Regulation of contractile activity in cardiac muscle is dependent upon a cooperative interaction between thick and thin filament sarcomeric proteins. Tropomyosin (TM), an essential thin filament protein, interacts with troponin (Tn) and regulates muscle contraction in a Ca2+- dependent manner. Although several in vitro studies have defined the role of specific TM regions, this may not accurately reflect the biological functions that occur in the in vivo situation. The long-term objective of this proposal is to comprehend the relative importance of TM in the cooperative interaction process that is essential for cardiac muscle dynamics. Using transgenic mice and mutagenesis of myofilament proteins as tools, we will test the central hypothesis that perturbation of stoichiometry of sarcomeric TM modifies cardiac muscle contractility which involves both allosteric control by Ca2+ and cooperative interactions among all of myofilament proteins. Using our beta-TM transgenic mouse model, we have recently demonstrated that altering the ratio of alpha- and beta-TM leads to physiological changes in myocardial relaxation and Ca2+ handling of myofilaments. Our basic experimental approach will be to overexpress mutant alpha-TM in the murine heart, and to determine whether these alterations in alpha-TM affect the interactions to actin and Tn complex, thereby changing thin filament activation.
The specific aims address the following questions: (1) what specific amino acids determine differential physiological activity between alpha- and beta-TM?; (2) what is the mechanism by which TM protein modulates thin filament activation?; and (3) what is the mechanism that causes abnormal cardiovascular function in transgenic mice that have a genetically modified sarcomere? This proposal defines the effect of overexpression of genetically modified striated muscle alpha-TM on cardiac muscle function at molecular, physiological, morphological and biochemical levels.
The specific aims of this proposal are: (1) delineate the molecular, physiological, and morphological effects which result from overexpression of mutant alpha-TM protein; and (2) determine the biochemical and cellular basis of functional consequences of the mutant alpha-TM protein. The cardiac alpha-myosin heavy chain (MHC) promoter will be used to express the mutant alpha-TM cDNAs in the murine heart. This study will provide biological information for understanding the role of TM in cardiac muscle contraction and in its interaction with other thin filament proteins. Cardiovascular studies using transgenic mouse model will provide explicit information on the heart function during both normal and diseased states.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
First Independent Research Support & Transition (FIRST) Awards (R29)
Project #
7R29HL060758-03
Application #
6184639
Study Section
Cardiovascular and Pulmonary Research A Study Section (CVA)
Project Start
1998-08-11
Project End
2003-07-31
Budget Start
2000-08-01
Budget End
2001-07-31
Support Year
3
Fiscal Year
2000
Total Cost
$143,387
Indirect Cost
Name
Texas A&M University
Department
Physiology
Type
Schools of Medicine
DUNS #
City
College Station
State
TX
Country
United States
Zip Code
77845
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Sarin, Vandana; Gaffin, Robert D; Meininger, Gerald A et al. (2005) Arginine-glycine-aspartic acid (RGD)-containing peptides inhibit the force production of mouse papillary muscle bundles via alpha 5 beta 1 integrin. J Physiol 564:603-17
Gaffin, Robert D; Tong, Carl W; Zawieja, David C et al. (2004) Charged residue alterations in the inner-core domain and carboxy-terminus of alpha-tropomyosin differentially affect mouse cardiac muscle contractility. J Physiol 561:777-91
Gaffin, Robert D; Gokulan, Kuppan; Sacchettini, James C et al. (2004) Charged residue changes in the carboxy-terminus of alpha-tropomyosin alter mouse cardiac muscle contractility. J Physiol 556:531-43
Tong, Carl W; Gaffin, Robert D; Zawieja, David C et al. (2004) Roles of phosphorylation of myosin binding protein-C and troponin I in mouse cardiac muscle twitch dynamics. J Physiol 558:927-41
Tong, C W; Kolomenskii, A; Lioubimov, V A et al. (2001) Measurements of the cross-bridge attachment/detachment process within intact sarcomeres by surface plasmon resonance. Biochemistry 40:13915-24