Murali Chandra - US grants

DESCRIPTION (provided by applicant): Despite the facts that troponin T (TnT) is the only subunit of the troponin (Tn) complex that is clearly known to interact strongly with Tropomyosin (Tm) and that it has close proximity to half of the actin monomers in the functional unit and is closely associated with troponin I (TnI), how this pivotally positioned protein modulates cardiac function is not understood. The amino terminus (N) the carboxyl terminus (C) of rat fast skeletal TnT (fsTnT) are important for modulating kinetic rates of transition between 'on' and 'off' states of thin filaments, and for Ca 2+ regulation of Tm movement on the actin filament, respectively. The corresponding regions in rat cardiac TnT (cTnT) differ considerably, which indicates that Ca2+ regulation of thin filament activation in cardiac muscle is different. Our hypothesis is that the unique structural features of cTnT underlie the molecular mechanism(s) by which the Ca2+ activation is so exquisitely modulated in cardiac muscle. Specific Aims 1 and 4 will address the questions of how the N- and C- domains of cTnT control the dynamics of transitions from non-force bearing to force-beating myosin cross-bridge states in cardiac muscle, and how the special features of length-dependent activation of cardiac muscle are due to unique structural features of cTnT. Specific aims 2 and 3 will focus on how the cooperative activation of cardiac muscle differs from that in fast skeletal muscle because of unique structural features in both the N and C termini of cTnT. This proposal exploits differences in rat cTnT and rat fsTnT in order to provide novel insights about the mechanisms underlying the unique aspects Ca 2+ and length-dependent regulation of cardiac muscle. Furthermore, FHC-related cTnT mutants will be used to understand how modifications to the N and C termini of cTnT contribute to cardiac disease. Methods include measurement of Ca 2+ dependent force, ATPase activity, rate of force redevelopment (ktr), and myofiber dynamic stiffness in rat cardiac fiber bundles reconstituted with recombinant cTnT-fsTnT chimeras. Interactions between Tn subunits and Tm will be measured by using steady-state fluorescence assays. We will use mathematical model ofmyofilament mechano-dynamics to understand how changes in myofilament regulation by cTnT are expressed as changes in global myofilament mechano-dynamics. We will use a novel method for measuring Ca 2+ binding kinetics in a fully regulated thin filament system. This proposal aims to provide new and diverse insight into the molecular mechanism by which myofilament response to Ca 2+ is so exquisitely modulated in cardiac muscle.

DESCRIPTION (provided by applicant): The lack of knowledge about the interactions among cardiac contractile regulatory proteins represents an important problem because it not only limits our understanding of how such interactions affect cardiac thin- filament activation, but it also precludes an understanding of mechanisms underlying many forms of heart disease. Cardiac muscle contraction depends on coordinated interactions among contractile regulatory proteins, which include cardiac troponin C (cTnC), troponin T (cTnT), troponin I (cTnI), and tropomyosin (Tm). Coordinated interactions among these proteins play key roles during contraction via Ca2+-, strong crossbridge (XB)-, and sarcomere length (SL)-mediated activation of thin filaments. However, very little is known about how cTnT influences the actions of Tm, cTnI, and cTnC to modulate Ca2+-, SL-, and XB- activation of cardiac thin filaments. Our long-term goal, therefore, is to determine how structural differences in contractile regulatory proteins determine Ca2+-, SL-, and strong XB-mediated activation of cardiac muscle contraction, and how they are altered in heart disease. The overall objective of this proposal is to determine how cTnT interacts with Tm, cTnI, and cTnC to modulate cardiac thin-filament activation by Ca2+, SL, and strong XB. Our hypothesis is that the structural features of the tail (cT1) and the head (cT2) domains of cTnT are the key determinants of functional features of cardiac thin-filament activation. To test our hypo- thesis, we will measure force/ATPase, rate of tension redevelopment and myofiber dynamic stiffness in re- constituted cardiac muscle fibers. Further, complementary studies such as Ca2+ binding kinetics measurements and quantitative mathematical modeling will be performed. Specific Aim 1 will determine how cT1 modulates the dynamics of strong XB recruitment during cardiac thin-filament activation. This aim will be accomplished by determining: the specific region of cT1 that affects thin-filament activation;how changes in the overlapping ends of contiguous Tm impact cT1 effects on cardiac thin-filament activation;and how cT1 effects on thin-filament activation are modified by myosin isoforms. Specific Aim 2 will determine how cTnT modulates the cooperative feedback of strong XB on conformational changes in cTnC during cardiac thin- filament activation. In this aim, we will determine how key regions of cT2 modulate Ca2+- and XB-induced changes in cTnC structure. Specific Aim 3 will determine how interactions between cTnT and cTnI mediate the feedback effect of strong XB on cardiac thin-filament activation. This aim will be accomplished by determining: how cT2-cTnI interactions modulate cardiac thin-filament activation and how differences in myosin isoforms alter cT2-cTnI effect on thin-filament activation. The expected outcome from our comprehensive and multidisciplinary approach will have a positive impact because it will significantly advance our understanding of the molecular mechanisms underlying cardiac thin-filament activation. Our study will lay a strong foundation for developing novel pharmacological strategies aimed at improving cardiac function in diseased hearts. PUBLIC HEALTH RELEVANCE The proposed research is relevant to public health because the goal of this proposal is to determine how structural differences in thin-filament contractile regulatory proteins determine Ca2+-, strong crossbridge-, and sarcomere length-dependent activation of cardiac muscle contraction, and how such interactions are altered in heart disease. The proposed research is an important area of cardiac muscle physiology because of its potential to contribute significantly to deeper understanding of the molecular mechanisms underlying many forms of human heart disease.

Muscle cells express a number of small stress responsive proteins called small heat shock proteins that are known to be critical to cell survival, growth, and repair. However, how these proteins accomplish their tasks is not understood. Proposed studies will examine the regulation and function of these proteins in muscle cells responding to low oxygen conditions that occur during injury, exercise and hibernation. The work will be conducted using zebrafish larvae as a model system because they can be used to study the organization and function of fluorescently labeled proteins in living tissues. Data resulting from these studies are expected to provide novel insight into the mechanisms underlying how muscle cells respond to stress. Such insights have implications for understanding or modifying the limits of muscle performance, engineering, adaptation and repair in a wide variety of organisms. In addition to the new information generated, the project will also benefit society by promoting teaching, training and learning, and by broadening the participation of underrepresented groups in science. Outreach educational workshops with area high schools will be developed and strengthened through conducting hands-on research projects with zebrafish larvae. Student conceptual understanding will be assessed through the use of pre-and post-workshop evaluations. Opportunities will also be created for high school students to visit and work in laboratories of active investigators at Washington State University and the neighboring University of Idaho. A summer internship will be provided annually to area high school science teachers who will learn methods for zebrafish husbandry and analysis of zebrafish embryonic development applicable to a K-12 classroom environment.

The overall goal of the studies is to test the hypothesis that the heat shock inducible transcription factor, HSF1, regulates expression of small heat shock proteins, which dynamically associate with cytoskeletal filaments in a phosphorylation-dependent manner in hypoxic muscle cells to preserve cytoskeletal integrity and muscle function. The proposal includes three specific aims. First the role of HSF1 in regulating muscle specific small heat shock protein expression in response to hypoxia will be assessed. To accomplish this, the function of transcription factor HSF1 will be manipulated and effects on hypoxia inducible small heat shock protein expression analyzed. Mutagenesis of small heat shock protein promoters will also be conducted to identify motifs required for hypoxia-induced expression. Second, phosphorylation-dependent interaction kinetics of small heat shock proteins with the cytoskeleton will be analyzed in hypoxic muscle cells. Mutagenesis of serine/threonine residues will be conducted and effects on hypoxia inducible phosphorylation established. Dynamic interactions of wild-type and phosphorylation site mutant heat shock proteins with the cytoskeleton and each other will be examined in control and hypoxic muscles. Finally, small heat shock protein expression will be altered in muscles, and cytoarchitecture and function of muscle cells responding to hypoxia will be assessed. To accomplish this aim, zebrafish lines that over-, and under-express small heat shock proteins in skeletal muscle will be created. Muscle cell architecture and function will be assayed in larvae and adults under control conditions and after exposure to low oxygen levels.