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.
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.
|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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