Many known and unknown ligands for G protein coupled receptors, released in the heart in response to stress, converge on RhoA activation, making it a nodal point in determining cardiomyocyte responses. The overall goal of this proposal is to demonstrate that activation of RhoA signaling provides cardioprotection against ischemic injury and to elucidate the molecular mechanisms by which this occurs. The specific focus is on the cardiomyocyte and an early time window, distinct from later effects of RhoA signaling pathways on fibroblasts and leukocytes, which may in fact be deleterious. The proposal examines direct RhoA targets, early genetic responses and mitochondrial involvement in cardioprotection by RhoA. The proposed work is divided into three Specific Aims.
Aim #1 characterizes several lines of transgenic mice expressing constitutively active RhoA in an inducible cardiac specific manner, comparing the ability of RhoA expression to provide protection against ischemia/reperfusion (I/R) injury in the ex vivo perfused heart, and extending this work to examine contractile function and susceptibility to I/R injury, inflammation and apoptotic gene expression in vivo. Studies using mice in which the upstream regulator of RhoA, G112, is deleted, are also proposed as are studies using RhoA knockout mice.
Aim #2 examines mediators of RhoA induced cardioprotection. One pathway examined is the activation of FAK and Akt through Rho kinase (ROCK). Two novel RhoA targets, the matricellular protein CCN1, which works through integrin signaling, and PLC epsilon which activates PKC epsilon and PKD, are also examined for their role as downstream effectors of RhoA signaling and mediators of protection in the heart. Finally three RhoA regulated transcription factors (cJun, NFKB, MRTF-A) are considered as targets of RhoA in the heart. Experiments are designed to determine if these pathways are differentially activated in tTA control vs. RhoA TG mice, and during ex vivo or in vivo I/R, and to examine the protective roles of these pathways using pharmacological inhibitors or, for PLC epsilon and CCN1, knockout mice.
Aim #3 considers a unique aspect of RhoA function in the heart i.e. its localization at mitochondria, as demonstrated by its presence in the mitochondrial heart proteome and by subcellular fractionation studies. Questions addressed include whether activation of RhoA alters its appearance at mitochondria, whether RhoA affects mitochondrial permeability transition (MPT) and associates with MPT components, whether mitochondrial structure and localization, as detected by electron microscopy, differ when RhoA at mitochondria is increased, whether RhoA associates with mitochondrial fission/fusion proteins or motility regulators, and whether RhoA affects mitochondrial metabolism. The significance of this research is that it could provide evidence that activation of RhoA and its novel effectors, at early times following ischemia reperfusion, are therapeutic target for preventing ischemic damage and improving contractile recovery of the heart.

Public Health Relevance

Ischemic heart disease, characterized by loss of blood flow and oxygenation, is a major cause of death worldwide. Current treatment of heart attacks involve restoration of blood flow and this reperfusion provides both protective and damaging signals. We are exploring a novel protective signal, the activation of RhoA and its downstream targets, which has the potential to be used as a therapeutic strategy to diminish loss of cardiac tissue and contractile function during reperfusion.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Method to Extend Research in Time (MERIT) Award (R37)
Project #
5R37HL028143-29
Application #
8459346
Study Section
Myocardial Ischemia and Metabolism Study Section (MIM)
Program Officer
Schwartz, Lisa
Project Start
1982-08-01
Project End
2015-03-31
Budget Start
2013-04-01
Budget End
2014-03-31
Support Year
29
Fiscal Year
2013
Total Cost
$364,033
Indirect Cost
$128,413
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
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