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Dissertation - Jian Wu (submitted).pdf (4.43 MB)

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Role of Phosphoinositide 3-Kinase a (PI3Ka) in Ouabain-induced Cardiac Signaling and Hypertrophy

Wu, Jian
http://rave.ohiolink.edu/etdc/view?acc_num=mco1384283312

Abstract Details

2013, Doctor of Philosophy (PhD), University of Toledo, College of Medicine.
Rationale: Cardiac glycosides including digoxin and ouabain have a long history in heart failure therapy. They bind to the membrane receptor Na/K-ATPase (NKA) and inhibit this enzyme, therefore produce positive inotropic effect through Na/Ca exchanger in the heart. However, the “positive inotropic effect” of cardiac glycosides by inhibiting the NKA could not fully explain the clinical benefits (decreasing hospitalization and mortality; improving the heart failure’s symptoms). Most of other positive inotropic drugs have the propensity to increase mortality. Phosphoinositide 3-Kinase a (PI3Ka) and PI3K¿ are two major PI3K isoforms in the heart and play distinct roles: PI3Ka involves in exercise- and receptor tyrosine kinase (RTK)- induced hypertrophy; however, PI3K¿ participates in G protein-coupled receptor (GPCR)- induced pathological hypertrophy. Activation of PI3Ka was reported to antagonize pathological hypertrophy. Our laboratory has previously shown that ouabain activates PI3Ka in cultured mouse/rat cardiomyocytes and induces protein synthesis. In this study, we assessed the hypothesis that ouabain-induced PI3Ka activation leads to physiological hypertrophy and antagonizes pressure-overload-induced pathological hypertrophy and heart failure. Methods/Results: Mice with striated muscle-specific p85 subunit of PI3K knockout (p85-KO) were generated by Cre-Loxp system. In vitro: Adult mouse cardiomyocytes from p85-KO and WT mice were isolated and cultured. Western blot showed that ouabain induced a time-dependent Akt/mTOR phosphorylation in the presence of PI3Ka. Co-immunoprecipitation (co-IP) and lipid kinase assay suggested that ouabain activated PI3Ka by regulating the molecular complex of NKA a1-p85-PTEN. In contrast, ouabain did not activate PI3K¿ regardless of the presence of PI3Ka. 3H-leucine incorporation assay and fetal gene real-time PCR experiments indicated that ouabain caused physiological hypertrophy in cardiomyocytes from WT mice; and it antagonized endothelin-1(ET-1) induced pathological hypertrophy. When lacking of ouabain-induced PI3K/Akt signaling, ouabain-induced ERK activation resulted in cell death in p85-KO cardiomyocytes. In vivo: WT and p85-KO mice were subjected to transverse aortic constriction (TAC) or sham surgery for 8 weeks. Chronic infusion of ouabain (50 µg/kg/day) was conducted with osmotic minipump for 4 weeks after surgery. TAC-induced pathological hypertrophy indicated by increase of wall thickness, heart mass, fetal gene expressions in both WT and p85-KO mouse hearts were revealed by echocardiography and real-time PCR; ouabain infusion attenuated the intensity of TAC-induced hypertrophy and fetal gene reintroduction in WT hearts, but not in p85-KO hearts. Cardiac functions (e.g., fraction shortening, myocardium performance index) assessed by echocardiography, were decreased dramatically after 4 weeks of TAC; ouabain partially preserved the cardiac functions and reduced the myocardial fibrosis in WT mice, but not in p85-KO mice. TAC also greatly decreased the protein and mRNA expression of NKA a2 in the WT mouse myocardium assayed by Western blot and real-time PCR; ouabain infusion preserved protein expression of the myocardial NKA a2 in WT mice. Conclusion: Ouabain activates PI3Ka/Akt signaling and leads to physiological hypertrophy in cultured cardiomyocytes. Chronic infusion of ouabain lessens TAC-induced cardiac hypertrophy and improves cardiac functions, also preserves NKA a2 protein reduction in WT hearts. In the absence of PI3Ka, ouabain loses the beneficial effects in cardiac hypertrophy and heart failure. Targeting of activation of PI3Ka and preservation of NKA a2 expression could be a strategy for drug development on heart failure therapy.
Lijun Liu (Committee Chair)
Amin Askari (Committee Member)
William Maltese (Committee Member)
Andrew Beavis (Committee Member)
Sandrine Pierre (Committee Member)
Ivana de la Serna (Committee Member)
140 p.
Biochemistry; Biomedical Research; Health Sciences; Medicine; Molecular Biology; Physiology

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Citations

  • Wu, J. (2013). Role of Phosphoinositide 3-Kinase a (PI3Ka) in Ouabain-induced Cardiac Signaling and Hypertrophy [Doctoral dissertation, University of Toledo]. OhioLINK Electronic Theses and Dissertations Center. http://rave.ohiolink.edu/etdc/view?acc_num=mco1384283312

    APA Style (7th edition)

  • Wu, Jian. Role of Phosphoinositide 3-Kinase a (PI3Ka) in Ouabain-induced Cardiac Signaling and Hypertrophy . 2013. University of Toledo, Doctoral dissertation. OhioLINK Electronic Theses and Dissertations Center, http://rave.ohiolink.edu/etdc/view?acc_num=mco1384283312.

    MLA Style (8th edition)

  • Wu, Jian. "Role of Phosphoinositide 3-Kinase a (PI3Ka) in Ouabain-induced Cardiac Signaling and Hypertrophy ." Doctoral dissertation, University of Toledo, 2013. http://rave.ohiolink.edu/etdc/view?acc_num=mco1384283312

    Chicago Manual of Style (17th edition)

mco1384283312
1,042
© 2013, some rights reserved.Creative Commons License Role of Phosphoinositide 3-Kinase a (PI3Ka) in Ouabain-induced Cardiac Signaling and Hypertrophy by Jian Wu is licensed under a Creative Commons Attribution-NonCommercial-NoDerivs 3.0 Unported License. Based on a work at etd.ohiolink.edu.
This open access ETD is published by University of Toledo Health Science Campus and OhioLINK.