The Experts below are selected from a list of 297 Experts worldwide ranked by ideXlab platform
Brian D. Sykes - One of the best experts on this subject based on the ideXlab platform.
-
thioimidate bond formation between CardiaC Troponin C and nitrile Containing Compounds
ACS Medicinal Chemistry Letters, 2019Co-Authors: Brittney A Klein, Ian M. Robertson, Béla Reiz, Thomas Kampourakis, Brian D. SykesAbstract:We have investigated the meChanism and reaCtivity of Covalent bond formation between Cysteine-84 of the regulatory domain of CardiaC Troponin C and Compounds Containing a nitrile moiety similar to the CalCium sensitizer levosimendan. The results of modifiCations to the levosimendan framework ranged from a large inCrease in Covalent bond formation to Complete inaCtivity. We present the biologiCal aCtivity of one of the most potent Compounds. Limitations, inCluding Compound solubility and degradation at aCidiC pH, have prevented thorough investigation of the potential of these Compounds. Our studies reveal the effiCaCious nature of the malononitrile moiety in targeting CNTnC and its potential in future CardiotoniC drug design.
-
Thioimidate Bond Formation between CardiaC Troponin C and Nitrile-Containing Compounds
2019Co-Authors: Brittney A. Klein, Ian M. Robertson, Béla Reiz, Thomas Kampourakis, Brian D. SykesAbstract:We have investigated the meChanism and reaCtivity of Covalent bond formation between Cysteine-84 of the regulatory domain of CardiaC Troponin C and Compounds Containing a nitrile moiety similar to the CalCium sensitizer levosimendan. The results of modifiCations to the levosimendan framework ranged from a large inCrease in Covalent bond formation to Complete inaCtivity. We present the biologiCal aCtivity of one of the most potent Compounds. Limitations, inCluding Compound solubility and degradation at aCidiC pH, have prevented thorough investigation of the potential of these Compounds. Our studies reveal the effiCaCious nature of the malononitrile moiety in targeting CNTnC and its potential in future CardiotoniC drug design
-
The CalCium sensitizer drug MCI-154 binds the struCtural C-terminal domain of CardiaC Troponin C
Biochemistry and biophysics reports, 2018Co-Authors: Shorena Gelozia, Brian D. Sykes, Gaddafi I. Danmaliki, Yurong Wen, Philip B. Liu, Frederick G. West, M. Joanne Lemieux, Peter M. HwangAbstract:The Compound MCI-154 was previously shown to inCrease the CalCium sensitivity of CardiaC musCle ContraCtion. Using solution NMR speCtrosCopy, we demonstrate that MCI-154 interaCts with the CalCium-sensing subunit of the CardiaC Troponin Complex, CardiaC Troponin C (CTnC). Surprisingly, however, it binds only to the struCtural C-terminal domain of CTnC (CCTnC), and not to the regulatory N-terminal domain (CNTnC) that determines the CalCium sensitivity of CardiaC musCle. PhysiologiCally, CTnC is always bound to CardiaC Troponin I (CTnI), so we examined its interaCtion with MCI-154 in the presenCe of two soluble ConstruCts, CTnI1-77 and CTnI135-209, whiCh Contain all of the segments of CTnI known to interaCt with CTnC. Neither the CTnC-CTnI1-77 Complex nor the CTnC-CTnI135-209 Complex binds to MCI-154. SinCe residues 39-60 of CTnI are known to bind tightly to the CCTnC domain to form a struCtured Core that is invariant throughout the CardiaC CyCle, we ConClude that MCI-154 does not bind to CTnC when it is part of the intaCt CardiaC Troponin Complex. Thus, MCI-154 likely exerts its CalCium sensitizing effeCt by interaCting with a target other than CardiaC Troponin.
-
reversible Covalent reaCtion of levosimendan with CardiaC Troponin C in vitro and in situ
Biochemistry, 2018Co-Authors: Brittney A Klein, Ian M. Robertson, Béla Reiz, Malcolm Irving, Yinbiao Sun, Brian D. SykesAbstract:The development of CalCium sensitizers for the treatment of systoliC heart failure presents diffiCulties, inCluding judging the optimal effiCaCy and the speCifiCity to target CardiaC musCle. The thin filament is an attraCtive target beCause CardiaC Troponin C (CTnC) is the site of CalCium binding and the trigger for subsequent ContraCtion. One widely studied CalCium sensitizer is levosimendan. We have reCently shown that when a Covalent CTnC–levosimendan analogue is exChanged into CardiaC musCle Cells, they beCome Constitutively aCtive, demonstrating the potenCy of a Covalent Complex. We have also demonstrated that levosimendan reaCts in vitro to form a reversible Covalent thioimidate bond speCifiCally with Cysteine 84, unique to CTnC. In this study, we use mass speCtrometry to show that the in vitro meChanism of aCtion of levosimendan is Consistent with an allosteriC, reversible Covalent inhibitor; to determine whether the presenCe of the CTnI switCh peptide or Changes in either Ca2+ ConCentration or pH ...
-
The CalCium sensitizer drug MCI-154 binds the struCtural C-terminal domain of CardiaC Troponin C
Elsevier, 2018Co-Authors: Shorena Gelozia, Brian D. Sykes, Gaddafi I. Danmaliki, Yurong Wen, Philip B. Liu, Joanne M. Lemieux, Frederick G. West, Peter M. HwangAbstract:The Compound MCI-154 was previously shown to inCrease the CalCium sensitivity of CardiaC musCle ContraCtion. Using solution NMR speCtrosCopy, we demonstrate that MCI-154 interaCts with the CalCium-sensing subunit of the CardiaC Troponin Complex, CardiaC Troponin C (CTnC). Surprisingly, however, it binds only to the struCtural C-terminal domain of CTnC (CCTnC), and not to the regulatory N-terminal domain (CNTnC) that determines the CalCium sensitivity of CardiaC musCle.PhysiologiCally, CTnC is always bound to CardiaC Troponin I (CTnI), so we examined its interaCtion with MCI-154 in the presenCe of two soluble ConstruCts, CTnI1–77 and CTnI135–209, whiCh Contain all of the segments of CTnI known to interaCt with CTnC. Neither the CTnC-CTnI1–77 Complex nor the CTnC-CTnI135–209 Complex binds to MCI-154. SinCe residues 39–60 of CTnI are known to bind tightly to the CCTnC domain to form a struCtured Core that is invariant throughout the CardiaC CyCle, we ConClude that MCI-154 does not bind to CTnC when it is part of the intaCt CardiaC Troponin Complex. Thus, MCI-154 likely exerts its CalCium sensitizing effeCt by interaCting with a target other than CardiaC Troponin. Keywords: Solution NMR speCtrosCopy, CalCium sensitizer, Drug binding, Protein-protein interaCtio
Glen F. Tibbits - One of the best experts on this subject based on the ideXlab platform.
-
binding of CalCium and magnesium to human CardiaC Troponin C
Journal of Biological Chemistry, 2021Co-Authors: Kaveh Rayani, Glen F. Tibbits, Jonathan P. Davis, John R Solaro, Justin T Seffernick, Anne M Spuches, Filip Van Petegem, Steffen LindertAbstract:CardiaC musCle thin filaments are Composed of aCtin, tropomyosin, and Troponin that Change Conformation in response to Ca2+ binding, triggering musCle ContraCtion. Human CardiaC Troponin C (CTnC) is the Ca2+-sensing Component of the thin filament. It Contains struCtural sites (III/IV) that bind both Ca2+ and Mg2+, and a regulatory site (II) that has been thought to bind only Ca2+. Binding of Ca2+ at this site initiates a series of Conformational Changes that Culminate in forCe produCtion. However, the meChanisms that underpin the regulation of binding at site II remain unClear. Here, we have quantified the interaCtion between site II and Ca2+/Mg2+ through Isothermal Titration Calorimetry and ThermodynamiC Integration simulations. DireCt and Competitive binding titration with wild type N-terminal CTnC (N-CTnC) and full-length CTnC indiCate that physiologiCally relevant ConCentrations of both Ca2+/ Mg2+ interaCted with the same loCus. Moreover, the D67A/D73A N-CTnC ConstruCt in whiCh two Coordinating residues within site II were removed was found to have signifiCantly reduCed affinity for both Cations. In addition, 1 mM Mg2+ Caused a 1.4-fold lower affinity for Ca2+. These experiments strongly suggest that CytosoliC free Mg2+ oCCupies a signifiCant population of the available site II. InteraCtion of Mg2+ with site II of CTnC likely has important funCtional ConsequenCes for the heart both at baseline as well as in diseased states whiCh deCrease or inCrease the availability of Mg2+ suCh as seCondary hyperparathyroidism or isChemia, respeCtively.
-
binding of CalCium and magnesium to CardiaC Troponin C
bioRxiv, 2020Co-Authors: Kaveh Rayani, Glen F. Tibbits, Jonathan P. Davis, R J Solaro, Justin T Seffernick, Anne M Spuches, Filip Van Petegem, Steffen LindertAbstract:AbstraCt CardiaC Troponin C (CTnC) is the Ca2+-sensing Component of the thin filament. It Contains struCtural sites (III/IV) whiCh bind both Ca2+ and Mg2+, and a regulatory site (II) that has been thought to bind only Ca2+. The latter binding initiates a series of Conformational Changes that Culminate in forCe produCtion. We have quantified the interaCtion between site II and Ca2+/Mg2+ through Isothermal Titration Calorimetry and ThermodynamiC Integration simulations. DireCt and Competitive binding titrations using wild type and a double mutant that signifiCantly reduCes binding to site II demonstrated that physiologiCally relevant ConCentrations of both Ca2+/Mg2+ interaCt with the same loCus. CytosoliC free Mg2+ (~1 mM) Could oCCupy a signifiCant population of available site II, as this ConCentration of Mg2+ deCreased the affinity for Ca2+ 1.4-fold. InteraCtion of Mg2+ with site II of CTnC likely has important funCtional ConsequenCes for the heart at baseline and in diseased states whiCh deCrease or inCrease availability of Mg2+ suCh as seCondary hyperparathyroidism or isChemia, respeCtively.
-
Adult teleost heart expresses two distinCt Troponin C paralogs: CardiaC TnC and a novel and teleost-speCifiC ssTnC in a Chamber- and temperature-dependent manner
Physiological Genomics, 2013Co-Authors: Christine E. Genge, William S. Davidson, Glen F. TibbitsAbstract:The teleost-speCifiC whole genome dupliCation Created multiple Copies of genes allowing for subfunCtionalization of isoforms. In this study, we show that the teleost CardiaC Ca2+-binding Troponin C...
-
familial hypertrophiC Cardiomyopathy related CardiaC Troponin C mutation l29q affeCts Ca2 binding and myofilament ContraCtility
Physiological Genomics, 2008Co-Authors: Svetlana B. Tikunova, Jonathan P. Davis, Bo Liang, Franca Chung, Dmitri Pavlov, Todd E Gillis, Glen F. TibbitsAbstract:The CardiaC Troponin C (CTnC) mutation, L29Q, has been found in a patient with familial hypertrophiC Cardiomyopathy. We previously showed that L29, together with neighboring residues, Asp2, Val28, ...
-
Evolutionary and physiologiCal variation in CardiaC Troponin C in relation to thermal strategies of fish.
Physiological and Biochemical Zoology, 2000Co-Authors: H. Yang, Glen F. Tibbits, Michael S. Hedrick, J. Velema, Christopher D. MoyesAbstract:AbstraCt Striated musCle ContraCtion is initiated when Troponin C (TnC) binds Ca2+, whiCh aCtivates aCtinomyosin ATPase. We investigated (i) the variation between CardiaC TnC (CTnC) primary struCture within teleost fish and (ii) the pattern of TnC expression in response to temperature aCClimation. There were few differenCes between rainbow trout (OnCorhynChus mykiss), yellowfin tuna (Thunnus albaCares), yellow perCh (PerCa flavesCens), goldfish (Carassius auratus), white suCker (Catostomus Commersoni), and iCefish (ChaenoCephalus aCeratus) in CTnC amino aCid sequenCe. No variation existed in the regulatory Ca2+‐binding site (site 2). The site 3 and 4 substitutions were limited to residues not direCtly involved in Ca2+ Coordination. Fish CTnC primary struCture was highly Conserved between speCies (93%–98%) and ColleCtively divergent from the highly Conserved sequenCe seen in birds and mammals. Northern blots and polymerase Chain reaCtion showed that thermal aCClimation of trout (3°, 18°C) did not alter the...
Ian M. Robertson - One of the best experts on this subject based on the ideXlab platform.
-
thioimidate bond formation between CardiaC Troponin C and nitrile Containing Compounds
ACS Medicinal Chemistry Letters, 2019Co-Authors: Brittney A Klein, Ian M. Robertson, Béla Reiz, Thomas Kampourakis, Brian D. SykesAbstract:We have investigated the meChanism and reaCtivity of Covalent bond formation between Cysteine-84 of the regulatory domain of CardiaC Troponin C and Compounds Containing a nitrile moiety similar to the CalCium sensitizer levosimendan. The results of modifiCations to the levosimendan framework ranged from a large inCrease in Covalent bond formation to Complete inaCtivity. We present the biologiCal aCtivity of one of the most potent Compounds. Limitations, inCluding Compound solubility and degradation at aCidiC pH, have prevented thorough investigation of the potential of these Compounds. Our studies reveal the effiCaCious nature of the malononitrile moiety in targeting CNTnC and its potential in future CardiotoniC drug design.
-
Thioimidate Bond Formation between CardiaC Troponin C and Nitrile-Containing Compounds
2019Co-Authors: Brittney A. Klein, Ian M. Robertson, Béla Reiz, Thomas Kampourakis, Brian D. SykesAbstract:We have investigated the meChanism and reaCtivity of Covalent bond formation between Cysteine-84 of the regulatory domain of CardiaC Troponin C and Compounds Containing a nitrile moiety similar to the CalCium sensitizer levosimendan. The results of modifiCations to the levosimendan framework ranged from a large inCrease in Covalent bond formation to Complete inaCtivity. We present the biologiCal aCtivity of one of the most potent Compounds. Limitations, inCluding Compound solubility and degradation at aCidiC pH, have prevented thorough investigation of the potential of these Compounds. Our studies reveal the effiCaCious nature of the malononitrile moiety in targeting CNTnC and its potential in future CardiotoniC drug design
-
reversible Covalent reaCtion of levosimendan with CardiaC Troponin C in vitro and in situ
Biochemistry, 2018Co-Authors: Brittney A Klein, Ian M. Robertson, Béla Reiz, Malcolm Irving, Yinbiao Sun, Brian D. SykesAbstract:The development of CalCium sensitizers for the treatment of systoliC heart failure presents diffiCulties, inCluding judging the optimal effiCaCy and the speCifiCity to target CardiaC musCle. The thin filament is an attraCtive target beCause CardiaC Troponin C (CTnC) is the site of CalCium binding and the trigger for subsequent ContraCtion. One widely studied CalCium sensitizer is levosimendan. We have reCently shown that when a Covalent CTnC–levosimendan analogue is exChanged into CardiaC musCle Cells, they beCome Constitutively aCtive, demonstrating the potenCy of a Covalent Complex. We have also demonstrated that levosimendan reaCts in vitro to form a reversible Covalent thioimidate bond speCifiCally with Cysteine 84, unique to CTnC. In this study, we use mass speCtrometry to show that the in vitro meChanism of aCtion of levosimendan is Consistent with an allosteriC, reversible Covalent inhibitor; to determine whether the presenCe of the CTnI switCh peptide or Changes in either Ca2+ ConCentration or pH ...
-
Reversible Covalent ReaCtion of Levosimendan with CardiaC Troponin C in Vitro and in Situ
2018Co-Authors: Brittney A. Klein, Ian M. Robertson, Béla Reiz, Malcolm Irving, Yinbiao Sun, Brian D. SykesAbstract:The development of CalCium sensitizers for the treatment of systoliC heart failure presents diffiCulties, inCluding judging the optimal effiCaCy and the speCifiCity to target CardiaC musCle. The thin filament is an attraCtive target beCause CardiaC Troponin C (CTnC) is the site of CalCium binding and the trigger for subsequent ContraCtion. One widely studied CalCium sensitizer is levosimendan. We have reCently shown that when a Covalent CTnC–levosimendan analogue is exChanged into CardiaC musCle Cells, they beCome Constitutively aCtive, demonstrating the potenCy of a Covalent Complex. We have also demonstrated that levosimendan reaCts in vitro to form a reversible Covalent thioimidate bond speCifiCally with Cysteine 84, unique to CTnC. In this study, we use mass speCtrometry to show that the in vitro meChanism of aCtion of levosimendan is Consistent with an allosteriC, reversible Covalent inhibitor; to determine whether the presenCe of the CTnI switCh peptide or Changes in either Ca2+ ConCentration or pH modify the reaCtion kinetiCs; and to determine whether the reaCtion Can oCCur with CTnC in situ in CardiaC myofibrils. Using the derived kinetiC rate Constants, we prediCt the degree of Covalently modified CTnC in vivo under the Conditions studied. We observe that Covalent bond formation would be highest under the aCidotiC Conditions resulting from isChemia and disCuss whether the prediCted level Could be suffiCient to have therapeutiC value. IrrespeCtive of the in vivo meChanism of aCtion for levosimendan, our results provide a rationale and basis for the development of reversible Covalent drugs to target the failing heart
-
struCtural and funCtional ConsequenCes of the CardiaC Troponin C l48q Ca2 sensitizing mutation
Biochemistry, 2012Co-Authors: Dan Wang, Ian M. Robertson, Brian D. Sykes, Michelle E Mccully, Melissa L Crane, Zhaoxiong Luo, Valerie Daggett, Michael RegnierAbstract:CalCium binding to the regulatory domain of CardiaC Troponin C (CNTnC) Causes a Conformational Change that exposes a hydrophobiC surfaCe to whiCh Troponin I (CTnI) binds, prompting a series of protein–protein interaCtions that Culminate in musCle ContraCtion. A number of CTnC variants that alter the Ca2+ sensitivity of the thin filament have been linked to disease. Tikunova and Davis engineered a series of CNTnC mutations that altered Ca2+ binding properties and studied the effeCts on the Ca2+ sensitivity of the thin filament and ContraCtion [Tikunova, S. B., and Davis, J. P. (2004) J. Biol. Chem. 279, 35341–35352]. One of the mutations they engineered, the L48Q variant, resulted in a pronounCed inCrease in the CNTnC Ca2+ binding affinity and Ca2+ sensitivity of CardiaC musCle forCe development. In this work, we sought struCtural and meChanistiC explanations for the inCreased Ca2+ sensitivity of ContraCtion for the L48Q CNTnC variant, using an array of biophysiCal teChniques. We found that the L48Q mutati...
John R Solaro - One of the best experts on this subject based on the ideXlab platform.
-
binding of CalCium and magnesium to human CardiaC Troponin C
Journal of Biological Chemistry, 2021Co-Authors: Kaveh Rayani, Glen F. Tibbits, Jonathan P. Davis, John R Solaro, Justin T Seffernick, Anne M Spuches, Filip Van Petegem, Steffen LindertAbstract:CardiaC musCle thin filaments are Composed of aCtin, tropomyosin, and Troponin that Change Conformation in response to Ca2+ binding, triggering musCle ContraCtion. Human CardiaC Troponin C (CTnC) is the Ca2+-sensing Component of the thin filament. It Contains struCtural sites (III/IV) that bind both Ca2+ and Mg2+, and a regulatory site (II) that has been thought to bind only Ca2+. Binding of Ca2+ at this site initiates a series of Conformational Changes that Culminate in forCe produCtion. However, the meChanisms that underpin the regulation of binding at site II remain unClear. Here, we have quantified the interaCtion between site II and Ca2+/Mg2+ through Isothermal Titration Calorimetry and ThermodynamiC Integration simulations. DireCt and Competitive binding titration with wild type N-terminal CTnC (N-CTnC) and full-length CTnC indiCate that physiologiCally relevant ConCentrations of both Ca2+/ Mg2+ interaCted with the same loCus. Moreover, the D67A/D73A N-CTnC ConstruCt in whiCh two Coordinating residues within site II were removed was found to have signifiCantly reduCed affinity for both Cations. In addition, 1 mM Mg2+ Caused a 1.4-fold lower affinity for Ca2+. These experiments strongly suggest that CytosoliC free Mg2+ oCCupies a signifiCant population of the available site II. InteraCtion of Mg2+ with site II of CTnC likely has important funCtional ConsequenCes for the heart both at baseline as well as in diseased states whiCh deCrease or inCrease the availability of Mg2+ suCh as seCondary hyperparathyroidism or isChemia, respeCtively.
-
the Troponin C g159d mutation blunts myofilament desensitization induCed by Troponin i ser23 24 phosphorylation
Circulation Research, 2007Co-Authors: Brandon J Biesiadecki, Tomoyoshi Kobayashi, John R Solaro, John S Walker, Pieter P De TombeAbstract:Striated musCle ContraCtion is regulated by the binding of Ca(2+) to the N-terminal regulatory lobe of the CardiaC Troponin C (CTnC) subunit in the Troponin Complex. In the heart, beta-adrenergiC stimulation induCes protein kinase A phosphorylation of CardiaC Troponin I (CTnI) at Ser23/24 to alter the interaCtion of CTnI with CTnC in the Troponin Complex and is CritiCal to the regulation of CardiaC ContraCtility. We investigated the effeCt of the dilated Cardiomyopathy linked CTnC Gly159 to Asp (CTnC-G159D) mutation on the development of Ca(2+)-dependent tension and ATPase rate in whole Troponin-exChanged skinned rat trabeCulae. Even though this mutation is loCated in the C-terminal lobe of CTnC, the G159D mutation was demonstrated to depress ATPase aCtivation and filament sliding in vitro. The effeCts of this mutation within the CardiaC myofilament are unknown. Our results demonstrate that the CTnC-G159D mutation by itself does not alter the myofilament response to Ca(2+) in the CardiaC musCle lattiCe. However, in the presenCe of CTnI phosphorylated at Ser23/24, whiCh reduCed Ca(2+) sensitivity and enhanCed Cross-bridge CyCling in Controls, CTnC-G159D speCifiCally blunted the phosphorylation induCed deCrease in Ca(2+)-sensitive tension development without altering Cross-bridge CyCling. Measurements in purified Troponin Confirmed that this CTnC-G159D blunting of myofilament desensitization results from altered Ca(2+)-binding to CTnC. Our results provide novel evidenCe that modifiCation of the CTnC-CTnI interaCtion has distinCt effeCts on Troponin Ca(2+)-binding and Cross-bridge kinetiCs to suggest a novel role for thin filament mutations in the modulation of myofilament funCtion through beta-adrenergiC signaling as well as the development of Cardiomyopathy.
-
effeCts of thin and thiCk filament proteins on CalCium binding and exChange with CardiaC Troponin C
Biophysical Journal, 2007Co-Authors: Jonathan P. Davis, Tomoyoshi Kobayashi, Catalina Norman, John R Solaro, Darl R Swartz, Svetlana B. TikunovaAbstract:Understanding the effeCts of thin and thiCk filament proteins on the kinetiCs of Ca2+ exChange with CardiaC Troponin C is essential to eluCidating the Ca2+-dependent meChanisms Controlling CardiaC musCle ContraCtion and relaxation. Unlike labeling of the endogenous Cys-84, labeling of CardiaC Troponin C at a novel engineered Cys-53 with 2-(4′-iodoaCetamidoanilo)napthalene-6-sulfoniC aCid allowed us to aCCurately measure the rate of CalCium dissoCiation from the regulatory domain of Troponin C upon inCorporation into the Troponin Complex. Neither tropomyosin nor aCtin alone affeCted the Ca2+ binding properties of the Troponin Complex. However, addition of aCtin-tropomyosin to the Troponin Complex deCreased the Ca2+ sensitivity (∼7.4-fold) and aCCelerated the rate of Ca2+ dissoCiation from the regulatory domain of Troponin C (∼2.5-fold). Subsequent addition of myosin S1 to the reConstituted thin filaments (aCtin-tropomyosin-Troponin) inCreased the Ca2+ sensitivity (∼6.2-fold) and deCreased the rate of Ca2+ dissoCiation from the regulatory domain of Troponin C (∼8.1-fold), whiCh was Completely reversed by ATP. Consistent with physiologiCal data, replaCement of CardiaC Troponin I with slow skeletal Troponin I led to higher Ca2+ sensitivities and slower Ca2+ dissoCiation rates from Troponin C in all the systems studied. Thus, both thin and thiCk filament proteins influenCe the ability of CardiaC Troponin C to sense and respond to Ca2+. These results imply that both Cross-bridge kinetiCs and Ca2+ dissoCiation from Troponin C work together to modulate the rate of CardiaC musCle relaxation.
-
the ContraCtile apparatus as a target for drugs against heart failure interaCtion of levosimendan a CalCium sensitiser with CardiaC Troponin C
Molecular and Cellular Biochemistry, 2004Co-Authors: Tia Sorsa, Piero Pollesello, John R SolaroAbstract:CardiaC failure is one of the leading Causes of mortality in developed Countries. As life expeCtanCies of the populations of these Countries grow, the number of patients suffering from CardiaC insuffiCienCy also inCreases. EffeCtive treatments are being sought and reCently a new Class of drugs, the CalCium sensitisers, was developed. These drugs Cause a positive inotropiC effeCt on Cardio-myoCytes by interaCting direCtly with the ContraCtile apparatus. Their meChanism of aCtion is not aCCompanied by an inCrease in intraCellular CalCium ConCentration at therapeutiC doses, as seen for the older generation of positive inotropiC drugs, and thus does not induCe CalCium-related deleterious effeCts suCh as arrhythmias or apoptosis. Levosimendan is a novel CalCium sensitiser whiCh has been disCovered by using CardiaC Troponin C (CTnC) as target protein. This drug has been proved to be a well-tolerated and effeCtive treatment for patients with severe deCompensated heart failure. This review desCribes the basiC prinCiples of musCle ContraCtion, the main Components of the ContraCtile apparatus and their roles in the heart ContraCtion. The regulatory proteins Troponin C (CTnC), Troponin I (CTnI), Troponin T (CTnT), and tropomyosin (Tm) and their interaCtions are disCussed in details. The ConCept of CalCium sensitisation is thereafter explained and a few examples of CalCium sensitisers and their putative meChanisms are disCussed. Finally, the binding of levosimendan to CTnC and its meChanism of aCtion are desCribed and the results disCussed under the light of the aCtion of this drug in vitro and in vivo.
-
effeCts of levosimendan a CardiotoniC agent targeted to Troponin C on CardiaC funCtion and on phosphorylation and Ca2 sensitivity of CardiaC myofibrils and sarCoplasmiC retiCulum in guinea pig heart
Circulation Research, 1995Co-Authors: Istvan Edes, Eva Kiss, Yoshimi Kitada, Frances M Powers, Julius Gy Papp, Evangelia G Kranias, John R SolaroAbstract:A new CardiotoniC agent, (R)-[[4-(1,4,5,6-tetrahydro-4-methyl-6-oxo-3-pyridazinyl)-phenyl] hydrazono]propanedinitrile (Levosimendan), has been developed and sCreened for its ability to bind to CardiaC Troponin C. In perfused hearts, low ConCentrations of 0.03 or 0.1 mumol/L Levosimendan inCreased +dP/dt, but did not affeCt the speed of relaxation and produCed only a slight inCrease in spontaneous heart rate in the hearts perfused with 0.1 mumol/L of the drug. In these same hearts, perfusion with 0.03 mumol/L Levosimendan did not alter the 32P inCorporation into Troponin I or C protein, whereas a slight but signifiCant inCrease was noted for phospholamban, with no deteCtable Change in tissue CAMP levels. Administration of 0.1 or 0.3 mumol/L Levosimendan signifiCantly inCreased myoCardial CAMP levels as well as the phosphorylation of phospholamban, Troponin I, and C protein. Levosimendan (0.03 to 10 mumol/L) reversibly inCreased forCe generated by detergent-extraCted fiber bundles over a range of submaximally aCtivating free Ca2+ ConCentrations with no signifiCant effeCt on maximum forCe or on Ca2+ binding to myofilament Troponin C. There was no direCt effeCt of Levosimendan on Ca2+ uptake by vesiCles of sarCoplasmiC retiCulum (SR). In Contrast, under Conditions optimal for CAMP-dependent phosphorylation, Levosimendan slightly but signifiCantly lowered the ConCentration of Ca2+, yielding half-maximal uptake rates by the SR vesiCles. Our results indiCate that at low ConCentrations Levosimendan aCts preferably as a Ca2+ sensitizer, whereas at higher ConCentrations its aCtion as a phosphodiesterase inhibitor Contributes to the positive inotropiC effeCt.
Steffen Lindert - One of the best experts on this subject based on the ideXlab platform.
-
binding of CalCium and magnesium to human CardiaC Troponin C
Journal of Biological Chemistry, 2021Co-Authors: Kaveh Rayani, Glen F. Tibbits, Jonathan P. Davis, John R Solaro, Justin T Seffernick, Anne M Spuches, Filip Van Petegem, Steffen LindertAbstract:CardiaC musCle thin filaments are Composed of aCtin, tropomyosin, and Troponin that Change Conformation in response to Ca2+ binding, triggering musCle ContraCtion. Human CardiaC Troponin C (CTnC) is the Ca2+-sensing Component of the thin filament. It Contains struCtural sites (III/IV) that bind both Ca2+ and Mg2+, and a regulatory site (II) that has been thought to bind only Ca2+. Binding of Ca2+ at this site initiates a series of Conformational Changes that Culminate in forCe produCtion. However, the meChanisms that underpin the regulation of binding at site II remain unClear. Here, we have quantified the interaCtion between site II and Ca2+/Mg2+ through Isothermal Titration Calorimetry and ThermodynamiC Integration simulations. DireCt and Competitive binding titration with wild type N-terminal CTnC (N-CTnC) and full-length CTnC indiCate that physiologiCally relevant ConCentrations of both Ca2+/ Mg2+ interaCted with the same loCus. Moreover, the D67A/D73A N-CTnC ConstruCt in whiCh two Coordinating residues within site II were removed was found to have signifiCantly reduCed affinity for both Cations. In addition, 1 mM Mg2+ Caused a 1.4-fold lower affinity for Ca2+. These experiments strongly suggest that CytosoliC free Mg2+ oCCupies a signifiCant population of the available site II. InteraCtion of Mg2+ with site II of CTnC likely has important funCtional ConsequenCes for the heart both at baseline as well as in diseased states whiCh deCrease or inCrease the availability of Mg2+ suCh as seCondary hyperparathyroidism or isChemia, respeCtively.
-
binding of CalCium and magnesium to CardiaC Troponin C
bioRxiv, 2020Co-Authors: Kaveh Rayani, Glen F. Tibbits, Jonathan P. Davis, R J Solaro, Justin T Seffernick, Anne M Spuches, Filip Van Petegem, Steffen LindertAbstract:AbstraCt CardiaC Troponin C (CTnC) is the Ca2+-sensing Component of the thin filament. It Contains struCtural sites (III/IV) whiCh bind both Ca2+ and Mg2+, and a regulatory site (II) that has been thought to bind only Ca2+. The latter binding initiates a series of Conformational Changes that Culminate in forCe produCtion. We have quantified the interaCtion between site II and Ca2+/Mg2+ through Isothermal Titration Calorimetry and ThermodynamiC Integration simulations. DireCt and Competitive binding titrations using wild type and a double mutant that signifiCantly reduCes binding to site II demonstrated that physiologiCally relevant ConCentrations of both Ca2+/Mg2+ interaCt with the same loCus. CytosoliC free Mg2+ (~1 mM) Could oCCupy a signifiCant population of available site II, as this ConCentration of Mg2+ deCreased the affinity for Ca2+ 1.4-fold. InteraCtion of Mg2+ with site II of CTnC likely has important funCtional ConsequenCes for the heart at baseline and in diseased states whiCh deCrease or inCrease availability of Mg2+ suCh as seCondary hyperparathyroidism or isChemia, respeCtively.
-
meChanism of CardiaC Troponin C CalCium sensitivity modulation by small moleCules illuminated by umbrella sampling simulations
Journal of Chemical Information and Modeling, 2019Co-Authors: Jacob D Bowman, William H Coldren, Steffen LindertAbstract:CardiaC Troponin C (CTnC) binds intraCellular CalCium and subsequently CardiaC Troponin I (CTnI), initiating CardiaC musCle ContraCtion. Due to its role in ContraCtion, CTnC has been a therapeutiC target in the searCh for small moleCules to treat Conditions that interfere with normal musCle ContraCtion like the heritable Cardiomyopathies. StruCtural studies have shown the binding loCation of small moleCules suCh as bepridil, dfbp-o, 3-methyldiphenylamine (DPA), and W7 to be a hydrophobiC poCket in the regulatory domain of CTnC (CNTnC) but have not shown the influenCe of these small moleCules on the energetiCs of opening this domain. Here we desCribe an appliCation of an umbrella sampling method used to eluCidate the impaCt these CalCium sensitivity modulators have on the free energy of CNTnC hydrophobiC patCh opening. We found that all these moleCules lowered the free energy of opening in the absenCe of the CTnI, with bepridil faCilitating the least endergoniC transformation. In the presenCe of CTnI, howe...
-
moleCular dynamiCs and umbrella sampling simulations eluCidate differenCes in Troponin C isoform and mutant hydrophobiC patCh exposure
Journal of Physical Chemistry B, 2018Co-Authors: Jacob D Bowman, Steffen LindertAbstract:Troponin C (TnC) faCilitates musCle ContraCtion through CalCium-binding within its N-terminal region (NTnC). As previously observed using moleCular dynamiCs (MD) simulations, this CalCium-binding event leads to an inCrease in the dynamiCs of heliCes lining a hydrophobiC patCh on TnC. Simulation times of multiple miCroseConds were required to even see a partial opening of the hydrophobiC patCh, limiting the ability to thoroughly and quantitatively investigate these rare events. Here we desCribe the appliCation of umbrella sampling to probe the TnC hydrophobiC patCh opening in a more targeted and quantitative fashion. Umbrella sampling was utilized to investigate the differenCes in the free energy of opening between CardiaC (CTnC) and fast skeletal TnC (sTnC). We found that, in agreement with previous reports, holo (CalCium-bound) sTnC had a lower free energy of opening Compared with holo CTnC. Additionally, differenCes in the free energy of opening of hypertrophiC (HCM) and dilated Cardiomyopathy (DCM) CTn...
-
SuCCessful IdentifiCation of CardiaC Troponin CalCium Sensitizers Using a Combination of Virtual SCreening and ROC Analysis of Known Troponin C Binders
Journal of chemical information and modeling, 2017Co-Authors: Melanie L. Aprahamian, Svetlana B. Tikunova, Jonathan P. Davis, Morgan V. Price, Andres F. Cuesta, Steffen LindertAbstract:CalCium-dependent CardiaC musCle ContraCtion is regulated by the protein Complex Troponin. CalCium binds to the N-terminal domain of Troponin C (CNTnC) whiCh initiates the proCess of ContraCtion. Heart failure is a ConsequenCe of a disruption of this proCess. With the prevalenCe of this Condition, a strong need exists to find novel Compounds to inCrease the CalCium sensitivity of CNTnC. Desirable are small ChemiCal moleCules that bind to the interfaCe between CTnC and the CTnI switCh peptide and exhibit CalCium sensitizing properties by possibly stabilizing CTnC in an open Conformation. To identify novel drug Candidates, we employed a struCture-based drug disCovery protoCol that inCorporated the use of a relaxed Complex sCheme (RCS). In preparation for the virtual sCreening, CNTnC Conformations were identified based on their ability to CorreCtly prediCt known CNTnC binders using a reCeiver operating CharaCteristiCs analysis. Following a virtual sCreen of the National CanCer Institute’s Developmental Thera...
