Cardiac contraction and relaxation kinetics are modulated continuously during life and can be altered in disease. It is known that both external and intracellular factors impact the contractile kinetics of the heart. We believe an under-appreciated element that can influence these kinetics is the Ca2+ exchange rate of troponin C (TnC). The goal of this proposal is to delineate the role of TnC in cardiac muscle contraction and relaxation kinetics. It is generally assumed that TnC equilibrates rapidly with Ca2+ and thus has no influence on the kinetics of cardiac muscle contraction or relaxation. However, our published and preliminary data demonstrate that 1) TnC Ca2+ binding properties influence the rate of cardiac muscle contraction, 2) Ca2+ dissociation from TnC is not rapid in more physiologically relevant biochemical systems (reconstituted thin filaments and myofibrils) and is similar to the rate of cardiac muscle relaxation, 3) physiological and pathophysiological modifications of TnI and TnT associated with accelerated or slowed cardiac muscle relaxation likewise accelerate or slow the rates of Ca2+ dissociation from TnC, and 4) the aberrant Ca2+ binding properties of the disease associated proteins can be corrected in biochemical and physiological systems by specifically engineered TnCs.
Specific Aim I will test the hypothesis that the rates of Ca2+ exchange with TnC are altered by TnI and TnT modifications that are associated with altered cardiac muscle contraction and relaxation kinetics. Ca2+ exchange rates will be monitored in physiologically relevant biochemical systems of reconstituted thin filaments and in rat ventricular myofibrils utilizing a stopped-flow apparatus and our novel fluorescent TnC. We will utilize TnI and TnT constructs that: 1) mimic PKA and PKC phosphorylation, 2) are associated with ischemia-reperfusion injury and 3) are associated with familial hypertrophic, restrictive and dilated cardiomyopathies (HCM, RCM and DCM, respectively).
Specific Aim II will elucidate and modulate the molecular mechanism(s) that influence the rates of Ca2+ exchange with TnC. We hypothesize that there are two fundamental mechanisms that influence the rates of Ca2+ exchange with TnC: 1) the intrinsic Ca2+ binding properties of TnC and 2) the ability of the regulatory domain of TnC to bind TnI. We have developed a novel competition assay and a fluorescence resonance energy transfer (FRET) system that can probe the critical TnC-TnI interactions.
Specific Aim III will test the hypothesis that the rates of Ca2+ exchange with TnC influences the rates of cardiac muscle contraction and relaxation. Relaxation will be induced by photolysis of the caged Ca2+ chelator diazo-2, whereas the kinetics of contraction will be assessed by measuring the rate of force redevelopment. Furthermore, we will test if specifically engineered TnC constructs can correct the aberrant contractile kinetics of the disease associated TnI and TnT modifications. The data obtained will elucidate the role of TnC in cardiac muscle contraction and relaxation kinetics, which will translate to novel hypothesis driven treatment strategies to combat systolic and diastolic dysfunction.

Public Health Relevance

The significance of this research is multifold and will: 1) provide insight into the mechanisms that control the Ca2+ binding and exchange kinetics of the cardiac thin filament, 2) provide a clearer understanding of how these mechanisms are altered in physiological and pathophysiological states, 3) determine if rationally engineered TnC constructs can correct the aberrant biochemical and physiological Ca2+ binding and exchange kinetics of the thin filament, 4) directly test whether the Ca2+ binding and exchange kinetics of the thin filament modulate the rates of cardiac muscle contraction and relaxation and 5) facilitate the design of therapeutic agents targeted against cardiac systolic and diastolic dysfunctions.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Project (R01)
Project #
1R01HL091986-01A2
Application #
7984700
Study Section
Cardiac Contractility, Hypertrophy, and Failure Study Section (CCHF)
Program Officer
Adhikari, Bishow B
Project Start
2010-08-01
Project End
2015-04-30
Budget Start
2010-08-01
Budget End
2011-04-30
Support Year
1
Fiscal Year
2010
Total Cost
$381,250
Indirect Cost
Name
Ohio State University
Department
Physiology
Type
Schools of Medicine
DUNS #
832127323
City
Columbus
State
OH
Country
United States
Zip Code
43210
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Zhang, Bo; Davis, Jonathan P; Ziolo, Mark T (2015) Cardiac Catheterization in Mice to Measure the Pressure Volume Relationship: Investigating the Bowditch Effect. J Vis Exp :e52618
Salhi, Hussam E; Walton, Shane D; Hassel, Nathan C et al. (2014) Cardiac troponin I tyrosine 26 phosphorylation decreases myofilament Ca2+ sensitivity and accelerates deactivation. J Mol Cell Cardiol 76:257-64
Nixon, Benjamin R; Walton, Shane D; Zhang, Bo et al. (2014) Combined troponin I Ser-150 and Ser-23/24 phosphorylation sustains thin filament Ca(2+) sensitivity and accelerates deactivation in an acidic environment. J Mol Cell Cardiol 72:177-85
Biesiadecki, Brandon J; Davis, Jonathan P; Ziolo, Mark T et al. (2014) Tri-modal regulation of cardiac muscle relaxation; intracellular calcium decline, thin filament deactivation, and cross-bridge cycling kinetics. Biophys Rev 6:273-289
Liu, Bin; Lopez, Joseph J; Biesiadecki, Brandon J et al. (2014) Protein kinase C phosphomimetics alter thin filament Ca2+ binding properties. PLoS One 9:e86279
Li, Alison Y; Stevens, Charles M; Liang, Bo et al. (2013) Familial hypertrophic cardiomyopathy related cardiac troponin C L29Q mutation alters length-dependent activation and functional effects of phosphomimetic troponin I*. PLoS One 8:e79363
Nixon, Benjamin R; Liu, Bin; Scellini, Beatrice et al. (2013) Tropomyosin Ser-283 pseudo-phosphorylation slows myofibril relaxation. Arch Biochem Biophys 535:30-8
Torres, Carlos A A; Varian, Kenneth D; Canan, Cynthia H et al. (2013) The positive inotropic effect of pyruvate involves an increase in myofilament calcium sensitivity. PLoS One 8:e63608

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