We identified Rho kinase ROCK1 as caspase-3 target in human failing hearts. The cleavage resulted in a constitutively active Rho kinase, ROCK?1. We also demonstrated that genetic deletion of ROCK1 inhibited stress-induced cardiac fibrosis. However, the function of ROCK?1 and molecular signaling linking ROCK1 to cardiac fibrosis remain obscure. The goal of the study is to determine the fibrogenic role of ROCK?1 and investigate molecular mechanism of ROCK?1-mediated cardiac fibrosis. To address these questions, we generated transgenic mice expressing ROCK?1 in heart to mimic human heart disease. Overt cardiac fibrosis was observed with marked upregulation of TGF?1 in the transgenic mice. Since activation of Rho kinase increased SRF activity, we assessed the transgenic heart and found manifest increase in SRF activity. Our preliminary results suggested SRF as a potential regulator of TGF?1. We also found robust increases in NF-?B expression and activity in the mice. Therefore, the central hypothesis is that constitutive activation of ROCK1 in cardiomyocytes is sufficient to result in cardiac fibrosis by upregulating TGF? signaling and other pro-fibrotic cytokines through activation of SRF and NF-?B, respectively.
Three aims will be completed.
Aim I is to determine the pro-fibrotic effect of ROCK?1 in intact heart. The transgenic mice will be studied under basal and stress challenging conditions. The fibrogenic phenotype will be determined in two mouse lines with high and low expression level of ROCK?1. A rescue experiment by Rho kinase inhibitor will be conducted.
Aim II is to elucidate the molecular mechanisms of ROCK?1-mediated cardiac fibrosis. The signaling pathway linked between Rho kinase and fibrotic response has been proposed with a considerable amount of preliminary data. The proposed mechanism includes the upregulation of TGF?1 and NF-?B-mediated cytokines. The hypothesis will be tested in vitro in cardiomyocytes and in vivo in the transgenic mice. To investigate SRF-directed TGF?1 regulation, the identified cis elements in TGF?1 promoter/enhancer region will be verified by extensive experiments including 1) luciferase, EMSA and CHIP assay;2) through transgenic mice expressing lacZ driven by either the wild or mutant cis elements;3) by analyzing expression of TGF?1 in SRF null mouse heart, where the decrease in TGF?1 level is expected.
Aim III is to determine if the loss of endogenous ROCK1 inhibitor, Rnd3, recapitulates ROCK?1-mediated fibrotic cardiomyopathy. The fibrotic phenotype, Rho kinase activity, TGF? and NF-?B signaling will be assessed under normal and stress challenging conditions. The outcome of the proposal will be to establish links between the activation of Rho kinase, TGF?1 and NF-?B signaling in fibrotic cardiac remodeling. The innovation of the proposal includes 1) demonstration of fibrogenic role of ROCK?1;2) elucidation of the ROCK1->SRF->TGF?1->fibrosis and the ROCK1->NF-?B->cytokines->fibrosis signaling pathways;3) revelation the role of Rnd3 in cardiac remodeling;and 4) implication of manipulating Rho kinase activity and caspase cleavage as candidate therapeutic targets.

Public Health Relevance

The study is to determine the fibrogenic role of the constitutively active ROCK?1 in intact heart, and to elucidate the molecular mechanism involved in Rho kinase-mediated cardiac fibrosis. The genetic-manipulated murine mice are applied.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Project (R01)
Project #
5R01HL102314-04
Application #
8499400
Study Section
Myocardial Ischemia and Metabolism Study Section (MIM)
Program Officer
Adhikari, Bishow B
Project Start
2010-05-01
Project End
2015-04-30
Budget Start
2013-05-01
Budget End
2014-04-30
Support Year
4
Fiscal Year
2013
Total Cost
$345,183
Indirect Cost
$109,563
Name
Texas A&M University
Department
Biochemistry
Type
Schools of Medicine
DUNS #
835607441
City
College Station
State
TX
Country
United States
Zip Code
77845
Yang, Xiangsheng; Wang, Tiannan; Lin, Xi et al. (2015) Genetic deletion of Rnd3/RhoE results in mouse heart calcium leakage through upregulation of protein kinase A signaling. Circ Res 116:e1-e10
Yue, X; Yang, X; Lin, X et al. (2014) Rnd3 haploinsufficient mice are predisposed to hemodynamic stress and develop apoptotic cardiomyopathy with heart failure. Cell Death Dis 5:e1284
Wang, Xiaohong; Huang, Wei; Yang, Yang et al. (2014) Loss of duplexmiR-223 (5p and 3p) aggravates myocardial depression and mortality in polymicrobial sepsis. Biochim Biophys Acta 1842:701-11
Wang, Xiaohong; Huang, Wei; Liu, Guansheng et al. (2014) Cardiomyocytes mediate anti-angiogenesis in type 2 diabetic rats through the exosomal transfer of miR-320 into endothelial cells. J Mol Cell Cardiol 74:139-50
Lin, Xi; Liu, Baohui; Yang, Xiangsheng et al. (2013) Genetic deletion of Rnd3 results in aqueductal stenosis leading to hydrocephalus through up-regulation of Notch signaling. Proc Natl Acad Sci U S A 110:8236-41
Li, Qi; Guo, Junli; Lin, Xi et al. (2013) An intragenic SRF-dependent regulatory motif directs cardiac-specific microRNA-1-1/133a-2 expression. PLoS One 8:e75470
Zhang, Xiaowei; Wang, Xiaohong; Zhu, Hongyan et al. (2012) Hsp20 functions as a novel cardiokine in promoting angiogenesis via activation of VEGFR2. PLoS One 7:e32765
Lin, Xi; Yang, Xiangsheng; Li, Qi et al. (2012) Protein tyrosine phosphatase-like A regulates myoblast proliferation and differentiation through MyoG and the cell cycling signaling pathway. Mol Cell Biol 32:297-308
Wong, Jerry; Zhang, Jingchun; Yanagawa, Bobby et al. (2012) Cleavage of serum response factor mediated by enteroviral protease 2A contributes to impaired cardiac function. Cell Res 22:360-71
Kim, Eun Young; Chen, Li; Ma, Yanlin et al. (2012) Enhanced desumoylation in murine hearts by overexpressed SENP2 leads to congenital heart defects and cardiac dysfunction. J Mol Cell Cardiol 52:638-49

Showing the most recent 10 out of 13 publications