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.
