Ischemic and pharmacologic preconditioning (PC) constitute the most powerful protection of the heart from ischemia/reperfusion (I/R) injury;however, the detailed molecular mechanisms underlying cardioprotection are still being defined. There is a general consensus that mitochondria are the final effectors of cardioprotective signaling regimes, and hexokinase (HK) has been suggested by multiple groups to regulate the mitochondrial permeability transition (MPT). Though the association of HK with voltage-dependent anion channels (VDAC) was elucidated over 10 years ago, two fundamental questions regarding the physiologic consequences of this interaction have remained unanswered, and consequently, have stalled the progression of the cardioprotection field. First, is the dissociation of HK from cardiac mitochondria a molecular trigger of cell death? That is, does HK dissociation from mitochondria precede all other cell death events [e.g., MPT, DY loss, and cytochrome C (cyto C) release]? Second, what are the unknown molecular players that stabilize the HK-VDAC interaction and impart its unique cardioprotective properties? Unequivocal answers to these questions have been unattainable due to the lack of technologies for (i) temporal profiling of the spatial distribution of HK in relation to MPT, ?? loss, and cyto release in live cardiomyocytes, and (ii) quantifying the molecular constituents of the HK-VDAC complex and deciphering their stoichiometry. In view of these challenges, our program has tailored state-of-the-art live-cell imaging and quantitative proteomic innovations to comprehensively delineate the dynamics of HK-induced cardioprotection on a biological timescale. We hypothesize that HK is a core regulator of cardioprotection, common to multiple models of injury and preconditioning. We will employ real-time imaging in live myocytes to define the temporal profile of the molecular events during injury (Aim 1);we will use an extensive biochemical and genetic toolbox to delineate the molecular paradigm of HK interaction with mitochondria as well as its physiological consequences mediating cardioprotection (Aim 2);we will quantitatively define the proteome dynamics and molecular stoichiometry of HK interaction with VDAC;characterize isoform-selective changes in assembly of the HK- VDAC interactome;and identify candidate proteins essential to stabilize the HK interaction with VDAC during cardioprotection (Aim 3);and we will use cardiac gene delivery of HK constructs or other molecular candidates identified in Aims 1-3, to test an in vivo gene therapy strategy to protect adult rats from I/R injury (Aim 4). The proposed investigations promise conceptual, technological, and methodological innovations. We will leverage close collaborations with the UCLA NHLBI Proteomics Center for immediate and efficient translation of knowledge obtained in cell/animal models to clinical studies. The success of the proposed investigations will undoubtedly propel the field of cardioprotection forward.

Public Health Relevance

Since heart disease is the major cause of death in industrialized societies, understanding how to protect the heart from injury has major implications for the health care mission of the NIH/NHLBI and for our society as a whole. To facilitate this understanding, this interdisciplinary multi-PI project will integrate biophysics, imaging, genetics, proteomics as well as systems approach to learn how mitochondria, the energy- producing organelle of heart cells, avoid becoming injured when exposed to ischemia/reperfusion, or heart attack in common parlance. These insights may suggest novel therapies to protect the heart from injury during heart attacks, and thereby reduce the mortality of this leading cause of death in the U.S.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Project (R01)
Project #
1R01HL117385-01A1
Application #
8600146
Study Section
Special Emphasis Panel (ZRG1-CVRS-K (02))
Program Officer
Wong, Renee P
Project Start
2013-08-12
Project End
2017-05-31
Budget Start
2013-08-12
Budget End
2014-05-31
Support Year
1
Fiscal Year
2013
Total Cost
$461,901
Indirect Cost
$161,965
Name
University of California Los Angeles
Department
Internal Medicine/Medicine
Type
Schools of Medicine
DUNS #
092530369
City
Los Angeles
State
CA
Country
United States
Zip Code
90095