Striated muscle contraction is dependent upon a cooperative interaction between thick and thin filament sarcomeric proteins. Tropomyosin (TM) plays an essential regulatory role in the sarcomere through its binding to actin and the troponin T (TnT) complex. Our long-term objective is to understand the importance of striated muscle TM isoforms and their post translational modifications in the myofilaments'response to mechanical and biochemical activity in normal and diseased cardiac muscle. This is important for biomedical research since mutations in TM cause hypertrophic and dilated cardiomyopathies that can lead to heart failure. Recent studies in our laboratory demonstrate that TnT binding domains in TM play a significant role in modifying sarcomeric performance. In this proposal, we will extend this work through an examination of the troponin binding domains of the b- and g-TM molecules, coupled with an examination of other regions within TM that modify thin filament function using both in vivo and in vitro approaches. To complement our TM structure-function analyses, we will also develop mouse models to determine the functional importance of TM phosphorylation in the heart.
The Specific Aims of this project are: c. 1. To ascertain the in vivo functional significance of TM isoform-specific amino acids in sarcomeric performance. We hypothesize that substitution of native a-TM for b-TM amino acids will decrease rates of contraction and relaxation, whereas g-TM substitution will increase these rates of cardiac performance. 2. To assess the in vitro functional significance of TM isoform specific amino acids. We will use in vitro biochemical studies to determine how a-, b-, and g-TM isoform-specific amino acid sequences affect binding to actin + troponin, influence actomyosin MgATPase activity, and affect the myosin S1-induced binding of TM to actin. 3. To determine the functional significance of TM phosphorylation. The focus of this aim is to test the hypothesis that genetically-designed mutations in TM that mimic phosphorylation or inhibit this process affect cardiac development and sarcomeric function. These experiments will be conducted using transgenic mice and thus provide unique in vivo models for addressing this significant research area.
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