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Calcium binding to troponin C (TnC) activates striated muscle contraction by removing TnI (troponin I) from its inhibitory site on actin. Troponin T (TnT) links TnI with tropomyosin, causing tropomyosin to move from an inhibitory position on actin to an activating position. Positive charges within the C-terminal region of human cardiac TnT limit Ca2+ activation. We now show that the positively charged region of TnT has an even larger impact on skeletal muscle regulation. We prepared one variant of human skeletal TnT that had the C-terminal 16 residues truncated (Δ16) and another with an added C-terminal Cys residue and Ala substituted for the last 6 basic residues (251C-HAHA). Both mutants reduced (based on S1 binding kinetics) or eliminated (based on acrylodan-tropomyosin fluorescence) the first inactive state of actin at

Striated muscle contraction is regulated by the translocation of troponin-tropomyosin strands over the thin filament surface. Relaxation relies partly on highly-favorable, conformation-dependent electrostatic contacts between actin and tropomyosin, which position tropomyosin such that it impedes actomyosin associations. Impaired relaxation and hypercontractile properties are hallmarks of various muscle disorders. The α-cardiac actin M305L hypertrophic cardiomyopathy-causing mutation lies near residues that help confine tropomyosin to an inhibitory position along thin filaments. Here, we investigate M305L actin in vivo, in vitro, and in silico to resolve emergent pathological properties and disease mechanisms. Our data suggest the mutation reduces actin flexibility and distorts the actin-tropomyosin electrostatic energy landscape that, in muscle, result in aberrant contractile inhibition and excessive force. Thus, actin flexibility may be required to establish and maintain interfacial contacts with tropomyosin as well as facilitate its movement over distinct actin surface features and is, therefore, likely necessary for proper regulation of contraction., The α-cardiac actin M305L hypertrophic cardiomyopathy-causing mutation is located near residues that help confine tropomyosin to an inhibitory position along thin filaments. Here the authors assessed M305L actin in vivo, in vitro, and in silico to characterize emergent pathological properties and define the mechanistic basis of disease.

The troponin complex regulates the Ca(2+) activation of myofilaments during striated muscle contraction and relaxation. Troponin genes emerged 500–700 million years ago during early animal evolution. Troponin T (TnT) is the thin-filament–anchoring subunit of troponin. Vertebrate and invertebrate TnTs have conserved core structures, reflecting conserved functions in regulating muscle contraction, and they also contain significantly diverged structures, reflecting muscle type- and species-specific adaptations. TnT in insects contains a highly-diverged structure consisting of a long glutamic acid–rich C-terminal extension of ∼70 residues with unknown function. We found here that C-terminally truncated Drosophila TnT (TpnT–CD70) retains binding of tropomyosin, troponin I, and troponin C, indicating a preserved core structure of TnT. However, the mutant TpnT(CD70) gene residing on the X chromosome resulted in lethality in male flies. We demonstrate that this X-linked mutation produces dominant-negative phenotypes, including decreased flying and climbing abilities, in heterozygous female flies. Immunoblot quantification with a TpnT-specific mAb indicated expression of TpnT–CD70 in vivo and normal stoichiometry of total TnT in myofilaments of heterozygous female flies. Light and EM examinations revealed primarily normal sarcomere structures in female heterozygous animals, whereas Z-band streaming could be observed in the jump muscle of these flies. Although TpnT–CD70-expressing flies exhibited lower resistance to cardiac stress, their hearts were significantly more tolerant to Ca(2+) overloading induced by high-frequency electrical pacing. Our findings suggest that the Glu-rich long C-terminal extension of insect TnT functions as a myofilament Ca(2+) buffer/reservoir and is potentially critical to the high-frequency asynchronous contraction of flight muscles.

Troponin (Tn) is composed of three subunits (TnI, TnC and TnT) that bind Ca2+ and regulate striated muscle contraction in vertebrates. TnT’s function has been extensively described in vertebrates, but its role has been obscure in molluscan muscles. Our previous work indicated that the TnC and TnI subunits work in adductor phasic muscle, but not in catch muscle. Here, we have characterized TnT from the Japanese bivalve pearl oyster Pinctada fucata to start to explain the function of Tn in molluscan muscle contraction. We determined the primary structure of the full-length TnT protein from the P. fucata adductor muscle (Pifuc-TnT), and found that it is composed of 316 amino acid residues with a predicted molecular mass of 37.4 kDa. Multiple sequence alignment showed that Pifuc-TnT has an extension of >60 residues at the C-terminus that are not present in vertebrate TnTs, including known TnTs from other mollusks. Pifuc-TnT gene structure predictions using Splign alignment of the cDNA generated in this study and genome sequences indicated that Pifuc-TnT consists of 13 exons. Start and stop codons are located in exons 2 and 12, respectively. Quantitative real-time PCR revealed that the Pifuc-TnT gene was predominantly expressed in adductor phasic muscle, weakly in adductor catch muscle, slightly in gill, and not at all in mantle and foot. These findings suggest that TnT plays a regulatory role in adductor phasic muscle contraction, but not in catch contraction. Isothermal titration calorimetry revealed that unlike vertebrate TnTs, Pifuc-TnT does not interact with P. fucata tropomyosin-1 nor with tropomyosin-2. These findings in P. fucata imply that Tn functions differently in molluscan muscle than it does in vertebrates.

Aberrant regulation of myocardial force production represents an early biomechanical defect associated with sarcomeric cardiomyopathies, but the molecular mechanisms remain poorly defined. Here, we evaluated the pathogenicity of a previously unreported sarcomeric gene variant identified in a pediatric patient with sporadic dilated cardiomyopathy, and we determined a molecular mechanism. Trio whole-exome sequencing revealed a de novo missense variant in TNNC1 that encodes a p.I4M substitution in the N-terminal helix of cardiac troponin C (cTnC). Reconstitution of this human cTnC variant into permeabilized porcine cardiac muscle preparations significantly decreases the magnitude and rate of isometric force generation at physiological Ca(2+)-activation levels. Computational modeling suggests that this inhibitory effect can be explained by a decrease in the rates of cross-bridge attachment and detachment. For the first time, we show that cardiac troponin T (cTnT), in part through its intrinsically disordered C terminus, directly binds to WT cTnC, and we find that this cardiomyopathic variant displays tighter binding to cTnT. Steady-state fluorescence and NMR spectroscopy studies suggest that this variant propagates perturbations in cTnC structural dynamics to distal regions of the molecule. We propose that the intrinsically disordered C terminus of cTnT directly interacts with the regulatory N-domain of cTnC to allosterically modulate Ca(2+) activation of force, perhaps by controlling the troponin I switching mechanism of striated muscle contraction. Alterations in cTnC–cTnT binding may compromise contractile performance and trigger pathological remodeling of the myocardium.

Recently, our understanding of the structural basis of troponin-tropomyosin’s Ca2+-triggered regulation of striated muscle contraction has advanced greatly, particularly via cryo-electron microscopy data. Compelling atomic models of troponin-tropomyosin-actin were published for both apo- and Ca2+-saturated states of the cardiac thin filament. Subsequent electron microscopy and computational analyses have supported and further elaborated the findings. Per cryo-electron microscopy, each troponin is highly extended and contacts both tropomyosin strands, which lie on opposite sides of the actin filament. In the apo-state characteristic of relaxed muscle, troponin and tropomyosin hinder strong myosin-actin binding in several different ways, apparently barricading the actin more substantially than does tropomyosin alone. The troponin core domain, the C-terminal third of TnI, and tropomyosin under the influence of a 64-residue helix of TnT located at the overlap of adjacent tropomyosins are all in positions that would hinder strong myosin binding to actin. In the Ca2+-saturated state, the TnI C-terminus dissociates from actin and binds in part to TnC; the core domain pivots significantly; the N-lobe of TnC binds specifically to actin and tropomyosin; and tropomyosin rotates partially away from myosin’s binding site on actin. At the overlap domain, Ca2+ causes much less tropomyosin movement, so a more inhibitory orientation persists. In the myosin-saturated state of the thin filament, there is a large additional shift in tropomyosin, with molecular interactions now identified between tropomyosin and both actin and myosin. A new era has arrived for investigation of the thin filament and for functional understandings that increasingly accommodate the recent structural results.

Troponin is the Ca2+ molecular switch that regulates striated muscle contraction. In the heart, troponin Ca2+ sensitivity is also modulated by the PKA-dependent phosphorylation of a unique 31-residue N-terminal extension region of the Troponin I subunit (NH2-TnI). However, the detailed mechanism for the propagation of the phosphorylation signal through Tn, which results in the enhancement of the myocardial relaxation rate, is difficult to examine within whole Tn. Several models exist for how phosphorylation modulates the troponin response in cardiac cells but these are mostly built from peptide-NMR studies and molecular dynamics simulations. Here we used a paramagnetic spin labeling approach to position and track the movement of the NH2-TnI region within whole Tn. Through paramagnetic relaxation enhancement (PRE)-NMR experiments, we show that the NH2-TnI region interacts with a broad surface area on the N-domain of the Troponin C subunit. This region includes the Ca2+ regulatory Site II and the TnI switch-binding site. Phosphorylation of the NH2-TnI both weakens and shifts this region to an adjacent site on TnC. Interspin EPR distances between NH2-TnI and TnC further reveal a phosphorylation induced re-orientation of the TnC N-domain under saturating Ca2+ conditions. We propose an allosteric model where phosphorylation triggered cooperative changes in both the interaction of the NH2-TnI region with TnC, and the re-orientation of the TnC interdomain orientation, together promote the release of the TnI switch-peptide. Enhancement of the myocardial relaxation rate then occurs. Knowledge of this unique role of phosphorylation in whole Tn is important for understanding pathological processes affecting the heart.

Troponin is a complex of three proteins (troponin I, troponin C, and troponin T) that binds Ca2+ and is a thin filament-associated regulator of vertebrate striated muscle contraction. The function of troponin I (TnI) in vertebrates has been extensively characterized, but its role in molluscan muscles has not yet been elucidated. Our previous work suggested that the troponin C subunit has a role in adductor phasic muscle but not in catch muscle. Here, we investigated the molecular characteristics of TnI from the bivalve Japanese pearl oyster, Pinctada fucata to aid the elucidation of the function of molluscan muscle troponin. We determined the primary structure of the full-length TnI protein from the P. fucata adductor muscle (Pifuc-TnI) and found that it is composed of 286 amino acid residues with a predicted molecular weight of 33,737. Motif structure predictions and multiple sequence alignments revealed that Pifuc-TnI has a 138 residue extension at its N-terminus compared with rabbit TnI. This is analogous to characterized TnIs from other mollusks. However, unlike scallop TnI, Pifuc-TnI is predicted to contain two cAMP-dependent protein kinase phosphorylation sites, at residues 39 - 45 (RRGTEDD) and 145 - 151 (KKKSKRK). Phylogenetic analysis indicated that Pifuc-TnI and molluscan TnIs were grouped into the same clade. Pifuc-TnI gene structure predictions using Splign alignment of our obtained cDNA and genome sequences indicated that Pifuc-TnI consists of fifteen exons, with the start and stop codons located in exon 2 and exon 11, respectively. Using quantitative real-time PCR, we determined that the Pifuc-TnI gene is predominantly expressed in adductor phasic muscle, weakly in adductor catch muscle, and is not expressed in the gill, mantle or foot. These findings suggest that TnI, as a component of the troponin complex, plays a regulatory role in adductor phasic muscle contraction, but not in catch contraction.

Troponin is a thin filament-associated regulator of vertebrate striated muscle contraction. Troponin changes its structure upon Ca2+ binding to troponin C, one of the subunits of troponin, allowing myosin to interact with actin. We recently elucidated the molecular characteristics of the Japanese pearl oyster Pinctada fucata troponin C (Pifuc-TnC), revealing the possibilities that Pifuc-TnC and vertebrate muscle TnC play dissimilar roles in muscle contraction. Pifuc-TnC has four EF-hand motifs, but, unlike vertebrate TnC, only one (site IV) was predicted to bind Ca2+. To confirm the number of Ca2+-binding sites in Pifuc-TnC and whether Ca2+ binding induces a conformational change, we purified the full-length protein and a variant, Pifuc-TnC-E142Q (that has a mutation in the predicted Ca2+-binding site of site IV), following their expression in laboratory E. coli. Isothermal titration calorimetry demonstrated Ca2+ binding to Pifuc-TnC, whereas Pifuc-TnC-E142Q was unable to bind Ca2+, confirming that site IV is the only Ca2+-binding site in Pifuc-TnC. Pifuc-TnC eluted in a later fraction from a gel filtration column in the presence of Ca2+ compared with the condition when Ca2+ was absent. In contrast, the elution profiles of Pifuc-TnC-E142Q were equivalent in both the presence and absence of Ca2+, suggesting that Ca2+ binding to Pifuc-TnC induces a conformational change that delays its elution from the column. UV-absorption spectral analysis revealed that binding of Ca2+ to Pifuc-TnC caused an increase in absorption at a wavelength of approximately 250 nm, possibly because phenylalanine residues had been exposed on the surface of the molecule as a result of a conformational change. Differential scanning calorimetric analyses of Pifuc-TnC showed aggregation in the presence of Ca2+ in accordance with an increase of temperature, but no aggregation was seen in the absence of Ca2+. In combination, these findings suggest that Ca2+ binding to site IV induces a conformational change in Pifuc-TnC.

Many genetic mutations in sarcomeric proteins, including the cardiac myosin regulatory light chain (RLC) encoded by the MYL2 gene, have been implicated in familial cardiomyopathies. Yet, the molecular mechanisms by which these mutant proteins regulate cardiac muscle mechanics in health and disease remain poorly understood. Evidence has been accumulating that RLC phosphorylation has an influential role in striated muscle contraction and, in addition to the conventional modulation via Ca2+ binding to troponin C, it can regulate cardiac muscle function. In this review, we focus on RLC mutations that have been reported to cause cardiomyopathy phenotypes via compromised RLC phosphorylation and elaborate on pseudo-phosphorylation rescue mechanisms. This new methodology has been discussed as an emerging exploratory tool to understand the role of phosphorylation as well as a genetic modality to prevent/rescue cardiomyopathy phenotypes. Finally, we summarize structural effects post-phosphorylation, a phenomenon that leads to an ordered shift in the myosin S1 and RLC conformational equilibrium between two distinct states.

The troponin complex plays a central role in regulating the contraction and relaxation of striated muscles. Among the three protein subunits of troponin, the calcium receptor subunit, TnC, belongs to the calmodulin family of calcium signaling proteins whereas the inhibitory subunit, TnI, and tropomyosin-binding/thin filament-anchoring subunit, TnT, are striated muscle-specific regulatory proteins. TnI and TnT emerged early in bilateral symmetric invertebrate animals and have co-evolved during the 500-700 million years of muscle evolution. To understand the divergence as well as conservation of the structures of TnI and TnT in invertebrate and vertebrate organisms adds novel insights into the structure-function relationship of troponin and the muscle type isoforms of TnI and TnT. Based on the significant growth of genomic database of multiple species in the past decade, this focused review studied the primary structure features of invertebrate troponin subunits in comparisons with the vertebrate counterparts. The evolutionary data demonstrate valuable information for a better understanding of the thin filament regulation of striated muscle contractility in health and diseases.

Inhibition of striated muscle contraction at resting Ca(2+) depends on the C-terminal half of troponin I (TnI) in thin filaments. Much focus has been on a short inhibitory peptide (Ip) sequence within TnI, but structural studies and identification of disease-associated mutations broadened emphasis to include a larger mobile domain (Md) sequence at the C-terminus of TnI. For Md to function effectively in muscle relaxation, tight mechanical coupling to troponin's core-and thus tropomyosin-is presumably needed. We generated recombinant, human cardiac troponins containing one of two TnI constructs: either an 8-amino acid linker between Md and the rest of troponin (cTnILink8), or an Md deletion (cTnI1-163). Motility assays revealed that Ca(2+)-sensitivity of reconstituted thin filament sliding was markedly increased with cTnILink8 (∼0.9 pCa unit leftward shift of speed-pCa relation compared to WT), and increased further when Md was missing entirely (∼1.4 pCa unit shift). Cardiac Tn's ability to turn off filament sliding at diastolic Ca(2+) was mostly (61%), but not completely eliminated with cTnI1-163. TnI's Md is required for full inhibition of unloaded filament sliding, although other portions of troponin-presumably including Ip-are also necessary. We also confirm that TnI's Md is not responsible for superactivation of actomyosin cycling by troponin.

Estrogen (E2) and polyunsaturated fatty acids (n-3PUFA) supplements independently support general wellbeing and enhance muscle regeneration in-vivo and myotube formation in-vitro. However, the combined effect of E2 and n-3PUFA on myoblast differentiation is not known. The purpose of the study was to identify whether E2 and n-3PUFA possess a synergistic effect on in-vitro myogenesis. Mouse C2C12 myoblasts, a reliable model to reiterate myogenic events in-vitro, were treated with 10nM E2 and 50&mu, M eicosapentaenoic acid (EPA) independently or combined, for 0&ndash, 24 h or 0&ndash, 120 h during differentiation. Immunofluorescence, targeted qPCR and next generation sequencing (NGS) were used to characterize morphological changes and differential expression of key genes involved in the regulation of myogenesis and muscle function pathways. E2 increased estrogen receptor &alpha, (Er&alpha, ) and the expression of the mitogen-activated protein kinase 11 (Mapk11) within 1 h of treatment and improved myoblast differentiation and myotube formation. A significant reduction (p &lt, 0.001) in myotube formation and in the expression of myogenic regulatory factors Mrfs (MyoD, Myog and Myh1) and the myoblast fusion related gene, Tmem8c, was observed in the presence of EPA and the combined E2/EPA treatment. Additionally, EPA treatment at 48 h of differentiation inhibited the majority of genes associated with the myogenic and striated muscle contraction pathways. In conclusion, EPA and E2 had no synergistic effect on myotube formation in-vitro. Independently, EPA inhibited myoblast differentiation and overrides the stimulatory effect of E2 when used in combination with E2.

Activation of striated muscle contraction occurs in response to Ca(2+) binding to troponin C. The resulting reorganization of troponin repositions tropomyosin on actin and permits activation of myosin-catalyzed ATP hydrolysis. It now appears that the C-terminal 14 amino acids of cardiac troponin T (TnT) control the level of activity at both low and high Ca(2+). We made a series of C-terminal truncation mutants of human cardiac troponin T, isoform 2, to determine if the same residues of TnT are involved in the low and high Ca(2+) effects. We measured the effect of these mutations on the normalized ATPase activity at saturating Ca(2+). Changes in acrylodan tropomyosin fluorescence and the degree of Ca(2+) stimulation of the rate of binding of rigor myosin subfragment 1 to pyrene-labeled actin-tropomyosin-troponin were measured at low Ca(2+). These measurements define the distribution of actin-tropomyosin-troponin among the three regulatory states. Residues SKTR and GRWK of TnT were required for the functioning of TnT at both low and high Ca(2+). Thus, the effects on forming the inactive B-state and in retarding formation of the active M-state require the same regions of TnT. We also observed that the rate of binding of rigor subfragment 1 to pyrene-labeled regulated actin at saturating Ca(2+) was higher for the truncation mutants than for wild-type TnT. This violated an assumption necessary for determining the B-state population by this kinetic method.

Activation of striated muscle contraction occurs in response to Ca2+binding to troponin C (TnC). The resulting reorganization of troponin repositions tropomyosin on actin and permits activation of myosin catalyzed ATP hydrolysis. It now appears that the levels of activity at both low and saturating Ca2+are modulated by the C-terminal 14 amino acids of cardiac troponin T (TnT). We made a series of mutants of human cardiac troponin T, isoform 2, with deletions from the C-terminal end: Δ4, Δ6, Δ8, Δ10 and Δ14. We measured the effect of these mutations on the normalized ATPase activity at saturating Ca2+, the change in acrylodan tropomyosin fluorescence at low Ca2+, and the degree of Ca2+stimulation of the rate of binding of rigor myosin S1 to pyrene-labeled actin-tropomyosin-troponin. Together, these measurements define the distribution of actin-tropomyosin-troponin among the 3 regulatory states. Results from rates of rigor S1 binding deviated from other measurements when > 8 residues of TnT were deleted. That deviation was due to increased rates of binding of rigor S1 to pyrene-labeled actin with truncated TnT at saturating Ca2+. Such behavior violated a key assumption in the determination of the B state by this method. Nevertheless, all methods show that as residues were removed from the C-terminus of TnT there was approximately a proportional loss of the inactive B state at low Ca2+and an increase in the active M state at saturating Ca2+. Most of the C-terminal 14 residues of human cardiac troponin T are essential for forming the inactive B state at low Ca2+and for limiting the formation of the active M state at saturating Ca2+.

SUMMARY Striated muscle contraction is regulated by the movement of tropomyosin over the thin filament surface, which blocks or exposes myosin binding sites on actin. Findings suggest that electrostatic contacts, particularly those between K326, K328, and R147 on actin and tropomyosin, establish an energetically favorable F-actin-tropomyosin configuration, with tropomyosin positioned in a location that impedes actomyosin associations and promotes relaxation. Here, we provide data that directly support a vital role for these actin residues, termed the A-triad, in tropomyosin positioning in intact functioning muscle. By examining the effects of an A295S α-cardiac actin hypertrophic cardiomyopathy-causing mutation, over a range of increasingly complex in silico, in vitro, and in vivo Drosophila muscle models, we propose that subtle A-triad-tropomyosin perturbation can destabilize thin filament regulation, which leads to hypercontractility and triggers disease. Our efforts increase understanding of basic thin filament biology and help unravel the mechanistic basis of a complex cardiac disorder., In Brief Viswanathan et al. demonstrate that the conserved actin A-triad, composed of K326, K328, and R147, normally biases tropomyosin to a position that impedes actomyosin associations along resting striated muscle thin filaments. The proximally located actin A295S hypertrophic cardiomyopathy mutation distorts A-triad-tropomyosin associations, which promotes contractile disinhibition, hypercontraction, and disease pathogenesis.

Striated muscle contraction is regulated by the actin-associated proteins tropomyosin and troponin. The extent of activation of myosin ATPase activity is lowest in the absence of both Ca(2+) and activating cross-bridges (i.e., S1-ADP or rigor S1). Binding of activating species of myosin to actin at a saturating Ca(2+) concentration stabilizes the most active state (M state) of the actin–tropomyosin–troponin complex (regulated actin). Ca(2+) binding alone produces partial stabilization of the active state. The extent of stabilization at a saturating Ca(2+) concentration depends on the isoform of the troponin subunits, the phosphorylation state of troponin, and, in the case of cardiac muscle, the presence of hypertrophic cardiomyopathy-producing mutants of troponin T and troponin I. Cardiac dysfunction is also associated with mutations of troponin C (TnC). Troponin C mutants A8V, C84Y, and D145E increase the Ca(2+) sensitivity of ATPase activity. We show that these mutants change the distribution of regulated actin states. The A8V and C84Y TnC mutants decreased the inactive B state distribution slightly at low Ca(2+) concentrations, but the D145E mutants had no effect on that state. All TnC mutants increased the level of the active M state compared to that of the wild type, at a saturating Ca(2+) concentration. Troponin complexes that contained two mutations that stabilize the active M state, A8V TnC and Δ14 TnT, appeared to be completely in the active state in the presence of only Ca(2+). Because Ca(2+) gives full activation, in this situation, troponin must be capable of positioning tropomyosin in the active M state without the need for rigor myosin binding.

Summary Ryanodine receptors (RyRs) release Ca 2+ to initiate striated muscle contraction. Three highly divergent regions (DRs) in the RyR protein sequence (DR1, DR2, and DR3) may confer isoform-specific functional properties to the RyRs. We used cell-based fluorescence resonance energy transfer (FRET) measurements to localize these DRs to the cryoelectron microscopic (cryo-EM) map of the skeletal muscle RyR isoform (RyR1). FRET donors were targeted to RyR1 using five different FKBP12.6 variants labeled with Alexa Fluor 488. FRET was then measured to the FRET acceptors, Cy3NTA or Cy5NTA, targeted to decahistidine tags introduced within the DRs. DR2 and DR3 were localized to separate positions within the "clamp" region of the RyR1 cryo-EM map, which is presumed to interface with Ca v 1.1. DR1 was localized to the "handle" region, near the regulatory calmodulin-binding site on the RyR. These localizations provide insights into the roles of DRs in RyR allosteric regulation during excitation contraction coupling.

Troponin C (TnC) is the calcium-binding subunit of the troponin complex responsible for initiating striated muscle contraction in response to calcium influx. In the skeletal TnC isoform, calcium binding induces a structural change in the regulatory N-domain of TnC that involves a transition from a closed to open structural state and accompanying exposure of a large hydrophobic patch for troponin I (TnI) to subsequently bind. However, little is understood about how calcium primes the N-domain of the cardiac isoform (cTnC) for interaction with the TnI subunit as the open conformation of the regulatory domain of cTnC has been observed only in the presence of bound TnI. Here we use paramagnetic relaxation enhancement (PRE) to characterize the closed to open transition of isolated cTnC in solution, a process that cannot be observed by traditional nuclear magnetic resonance methods. Our PRE data from four spin-labeled monocysteine constructs of isolated cTnC reveal that calcium binding triggers movement of the N-domain helices toward an open state. Fitting of the PRE data to a closed to open transition model reveals the presence of a small population of cTnC molecules in the absence of calcium that possess an open conformation, the level of which increases substantially upon Ca(2+) binding. These data support a model in which calcium binding creates a dynamic equilibrium between the closed and open structural states to prime cTnC for interaction with its target peptide. We also used PRE data to assess the structural effects of a familial hypertrophic cardiomyopathy point mutation located within the N-domain of cTnC (A8V). The PRE data show that the Ca(2+) switch mechanism is perturbed by the A8V mutation, resulting in a more open N-domain conformation in both the apo and holo states.

Troponin T (TnT) is the tropomyosin (Tm)-binding and thin filament-anchoring subunit of troponin and plays a central role in striated muscle contraction. A nonsense mutation in exon 11 of the TNNT1 gene encoding slow skeletal muscle troponin T (ssTnT) truncating the polypeptide chain at Glu(180) causes a lethal recessive nemaline myopathy (NM) in the Amish (ANM). More TNNT1 NM mutations have been reported recently with similar recessive phenotypes. A nonsense mutation in exon 9 causes truncation at Ser(108), and a splicing site mutation causes truncation at Leu(203). Another splicing site mutation causes an internal deletion of the 39 exon 8-encoded amino acids. We engineered and characterized these ssTnT mutants to demonstrate that the Ser(108) truncation exhibits a Tm binding affinity lower than that of the ANM Glu(180) truncation, indicating a partial loss of Tm-binding site 1. Despite the presence of Tm-binding sites 1 and 2, ssTnT truncated at Leu(203) binds Tm with decreased affinity, consistent with its recessive NM phenotype and the requirement of troponin complex formation for high-affinity binding of TnT to Tm. The exon 8-deleted ssTnT has a partial loss of Tm-binding site 1 but retains high-affinity Tm-binding site 2. However, exon 8-deleted ssTnT exhibits a dramatically diminished Tm binding affinity, indicating a long-range conformational effect of this middle region deletion. Predicted from the TnT structure-function relationship, removal of the N-terminal variable region partially rescued this negative impact. These novel findings lay a foundation for understanding the pathogenesis of TNNT1 myopathies and provide insights into the development of targeted treatment.

End-to-end connections between adjacent tropomyosin molecules along the muscle thin filament allow long-range conformational rearrangement of the multicomponent filament structure. This process is influenced by Ca(2+) and the troponin regulatory complexes, as well as by myosin crossbridge heads that bind to and activate the filament. Access of myosin crossbridges onto actin is gated by tropomyosin, and in the case of striated muscle filaments, troponin acts as a gatekeeper. The resulting tropomyosin-troponin-myosin on-off switching mechanism that controls muscle contractility is a complex cooperative and dynamic system with highly nonlinear behavior. Here, we review key information that leads us to view tropomyosin as central to the communication pathway that coordinates the multifaceted effectors that modulate and tune striated muscle contraction. We posit that an understanding of this communication pathway provides a framework for more in-depth mechanistic characterization of myopathy-associated mutational perturbations currently under investigation by many research groups.

Striated muscle contraction is regulated primarily through the action of tropomyosin and troponin that are bound to actin. Activation requires Ca(2+) binding to troponin and/or binding of high affinity myosin complexes to actin. Mutations within components of the regulatory complex may lead to familial cardiomyopathies and myopathies. In several cases examined, either physiological or pathological changes in troponin alter the distribution among states of actin-tropomyosin-troponin that differ in their abilities to stimulate myosin ATPase activity. These observations open possibilities for managing disorders of the troponin complex. Furthermore, analyses of mutant forms of troponin give insights into the regulation of striated muscle contraction.

Troponin is well known as a Ca 2+ -dependent regulator of striated muscle contraction and it has been generally accepted that troponin functions as an inhibitor of muscle contraction or actin–myosin interaction at low Ca 2+ concentrations, and Ca 2+ at higher concentrations removes the inhibitory action of troponin. Recently, however, troponin became detectable in non-striated muscles of several invertebrates and in addition, unique troponin that functions as a Ca 2+ -dependent activator of muscle contraction has been detected in protochordate animals, although troponin in vertebrate striated muscle is known as an inhibitor of the contraction in the absence of a Ca 2+ . Further studies on troponin in invertebrate muscle, especially in non-striated muscle, would provide new insight into the evolution of regulatory systems for muscle contraction and diverse function of troponin and related proteins. The methodology used for preparation and characterization of functional properties of protochordate striated and smooth muscles will be helpful for further studies of troponin in other invertebrate animals.

Conditional deletion of Mbtps1 (cKO) protease in bone osteocytes leads to an age-related increase in mass (12%) and in contractile force (30%) in adult slow twitch soleus muscles (SOL) with no effect on fast twitch extensor digitorum longus muscles. Surprisingly, bone from 10-12-month-old cKO animals was indistinguishable from controls in size, density, and morphology except for a 25% increase in stiffness. cKO SOL exhibited increased expression of Pax7, Myog, Myod1, Notch, and Myh3 and 6-fold more centralized nuclei, characteristics of postnatal regenerating muscle, but only in type I myosin heavy chain-expressing cells. Increased expression of gene pathways mediating EGF receptor signaling, circadian exercise, striated muscle contraction, and lipid and carbohydrate oxidative metabolism were also observed in cKO SOL. This muscle phenotype was not observed in 3-month-old mice. Although Mbtps1 mRNA and protein expression was reduced in cKO bone osteocytes, no differences in Mbtps1 or cre recombinase expression were observed in cKO SOL, explaining this age-related phenotype. Understanding bone-muscle cross-talk may provide a fresh and novel approach to prevention and treatment of age-related muscle loss.

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 Ca2+ 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 Ca2+-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 Ca2+ 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 Ca2+-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 Ca2+ regulation of muscle thin filament through interaction with tropomyosin. Structured digital abstract • McTnT binds to Tropomyosin by enzyme linked immunosorbent assay (View interaction) • McTnI binds to TnC by enzyme linked immunosorbent assay (View interaction) • Tropomyosin physically interacts with McTnT and McTnI by enzyme linked immunosorbent assay (View interaction) • McTnI binds to TnT by enzyme linked immunosorbent assay (View interaction)

Troponin T (TnT) and troponin I (TnI) are two evolutionarily and functionally linked subunits of the troponin complex that regulates striated muscle contraction. We previously reported a single amino acid substitution in the highly conserved TnT-binding helix of cardiac TnI (cTnI) in wild turkey hearts in concurrence with an abnormally spliced myopathic cardiac TnT (cTnT) (Biesiadecki, B. J., Schneider, K. L., Yu, Z. B., Chong, S. M., and Jin, J. P. (2004) J. Biol. Chem. 279, 13825-13832). To investigate the functional effect of this cTnI mutation and its potential value in compensating for the cTnT abnormality, we developed transgenic mice expressing the mutant cTnI (K118C) in the heart with or without the deletion of the endogenous cTnI gene to mimic the homozygote and heterozygote of wild turkeys. Double and triple transgenic mice were created by crossing the cTnI-K118C lines with transgenic mice overexpressing the myopathic cTnT (exon 7 deletion). Functional studies of ex vivo working hearts found that cTnI-K118C alone had a dominantly negative effect on diastolic function and blunted the inotropic responses of cardiac muscle to beta-adrenergic stimuli without abolishing the protein kinase A-dependent phosphorylation of cTnI. When co-expressed with the cTnT mutation, cTnI-K118C corrected the significant depression of systolic function caused by cTnT exon 7 deletion, and the co-existence of exon 7-deleted cTnT minimized the diastolic abnormality of cTnI-K118C. Characterization of this naturally selected pair of mutually rescuing mutations demonstrated that TnI-TnT interaction is a critical link in the Ca(2+) signaling and beta-adrenergic regulation in cardiac muscle, suggesting a potential target for the treatment of troponin cardiomyopathies and heart failure.

Over the 40 years since its discovery, many studies have focused on understanding the role of troponin as a myofilament based molecular switch in regulating the Ca(2+)-dependent activation of striated muscle contraction. Recently, studies have explored the role of cardiac troponin as a target for cardiotonic agents. These drugs are clinically useful for treating heart failure, a condition in which the heart is no longer able to pump enough blood to other organs. These agents act via a mechanism that modulates the Ca(2+)-sensitivity of troponin; such a mode of action is therapeutically desirable because intracellular Ca(2+) concentration is not perturbed, preserving the regulation of other Ca(2+)-based signaling pathways. This review describes molecular details of the interaction of cardiac troponin with a variety of cardiotonic drugs. We present recent structural work that has identified the docking sites of several cardiotonic drugs in the troponin C-troponin I interface and discuss their relevance in the design of troponin based drugs for the treatment of heart disease.

Troponin (Tn), in association with tropomyosin (Tm), plays a central role in the calcium regulation of striated muscle contraction. Fluorescence resonance energy transfer (FRET) between probes attached to the Tn subunits (TnC, TnI, TnT) and to Tm was measured to study the spatial relationship between Tn and Tm on the thin filament. We generated single-cysteine mutants of rabbit skeletal muscle alpha-Tm, TnI and the beta-TnT 25-kDa fragment. The energy donor was attached to a single-cysteine residue at position 60, 73, 127, 159, 200 or 250 on TnT, at 98 on TnC and at 1, 9, 133 or 181 on TnI, while the energy acceptor was located at 13, 146, 160, 174, 190, 209, 230, 271 or 279 on Tm. FRET analysis showed a distinct Ca(2+)-induced conformational change of the Tm-Tn complex and revealed that TnT60 and TnT73 were closer to Tm13 than Tm279, indicating that the elongated N-terminal region of TnT extends beyond the beginning of the next Tm molecule on the actin filament. Using the atomic coordinates of the crystal structures of Tm and the Tn core domain, we searched for the disposition and orientation of these structures by minimizing the deviations of the calculated FRET efficiencies from the observed FRET efficiencies in order to construct atomic models of the Tn-Tm complex with and without bound Ca(2+). In the best-fit models, the Tn core domain is located on residues 160-200 of Tm, with the arrowhead-shaped I-T arm tilting toward the C-terminus of Tm. The angle between the Tm axis and the long axis of TnC is approximately 75 degrees and approximately 85 degrees with and without bound Ca(2+), respectively. The models indicate that the long axis of TnC is perpendicular to the thin filament without bound Ca(2+), and that TnC and the I-T arm tilt toward the filament axis and rotate around the Tm axis by approximately 20 degrees upon Ca(2+) binding.

ca2+ sensitivity of striated muscle contraction is modulated by a number of factors, the mechanism by which force is altered by Ca2+ appears to be complex. If we focus on the level of the regulatory, heterotrimeric troponin complex (Tn = TnC·TnI·TnT), not only an altered Ca2+ affinity of troponin

Striated muscle contraction is regulated by an interaction network connecting the effects of troponin, Ca(2+), and myosin-heads to the azimuthal positioning of tropomyosin along thin filaments. Many missense mutations, located at the actin-tropomyosin interface, however, reset the regulatory switching mechanism either by weakening or strengthening residue-specific interactions, leading to hyper- or hypo-contractile pathologies. Here, we compute energy landscapes for the actin-tropomyosin interface and quantify contributions of single amino acid residues to actin-tropomyosin binding. The method is a useful tool to assess effects of actin and tropomyosin mutations, potentially relating initial stages of myopathy to alterations in thin filament stability and regulation. Landscapes for mutant filaments linked to hyper-contractility provide a simple picture that describes a decrease in actin-tropomyosin interaction energy. Destabilizing the blocked (relaxed)-state parallels previously noted enhanced Ca(2+)-sensitivity conferred by these mutants. Energy landscapes also identify post-translational modifications that can rescue regulatory imbalances. For example, cardiomyopathy-associated E62Q tropomyosin mutation weakens actin-tropomyosin interaction, but phosphorylation of neighboring S61 rescues the binding-deficit, results confirmed experimentally by in vitro motility assays. Unlike results on hyper-contractility-related mutants, landscapes for tropomyosin mutants tied to hypo-contractility do not present a straightforward picture. These mutations may affect other components of the regulatory network, e.g., troponin-tropomyosin signaling.

Troponin senses Ca 2+ to regulate contraction in striated muscle. Structures of skeletal muscle troponin composed of TnC (the sensor), TnI (the regulator), and TnT (the link to the muscle thin filament) have been determined. The structure of troponin in the Ca 2+ -activated state features a nearly twofold symmetrical assembly of TnI and TnT subunits penetrated asymmetrically by the dumbbell-shaped TnC subunit. Ca ions are thought to regulate contraction by controlling the presentation to and withdrawal of the TnI inhibitory segment from the thin filament. Here, we show that the rigid central helix of the sensor binds the inhibitory segment of TnI in the Ca 2+ -activated state. Comparison of crystal structures of troponin in the Ca 2+ -activated state at 3.0 Å resolution and in the Ca 2+ -free state at 7.0 Å resolution shows that the long framework helices of TnI and TnT, presumed to be a Ca 2+ -independent structural domain of troponin are unchanged. Loss of Ca ions causes the rigid central helix of the sensor to collapse and to release the inhibitory segment of TnI. The inhibitory segment of TnI changes conformation from an extended loop in the presence of Ca 2+ to a short α-helix in its absence. We also show that Anapoe, a detergent molecule, increases the contractile force of muscle fibers and binds specifically, together with the TnI switch helix, in a hydrophobic pocket of TnC upon activation by Ca ions.

Several striated muscle myopathies have been directly linked to mutations in contractile and associated proteins. Troponin T (TnT) is one of the three subunits that form troponin (Tn) which together with tropomyosin is responsible for the regulation of striated muscle contraction. All three subunits of cardiac Tn as well as tropomyosin have been associated with hypertrophic cardiomyopathy (HCM). However, TnT accounts for most of the mutations that cause HCM in these regulatory proteins. To date 30 mutations have been identified in the cardiac TnT (CTnT) gene that results in familial HCM (FHC). The CTnT gene has also been associated with familial dilated cardiomyopathy (DCM). CTnT deficiency is lethal due to impaired cardiac development. A recessive nonsense mutation in the gene encoding slow skeletal TnT has been associated with an unusual, severe form of nemaline myopathy among the Old Order Amish. How each mutation leads to the diverse clinical symptoms associated with FHC, DCM or nemaline myopathy is unclear. However, the use of animal model systems, in particular transgenic mice, has significantly increased our knowledge of normal and myopathic muscle physiology. In this review, we focus on the role of TnT in muscle physiology and disease.

In vertebrates, three troponin T (TnT) genes, cardiac TnT (cTnT), skeletal muscle fast-twitch TnT (fTnT), and slow-twitch TnT (sTnT), have evolved for the regulation of striated muscle contraction. To understand the mechanism for muscle fiber-specific expression of the TnT genes, we compared their expression patterns during mouse development. Our data revealed that the TnT expression in the developing embryo was not as restricted as that in the adult. In addition to a strong expression in the developing heart beginning at day 7.5 p.c (postcoitum), the cTnT transcript was detected at later stages in some skeletal muscles, where beginning at day 11.75 p.c. the fTnT and sTnT genes were also expressed. Only sTnT but not fTnT was found transiently in the developing heart. At day 13.5 p.c., expressions of all three genes were detected in the developing tongue and this co-expression continued to day 16.5 p.c. with the fTnT isoform being predominant. At this stage, overlapping and distinct expression patterns of both sTnT and fTnT genes were also evident in many developing skeletal muscles. These data suggest that different muscles during development undergo a complex change in TnT isoforms resulting in different contractile properties. Unexpectedly, the cTnT transcript was persistently found in the developing bladder, where presumably smooth muscle is present. In transgenic mice, expression of a LacZ gene driven by a rat cTnT promoter (−497 to +192 bp) was very similar to that of the endogenous cTnT gene, suggesting that this promoter contained regulatory elements sufficient for the control of tissue-specific cTnT expression during development. Anat Rec 263:72–84, 2001. © 2001 Wiley-Liss, Inc.

Contraction of skeletal and cardiac muscles is regulated by Ca2+ through a specific Ca(2+)-receptive protein, troponin, regularly distributed along the thin filaments. This protein consists of three different subunits, troponins C, I and T. In this article, studies on the structural and biochemical aspects of the molecular mechanisms of Ca(2+)-regulation were first reviewed with particular reference to the regulatory role of troponin T. Several properties of the isoforms of troponin from fast and slow skeletal and cardiac muscles were discussed, based on the findings obtained by the use of troponin-exchange techniques under physiological conditions. Recent findings on the functional consequence of mutations in human cardiac troponins T and I found in familial hypertrophic cardiomyopathy were also presented. The results clarified the increase in Ca(2+)-sensitivity of contraction to be the critical consequence due to this genetic disorder.

Troponin T (TnT) plays an allosteric signal transduction role in the actin thin-filament-based Ca(2+)-regulation of striated muscle contraction. Developmentally regulated alternative RNA splicing produces TnT isoforms differing in their NH(2)-terminal structure. Physical property variations of the NH(2)-terminal hypervariable region of TnT may have a role in tuning the Ca(2+)-sensitivity and overall cooperativity of the muscle. We have previously demonstrated that metal ion or monoclonal antibody binding to the NH(2)-terminal region can modulate the epitopic conformation and troponin I and tropomyosin binding affinity of TnT. To further establish the molecular basis of this conformational and functional modulation, we have characterized the NH(2)-terminal variable region-originated secondary conformational effect in TnT using fluorescence spectral analysis. The chicken fast skeletal muscle TnT isoform, TnT8e16, containing a cluster of transition-metal ion binding sites (Tx) in the NH(2)-terminal variable region was used in this study. TnT8e16 was titrated for Cu(II) binding-induced changes in fluorescence intensity and anisotropy of the COOH-domain Trp residues (W234, W236, and W285), which demonstrated considerable environmental sensitivity in TnT denaturation studies. Nonlinear Stern-Volmer plots of Trp quenching indicated a metal ion binding-induced conformational change in TnT. Fluorescence anisotropy changes upon metal ion binding indicated a decrease in the mobility of the Trp residues and an increase in the flexibility of fluorescein-labeled Cys263 in the COOH domain. These data support a model that the alternatively spliced NH(2)-terminal variable region of TnT modulates conformation and flexibility of other domains of the protein.

Troponin T (TnT) is an essential protein in the transduction of the Ca 2+ -binding signal that triggers striated muscle contraction. Functional diversity among various TnT isoforms found in cardiac and skeletal muscles has been correlated with the sequence heterogeneity at the amino (N-) and the carboxyl (C-) terminal regions. The most striking difference between cardiac TnT (cTnT) and skeletal TnT (sTnT) is that cTnT has an extended N-terminus, which is rich in negatively charged amino acids. To investigate the role of this region in cTnT, we deleted the first 76 amino acids in rat cTnT (cTnT 77–289 ) by site-directed mutagenesis. We exchanged the native troponin complex in rat cardiac myofibrillar preparations and detergent skinned cardiac fiber bundles by treatment with excess cTnT or cTnT 77–289 . After reconstituting the cTnT 77–289 containing myofibrils with cardiac troponin I–cardiac troponin C (cTnI–cTnC), the MgATPase activity was 70% of the cTnT treated myofibrils in the relaxed state and 83% of the cTnT treated myofibrils in the maximal Ca 2+ -activated state. These observations were supported by force measurements in which cTnT and cTnT 77–289 were exchanged into skinned fiber bundles. Prior to reconstitution with cTnI–cTnC, the Ca 2+ -independent maximal force developed by the cTnT 77–289 containing fiber was 45% of the force developed by the cTnT containing fiber. After reconstituting with cTnI–cTnC, the Ca 2+ -activated maximal force of the cTnT 77–289 containing fiber was 62% of the force developed by the cTnT containing +cTnI–cTnC reconstituted fiber. In both assays, no significant changes in the normalized Ca 2+ -activity relation or in co-operativity were observed. Fluorescence experiments using pyrene-labeled Tm demonstrated that the binding of cTnT 77–289 to Tm was 3–4 fold stronger than that of cTnT. Our results suggest that strong interactions between cTnT 77–289 and Tm stabilize cardiac myofilaments in a sub-maximally activated state. Our findings also indicate that the N-terminus of cTnT is essential for maximal activation of cardiac myofilaments.

Ca2+ regulation of vertebrate striated muscle contraction is initiated by conformational changes in the N-terminal, regulatory domain of the Ca2+-binding protein troponin C (TnC), altering the interaction of TnC with the other subunits of troponin complex, TnI and TnT. We have investigated the role of acidic amino acid residues in the N-terminal, regulatory domain of TnC in binding to the inhibitory region (residues 96–116) of TnI. We constructed three double mutants of TnC (E53A/E54A, E60A/E61A and E85A/D86A), in which pairs of acidic amino acid residues were replaced by neutral alanines, and measured their affinities for synthetic inhibitory peptides. These peptides had the same amino acid sequence as TnI segments 95–116, 95–119 or 95–124, except that the natural Phe-100 of TnI was replaced by a tryptophan residue. Significant Ca2+-dependent increases in the affinities of the two longer peptides, but not the shortest one, to TnC could be detected by changes in Trp fluorescence. In the presence of Ca2+, all the mutant TnCs showed about the same affinity as wild-type TnC for the inhibitory peptides. In the presence of Mg2+ and EGTA, the N-terminal, regulatory Ca2+-binding sites of TnC are unoccupied. Under these conditions, the affinity of TnC(E85A/D86A) for inhibitory peptides was about half that of wild-type TnC, while the other two mutants had about the same affinity. These results imply a Ca2+-dependent change in the interaction of TnC Glu-85 and/or Asp-86 with residues (117–124) on the C-terminal side of the inhibitory region of TnI. Since Glu-85 and/or Asp-86 of TnC have also been demonstrated to be involved in Ca2+-dependent regulation through interaction with TnT, this region of TnC must be critical for troponin function.

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.

Tropomyosin, a coiled-coil dimer, stabilizes actin filaments and is central to the control of calcium-regulated striated muscle contraction. Striated muscle-specific alpha-tropomyosin is the predominant isoform in cardiac muscle, with low levels of beta-tropomyosin restricted to fetal development in the mouse. To understand the functional role of various tropomyosin isoforms during myofilament activation and regulation in the intact sarcomere, we generated transgenic mice that overexpress striated muscle-specific beta-tropomyosin in the adult heart. Our earlier results succinctly demonstrate that overexpression of beta-tropomyosin in the hearts of transgenic mice decreases endogeneous alpha-tropomyosin levels while altering diastolic function of the myocardium. To explore further the significance of altering the alpha- to beta-tropomyosin isoform ratio in developing murine myocardium, we generated transgenic mice which express beta-tropomyosin at high levels in the heart. The data show that higher levels of beta-tropomyosin expression are lethal with death ensuing between 10-14 days postnatally. A detailed histological analysis demonstrates that the hearts of these mice exhibit several pathological abnormalities, including thrombus formation in the lumen of both atria and in the subendocardium of the left ventricle. Other changes include atrial enlargement and fibrosis, and diffuse myocytolysis, Physiological analyses using ventricular muscle strip preparations from these mice reveal that both myocardial contraction and relaxation parameters are severely impaired. Thus, these results firmly demonstrate an essential difference in tropomyosin isoform function in physiologically regulating cardiac performance.

Troponin T (TnT), a thin filament myofibrillar protein, is essential for the Ca 2+ regulation of striated muscle contraction in vertebrates, both in vivo and in vitro . To understand the role of TnT in this process, its interaction with two other troponin components, troponin I (TnI) and troponin C (TnC) was examined by using the yeast two hybrid system, which is a genetic approach to detect protein-protein interactions. Computer assisted analysis of phylogenetically distant TnT amino acid sequences unveiled a highly conserved protein domain that is characterized by a heptad repeat (HR) motif with a potential for α-helical coiled coil formation. A similar, potentially coiled coil forming domain is also conserved in all known TnI sequences. These protein motifs appeared to be the regions where TnI-TnT interaction may take place. Deletions and point mutations in TnT, which disrupted its HR motif, severely reduced or abolished TnI binding, but binding to TnC was not affected, indicating that the TnT-TnI and TnT-TnC binary interactions can be uncoupled. Remarkably, the truncated fragments of TnT and TnI in which the HR motifs were retained showed binary interaction in the yeast two hybrid system. It was also observed that the formation of the TnT-TnI heterodimers is favored over the homodimers TnT-TnT and TnI-TnI. These results indicate that the evolutionarily conserved HR motifs may play a role in TnT-TnI dimerization, presumably through the formation of α-helical coiled coils.

Troponin T (TnT) is present in striated muscle of vertebrates and invertebrates as a group of homologous proteins with molecular weights usually in the 31–36kDa range. It occupies a unique role in the regulatory protein system in that it interacts with TnC and TnI of the troponin complex and the proteins of the myofibrillar thin filament, tropomyosin and actin. In the myofibril the molecule is about 18nm long and for much its length interacts with tropomyosin. The ability of TnT to form a complex with tropomyosin is responsible for locating the troponin complex with a periodicity of 38.5nm along the thin filament of the myofibril. In addition to its structural role, TnT has the important function of transforming the TnI–TnC complex into a system, the inhibitory activity of which, on the tropomyosin–actomyosin MgATPase of the myofibril, becomes sensitive to calcium ions. Different genes control the expression of TnT in fast skeletal, slow skeletal and cardiac muscles. In all muscles, and particularly in fast skeletal, alternative splicing of mRNA produces a series of isoforms in a developmentally regulated manner. In consequence TnT exists in many more isoforms than any of the other thin filament proteins, the TnT superfamily. Despite the general homology of TnT isoforms, this alternative splicing leads to variable regions close to the N-␣and C-termini. As the isoforms have slightly different effects on the calcium sensitivity of the actomyosin MgATPase, modulation of the contractile response to calcium can occur during development and in different muscle types. TnT has recently aroused clinical interest in its potential for detecting myocardial damage and the association of mutations in the cardiac isoform with hypertrophic cardiomyopathy.

Troponin (Tn), the complex of three subunits (TnC, TnI and TnT), plays a key role in calcium-dependent regulation of striated muscle contraction. In order to elucidate the interactions between the Tn subunits, we have determined the crystal structure of TnC in complex with the N-terminal fragment of TnI (TnI1-47) at 2.3-angstrom resolution. The TnC/TnI1-47 structure implies a mechanism of how Tn regulate the muscle contraction and suggests a unique a-helical regulatory TnI segment, which binds to the N-lobe of TnC in its calcium-bound comformation.

Troponin (Tn), consisting of three subunits, TnT, TnC, and TnI, plays a crucial role in the calcium-dependent regulation of vertebrate striated muscle contraction. In the present study, we have applied limited proteolysis to the Tn complex in order to study domain structures and to detect conformational differences of Tn under different conditions. We found that both TnT and TnI were susceptible to chymotryptic digestion: while TnT was cleaved into TnT-(1-158)-peptide and TnT-(159-259)-peptide irrespective of Ca2+ concentration, the cleavage sites of TnI were dependent on the Ca2+ occupancy of TnC. In addition, we characterized the effects of depletion of the C-terminal part of TnI on acto-S1 ATPase activity. The TnT-(159-259)-peptide-TnC-TnICa-frag complex [TnICa-frag = (TnI-(1-134 and 1-140)-peptide], which was produced in the presence of CaCl2 and MgCl2, retains both the activating and inhibitory capabilities of whole Tn on the acto-S1 ATPase activity, while TnT-(159-259)-peptide-TnC-TnIMg-frag complex [TnIMg-frag = (TnI-(1-116)-peptide], which was obtained in the presence of MgCl2 and EGTA, lost its ability to activate acto-S1 ATPase activity. Our results indicate that residues 117-134 or 117-140 of TnI undergo structural changes upon Ca(2+)-binding to the regulatory sites of TnC and are necessary for the Ca(2+)-dependent inhibitory action of the Tn complex on acto-S1 ATPase activity. We also showed that residues 135-181 or 141-181 of TnI are involved in the interaction of Tn with the tropomyosin-actin filament.

The striated muscle thin filament comprises actin, tropomyosin, and troponin. The Tn complex consists of three subunits, troponin C (TnC), troponin I (TnI), and troponin T (TnT). TnT may serve as a bridge between the Ca2+ sensor (TnC) and the actin filament. In the short helix preceding the IT-arm region, H1(T2), there are known dilated cardiomyopathy-linked mutations (among them R205L). Thus we hypothesized that there is an element in this short helix that plays an important role in regulating the muscle contraction, especially in Ca2+ activation. We mutated Arg-205 and several other amino acid residues within and near the H1(T2) helix. Utilizing an alanine replacement method to compare the effects of the mutations, the biochemical and mechanical impact on the actomyosin interaction was assessed by solution ATPase activity assay, an in vitro motility assay, and Ca2+ binding measurements. Ca2+ activation was markedly impaired by a point mutation of the highly conserved basic residue R205A, residing in the short helix H1(T2) of cTnT, whereas the mutations to nearby residues exhibited little effect on function. Interestingly, rigor activation was unchanged between the wild type and R205A TnT. In addition to the reduction in Ca2+ sensitivity observed in Ca2+ binding to the thin filament, myosin S1-ADP binding to the thin filament was significantly affected by the same mutation, which was also supported by a series of S1 concentration-dependent ATPase assays. These suggest that the R205A mutation alters function through reduction in the nature of cooperative binding of S1. Background: Striated muscle contraction is regulated by Ca2+ binding to troponin. Results: A mutation at position 205 of cardiac troponin T drastically attenuates the Ca2+ activation of the thin filament by altering its properties. Conclusion: The main effect of this mutation is on the cooperativity in the thin filament activation. Significance: Our study sheds light on how mutations in troponin T affect the thin filament regulation.

Cardiac and skeletal muscle contraction are activated by Ca2+ binding to specific regulatory sites on the striated muscle thin filament. The thin filament is a large allosteric assembly, containing multiple copies of actin, tropomyosin, and the three troponin subunits (troponin C, troponin I, and troponin T). This review describes recent developments in understanding the structure and dynamics of these proteins and how they function to regulate contraction. Emphasis is placed on the cardiac thin filament and on the features that distinguish it from the skeletal muscle system. The discussion also emphasizes current knowledge of the protein-protein interactions involved in the cooperative processes of thin filament assembly and regulation.

Mild proteolytic cleavage of the troponin complex yields TnT1, the N-terminal fragment of troponin T, and TnT2IC, a complex of the C-terminal fragment of troponin T (TnT2) with troponin I (TnI) and troponin C (TnC) [Morris, E. P., & Lehrer, S. S. (1984) Biochemistry 23, 2214-2220]. Both TnT1 and TnT2IC bind tightly to the tropomyosin.actin (Tm.actin) thin filament and influence the interaction of myosin subfragment 1 (S1) with Tm.actin. TnT1 does not affect the rate of S1 binding to Tm.actin but does increase the cooperativity with which S1 "turns on" Tm.actin, monitored by the excimer fluorescence of a pyrene label attached to Cys 190 of Tm [Geeves, M.A., & Lehrer, S. S. (1994) Biophys. J. 67, 273-282]. The apparent cooperative unit size of Tm.actin is increased from 6 to 9 by TnT1 and to 12 by whole troponin. In contrast, TnT2IC has no effect on the cooperativity of Tm.actin but does make the apparent S1-binding rate constant, kapp, Ca(2+)-sensitive; i.e., in the absence of Ca2+, kapp is reduced 2-3-fold by both TnT2IC and whole troponin. Thus, the N- and C-terminal regions of TnT appear to act independently in modulating effects of S1 binding to the Tm.actin thin filament that are important in regulation.

Troponin (Tn), containing three subunits: Ca2+ binding (TnC), inhibitory (TnI), and tropomyosin binding (TnT), plays a crucial role in the Ca2+ regulation of vertebrate striated muscle contraction. These three subunits function by interacting with each other and with the other thin filament proteins. Previous studies suggested that the primary role of TnT is to anchor the TnI.TnC complex to the thin filament, primarily through its interactions with TnI and tropomyosin. We propose here a new role for TnT. Our results indicate that, when TnT is combined with the TnI.TnC complex, there is an activation of actomyosin ATPase that is Ca(2+)-dependent. To determine whether the latter results from a direct effect of TnC on TnT or indirectly from an effect of TnC on TnI which is transmitted to TnT, we prepared a deletion mutant (deletion of residues 1-57) of TnI, TnId57 (Sheng et al. (1992) J. Biol. Chem. 267, 25407-25413), which interacts with TnC but not TnT. Both wild type (TnI.TnC.TnT) and mutant (TnId57.TnC.TnT) Tn complexes demonstrated equivalent activity in the Ca2+ regulation of actomyosin-S1 ATPase activity. Similarly, both TnI and TnId57 could equally reconstitute TnI-depleted skinned muscle fibers. Therefore, since TnId57 does not interact with TnT, these results suggest that TnT reconstitutes native Ca2+ sensitivity via direct interaction with TnC. Thus Ca2+ binding to TnC would have a dual role: 1) release of the ATPase inhibition by TnI and 2) activation of the ATPase through interaction with TnT.

In past decades, several investigators have characterized deficiencies in striated muscle contraction in murine models of Duchenne muscular dystrophy. We have identified in multiple studies potential beneficial therapeutic combinations of ACE inhibitors and anti-mineralocorticoids. There has been an almost exclusive focus on the force deficits in tetanic contractions of extensor digitorum longus (EDL) and diaphragm muscles from these models, as well as their susceptibility to fatigue from repeated contractions. However the force of contraction is not of sole importance. The kinetics of twitch contractions and baseline tension both are critically important parameters that can impact dysfunction. Here we show EDL and diaphragm muscles from dystrophin deficient (mdx) mice have significantly slower kinetic parameters of contraction and relaxation than their wild-type counterparts. We have also found that treatment with Lisinopril and Spironolactone can significantly alter these parameters, and in some cases, back to near-wild type values. Additionally, we investigated kinetic changes in another dystrophin deficient model that is also haplosufficient for compensating utrophin treated with Eplerenone. By calculating the impulse of twitch contractions, we can not only determine work being performed, but also the magnitude of previously mentioned kinetic parameters on this computation. We illustrate that the kinetic information in contractile measurements can further elucidate our understanding of both underlying pathology, as well as serve as quantitative markers of treatment effectiveness.

The contraction of striated muscle (represented by vertebrate skeletal and cardiac muscles) is powered by actin-activated myosin II ATPase. The contractile machinery in striated muscles is the numerous myofibrils that consist of serially connected contractile units called sarcomeres. A sarcomere is composed of overlapping myosin thick filaments and actin thin filaments. In vertebrate striated muscles, contraction is generated from ATP hydrolysis during actomyosin cross-bridge cycling that is regulated by intracellular Ca2+ transient via the thin filament-associated proteins troponin and tropomyosin (1). Residing at ~37 nm intervals along the thin filament of F-actin-tropomyosin double helices (2, 3, 4), the troponin complex consists of three protein subunits: The Ca2+-binding subunit troponin C (TnC1), the actomyosin ATPase inhibiting subunit troponin I (TnI), and the tropomyosin-binding subunit troponin T (TnT) (5). The name of TnT was based on the original ultracentrifugation and co-crystallization studies that verified TnT interaction with tropomyosin as the key subunit holding the troponin complex on the thin filament (6). To convert the cellular signal of cytosolic Ca2+ transient originated from sarcolemmal electrical activity to myofilament movements during each excitation-contraction-relaxation cycle, troponin functions through cooperative interactions among the three subunits and with tropomyosin (1, 7). While TnC, a relative of the calmodulin gene family, functions as the Ca2+ receptor in the thin filament regulatory system, TnI and TnT are striated muscle-specific proteins encoded by closely linked genes and have co-evolved into three pairs of fiber type-specific isoforms (8, 9). In addition to anchoring the troponin complex to the thin filament, TnT directly interacts with all key components in the thin filament regulatory system to play an organizer role in the troponin complex [7, 10]. Through isoform gene regulation, alternative RNA splicing, and posttranslational modifications, structural and functional variations of TnT modulate striated muscle contraction and relaxation. To help understanding the structure-function relationship of TnT in physiological and disease conditions, this review focuses on the evolution of muscle type-specific TnT isoform genes, the multiple alternative splice forms, the developmental regulation of isoform expression and alternative splicing, and the posttranslational modifications in physiological and pathophysiological adaptations. For background information, comprehensive summaries of striated muscle thin filament regulation and the functions of TnC, TnI and tropomyosin can be found in several previous review articles (1, 7, 8, 10, 11, 12, 13, 14).

Troponin T (TnT) binds to tropomyosin (Tm) to anchor the troponin complex in the thin filament, and it thus serves as a vital link in the Ca(2+) regulation of striated muscle contraction. Pioneer work three decades ago determined that the T1 and T2 chymotryptic fragments of TnT each contains a Tm-binding site. A more precise localization of the two Tm-binding sites of TnT remains to be determined. In the present study, we tested serial deletion constructs of TnT and carried out monoclonal antibody competition experiments to show that the T1 region Tm-binding site involves mainly a 39 amino acids segment in the N-terminal portion of the conserved middle region of TnT. We further employed another set of TnT fragments to locate the T2 region Tm-binding site to a segment of 25 amino acids near the beginning of the T2 fragment. The localization of the two Tm-binding sites of TnT provided new information for the structure-function relationship of TnT and the anchoring of troponin complex on muscle thin filament.