- Email: [email protected]
Designing a High Affinity Cardiac Troponin Activator
Tuesday, March 1, 2016 Ca2þ-sensitivity, but unable to relax adequately resulting in diastolic dysfunction, followed by HCM development. 2289-Pos Board B433 HCM Associated Cardiac Troponin I Mutations Alter Cardiac Troponin Function, Contractile Properties and Modulation by PKA Mediated Phosphorylation Yuanhua Cheng1, Lindert Steffen2, An-Yue Tu1, Maria V. Razumova1, Luping Xie1, Lucas Oxenford1, Andrew D. McCulloch3, J Andrew McCammon3, Michael Regnier1. 1 Department of Bioengineering, University of Washington, Seattle, WA, USA, 2The Ohio State University, Columbus, OH, USA, 3University of California San Diego, San Diego, CA, USA. We recently reported on the altered structure-function relationship for two HCM-associated cardiac troponin I (cTnI) mutations, R146G and R21C (located in the inhibitory-peptide and cardiac-specific N-terminus of cTnI, respectively), and how this resulted in the cardiac dysfunction. Both mutations significantly increased Ca2þ binding affinity to cTn (KCa) and the affinity of cTnC for cTnI (KC-I). In isolated myofibrils, both mutations increased Ca2þ sensitivity of tension (pCa50) while maximal tension (TMAX) was maintained. PKA phosphorylation of cTnI resulted in decreased pCa50 for cTnIWT exchanged myofibrils, but not for either mutation. PKA phosphorylation accelerated the early, slow-phase relaxation for cTnIWT myofibrils, especially at Ca2þ-levels that the heart operates in-vivo. Importantly, this effect was blunted for cTnIR146G and cTnIR21C exchanged myofibrils, as was PKA-mediated reduction in KC-I. Molecular-dynamics simulations of cardiac troponin (cTn) suggested both mutations inhibit formation of intra-subunit contacts between the N-terminus and the inhibitory-peptide of cTnI that is normally seen with WT-cTn upon PKA phosphorylation, suggesting this may be the mechanism of disrupting modulation of contractile properties for these mutants. In ongoing studies, we are examining two additional HCM-associated mutations, cTnIP83S or cTnIA158V, located in the I-T arm and switch-peptide of cTnI, respectively. Both mutations also significantly increased KCa and KC-I, and blunt the PKA effects on KC-I, similar to cTnIR146G and cTnIR21C. Preliminary kinetics/mechanics results indicated that TMAX was also maintained for cTnIP83S and cTnIA158V exchanged myofibrils, but pCa50 was significantly increased only for cTnIA158V myofibrils. Additionally, preliminary measurements suggest these mutants also blunt the ability of PKA phosphorylation to decrease pCa50 and accelerate the early, slow-phase relaxation. We are currently performing molecular-dynamics simulations of cTn to understand the structural basis of these effects for both mutations. 2290-Pos Board B434 The Role of Calcium Affinity and C-I Interaction in Length-Dependent Activation Jordan M. Klaiman1, Maria V. Razumova1, Joseph D. Powers1, Cameron W. Turtle1, Farid Moussavi-Harami2, Todd E. Gillis3, Michael Reniger1. 1 Bioengineering, University of Washington, Seattle, WA, USA, 2Internal Medicine, University of Washington, Seattle, WA, USA, 3Integrative Biology, University of Guelph, Guelph, ON, Canada. The Frank-Starling response is an intrinsic property of cardiac muscle that helps to modulate cardiac output on a beat-to-beat basis. At the cellular level an increase in pressure during ventricle loading stretches the myocytes in the ventricle increasing sarcomere length (SL) and results in increased Ca2þ sensitivity and force of contraction. This mechanism is known as length-dependent activation (LDA), and multiple components of the sarcomere are believed to be involved in modulating this response. This includes changes in myofilament lattice spacing and/or myosin crossbridge orientation, affecting the probability of myosin binding to actin at longer SLs. Previous work from our lab, using the L48Q-cTnC variant, demonstrated that the inherent properties of cardiac troponin C (cTnC) maybe important in determining LDA. LDA is measured as a difference between pCa50 in force-pCa relationship at SL=2.3 and 2.0 in demembranated rat trabecula after a passive exchange of troponin complexes containing cTnC variants. Incorporation of L48Q-cTnC variant (with higher Ca2þbinding affinity and increased strength of cTnC-cTnI interaction, denoted KCa and KC-I, respectively) into rat cardiac trabeculae diminished LDA, DpCa50= ~0.03 compared to ~0.1 for WT-cTn. Alternatively, L57Q-cTnC (which decreases KCa and KC-I) increases LDA, DpCa50= ~0.2. The aim of the current study is to isolate changes in Ca2þ binding affinity of cTnC vs. C-I interaction on LDA using a series of cTnC variants that affect KCa and/ or KC-I, as determined using fluorescently labeled cTnC. Our initial findings suggest that salmonid cTnC (ScTnC) with higher KCa, increases LDA (DpCa50= ~0.2), in contrast to effects of L48Q-cTnC. In ongoing experiments
465a
we are examining the strength of ScTnC-cTnI interaction and investigating the changes in LDA caused by L48A-cTnC, which does not alter KCa but decreases KC-I. 2291-Pos Board B435 Sarcomere Length Dependent Effects on Ca2D-Induced Troponin Regulation within Chemically Skinned Cardiac Muscle Fibers King-Lun Li1, R. John Solaro2, Wenji Dong1. 1 School of Chemical Engineering and Bioengineering, Washington State University, Pullman, WA, USA, 2The Department of Physiology and Biophysics, Center for Cardiovascular Research, College of Medicine, University of Illinois at Chicago, Chicago, IL, USA. Sarcomere length dependent activation (LDA) of myocardial force development is the cellular basis underlying the Frank-Starling law of the heart, but it is still elusive how sarcomere detects the SL changes and converts it into altered activation of thin filament. Among the myofilament proteins, troponin and myosin are two key components that are likely involved in the LDA. The two components are functionally linked through Ca2þ activation and cross-bridge feedback. Although the active cross-bridge feedback has been strongly implicated in LDA, there was no evidence linking sarcomere length changes and troponin regulation until our recent study showing that Ca2þcTnC interaction in cardiac muscle can be modulated by sarcomere length through cross-bridge feedback [Biophysical Journal, 107(3), 682-93 (2014)]. In this study, we continue our efforts to understand the role of the C-domain of cTnI in LDA. Specifically, we used in situ time-resolved FRET measurements to determine how the switch region of cTnI is affected by Ca2þ, sarcomere length, and cross-bridge in skinned cardiac muscle fibers. To monitor the Ca2þ-induced structural transition of the switch region, skinned myocardial fibers were reconstituted with troponin complex containing FRET donor (AEDANS) modified cTnI(167C)AEDANS and acceptor (DDPM) modified cTnC(89C)DDPM. The measured FRET distance changes show that Ca2þ, strong cross-bridges and sarcomere length all influence the structural transition of the switching region of cTnI within myocardial fibers. The results provide a mechanism by which sarcomere length can modulate Ca2þ-troponin regulation via strong cross-bridge binding and suggest that the sarcomere length dependent cross-bridge effect plays an important role in the Frank-Starling law of the heart. 2292-Pos Board B436 Designing a High Affinity Cardiac Troponin Activator Fangze Cai1, Monica Li2, Sandra Pineda-Sanabria1, Shorena Gelozia3, Steffen Lindert4, J. Andrew McCammon5, Frederick West3, Brian Sykes1, Peter Hwang2. 1 Biochemistry, University of Alberta, Edmonton, AB, Canada, 2Medicine, University of Alberta, Edmonton, AB, Canada, 3Chemistry, University of Alberta, Edmonton, AB, Canada, 4Chemistry and Biochemistry, Ohio State University, Columbus, OH, USA, 5Chemistry and Biochemistry, University of California San Diego, San Diego, CA, USA. Heart failure is the end stage of almost all heart disease, characterized by an inability of the heart to pump enough blood to satisfy the needs of the body. Systolic heart failure is caused by impaired cardiac muscle contraction. In theory, an ideal drug for systolic heart failure would be a positive inotrope, which increases the contractility of heart muscle. However, no positive inotrope has been shown to improve mortality in patients with heart failure. We propose to develop a cardiac troponin activator, which would increase the contractile response of cardiac muscle without increasing the calcium currents entering and exiting the cell. All troponin-modulating drugs studied to-date bind to the interface between the regulatory N-terminal domain of troponin (NTnC) and the troponin I (TnI) switch region, so we developed a cardiac cNTnC-cTnI switch peptide chimera (cChimera) to use for drug screening and structural studies by NMR. We used bepridil as a starting point, because it was a calcium sensitizer with a high affinity for cNTnC-cTnI switch region (KD ~80 mM). We determined that the two aromatic rings of bepridil accounted for most of its binding affinity, and diphenylamine (DPA) appeared to be an excellent starting compound for further development. We obtained atomic resolution structures of cChimera using solution NMR, both alone and bound to the compound, 3-methyl diphenylamine (3-mDPA). The unsubstituted aromatic ring of 3-mDPA binds to an inner hydrophobic pocket adjacent to the central beta sheet of cNTnC. The methyl-substituted ring binds at the hydrophobic cNTnC-cTnI interface, causing minimal perturbation of cTnI binding. Our work shows that preserving the native interaction between cNTnC-cTnI is key to the development of a high affinity troponin activator.
465a
we are examining the strength of ScTnC-cTnI interaction and investigating the changes in LDA caused by L48A-cTnC, which does not alter KCa but decreases KC-I. 2291-Pos Board B435 Sarcomere Length Dependent Effects on Ca2D-Induced Troponin Regulation within Chemically Skinned Cardiac Muscle Fibers King-Lun Li1, R. John Solaro2, Wenji Dong1. 1 School of Chemical Engineering and Bioengineering, Washington State University, Pullman, WA, USA, 2The Department of Physiology and Biophysics, Center for Cardiovascular Research, College of Medicine, University of Illinois at Chicago, Chicago, IL, USA. Sarcomere length dependent activation (LDA) of myocardial force development is the cellular basis underlying the Frank-Starling law of the heart, but it is still elusive how sarcomere detects the SL changes and converts it into altered activation of thin filament. Among the myofilament proteins, troponin and myosin are two key components that are likely involved in the LDA. The two components are functionally linked through Ca2þ activation and cross-bridge feedback. Although the active cross-bridge feedback has been strongly implicated in LDA, there was no evidence linking sarcomere length changes and troponin regulation until our recent study showing that Ca2þcTnC interaction in cardiac muscle can be modulated by sarcomere length through cross-bridge feedback [Biophysical Journal, 107(3), 682-93 (2014)]. In this study, we continue our efforts to understand the role of the C-domain of cTnI in LDA. Specifically, we used in situ time-resolved FRET measurements to determine how the switch region of cTnI is affected by Ca2þ, sarcomere length, and cross-bridge in skinned cardiac muscle fibers. To monitor the Ca2þ-induced structural transition of the switch region, skinned myocardial fibers were reconstituted with troponin complex containing FRET donor (AEDANS) modified cTnI(167C)AEDANS and acceptor (DDPM) modified cTnC(89C)DDPM. The measured FRET distance changes show that Ca2þ, strong cross-bridges and sarcomere length all influence the structural transition of the switching region of cTnI within myocardial fibers. The results provide a mechanism by which sarcomere length can modulate Ca2þ-troponin regulation via strong cross-bridge binding and suggest that the sarcomere length dependent cross-bridge effect plays an important role in the Frank-Starling law of the heart. 2292-Pos Board B436 Designing a High Affinity Cardiac Troponin Activator Fangze Cai1, Monica Li2, Sandra Pineda-Sanabria1, Shorena Gelozia3, Steffen Lindert4, J. Andrew McCammon5, Frederick West3, Brian Sykes1, Peter Hwang2. 1 Biochemistry, University of Alberta, Edmonton, AB, Canada, 2Medicine, University of Alberta, Edmonton, AB, Canada, 3Chemistry, University of Alberta, Edmonton, AB, Canada, 4Chemistry and Biochemistry, Ohio State University, Columbus, OH, USA, 5Chemistry and Biochemistry, University of California San Diego, San Diego, CA, USA. Heart failure is the end stage of almost all heart disease, characterized by an inability of the heart to pump enough blood to satisfy the needs of the body. Systolic heart failure is caused by impaired cardiac muscle contraction. In theory, an ideal drug for systolic heart failure would be a positive inotrope, which increases the contractility of heart muscle. However, no positive inotrope has been shown to improve mortality in patients with heart failure. We propose to develop a cardiac troponin activator, which would increase the contractile response of cardiac muscle without increasing the calcium currents entering and exiting the cell. All troponin-modulating drugs studied to-date bind to the interface between the regulatory N-terminal domain of troponin (NTnC) and the troponin I (TnI) switch region, so we developed a cardiac cNTnC-cTnI switch peptide chimera (cChimera) to use for drug screening and structural studies by NMR. We used bepridil as a starting point, because it was a calcium sensitizer with a high affinity for cNTnC-cTnI switch region (KD ~80 mM). We determined that the two aromatic rings of bepridil accounted for most of its binding affinity, and diphenylamine (DPA) appeared to be an excellent starting compound for further development. We obtained atomic resolution structures of cChimera using solution NMR, both alone and bound to the compound, 3-methyl diphenylamine (3-mDPA). The unsubstituted aromatic ring of 3-mDPA binds to an inner hydrophobic pocket adjacent to the central beta sheet of cNTnC. The methyl-substituted ring binds at the hydrophobic cNTnC-cTnI interface, causing minimal perturbation of cTnI binding. Our work shows that preserving the native interaction between cNTnC-cTnI is key to the development of a high affinity troponin activator.