Ischemic heart disease (IHD) is a major epidemic throughout the developed world, however, the molecular mechanisms that lead to myocardial cell death in IHD are not totally understood. microRNAs (miRNAs) are short noncoding RNAs that regulate gene expression by binding to their target mRNA and causing degradation or translational inhibition. We recently showed that the levels of miRNA-210 (miR-210) are elevated in cardiomyocytes exposed to hypoxia. miR-210 response to hypoxia is attenuated in p53-/- mouse embryonic fibroblasts (MEF), implicating a role for p53 in the regulation of miR-210. However, it is not known what role miR-210 plays in cardiomyocyte response to hypoxia. Furthermore, although both miR-210 and hypoxia- inducible factor (HIF) are activated by hypoxia and HIF regulates miR-210 expression, the relative contribution of miR-210 to the biological effects of HIF activation is not known. In this proposal, we will elucidate the mechanism of miR-210 regulation by hypoxia and will determine the role of miR-210 in cardiomyocyte response to ischemia-reperfusion (I/R). Our central hypothesis is that miR-210 is regulated by p53 at the transcriptional level, reduces cardiomyocyte death in response to I/R, and mediates some of the physiological effects of HIF.
In Aim 1, we will determine whether miR-210 plays a role in cardiomyocyte cell death and ROS production in response to I/R. In addition to in vitro studies, we will also study miR-210 KO mice and mice with cardiac miR-210 knock down using a novel approach with gold conjugated nanoparticles. We will subject these mice to I/R followed by measurement of cell death and ischemic damage.
In Aim 2, we will determine the role of miR-210 in the biological effects of HIF activation. We will overexpress miR-210 in HIF11-/- MEFs, followed by measurement of various physiological parameters in response to hypoxia. We will also use cardiac specific Von Hippel-Lindau (VHL) knockout mice that have constitutively active HIF and display several abnormalities including atrial tumor formation, lipid accumulation and cardiomyopathy. We will cross VHL-/- mice with miR-210 knockout animals and will assess whether miR-210 knockout alters changes from chronic HIF activation in the heart. Finally, in Aim 3, we will elucidate the mechanism of miR-210 regulation by p53. We will assess whether p53 regulates miR-210 at the transcriptional level by measuring miR-210 precursor molecules in p53-/- MEFs and performing ChIP and nuclear run-on assays. Our proposal is novel and unique in several ways: 1) it will study the role of miR-210 in ischemic heart disease, 2) it will use a novel approach involving redox-sensitive probes to measure cellular ROS levels, 3) it will evaluate the relative contribution of miR-210 to HIF-mediated response in cardiomyocytes, 4) it will study a novel approach to introduce miR-210 silencing molecules into cardiomyocytes using gold-conjugated nanoparticles, and 5) it will use miR-210 and VHK KO mice. The proposal is based on a robust collaboration among investigators who are experts in their fields and each bring an entirely different skill set to the project. These lines of investigation promise to advance our knowledge of the molecular basis of ischemic-induced cell death.

Public Health Relevance

The major consequence of a heart attack is death of heart cells from lack of enough oxygen supply to the heart. As of now, there are no medicines that can protect heart cells from dying. We propose to study the role of a newly identified biological process called microRNA to reduce damage to the heart from a heart attack.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Project (R01)
Project #
5R01HL104181-04
Application #
8645703
Study Section
Cardiac Contractility, Hypertrophy, and Failure Study Section (CCHF)
Program Officer
Schwartz, Lisa
Project Start
2011-04-01
Project End
2015-03-31
Budget Start
2014-04-01
Budget End
2015-03-31
Support Year
4
Fiscal Year
2014
Total Cost
$368,920
Indirect Cost
$121,311
Name
Northwestern University Chicago
Department
Internal Medicine/Medicine
Type
Schools of Medicine
DUNS #
005436803
City
Chicago
State
IL
Country
United States
Zip Code
60611
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