Study The Role Of PTEN-PDK-Akt Signaling In Postnatal Heart Function

This thesis focus on the PTEN-PDK1-Akt signaling pathway’s role in mataining postnatal heart function. It has three parts:In part one, we study Akt’s upstream:PTEN and PDK1’s role in postnatal heart function. PTEN is a phosphotase and can inhibite PIP2changed to PIP3. PIP3can recruit Akt to the cell membrane and promote its phosphorylation to activate it. So PTEN is an inhibitor of Akt. PDK1is a kinase. It can phosphorylate Akt at its T308site. Other kinase such as mTORC2can phosphorylate Akt at its S473site. After phosphorylation of T308and S473, Akt is activated totally. Previous researches found that knocking out of PDK1using MCK-cre in cardiomyocytes and skeletal muscles cause dystrophy of cardiomyocytes and then heart failure. Deletion of PTEN using MCK-cre cause cadiomyocytes hypertrophy with out heart disfunction and the phosphorylation of Akt S473site increased. In this thesis, we used a-MHC-cre as tool mice, and this cre begin to express at postnatal day7in cardiomyocytes. Knocking out PDK1using this cre caused smaller cardiomyocytes compared with control littermates and this mice die at about2-3months old because of heart failure. Through TUNEL staining of PDK1konck out mice hearts at different time points, we found no apotosis cells until heart failure. So the initial phenotype of PDK1knock out mice is hypotrophy of cardiomyocytes. We also found that phosphorylation of Akt T308is decreased expectedly while the phosphorylation of Akt S473is increased unexpectedly by western blot. This result is in accordance with previous research. But what is the role of upregulation of S473phosphorylation is still unknown, we speculate that upregulation of S473phosphorylation can matain part of Akt’s activity and protect heart function. With this speculation, we made PDKl and Rictor double knock out mice. Rictor is a composition of mTORC2. Knocking out Rictor means damaging mTORC2that inhibits Akt S473phosphorylation. PDKl;Rictor double knock out mice die earlier than PDK1knock out mice. They can only survived20-30days. Through histological analysis, we found double konck out mice hearts are smaller than PDK1knock out mice. Through western blot we found phosphorylation of Akt S473is downregulated in double knock out mice. So in this experiment, we can conclude that phosphorylation of Akt S473can partially maintain heart function. Then whether upregulation of phosphorylation of Akt S473can rescue PDK1knock out mice’s phenotype? With this question, we knock out PDKl and PTEN using a-MHC-cre at the same time. We found that phosphorylation of Akt S473is increased in double knock out mice and double knock out mice can survive more than one year which is longer than PDKl knock out mice. The cardiomyocytes is larger than PDKl knock out mice but still smaller than control littermates. As PTEN have other downstreams targets other than Akt, we still can not exclude other downstream targets of PTEN rescue PDK1knock out mice phenotype. In order to check whether increased AktS473’s phosphorylation is the major rescue factor, we delete Akt1,PDKl,PTEN at the same time and found PTEN’s rescue effect was reversed. Triple knock out mice survived as long as PDK1knock out mice and cardiomyocytes size is almost the same as PDK1knock out mice. Triple knock out PDK1, PTEN, Rictor mice’s phenotype is severer than PDK1knock out mice. Survive time is shorter and cardiomyocytes size is smaller. Both of these experiments show that phosphorylation of AktS473is the major rescue factor.In this part, we found that deletion of PTEN in cardiomyocytes can rescue cardiomyocytes specific PDK1konck out mice’s heart failure through upregulating mTORC2mediated AktS473phosphorylation. AktS473’s phosphorylation can increase Akt’s activity and protect heart function. This finding can help to cure cardiomyopathy caused by decreased Akt activity. In the second part, we study Akt’s role to postnatal heart function. Akt family has three homologous:Akt1, Akt2, Akt3. The three proteins have complementary roles but play different major roles. Aktl regulates cell growth, Akt2regulates cell metabolism while Akt3regulates brain development. There were many researches about Akt and heart and found that Akt influences cardiomyocytes survive, size, calcium transition and metabolism. Previous researches about Akt and heart are almost using Aktl heart specific transgenic mouse as model. These mice have hypertrophic cardiomyopathy and heart failure finnally. In this part we used Akt knock out mouse model. At first we used α-MHC-cre to knock out Aktl and found no phenotype of these mice that can survive normally. We speculate that excess Akt2and Akt3maintain heart function. So we delete Akt2and found that Aktl,2double knock out mice’s cardiomyocytes were smaller than control littermates and die in1-2months because of heart failure. In order to clarify whether Akt3has roles in this process, we delete it in Akt1,2double knock out mice and found that Akt1,2,3triple knock out mice can only survive no more than20days. We choose postnatal day14as a time point to analysis phenotypes of Akt1F/F; Akt1F/F;aMHC-cre; AktlF/F;αMEC-cre;Akt2-/-;Akt1F/F;αMHC-cre;Akt2-/-;Akt3-/--mice. Through histological analysis we found that cardiomyocytes are more smaller as more Akt deletion and there were no apoptotic cells in mice hearts until heart failure. So the initial phenotype of Akt knock out mice is hypotrophic cardiomyocytes. Cell size is controlled by protein synthesis and protein degradation. As we know, GSK3β, mTORC1, S6K as downstream targets of Akt can regulate protein translation and influence protein synthesis. Foxo, another Akt target, can regulate protein degradation. So we checked phosphorylation leve of these proteins in Akt knock out mice hearts and found that these proteins’phosphorylation are decreased in Akt knock out mice and more Akt deletion more phosphorylation downregulation. This result means that protein synthesis is decreased while protein degradation is increased. We also checked the expression level of proteins controlling angiogenesis, such as Ang2, VEGF and eNOS in Akt knock out mice hearts and found no evident changes. In the future experiments we want to check cardiomyocytes’ glycometabolism and lipid metabolism.Through research of this part we found that heart specific deletion of Akt can decrease protein synthesis and increase protein degradation which cause hypotrophic cardiomyocytes and heart failure finnally. This process is Akt1, Akt2, and Akt3dosage-dependent.In the third part, we found a new target of Akt, Handl and study the role of its phosphorylation by Akt in postnatal heart function. Handl also called Hxtl, Thing1or eHandl, is abbreviation of heart and neural creast derivatives expressed1. Handl is a member of basic helix-loop-helix transcription factor family. As already known, its basic helix1domain can be phosphorylated which can influence its activity and it involved in many development process, such as heart and limb development and trophoblast differentiation. But researches about Handl’s role in postnatal heart function is still very rare. We only know that Handl heart specific transgenic mice have heart arrhythmia phenotype. Bioinformatics analysis show that Handl’s basic helix domain has Akt’s target sequences and it may be Akt’s target. In order to verify this speculation, we did many in vitro biochemical study and found that Handl’s T107and T109can be phosphorylated by Akt and phosphorylation influences its binding ability to its targets. So the transcription of its target can be influenced by Akt phosphorylation. In order to study physiological significance of phosphorylation of Hand1, we made heart specific Hand1mutan transgenic mice to mimic constitutively phosphorylation (Hand1T107D;T109D) and unphosphorylation (Hand1T107A;T109A) states. We found both of these transgenic mice have heart disfunction, hypertrophic and apoptotic cardiomyocytes, heart fibrosis and heart failue and sudden death finnally. Through gene chips analysis we found genes regulating cell metabolism is decreased while genes regulating cell growth is increased which means cell disfunction.Research of this part show that Akt can phosphorylate Handl and phosphorylation of it involved in heart remodeling. In summary, this thesis focus on the role of Akt signaling in postnatal heart function deeply and in detail. We found that Akt signaling major regulates postnatal cardiomyocytes size. Apoptosis and heart failure are secondary phenotype. Our research results can be used to guide prevention and treatment of heart diseases caused by disfunction of Akt signaling pathway in clinical cardiomyopathies.