The protein kinase Akt provides strong survival signals in cardiomyocytes, both chronically through altered gene programming, and more acutely through mechanisms that have not been fully elucidated. This proposal examines the hypothesis that activated Akt translocates to mitochondria and provides acute cardioprotection via its ability to phosphorylate hexokinase-II (HK-II) and enhance HK-II association with and inhibition of permeability transition pore (PT-pore) opening. Data I have generated in my published work show that Akt translocates to mitochondria upon acute activation by leukemia inhibitory factor (LIF), phosphorylates HK-II and prevents H202 and Ca2+induced PT-pore opening in neonatal rat ventricular myocytes or mitochondria isolated from adult mouse heart. Protective responses mediated by Akt are impaired by dissociation of HK-II.
Aim #1 of this proposal seeks to establish a requirement for mitochondrial HK-II and its phosphorylation in Akt-mediated protection against PT-pore opening. This will be tested using loss and gain of function approaches in which mitochondrial HK-II is decreased by an HK-II dissociating peptide, and increased by adenoviral expression of WT HK-II, a mutated HK-II lacking its mitochondrial binding motif or kinase dead HK-II. The requirement for HK-II phosphorylation will be tested by using HK-II mutated to either prevent or mimic phosphorylation at its putative Akt phosphorylation site. To extend these findings to the adult heart, Aim #2 will examine regulation of mitochondrial Akt and HK-II and the phosphorylation of HK-II using Langendorff perfused heart model and in vivo. The effects of HK-II dissociation from mitochondria and of TAT-fusion HK-II and mutants into the heart will be tested for their effects on ischemia/reperfusion (I/R) damage in perfused heart in the presence or absence of IGF-1 or in vivo.
In Aim #3, I will determine whether the recently discovered Akt-phosphatase, PHLPP-1, localizes to mitochondria and regulates mitochondrial Akt activity and thus mitochondrial HK-II/PT-pore opening. This will be examined by knockdown of PHLPP (using siRNA and PHLPP knock-out mice) and by increasing PHLPP-1 expression via adenoviral expression. These experiments will provide evidence for HK-II as a potential downstream target of Akt, and PHLPP as a potential regulator of Akt, and will suggest novel sites of intervention for inhibiting I/R induced mitochondria mediated cardiomyocyte cell death.

Public Health Relevance

Cardiomyocyte death plays a crucial role in heart disease and opening of the permeability transition pore (PT-pore) in mitochondria is a major executor of cell death. The protein kinase Akt provides strong survival signals in cardiomyocytes and the mechanisms by which Akt prevents cell death have not been fully elucidated. This proposal examines the hypotheses that activated Akt translocates to mitochondria and provides acute cardioprotection via its ability to phosphorylate hexokinase-II (HK-II) and that this protective signaling is regulated by a Akt phosphatase at mitochondria.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Project (R01)
Project #
5R01HL097037-04
Application #
8269124
Study Section
Myocardial Ischemia and Metabolism Study Section (MIM)
Program Officer
Schwartz, Lisa
Project Start
2009-08-01
Project End
2014-04-30
Budget Start
2012-05-01
Budget End
2013-04-30
Support Year
4
Fiscal Year
2012
Total Cost
$382,388
Indirect Cost
$134,888
Name
University of California San Diego
Department
Pharmacology
Type
Schools of Medicine
DUNS #
804355790
City
La Jolla
State
CA
Country
United States
Zip Code
92093
Zhao, Xia; Ding, Eric Y; Yu, Olivia M et al. (2014) Induction of the matricellular protein CCN1 through RhoA and MRTF-A contributes to ischemic cardioprotection. J Mol Cell Cardiol 75:152-61
Roberts, David J; Tan-Sah, Valerie P; Ding, Eric Y et al. (2014) Hexokinase-II positively regulates glucose starvation-induced autophagy through TORC1 inhibition. Mol Cell 53:521-33
Roberts, David J; Tan-Sah, Valerie P; Smith, Jeffery M et al. (2013) Akt phosphorylates HK-II at Thr-473 and increases mitochondrial HK-II association to protect cardiomyocytes. J Biol Chem 288:23798-806
Mishra, Shikha; Gray, Charles B B; Miyamoto, Shigeki et al. (2011) Location matters: clarifying the concept of nuclear and cytosolic CaMKII subtypes. Circ Res 109:1354-62
Miyamoto, Shigeki; Purcell, Nicole H; Smith, Jeffrey M et al. (2010) PHLPP-1 negatively regulates Akt activity and survival in the heart. Circ Res 107:476-84