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.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Project (R01)
Project #
5R01HL075643-03
Application #
7095881
Study Section
Cardiac Contractility, Hypertrophy, and Failure Study Section (CCHF)
Program Officer
Varghese, Jamie
Project Start
2004-09-01
Project End
2008-08-31
Budget Start
2006-09-01
Budget End
2007-08-31
Support Year
3
Fiscal Year
2006
Total Cost
$286,700
Indirect Cost
Name
Washington State University
Department
Veterinary Sciences
Type
Schools of Veterinary Medicine
DUNS #
041485301
City
Pullman
State
WA
Country
United States
Zip Code
99164
Reda, Sherif M; Chandra, Murali (2018) Cardiomyopathy mutation (F88L) in troponin T abolishes length dependency of myofilament Ca2+ sensitivity. J Gen Physiol 150:809-819
Michael, John Jeshurun; Chandra, Murali (2016) Interplay Between the Effects of Dilated Cardiomyopathy Mutation (R206L) and the Protein Kinase C Phosphomimic (T204E) of Rat Cardiac Troponin T Are Differently Modulated by ?- and ?-Myosin Heavy Chain Isoforms. J Am Heart Assoc 5:e002777
Kawai, Masataka; Karam, Tarek S; Michael, John Jeshurun et al. (2016) Comparison of elementary steps of the cross-bridge cycle in rat papillary muscle fibers expressing ?- and ?-myosin heavy chain with sinusoidal analysis. J Muscle Res Cell Motil 37:203-214
Gollapudi, Sampath K; Chandra, Murali (2016) The effect of cardiomyopathy mutation (R97L) in mouse cardiac troponin T on the muscle length-mediated recruitment of crossbridges is modified divergently by ?- and ?-myosin heavy chain. Arch Biochem Biophys 601:105-12
Michael, John Jeshurun; Gollapudi, Sampath K; Chandra, Murali (2016) Interplay between the effects of a Protein Kinase C phosphomimic (T204E) and a dilated cardiomyopathy mutation (K211? or R206W) in rat cardiac troponin T blunts the magnitude of muscle length-mediated crossbridge recruitment against the ?-myosin heavy cha J Muscle Res Cell Motil 37:83-93
Gollapudi, Sampath K; Tardiff, Jil C; Chandra, Murali (2015) The functional effect of dilated cardiomyopathy mutation (R144W) in mouse cardiac troponin T is differently affected by ?- and ?-myosin heavy chain isoforms. Am J Physiol Heart Circ Physiol 308:H884-93
Chandra, Vikram; Gollapudi, Sampath K; Chandra, Murali (2015) Rat cardiac troponin T mutation (F72L)-mediated impact on thin filament cooperativity is divergently modulated by ?- and ?-myosin heavy chain isoforms. Am J Physiol Heart Circ Physiol 309:H1260-70
Michael, John Jeshurun; Gollapudi, Sampath K; Chandra, Murali (2014) Effects of pseudo-phosphorylated rat cardiac troponin T are differently modulated by ?- and ?-myosin heavy chain isoforms. Basic Res Cardiol 109:442
Mamidi, Ranganath; Mallampalli, Sri Lakshmi; Wieczorek, David F et al. (2013) Identification of two new regions in the N-terminus of cardiac troponin T that have divergent effects on cardiac contractile function. J Physiol 591:1217-34
Ford, Steven J; Chandra, Murali (2013) Length-dependent effects on cardiac contractile dynamics are different in cardiac muscle containing ýý- or ýý-myosin heavy chain. Arch Biochem Biophys 535:3-13

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