The objective is to develop and characterize genetic mouse models of non-syndromic cardiac progeria (NSCP). The impetus for the studies is originated from the findings of NSCP in young patients with premature and progressive degenerative cardiovascular disease, characteristic of aging. Clinical and genetic studies have led us to advocate the doctrine that cardiac involvement in LMNA (Lamin A/C) mutations, referred to as cardiolaminopathy represents NSCP. We propose to develop and characterize mouse models expressing mutant LmnaD300N and LmnaR644C found in patients with progressive degenerative cardiovascular diseases. The p.D300N is associated with degenerative disease involving myocardium, conduction system, valves and the coronary arteries, while the p.R644C is the most common cause of cardiolaminopathy. We will use the recombineering (recombination-mediated genetic engineering) method to generate LmnaD300N, LmnaR644C and Lmna-wild type (WT), latter as a control, transgenic mice. Recombineering is a relatively novel and powerful method that enables efficient construction of the transgene vectors that are similar to the WT gene with the exception of the intended mutation/changes. The method utilizes homologous recombination in E. coli using recombination proteins provided from a lambda phage. The recombined transgene constructs are microinjected into fertilized single cell mouse embryos to produce genetic mouse models in which expression of the transgene is regulated by the regulatory sequences that govern expression of the endogenous gene. The models will genetically recapitulate the human genotype of Lmna mutations and also will enable analyzing involvement of multiple cardiac and non-cardiac cell types, including myocytes, fibroblasts and conduction system. The phenotype of aging is defined at the molecular level by induced expression of established markers of senescence, namely senescence-associated 2-galactosidase (S-A 2-Gal), p16INK4a (Cdkn2a), P19, p21WAF1/CIP1, telomere attrition and telomerase activity. At the cellular level we will assess trophic, replicative and reparative responses to agonists and at the organ level myocardial dysfunction and conduction defects. We will assess survival by Kaplan-Meier survival analysis;cardiac structure and function by echocardiography;and conduction defects and arrhythmias by electrocardiography. Likewise, we will analyze trophic (myocyte), proliferative and reparative (fibroblasts) responses of cells isolated from the hearts to angiotensin II (AT) and transforming growth factor 2 (TGF2). Moreover, we will quantify telomerase activity, telomere attrition and levels of p16INK4a, p21WAF1/CIP1, SA-2-Gal and markers of oxidative stress in the heart cardiac. The studies are expected to establish the proposed models as mouse models of NSCP and provide the opportunity to elucidate the fundamental mechanisms that govern cardiac senescence. The discoveries could facilitate prevention, attenuation and cure of cardiolaminopathy in humans.

Public Health Relevance

PROJECT NARRATIVE Clinical and genetic studies in human patients with premature degenerative cardiovascular diseases, characteristics of aging, have led us to advocate the doctrine that LMNA (Lamin A/C) mutations cause non- syndromic cardiac progeria. To test this hypothesis we propose to develop and characterize recombineered mouse models of specific LMNA mutations that are associated with the non-syndromic cardiac progeria. The models could afford the opportunity to delineate the molecular and cellular mechanisms of cardiolaminopathy and shed light into the mechanisms of cardiovascular senescence.

Agency
National Institute of Health (NIH)
Institute
National Institute on Aging (NIA)
Type
Exploratory/Developmental Grants (R21)
Project #
5R21AG038597-02
Application #
8249396
Study Section
Myocardial Ischemia and Metabolism Study Section (MIM)
Program Officer
Kohanski, Ronald A
Project Start
2011-04-01
Project End
2013-03-31
Budget Start
2012-04-01
Budget End
2013-03-31
Support Year
2
Fiscal Year
2012
Total Cost
$153,750
Indirect Cost
$51,250
Name
University of Texas Health Science Center Houston
Department
Type
Schools of Medicine
DUNS #
800771594
City
Houston
State
TX
Country
United States
Zip Code
77225
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