title: mTORC2 phosphorylation of Akt1: a possible mechanism for hydrogen sulfide-induced cardioprotection.
abstract: Hydrogen sulfide (H2S) is known to have cardiac protective effects through Akt activation. Akt acts as a 'central sensor' for myocyte survival or death; its activity is regulated by multiple kinases including PI3K, mTORC2, PDK1 and phosphatases including PTEN, PP2A and PHLPPL. Based on the previous finding that PI3K inhibitor LY294002 abolishes H2S-induced Akt phosphorylation and cardioprotection, it is accepted that PI3K is the mediator of H2S-induced Akt phosphorylation. However, LY294002 inhibits both PI3K and mTOR, and PI3K only recruits Akt to the membrane where Akt is phosphorylated by Akt kinases. We undertook a series of experiments to further evaluate the role of mTORC2, PDK1, PTEN, PP2A and PHLPPL in H2S-induced Akt phosphorylation and cardioprotection, which, we believe, has not been investigated before. Hearts from adult Sprague-Dawley rats were isolated and subjected to (i) normoxia, (ii) global ischemia and (iii) ischemia/reperfusion in the presence or absence of 50 µM of H2S donor NaHS. Cardiac mechanical function and lactate dehydrogenase (LDH) release were assessed. All hearts also were Western analyzed at the end of perfusion for Akt and a panel of appropriate Akt regulators and targets. Hearts pretreated with 50 µM NaHS had improved function at the end of reperfusion (Rate pressure product; 19±4×10(3) vs. 10±3×10(3) mmHg/min, p<0.05) and reduced cell injury (LDH release 19±10 vs. 170±87 mU/ml p<0.05) compared to untreated hearts. NaHS significantly increased phospho-Akt, phospho-mTOR, phospho-Bim and Bcl-2 in reperfused hearts (P<0.05). Furthermore using H9c2 cells we demonstrate that NaHS pretreatment reduces apoptosis following hypoxia/re-oxygenation. Importantly, PP242, a specific mTOR inhibitor, abolished both cardioprotection and protein phosphorylation in isolated heart and reduced apoptotic effects in H9c2 cells. Treating hearts with NaHS only during reperfusion produced less cardioprotection through a similar mechanism. These data suggest mTORC2 phosphorylation of Akt is a key mediator of H2S-induced cardioprotection in I/R.
authors.affiliation: Cardiovascular Research Institute, National University of Singapore, Singapore, Singapore.
authors.forename: Yue
authors.initials: Y
authors.lastname: Zhou
authors.affiliation: Department of Cardiology, Putuo Hospital, Shanghai, China.
authors.forename: Daying
authors.initials: D
authors.lastname: Wang
authors.affiliation: Department of Cardiology, Huashan Hospital, Fudan University, Shanghai, China.
authors.forename: Xiufang
authors.initials: X
authors.lastname: Gao
authors.affiliation: Cardiovascular Research Institute, National University of Singapore, Singapore, Singapore.
authors.forename: Karsheng
authors.initials: K
authors.lastname: Lew
authors.affiliation: Cardiovascular Research Institute, National University of Singapore, Singapore, Singapore.
authors.forename: Arthur Mark
authors.initials: AM
authors.lastname: Richards
authors.affiliation: Cardiovascular Research Institute, National University of Singapore, Singapore, Singapore.
authors.forename: Peipei
authors.initials: P
authors.lastname: Wang
mesh_terms: D000818:Animals; D002316:Cardiotonic Agents / Q000008:administration & dosage; D002867:Chromones / Q000008:administration & dosage; D006801:Humans; D006862:Hydrogen Sulfide / Q000008:administration & dosage*; D000076225:Mechanistic Target of Rapamycin Complex 2; D009025:Morpholines / Q000008:administration & dosage; D046912:Multiprotein Complexes / Q000378:metabolism*; D032342:Muscle Cells / Q000187:drug effects / Q000378:metabolism / Q000473:pathology; D009924:Organ Culture Techniques; D051059:PTEN Phosphohydrolase / Q000378:metabolism; D000081082:Phosphoinositide-3 Kinase Inhibitors; D010766:Phosphorylation / Q000187:drug effects; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D051057:Proto-Oncogene Proteins c-akt / Q000378:metabolism*; D000081382:Pyruvate Dehydrogenase Acetyl-Transferring Kinase; D051381:Rats; D015427:Reperfusion Injury / Q000188:drug therapy / Q000378:metabolism* / Q000503:physiopathology; D058570:TOR Serine-Threonine Kinases / Q000378:metabolism*
chemical_list: D002316:Cardiotonic Agents; D002867:Chromones; D009025:Morpholines; D046912:Multiprotein Complexes; C000631429:PDK1 protein, human; C000631434:Pdk1 protein, rat; D000081082:Phosphoinositide-3 Kinase Inhibitors; D000081382:Pyruvate Dehydrogenase Acetyl-Transferring Kinase; C085911:2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one; C494920:Akt1 protein, rat; D000076225:Mechanistic Target of Rapamycin Complex 2; D017346:Protein Serine-Threonine Kinases; D051057:Proto-Oncogene Proteins c-akt; D058570:TOR Serine-Threonine Kinases; D051059:PTEN Phosphohydrolase; C487216:Pten protein, rat; D006862:Hydrogen Sulfide