
Pitts, K.R., Derry, J.M., Kerkof, K., Lawrence, W.A., Toombs, C.F., 2008. Differentially regulated functional gene clusters identified during ischemia and reperfusion in isolated cardiac myocytes using coverslip hypoxia. J Pharmacol Toxicol Methods 57 (1), 42-51.
Quinsay, M.N., Thomas, R.L., Lee, Y., Gustafsson, A.B., 2010. Bnip3-mediated mitochondrial autophagy is independent of the mitochondrial permeability transition pore. Autophagy 6 (7), 855-62.
Rajaram, S., Baylink, D.J., Mohan, S., 1997. Insulin-like growth factor-binding proteins in serum and other biological fluids: Regulation and functions. Endocr Rev 18 (6), 801-31.
Ravikumar, B., Sarkar, S., Davies, J.E., Futter, M., Garcia-Arencibia, M., Green-Thompson, Z.W., Jimenez-Sanchez, M., Korolchuk, V.I., Lichtenberg, M., Luo, S., Massey, D.C., Menzies, F.M., Moreau, K., Narayanan, U., Renna, M., Siddiqi, F.H., Underwood, B.R., Winslow, A.R., Rubinsztein, D.C., 2010. Regulation of mammalian autophagy in physiology and pathophysiology. Physiol Rev 90 (4), 1383-435.
Ray, R., Chen, G., Vande Velde, C., Cizeau, J., Park, J.H., Reed, J.C., Gietz, R.D., Greenberg, A.H., 2000. Bnip3 heterodimerizes with bcl-2/bcl-x(l) and induces cell death independent of a bcl-2 homology 3 (bh3) domain at both mitochondrial and nonmitochondrial sites. J Biol Chem 275 (2), 1439-48.
Rosenfeldt, M.T., Ryan, K.M., 2009. The role of autophagy in tumour development and cancer therapy. Expert Rev Mol Med 11, e36.
Scott, R.C., Juhasz, G., Neufeld, T.P., 2007. Direct induction of autophagy by atg1 inhibits cell growth and induces apoptotic cell death. Curr Biol 17 (1), 1-11.
Semenza, G.L., 1999. Regulation of mammalian o2 homeostasis by hypoxia-inducible factor 1. Annu Rev Cell Dev Biol 15, 551-78.
Shaw, J., Kirshenbaum, L.A., 2008. Molecular regulation of autophagy and apoptosis during ischemic and non-ischemic cardiomyopathy. Autophagy 4 (4), 427-34.
Shimomura, H., Terasaki, F., Hayashi, T., Kitaura, Y., Isomura, T., Suma, H., 2001. Autophagic degeneration as a possible mechanism of myocardial cell death in dilated cardiomyopathy. Jpn Circ J 65 (11), 965-8.
Shintani, T., Klionsky, D.J., 2004. Autophagy in health and disease: A double-edged sword. Science 306 (5698), 990-5.
Simon, M.C., Liu, L., Barnhart, B.C., Young, R.M., 2008. Hypoxia-induced signaling in the cardiovascular system. Annu Rev Physiol 70, 51-71.
Srinivas, V., Zhang, L.P., Zhu, X.H., Caro, J., 1999. Characterization of an oxygen/redox-dependent degradation domain of hypoxia-inducible factor alpha (hif-alpha) proteins. Biochem Biophys Res Commun 260 (2), 557-61.
Takemura, G., Miyata, S., Kawase, Y., Okada, H., Maruyama, R., Fujiwara, H., 2006. Autophagic degeneration and death of cardiomyocytes in heart failure. Autophagy 2 (3), 212-4.
Tanaka, M., Ito, H., Adachi, S., Akimoto, H., Nishikawa, T., Kasajima, T., Marumo, F., Hiroe, M., 1994. Hypoxia induces apoptosis with enhanced expression of fas antigen messenger rna in cultured neonatal rat cardiomyocytes. Circ Res 75 (3), 426-33.
Todde, V., Veenhuis, M., Van Der Klei, I.J., 2009. Autophagy: Principles and significance in health and disease. Biochim Biophys Acta 1792 (1), 3-13.
Yan, L., Vatner, D.E., Kim, S.J., Ge, H., Masurekar, M., Massover, W.H., Yang, G., Matsui, Y., Sadoshima, J., Vatner, S.F., 2005. Autophagy in chronically ischemic myocardium. Proc Natl Acad Sci U S A 102 (39), 13807-12.
Yang, Y.P., Liang, Z.Q., Gu, Z.L., Qin, Z.H., 2005. Molecular mechanism and regulation of autophagy. Acta Pharmacol Sin 26 (12), 1421-34.
Yasuda, M., Theodorakis, P., Subramanian, T., Chinnadurai, G., 1998. Adenovirus e1b-19k/bcl-2 interacting protein bnip3 contains a bh3 domain and a mitochondrial targeting sequence. J Biol Chem 273 (20), 12415-21.
Yorimitsu, T., Klionsky, D.J., 2005. Autophagy: Molecular machinery for self-eating. Cell Death Differ 12 Suppl 2, 1542-52.
Yurkova, N., Shaw, J., Blackie, K., Weidman, D., Jayas, R., Flynn, B., Kirshenbaum, L.A., 2008. The cell cycle factor e2f-1 activates bnip3 and the intrinsic death pathway in ventricular myocytes. Circ Res 102 (4), 472-9.
Zhang, H., Bosch-Marce, M., Shimoda, L.A., Tan, Y.S., Baek, J.H., Wesley, J.B., Gonzalez, F.J., Semenza, G.L., 2008. Mitochondrial autophagy is an hif-1-dependent adaptive metabolic response to hypoxia. J Biol Chem 283 (16), 10892-903.
Zhao, J., Brault, J.J., Schild, A., Cao, P., Sandri, M., Schiaffino, S., Lecker, S.H., Goldberg, A.L., 2007. Foxo3 coordinately activates protein degradation by the autophagic/lysosomal and proteasomal pathways in atrophying muscle cells. Cell Metab 6 (6), 472-83.

Part II:
Abbate, A., Biondi-Zoccai, G.G., Bussani, R., Dobrina, A., Camilot, D., Feroce, F., Rossiello, R., Baldi, F., Silvestri, F., Biasucci, L.M., Baldi, A., 2003. Increased myocardial apoptosis in patients with unfavorable left ventricular remodeling and early symptomatic post-infarction heart failure. J Am Coll Cardiol 41 (5), 753-60.
Camper-Kirby, D., Welch, S., Walker, A., Shiraishi, I., Setchell, K.D., Schaefer, E., Kajstura, J., Anversa, P., Sussman, M.A., 2001. Myocardial akt activation and gender: Increased nuclear activity in females versus males. Circ Res 88 (10), 1020-7.
Chao, W., Matsui, T., Novikov, M.S., Tao, J., Li, L., Liu, H., Ahn, Y., Rosenzweig, A., 2003. Strategic advantages of insulin-like growth factor-i expression for cardioprotection. J Gene Med 5 (4), 277-86.
Coleman, K.M., Dutertre, M., El-Gharbawy, A., Rowan, B.G., Weigel, N.L., Smith, C.L., 2003. Mechanistic differences in the activation of estrogen receptor-alpha (er alpha)- and er beta-dependent gene expression by camp signaling pathway(s). J Biol Chem 278 (15), 12834-45.
Datta, S.R., Brunet, A., Greenberg, M.E., 1999. Cellular survival: A play in three akts. Genes Dev 13 (22), 2905-27.
Delaunay, F., Pettersson, K., Tujague, M., Gustafsson, J.A., 2000. Functional differences between the amino-terminal domains of estrogen receptors alpha and beta. Mol Pharmacol 58 (3), 584-90.
Fliegner, D., Schubert, C., Penkalla, A., Witt, H., Kararigas, G., Dworatzek, E., Staub, E., Martus, P., Ruiz Noppinger, P., Kintscher, U., Gustafsson, J.A., Regitz-Zagrosek, V., 2010. Female sex and estrogen receptor-beta attenuate cardiac remodeling and apoptosis in pressure overload. Am J Physiol Regul Integr Comp Physiol 298 (6), R1597-606.
Hale, S.L., Birnbaum, Y., Kloner, R.A., 1997. Estradiol, administered acutely, protects ischemic myocardium in both female and male rabbits. J Cardiovasc Pharmacol Ther 2 (1), 47-52.
Haynes, M.P., Sinha, D., Russell, K.S., Collinge, M., Fulton, D., Morales-Ruiz, M., Sessa, W.C., Bender, J.R., 2000. Membrane estrogen receptor engagement activates endothelial nitric oxide synthase via the pi3-kinase-akt pathway in human endothelial cells. Circ Res 87 (8), 677-82.
Hisamoto, K., Ohmichi, M., Kurachi, H., Hayakawa, J., Kanda, Y., Nishio, Y., Adachi, K., Tasaka, K., Miyoshi, E., Fujiwara, N., Taniguchi, N., Murata, Y., 2001. Estrogen induces the akt-dependent activation of endothelial nitric-oxide synthase in vascular endothelial cells. J Biol Chem 276 (5), 3459-67.
Kang, P.M., Izumo, S., 2000. Apoptosis and heart failure: A critical review of the literature. Circ Res 86 (11), 1107-13.
Liou, C.M., Yang, A.L., Kuo, C.H., Tin, H., Huang, C.Y., Lee, S.D., 2010. Effects of 17beta-estradiol on cardiac apoptosis in ovariectomized rats. Cell Biochem Funct 28 (6), 521-8.
Liu, C.J., Lo, J.F., Kuo, C.H., Chu, C.H., Chen, L.M., Tsai, F.J., Tsai, C.H., Tzang, B.S., Kuo, W.W., Huang, C.Y., 2009. Akt mediates 17beta-estradiol and/or estrogen receptor-alpha inhibition of lps-induced tumor necresis factor-alpha expression and myocardial cell apoptosis by suppressing the jnk1/2-nfkappab pathway. J Cell Mol Med 13 (9B), 3655-67.
Lopez, A.D., Murray, C.C., 1998. The global burden of disease, 1990-2020. Nat Med 4 (11), 1241-3.
Mosca, L., Barrett-Connor, E., Wenger, N.K., 2011. Sex/gender differences in cardiovascular disease prevention: What a difference a decade makes. Circulation 124 (19), 2145-54.
Nilsson, S., Gustafsson, J.A., 2011. Estrogen receptors: Therapies targeted to receptor subtypes. Clin Pharmacol Ther 89 (1), 44-55.
Node, K., Kitakaze, M., Kosaka, H., Minamino, T., Funaya, H., Hori, M., 1997. Amelioration of ischemia- and reperfusion-induced myocardial injury by 17beta-estradiol: Role of nitric oxide and calcium-activated potassium channels. Circulation 96 (6), 1953-63.
Olefsky, J.M., 2001. Nuclear receptor minireview series. J Biol Chem 276 (40), 36863-4.
Olivetti, G., Quaini, F., Sala, R., Lagrasta, C., Corradi, D., Bonacina, E., Gambert, S.R., Cigola, E., Anversa, P., 1996. Acute myocardial infarction in humans is associated with activation of programmed myocyte cell death in the surviving portion of the heart. J Mol Cell Cardiol 28 (9), 2005-16.
Osborne, C.K., 1998. Steroid hormone receptors in breast cancer management. Breast Cancer Res Treat 51 (3), 227-38.
Osborne, C.K., Zhao, H., Fuqua, S.A., 2000. Selective estrogen receptor modulators: Structure, function, and clinical use. J Clin Oncol 18 (17), 3172-86.
Palojoki, E., Saraste, A., Eriksson, A., Pulkki, K., Kallajoki, M., Voipio-Pulkki, L.M., Tikkanen, I., 2001. Cardiomyocyte apoptosis and ventricular remodeling after myocardial infarction in rats. Am J Physiol Heart Circ Physiol 280 (6), H2726-31.
Patten, R.D., Pourati, I., Aronovitz, M.J., Baur, J., Celestin, F., Chen, X., Michael, A., Haq, S., Nuedling, S., Grohe, C., Force, T., Mendelsohn, M.E., Karas, R.H., 2004. 17beta-estradiol reduces cardiomyocyte apoptosis in vivo and in vitro via activation of phospho-inositide-3 kinase/akt signaling. Circ Res 95 (7), 692-9.
Pearce, S.T., Jordan, V.C., 2004. The biological role of estrogen receptors alpha and beta in cancer. Crit Rev Oncol Hematol 50 (1), 3-22.
Pedram, A., Razandi, M., Lubahn, D., Liu, J., Vannan, M., Levin, E.R., 2008. Estrogen inhibits cardiac hypertrophy: Role of estrogen receptor-beta to inhibit calcineurin. Endocrinology 149 (7), 3361-9.
Rossouw, J.E., Anderson, G.L., Prentice, R.L., Lacroix, A.Z., Kooperberg, C., Stefanick, M.L., Jackson, R.D., Beresford, S.A., Howard, B.V., Johnson, K.C., Kotchen, J.M., Ockene, J., 2002. Risks and benefits of estrogen plus progestin in healthy postmenopausal women: Principal results from the women''s health initiative randomized controlled trial. JAMA 288 (3), 321-33.
Saraste, A., Pulkki, K., Kallajoki, M., Henriksen, K., Parvinen, M., Voipio-Pulkki, L.M., 1997. Apoptosis in human acute myocardial infarction. Circulation 95 (2), 320-3.
Simoncini, T., Hafezi-Moghadam, A., Brazil, D.P., Ley, K., Chin, W.W., Liao, J.K., 2000. Interaction of oestrogen receptor with the regulatory subunit of phosphatidylinositol-3-oh kinase. Nature 407 (6803), 538-41.
Tora, L., White, J., Brou, C., Tasset, D., Webster, N., Scheer, E., Chambon, P., 1989. The human estrogen receptor has two independent nonacidic transcriptional activation functions. Cell 59 (3), 477-87.
Tsai, M.J., O''malley, B.W., 1994. Molecular mechanisms of action of steroid/thyroid receptor superfamily members. Annu Rev Biochem 63, 451-86.
Van Eickels, M., Patten, R.D., Aronovitz, M.J., Alsheikh-Ali, A., Gostyla, K., Celestin, F., Grohe, C., Mendelsohn, M.E., Karas, R.H., 2003. 17-beta-estradiol increases cardiac remodeling and mortality in mice with myocardial infarction. J Am Coll Cardiol 41 (11), 2084-92.
Wu, C.H., Liu, J.Y., Wu, J.P., Hsieh, Y.H., Liu, C.J., Hwang, J.M., Lee, S.D., Chen, L.M., Chang, M.H., Kuo, W.W., Shyu, J.C., Tsai, J.H., Huang, C.Y., 2005. 17beta-estradiol reduces cardiac hypertrophy mediated through the up-regulation of pi3k/akt and the suppression of calcineurin/nf-at3 signaling pathways in rats. Life Sci 78 (4), 347-56.
Ylikorkala, O., 2004. Hrt as secondary prevention of cardiovascular disease. Maturitas 47 (4), 315-8.
Zhao, Z.Q., Nakamura, M., Wang, N.P., Wilcox, J.N., Shearer, S., Ronson, R.S., Guyton, R.A., Vinten-Johansen, J., 2000. Reperfusion induces myocardial apoptotic cell death. Cardiovasc Res 45 (3), 651-60.