Coupled expression of troponin T and troponin I isoforms in single skeletal muscle fibers correlates with contractility

Striated muscle contraction is powered by actin-activated myosin ATPase. This process is regulated by Ca(2+) via the troponin complex. Slow- and fast-twitch fibers of vertebrate skeletal muscle express type I and type II myosin, respectively, and these myosin isoenzymes confer different ATPase activities, contractile velocities, and force. Skeletal muscle troponin has also diverged into fast and slow isoforms, but their functional significance is not fully understood. To investigate the expression of troponin isoforms in mammalian skeletal muscle and their functional relationship to that of the myosin isoforms, we concomitantly studied myosin, troponin T (TnT), and troponin I (TnI) isoform contents and isometric contractile properties in single fibers of rat skeletal muscle. We characterized a large number of Triton X-100-skinned single fibers from soleus, diaphragm, gastrocnemius, and extensor digitorum longus muscles and selected fibers with combinations of a single myosin isoform and a single class (slow or fast) of the TnT and TnI isoforms to investigate their role in determining contractility. Types IIa, IIx, and IIb myosin fibers produced higher isometric force than that of type I fibers. Despite the polyploidy of adult skeletal muscle fibers, the expression of fast or slow isoforms of TnT and TnI is tightly coupled. Fibers containing slow troponin had higher Ca(2+) sensitivity than that of the fast troponin fibers, whereas fibers containing fast troponin showed a higher cooperativity of Ca(2+) activation than that of the slow troponin fibers. These results demonstrate distinct but coordinated regulation of troponin and myosin isoform expression in skeletal muscle and their contribution to the contractile properties of muscle.     

Ca(2+)- and S1-induced movement of troponin T on reconstituted skeletal muscle thin filaments observed by fluorescence energy transfer spectroscopy

Troponin T (TnT) is an essential component of troponin (Tn) for the Ca(2+)-regulation of vertebrate striated muscle contraction. TnT consists of an extended NH(2)-terminal domain that interacts with tropomyosin (Tm) and a globular COOH-terminal domain that interacts with Tm, troponin I (TnI), and troponin C (TnC). We have generated two mutants of a rabbit skeletal beta-TnT 25-kDa fragment (59-266) that have a unique cysteine at position 60 (N-terminal region) or 250 (C-terminal region). To understand the spatial rearrangement of TnT on the thin filament in response to Ca(2+) binding to TnC, we measured distances from Cys-60 and Cys-250 of TnT to Gln-41 and Cys-374 of F-actin on the reconstituted thin filament by using fluorescence resonance energy transfer (FRET). The distances from Cys-60 and Cys-250 of TnT to Gln-41 of F-actin were 39.5 and 30.0 A, respectively in the absence of Ca(2+), and increased by 2.6 and 5.8 A, respectively upon binding of Ca(2+) to TnC. The rigor binding of myosin subfragment 1 (S1) further increased these distances by 4 and 5 A respectively, when the thin filaments were fully decorated with S1. This indicates that not only the C-terminal but also the N-terminal region of TnT showed the Ca(2+)- and S1-induced movement, and the C-terminal region moved more than N-terminal region. In the absence of Ca(2+), the rigor S1 binding also increased the distances to the same extent as the presence of Ca(2+) when the thin filaments were fully decorated with S1. The addition of ATP completely reversed the changes in FRET induced by rigor S1 binding both in the presence and absence of Ca(2+). However, plots of the extent of S1-induced conformational change vs. molar ratio of S1 to actin showed hyperbolic curve in the presence of Ca(2+) but sigmoidal curve in the absence of Ca(2+). FRET measurement of the distances from Cys-60 and Cys-250 of TnT to Cys-374 of actin showed almost the same results as the case of Gln-41 of actin. The present FRET measurements demonstrated that not only TnI but also TnT change their positions on the thin filament corresponding to three states of thin filaments (relaxed, Ca(2+)-induced or closed, and S1-induced or open states).     

Structural basis for Ca2+-regulated muscle relaxation at interaction sites of troponin with actin and tropomyosin

Troponin and tropomyosin on actin filaments constitute a Ca2+-sensitive switch that regulates the contraction of vertebrate striated muscle through a series of conformational changes within the actin-based thin filament. Troponin consists of three subunits: an inhibitory subunit (TnI), a Ca2+-binding subunit (TnC), and a tropomyosin-binding subunit (TnT). Ca2+-binding to TnC is believed to weaken interactions between troponin and actin, and triggers a large conformational change of the troponin complex. However, the atomic details of the actin-binding sites of troponin have not been determined. Ternary troponin complexes have been reconstituted from recombinant chicken skeletal TnI, TnC, and TnT2 (the C-terminal region of TnT), among which only TnI was uniformly labelled with 15N and/or 13C. By applying NMR spectroscopy, the solution structures of a "mobile" actin-binding domain (approximately 6.1 kDa) in the troponin ternary complex (approximately 52 kDa) were determined. The mobile domain appears to tumble independently of the core domain of troponin. Ca2+-induced changes in the chemical shift and line shape suggested that its tumbling was more restricted at high Ca2+ concentrations. The atomic details of interactions between actin and the mobile domain of troponin were defined by docking the mobile domain into the cryo-electron microscopy (cryo-EM) density map of thin filament at low [Ca2+]. This allowed the determination of the 3D position of residue 133 of TnI, which has been an important landmark to incorporate the available information. This enabled unique docking of the entire globular head region of troponin into the thin filament cryo-EM map at a low Ca2+ concentration. The resultant atomic model suggests that troponin interacted electrostatically with actin and caused the shift of tropomyosin to achieve muscle relaxation. An important feature is that the coiled-coil region of troponin pushed tropomyosin at a low Ca2+ concentration. Moreover, the relationship between myosin and the mobile domain on actin filaments suggests that the latter works as a fail-safe latch.     

The role of the NH(2)- and COOH-terminal domains of the inhibitory region of troponin I in the regulation of skeletal muscle contraction.

The role of the inhibitory region of troponin (Tn) I in the regulation of skeletal muscle contraction was studied with three deletion mutants of its inhibitory region: 1) complete (TnI-(Delta96-116)), 2) the COOH-terminal domain (TnI-(Delta105-115)), and 3) the NH(2)-terminal domain (TnI-(Delta95-106)). Measurements of Ca(2+)-regulated force and relaxation were performed in skinned skeletal muscle fibers whose endogenous TnI (along with TnT and TnC) was displaced with high concentrations of added troponin T. Reconstitution of the Tn-displaced fibers with a TnI.TnC complex restored the Ca(2+) sensitivity of force; however, the levels of relaxation and force development varied. Relaxation of the fibers (pCa 8) was drastically impaired with two of the inhibitory region deletion mutants, TnI-(Delta96-116).TnC and TnI-(Delta105-115).TnC. The TnI-(Delta95-106).TnC mutant retained approximately 55% relaxation when reconstituted in the Tn-displaced fibers. Activation in skinned skeletal muscle fibers was enhanced with all TnI mutants compared with wild-type TnI. Interestingly, all three mutants of TnI increased the Ca(2+) sensitivity of contraction. None of the TnI deletion mutants, when reconstituted into Tn, could inhibit actin-tropomyosin-activated myosin ATPase in the absence of Ca(2+), and two of them (TnI-(Delta96-116) and TnI-(Delta105-115)) gave significant activation in the absence of Ca(2+). These results suggest that the COOH terminus of the inhibitory region of TnI (residues 105-115) is much more critical for the biological activity of TnI than the NH(2)-terminal region, consisting of residues 95-106. Presumably, the COOH-terminal domain of the inhibitory region of TnI is a part of the Ca(2+)-sensitive molecular switch during muscle contraction.     

Dephosphorylation specificities of protein phosphatase for cardiac troponin I, troponin T, and sites within troponin T

Protein dephosphorylation by protein phosphatase 1 (PP1), acting in concert with protein kinase C (PKC) and protein kinase A (PKA), is a pivotal regulatory mechanism of protein phosphorylation. Isolated rat cardiac myofibrils phosphorylated by PKC/PKA and dephosphorylated by PP1 were used in determining dephosphorylation specificities, Ca(2+)-stimulated Mg(2+)ATPase activities, and Ca(2+) sensitivities. In reconstituted troponin (Tn) complex, PP1 displayed distinct substrate specificity in dephosphorylation of TnT preferentially to TnI, in vitro. In situ phosphorylation of cardiomyocytes with calyculin A, a protein phosphatase inhibitor, resulted in an increase in the phosphorylation stiochiometry of TnT (0.3 to 0.5 (67%)), TnI (2.6 to 3.6 (38%)), and MLC2 (0.4 to 1.7 (325%)). These results further confirmed that though MLC2 is the preferred target substrate for protein phosphatase in the thick filament, the Tn complex (TnI and TnT) from thin filament and C-protein in the thick filament are also protein phosphatase substrates. Our in vitro dephosphorylation experiments revealed that while PP1 differentially dephosphorylated within TnT at multiple sites, TnI was uniformly dephosphorylated. Phosphopeptide maps from the in vitro experiments show that TnT phosphopeptides at spots 4A and 4B are much more resistant to PP1 dephosphorylation than other TnT phosphopeptides. Mg(2+)ATPase assays of myofibrils phosphorylated by PKC/PKA and dephosphorylated by PP1 delineated that while PKC and PKA phosphorylation decreased the Ca(2+)-stimulated Mg(2+)ATPase activities, dephosphorylation antagonistically restored it. PKC and PKA phosphorylation decreased Ca(2+) sensitivity to 3.6 microM and 5.0 microM respectively. However, dephosphorylation restored the Mg(2+)ATPase activity of PKC (99%) and PKA (95%), along with the Ca(2+) sensitivities (3.3 microM and 3.0 microM, respectively).     

Ca(2+)-dependent, myosin subfragment 1-induced proximity changes between actin and the inhibitory region of troponin I.

In order to help understand the spatial rearrangements of thin filament proteins during the regulation of muscle contraction, we used fluorescence resonance energy transfer (FRET) to measure Ca(2+)-dependent, myosin-induced changes in distances and fluorescence energy transfer efficiencies between actin and the inhibitory region of troponin I (TnI). We labeled the single Cys-117 of a mutant TnI with N-(iodoacetyl)-N'-(1-sulfo-5-naphthyl)ethylenediamine (IAEDANS) and Cys-374 of actin with 4-dimethylaminophenylazophenyl-4'-maleimide (DABmal). These fluorescent probes were used as donor and acceptor, respectively, for the FRET measurements. We reconstituted a troponin-tropomyosin (Tn-Tm) complex which contained the AEDANS-labeled mutant TnI, together with natural troponin T (TnT), troponin C (TnC) and tropomyosin (Tm) from rabbit fast skeletal muscle. Fluorescence titration of the AEDANS-labeled Tn-Tm complex with DABmal-labeled actin, in the presence and absence of Ca(2+), resulted in proportional, linear increases in energy transfer efficiency up to a 7:1 molar excess of actin over Tn-Tm. The distance between AEDANS on TnI Cys-117 and DABmal on actin Cys-374 increased from 37.9 A to 44.1 A when Ca(2+) bound to the regulatory sites of TnC. Titration of reconstituted thin filaments, containing AEDANS-labeled Tn-Tm and DABmal-labeled actin, with myosin subfragment 1 (S1) decreased the energy transfer efficiency, in both the presence and absence of Ca(2+). The maximum decrease occurred at well below stoichiometric levels of S1 binding to actin, showing a cooperative effect of S1 on the state of the thin filaments. S1:actin molar ratios of approximately 0.1 in the presence of Ca(2+), and approximately 0.3 in the absence of Ca(2+), were sufficient to cause a 50% reduction in normalized transfer efficiency. The distance between AEDANS on TnI Cys-117 and DABmal on actin Cys-374 increased by approximately 7 A in the presence of Ca(2+) and by approximately 2 A in the absence of Ca(2+) when S1 bound to actin. Our results suggest that TnI's interaction with actin inhibits actomyosin ATPase activity by modulating the equilibria among active and inactive states of the thin filament. Structural rearrangements caused by myosin S1 binding to the thin filament, as detected by FRET measurements, are consistent with the cooperative behavior of the thin filament proteins.     

Photocrosslinking of benzophenone-labeled single cysteine troponin I mutants to other thin filament proteins

The interaction sites of rabbit skeletal troponin I (TnI) with troponin C (TnC), troponin T (TnT), tropomyosin (Tm) and actin were mapped systematically using nine single cysteine residue TnI mutants with mutation sites at positions 6, 48, 64, 89, 104, 121, 133, 155 or 179 (TnI6, TnI48 etc.). Each mutant was labeled with the heterobifunctional photocrosslinker 4-maleimidobenzophenone (BP-Mal), and incorporated into the TnI.TnC binary complex, the TnI.TnC.TnT ternary troponin (Tn) complex, and the Tn.Tm.F-actin synthetic thin filament. Photocrosslinking reactions carried out in the presence and absence of Ca(2+) yielded the following results: (1) BP-TnI6 photocrosslinked primarily to TnC with a small degree of Ca(2+)-dependence in all the complex forms. (2) BP-TnI48, TnI64 and TnI89 photocrosslinked to TnT with no Ca(2+)-dependence. Photocrosslinking to TnC was reduced in the ternary versus the binary complex. BP-TnI89 also photocrosslinked to actin with higher yields in the absence of Ca(2+) than in its presence. (3) BP-TnI104 and TnI133 photocrosslinked to actin with much higher yields in the absence than in the presence of Ca(2+). (4) BP-TnI121 photocrosslinked to TnC with a small degree of Ca(2+)-dependence, and did not photocrosslink to actin. (5) BP-TnI155 and TnI179 photocrosslinked to TnC, TnT and actin, but all with low yields. All the labeled mutants photocrosslinked to TnC with varying degrees of Ca(2+)-dependence, and none to Tm. These results, along with those published allowed us to construct a structural and functional model of TnI in the Tn complex: in the presence of Ca(2+), residues 1-33 of TnI interact with the C-terminal domain hydrophobic cleft of TnC, approximately 48-89 with TnT, approximately 90-113 with TnC's central helix, approximately 114-125 with TnC's N-terminal domain hydrophobic cleft, and approximately 130-150 with TnC's A-helix. In the absence of Ca(2+), residues approximately 114-125 move out of TnC's N-terminal domain hydrophobic cleft and trigger the movements of residues approximately 89-113 and approximately 130-150 away from TnC and towards actin.     

Roles for the troponin tail domain in thin filament assembly and regulation. A deletional study of cardiac troponin T

Striated muscle contraction is regulated by Ca2+ binding to troponin, which has a globular domain and an elongated tail attributable to the NH2-terminal portion of the bovine cardiac troponin T (TnT) subunit. Truncation of the bovine cardiac troponin tail was investigated using recombinant TnT fragments and subunits TnI and TnC. Progressive truncation of the troponin tail caused progressively weaker binding of troponin-tropomyosin to actin and of troponin to actin-tropomyosin. A sharp drop-off in affinity occurred with NH2-terminal deletion of 119 rather than 94 residues. Deletion of 94 residues had no effect on Ca2+-activation of the myosin subfragment 1-thin filament MgATPase rate and did not eliminate cooperative effects of Ca2+ binding. Troponin tail peptide TnT1-153 strongly promoted tropomyosin binding to actin in the absence of TnI or TnC. The results show that the anchoring function of the troponin tail involves interactions with actin as well as with tropomyosin and has comparable importance in the presence or absence of Ca2+. Residues 95-153 are particularly important for anchoring, and residues 95-119 are crucial for function or local folding. Because striated muscle regulation involves switching among the conformational states of the thin filament, regulatory significance for the troponin tail may arise from its prominent contribution to the protein-protein interactions within these conformations.     

Characterization of troponin T dilated cardiomyopathy mutations in the fetal troponin isoform

The major goal of this study was to elucidate how troponin T (TnT) dilated cardiomyopathy (DCM) mutations in fetal TnT and fetal troponin affect the functional properties of the fetal heart that lead to infantile cardiomyopathy. The DCM mutations R141W and DeltaK210 were created in the TnT1 isoform, the primary isoform of cardiac TnT in the embryonic heart. In addition to a different TnT isoform, a different troponin I (TnI) isoform, slow skeletal TnI (ssTnI), is the dominant isoform in the embryonic heart. In skinned fiber studies, TnT1-wild-type (WT)-treated fibers reconstituted with cardiac TnI.troponin C (TnC) or ssTnI.TnC significantly increased Ca(2+) sensitivity of force development when compared with TnT3-WT-treated fibers at both pH 7.0 and pH 6.5. Porcine cardiac fibers treated with TnT1 that contained the DCM mutations (R141W and DeltaK210), when reconstituted with either cardiac TnI.TnC or ssTnI.TnC, significantly decreased Ca(2+) sensitivity of force development compared with TnT1-WT at both pH values. The R141W mutation, which showed no significant change in the Ca(2+) sensitivity of force development in the TnT3 isoform, caused a significant decrease in the TnT1 isoform. The DeltaK210 mutation caused a greater decrease in Ca(2+) sensitivity and maximal isometric force development compared with the R141W mutation in both the fetal and adult TnT isoforms. When complexed with cardiac TnI.TnC or ssTnI.TnC, both TnT1 DCM mutations strongly decreased maximal actomyosin ATPase activity as compared with TnT1-WT. Our results suggest that a decrease in maximal actomyosin ATPase activity in conjunction with decreased Ca(2+) sensitivity of force development may cause a severe DCM phenotype in infants with the mutations.     

The rates of switching movement of troponin T between three states of skeletal muscle thin filaments determined by fluorescence resonance energy transfer

Troponin (Tn) plays the key roles in the regulation of striated muscle contraction. Tn consists of three subunits (TnT, TnC, and TnI). In combination with the stopped-flow method, fluorescence resonance energy transfer between probes attached to Cys-60 or Cys-250 of TnT and Cys-374 of actin was measured to determine the rates of switching movement of the troponin tail domain (Cys-60) and of the TnT-TnI coiled-coil C terminus (Cys-250) between three states (relaxed, closed, and open) of the thin filament. When the free Ca(2+) concentration was rapidly changed, these domains moved with rates of approximately 450 and approximately 85 s(-1) at pH 7.0 on Ca(2+) up and down, respectively. When myosin subfragment 1 (S1) was dissociated from thin filaments by rapid mixing with ATP, these domains moved with a single rate constant of approximately 400 s(-1) in the presence and absence of Ca(2+). The light scattering measurements showed that ATP-induced S1 dissociation occurred with a rate constant >800 s(-1). When S1 was rapidly mixed with the thin filament, these domains moved with almost the same or slightly faster rates than those of S1 binding measured by light scattering. In most but not all aspects, the rates of movement of the troponin tail domain and of the TnT-TnI coiled-coil C terminus were very similar to those of certain TnI sites (N terminus, Cys-133, and C terminus) previously characterized (Shitaka, Y., Kimura, C., Iio, T., and Miki, M. (2004) Biochemistry 43, 10739-10747), suggesting that a series of conformational changes in the Tn complex during switching on or off process occurs synchronously.     

Calcium-regulated conformational change in the C-terminal end segment of troponin I and its binding to tropomyosin

The troponin complex plays an essential role in the thin filament regulation of striated muscle contraction. Of the three subunits of troponin, troponin I (TnI) is the actomyosin ATPase inhibitory subunit and its effect is released upon Ca(2+) binding to troponin C. The exon-8-encoded C-terminal end segment represented by the last 24 amino acids of cardiac TnI is highly conserved and is critical to the inhibitory function of troponin. Here, we investigated the function and calcium regulation of the C-terminal end segment of TnI. A TnI model molecule was labeled with Alexa Fluor 532 at a Cys engineered at the C-terminal end and used to reconstitute the tertiary troponin complex. A Ca(2+) -regulated conformational change in the C-terminus of TnI was shown by a sigmoid-shape fluorescence intensity titration curve similar to that of the CD calcium titration curve of troponin C. Such corresponding Ca(2+) responses are consistent with the function of troponin as a coordinated molecular switch. Reconstituted troponin complex containing a mini-troponin T lacking its two tropomyosin-binding sites showed a saturable binding to tropomyosin at pCa 9 but not at pCa 4. This Ca(2+) -regulated binding was diminished when the C-terminal 19 amino acids of cardiac TnI were removed. These results provided novel evidence for suggesting that the C-terminal end segment of TnI participates in the Ca(2+) regulation of muscle thin filament through interaction with tropomyosin.     

Conformational changes induced in troponin I by interaction with troponin T and actin/tropomyosin.

Troponin I (TnI) is the inhibitory component of the striated muscle Ca2+ regulatory protein troponin (Tn). The other two components of Tn are troponin C (TnC), the Ca2+-binding component, and troponin T (TnT), the tropomyosin-binding component. We have used limited chymotryptic digestion to probe the local conformation of TnI in the free state, the binary TnC*TnI complex, the ternary TnC*. TnI*TnT (Tn) complex, and in the reconstituted Tn*tropomyosin*F-actin filament. The digestion of TnI alone or in the TnC*TnI complex produced initially two major fragments via a cleavage of the peptide bond between Phe100 and Asp101 in the so-called inhibitory region. In the ternary Tn complex cleavage occurred at a new site between Leu140 and Lys141. In the absence of Ca2+ this was followed by digestion of the 1-140 fragment at Leu122 and Met116. In the reconstituted thin filament the same fragments as in the case of the ternary complex were produced, but the rate of digestion was slower in the absence than in the presence of Ca2+. These results indicate firstly that in both free TnI and TnI complexed with TnC there is an exposed and flexible site in the inhibitory region. Secondly, TnT affects the conformation of TnI in the inhibitory region and also in the region that contains the 140-141 bond. Thirdly, the 140-141 region of TnI is likely to interact with actin in the reconstituted thin filament when Ca2+ is absent. These findings are discussed in terms of the role of TnI in the mechanism of thin filament regulation, and in light of our previous results [Y. Luo, J.-L. Wu, J. Gergely, T. Tao, Biochemistry 36 (1997) 13449-13454] on the global conformation of TnI.     

Ca(2+)- and s1-induced movement of troponin T on mutant thin filaments reconstituted with functionally deficient mutant tropomyosin

The deletion mutant (D234Tm) of rabbit skeletal muscle alpha-tropomyosin, in which internal actin-binding pseudo-repeats 2, 3, and 4 are missing, inhibits the thin filament activated myosin-ATPase activity whether Ca(2+) ion is present or not [Landis et al. (1997) J. Biol. Chem. 272, 14051-14056]. Fluorescence resonance energy transfer (FRET) showed substantial changes in distances between Cys-60 or 250 of troponin T (TnT) and Gln-41 or Cys-374 of actin on wild-type thin filaments corresponding to three states of thin filaments [Kimura et al. (2002) J. Biochem. 132, 93-102]. Troponin T movement on mutant thin filaments reconstituted with D234Tm was compared with that on wild-type thin filaments to understand from which the functional deficiency of mutant thin filaments derives. The Ca(2+)-induced changes in distances between Cys-250 of TnT and Gln-41 or Cys-374 of F-actin were smaller on mutant thin filaments than on wild-type thin filaments. On the other hand, the distances between Cys-60 of TnT and Gln-41 or Cys-374 of F-actin on mutant thin filaments did not change at all regardless of whether Ca(2+) was present. Thus, FRET showed that the Ca(2+)-induced movement of TnT was severely impaired on mutant thin filaments. The rigor binding of myosin subfragment 1 (S1) increased the distances when the thin filaments were fully decorated with S1 in the presence and absence of Ca(2+). However, plots of the extent of S1-incuced movement of TnT against molar ratio of S1 to actin in the presence and absence of Ca(2+) showed that the S1-induced movement of TnT was also impaired on mutant thin filaments. The deficiency of TnT movement on mutant thin filaments causes the altered S1-induced movement of TnI, and mutant thin filaments consequently fail to activate the myosin-ATPase activity even in the presence of Ca(2+).     

Invited Review: pathophysiology of cardiac muscle contraction and relaxation as a result of alterations in thin filament regulation.

Cardiac muscle contraction depends on the tightly regulated interactions of thin and thick filament proteins of the contractile apparatus. Mutations of thin filament proteins (actin, tropomyosin, and troponin), causing familial hypertrophic cardiomyopathy (FHC), occur predominantly in evolutionarily conserved regions and induce various functional defects that impair the normal contractile mechanism. Dysfunctional properties observed with the FHC mutants include altered Ca(2+) sensitivity, changes in ATPase activity, changes in the force and velocity of contraction, and destabilization of the contractile complex. One apparent tendency observed in these thin filament mutations is an increase in the Ca(2+) sensitivity of force development. This trend in Ca(2+) sensitivity is probably induced by altering the cross-bridge kinetics and the Ca(2+) affinity of troponin C. These in vitro defects lead to a wide variety of in vivo cardiac abnormalities and phenotypes, some more severe than others and some resulting in sudden cardiac death.     

A modulatory role for the troponin T tail domain in thin filament regulation

In striated muscle the force generating acto-myosin interaction is sterically regulated by the thin filament proteins tropomyosin and troponin (Tn), with the position of tropomyosin modulated by calcium binding to troponin. Troponin itself consists of three subunits, TnI, TnC, and TnT, widely characterized as being responsible for separate aspects of the regulatory process. TnI, the inhibitory unit is released from actin upon calcium binding to TnC, while TnT performs a structural role forming a globular head region with the regulatory TnI- TnC complex with a tail anchoring it within the thin filament. We have examined the properties of TnT and the TnT(1) tail fragment (residues 1-158) upon reconstituted actin-tropomyosin filaments. Their regulatory effects have been characterized in both myosin S1 ATPase and S1 kinetic and equilibrium binding experiments. We show that both inhibit the actin-tropomyosin-activated S1 ATPase with TnT(1) producing a greater inhibitory effect. The S1 binding data show that this inhibition is not caused by the formation of the blocked B-state but by significant stabilization of the closed C-state with a 10-fold reduction in the C- to M-state equilibrium, K(T), for TnT(1). This suggests TnT has a modulatory as well as structural role, providing an explanation for its large number of alternative isoforms.     

Effects of thin and thick filament proteins on calcium binding and exchange with cardiac troponin C

Understanding the effects of thin and thick filament proteins on the kinetics of Ca(2+) exchange with cardiac troponin C is essential to elucidating the Ca(2+)-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 Ca(2+) binding properties of the troponin complex. However, addition of actin-tropomyosin to the troponin complex decreased the Ca(2+) sensitivity ( approximately 7.4-fold) and accelerated the rate of Ca(2+) dissociation from the regulatory domain of troponin C ( approximately 2.5-fold). Subsequent addition of myosin S1 to the reconstituted thin filaments (actin-tropomyosin-troponin) increased the Ca(2+) sensitivity ( approximately 6.2-fold) and decreased the rate of Ca(2+) dissociation from the regulatory domain of troponin C ( approximately 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 Ca(2+) sensitivities and slower Ca(2+) 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 Ca(2+). These results imply that both cross-bridge kinetics and Ca(2+) dissociation from troponin C work together to modulate the rate of cardiac muscle relaxation.     

Force-calcium relationship depends on myosin heavy chain and troponin isoforms in rat diaphragm muscle fibers.

The present study examined Ca(2+) sensitivity of diaphragm muscle (Dia(m)) fibers expressing different myosin heavy chain (MHC) isoforms. We hypothesized that Dia(m) fibers expressing the MHC(slow) isoform have greater Ca(2+) sensitivity than fibers expressing fast MHC isoforms and that this fiber-type difference in Ca(2+) sensitivity reflects the isoform composition of the troponin (Tn) complex (TnC, TnT, and TnI). Studies were performed in single Triton-X-permeabilized Dia(m) fibers. The Ca(2+) concentration at which 50% maximal force was generated (pCa(50)) was determined for each fiber. SDS-PAGE and Western analyses were used to determine the MHC and Tn isoform composition of single fibers. The pCa(50) for Dia(m) fibers expressing MHC(slow) was significantly greater than that of fibers expressing fast MHC isoforms, and this greater Ca(2+) sensitivity was associated with expression of slow isoforms of the Tn complex. However, some Dia(m) fibers expressing MHC(slow) contained the fast TnC isoform. These results suggest that the combination of TnT, TnI, and TnC isoforms may determine Ca(2+) sensitivity in Dia(m) fibers.     

Altered regulation of cardiac muscle contraction by troponin T mutations that cause familial hypertrophic cardiomyopathy

To study the effect of troponin (Tn) T mutations that cause familial hypertrophic cardiomyopathy (FHC) on cardiac muscle contraction, wild-type, and the following recombinant human cardiac TnT mutants were cloned and expressed: I79N, R92Q, F110I, E163K, R278C, and intron 16(G(1) --> A) (In16). These TnT FHC mutants were reconstituted into skinned cardiac muscle preparations and characterized for their effect on maximal steady state force activation, inhibition, and the Ca(2+) sensitivity of force development. Troponin complexes containing these mutants were tested for their ability to regulate actin-tropomyosin(Tm)-activated myosin-ATPase activity. TnT(R278C) and TnT(F110I) reconstituted preparations demonstrated dramatically increased Ca(2+) sensitivity of force development, while those with TnT(R92Q) and TnT(I79N) showed a moderate increase. The deletion mutant, TnT(In16), significantly decreased both the activation and the inhibition of force, and substantially decreased the activation and the inhibition of actin-Tm-activated myosin-ATPase activity. ATPase activation was also impaired by TnT(F110I), while its inhibition was reduced by TnT(R278C). The TnT(E163K) mutation had the smallest effect on the Ca(2+) sensitivity of force; however, it produced an elevated activation of the ATPase activity in reconstituted thin filaments. These observed changes in the Ca(2+) regulation of force development caused by these mutations would likely cause altered contractility and contribute to the development of FHC.     

A pH-sensitive interaction of troponin I with troponin C coupled with strongly binding cross-bridges in cardiac myofilament activation

Slow skeletal muscle troponin I (ssTnI) expressed predominantly in perinatal heart confers a marked resistance to acidic pH on Ca(2+) regulation of cardiac muscle contraction. To explore the molecular mechanism underlying this phenomenon, we investigated the roles of TnI isoforms (ssTnI and cardiac TnI (cTnI)) in the thin filament activation by strongly binding cross-bridges, by exchanging troponin subunits in cardiac permeabilized muscle fibers. Fetal cardiac muscle showed a marked resistance to acidic pH in activation of the thin filament by strongly binding cross-bridges compared to adult muscle. Exchanging ssTnI into adult fibers altered the pH sensitivity from adult to fetal type, indicating that ssTnI also confers a marked resistance to acidic pH on the cross-bridge-induced thin filament activation. However, the adult fibers containing ssTnI or cTnI but lacking TnC showed no pH sensitivity. These findings provide the first evidence for the coupling between strongly binding cross-bridges and a pH-sensitive interaction of TnI with TnC in cardiac muscle contraction, as a molecular basis of the mechanism conferring the differential pH sensitivity on Ca(2+) regulation.     

Cross-bridge versus thin filament contributions to the level and rate of force development in cardiac muscle

In striated muscle thin filament activation is initiated by Ca(2+) binding to troponin C and augmented by strong myosin binding to actin (cross-bridge formation). Several lines of evidence have led us to hypothesize that thin filament properties may limit the level and rate of force development in cardiac muscle at all levels of Ca(2+) activation. As a test of this hypothesis we varied the cross-bridge contribution to thin filament activation by substituting 2 deoxy-ATP (dATP; a strong cross-bridge augmenter) for ATP as the contractile substrate and compared steady-state force and stiffness, and the rate of force redevelopment (k(tr)) in demembranated rat cardiac trabeculae as [Ca(2+)] was varied. We also tested whether thin filament dynamics limits force development kinetics during maximal Ca(2+) activation by comparing the rate of force development (k(Ca)) after a step increase in [Ca(2+)] with photorelease of Ca(2+) from NP-EGTA to maximal k(tr), where Ca(2+) binding to thin filaments should be in (near) equilibrium during force redevelopment. dATP enhanced steady-state force and stiffness at all levels of Ca(2+) activation. At similar submaximal levels of steady-state force there was no increase in k(tr) with dATP, but k(tr) was enhanced at higher Ca(2+) concentrations, resulting in an extension (not elevation) of the k(tr)-force relationship. Interestingly, we found that maximal k(tr) was faster than k(Ca), and that dATP increased both by a similar amount. Our data suggest the dynamics of Ca(2+)-mediated thin filament activation limits the rate that force develops in rat cardiac muscle, even at saturating levels of Ca(2+).