The molecular interaction of six single-stranded DNA aptamers to cardiac troponin I revealed by docking and molecular dynamics simulation.

Cardiac troponin I (cTnI) is a cardiac biomarker for diagnosing ischemic heart disease and acute myocardial infarction. Current biochemical assays use antibodies (Abs) due to their high specificity and sensitivity. However, there are some limitations, such as the high-cost production of Abs due to complex instruments, reagents, and steps; the variability of Abs quality from batch to batch; the low stability at high temperatures; and the difficulty of chemical modification. Aptamer overcomes the limitations of antibodies, such as relatively lower cost, high reproducibility, high stability, and ease of being chemically modified. Aptamers are three-dimensional architectures of single-stranded RNA or DNA that bind to targets such as proteins. Six aptamers (Tro1-Tro6) with higher binding affinity than an antibody have been identified, but the molecular interaction has not been studied. In this study, six DNA aptamers were modeled and docked to cTnI protein. Molecular docking revealed that the interaction between all aptamer and cTnI happened in the similar cTnI region. The interaction between aptamer and cTnI involved hydrophobic interaction, hydrogen bonds, π-cation interactions, π-stack interactions, and salt-bridge formation. The calculated binding energy of all complexes was negative, which means that the complex formation was thermodynamically favorable. The electrostatic energy term was the main driving force of the interaction between all aptamer and cTnI. This study could be used to predict the behavior of further modified aptamer to improve aptamer performance.

Ternary BiOI/Bi2S3/Au Nanosheet Arrays as a Photoelectrochemical Signal Converter for the Detection of Cardiac Troponin I.

Nanosheet arrays with stable signal output have become promising photoactive materials for photoelectrochemical (PEC) immunosensors. However, an essential concern is the facile recombination of carriers in one-component nanoarrays, which cannot be readily prevented, ultimately resulting in weak photocurrent signals. In this study, an immunosensor using gold nanoparticle-anchored BiOI/Bi2S3 nanosheet arrays (BiOI/Bi2S3/Au) as a signal converter was fabricated for sensitive detection of cardiac troponin I (cTnI). The ternary nanosheet arrays were prepared by a simple method in which Bi2S3 was well-coated on the BiOI surface by in situ growth, whereas the addition of Au further improved the photoelectric conversion efficiency and could link more antibodies. The three-dimensional (3D) ordered sheet-like network array structure and BiOI/Bi2S3/Au ternary nanosheet arrays showed stable and high photoelectric signal output and no significant difference in signals across different batches under visible light excitation. The fabricated immunosensor has a sensitive response to the target detection marker cTnI in a wide linear range of 500 fg/mL to 50 ng/mL, and the detection limit was 32 fg/mL, demonstrating good stability and selectivity. This work not only shows the great application potential of ternary heterojunction arrays in the field of PEC immunosensors but also provides a useful exploration for improving the stability of immunosensors.

Enhancing the Electrochemiluminescence of Porphyrin via Crystalline Networks of Metal-Organic Frameworks for Sensitive Detection of Cardiac Troponin I.

Porphyrins easily aggregate due to unfavorable π-π accumulation, causing luminescent quenching in the aqueous phase and subsequently reducing luminescent efficiency. It is a feasible way to immobilize porphyrin molecules through metal-organic framework materials (MOFs). In this study, 5,10,15,20-tetrakis (4-carboxyphenyl) porphyrin (TCPP) was introduced into the metal-organic skeleton (PCN-224) as a ligand. The result showed that the electrochemiluminescence (ECL) and photoluminescence (PL) efficiency of the MOF skeleton was 8.2 and 6.5 times higher than TCPP, respectively. Impressively, the periodic distribution of porphyrin molecules in the MOF framework can overcome the bottleneck of porphyrin aggregation, resulting in the organic ligand TCPP participating in the electron transfer reaction. Herein, based on the PCN-224, a sandwich-type ECL immunosensor was constructed for the determination of cardiac troponin I (cTnI). It provided sensitive detection of cTnI in the range of 1 fg/mL to 10 ng/mL with a detection limit of 0.34 fg/mL. This work not only innovatively exploited a disaggregation ECL (DIECL) strategy via the crystalline framework of MOF to enhance the PL and ECL efficiency of porphyrin but also provided a promising ECL platform for the ultrasensitive monitoring of cTnI.

Umbrella Sampling Simulations of Cardiac Thin Filament Reveal Thermodynamic Consequences of Troponin I Inhibitory Peptide Mutations.

The cardiac thin filament comprises F-actin, tropomyosin, and troponin (cTn). cTn is composed of three subunits: troponin C (cTnC), troponin I (cTnI), and troponin T (cTnT). To computationally study the effect of the thin filament on cTn activation events, we employed targeted molecular dynamics followed by umbrella sampling using a model of the thin filament to measure the thermodynamics of cTn transition events. Our simulations revealed that the thin filament causes an increase in the free energy required to open the cTnC hydrophobic patch and causes a more favorable interaction between this region and the cTnI switch peptide. Mutations to the cTn complex can lead to cardiomyopathy, a collection of diseases that present clinically with symptoms of hypertrophy or dilation of the cardiac muscle, leading to impairment of the heart's ability to function normally and ultimately myocardial infarction or heart failure. Upon introduction of cardiomyopathic mutations to R145 of cTnI, we observed a general decrease in the free energy of opening the cTnC hydrophobic patch, which is on par with previous experimental results. These mutations also exhibited a decrease in electrostatic interactions between cTnI-R145 and actin-E334. After introduction of a small molecule to the wild-type cTnI-actin interface to intentionally disrupt intersubunit contacts, we successfully observed similar thermodynamic consequences and disruptions to the same protein-protein contacts as observed with the cardiomyopathic mutations. Computational studies utilizing the cTn complex in isolation would have been unable to observe these effects, highlighting the importance of using a more physiologically relevant thin-filament model to investigate the global consequences of cardiomyopathic mutations to the cTn complex.

Evaluation of cardiac troponin I as a predictor of death in critically ill cats.

BACKGROUND: Abnormally high serum cardiac troponin I (cTnI) concentration, reflecting leakage from or necrosis of cardiomyocytes, is a negative prognosticator for death in dogs. OBJECTIVES: To investigate in critically ill cats whether serum cTnI concentration is abnormally high, identify conditions associated with abnormally high cTnI concentrations, and evaluate cTnI as an independent prognosticator for death and a potential coprognosticator to the acute patient physiologic and laboratory evaluation (APPLE) score in cats. ANIMALS: One hundred nineteen cats admitted to intensive care units (ICU) and 13 healthy cats at 2 university teaching hospitals. METHODS: Prospective study. Clinical examinations were performed, APPLE scores calculated, and serum cTnI and serum amyloid A (SAA) measured within 24 hours after admission. Outcome was defined as death/euthanasia or survival to discharge, 28 and 90 days after ICU-admission. Prognostic capacity of cTnI, APPLE scores and models combining cTnI and scores were evaluated by receiver-operator-characteristic analyses. RESULTS: Median (IQR) serum cTnI concentration was higher in ill (0.63 [0.18-2.65] ng/mL) compared to healthy (0.015 [0.005-0.041] ng/mL) cats (P < .001) and higher in subgroups with structural cardiac disease (2.05 [0.54-16.59] ng/mL; P < .001) or SAA >5 mg/L (0.84 [0.23-2.81] ng/mL; P = .009) than in cats without these characteristics (0.45 [0.12-1.70] and 0.35 [0.015-0.96] ng/mL). The in-hospital case fatality rate was 29%. Neither serum cTnI concentration for all critically ill cats (area-under-the-curve 0.567 [95% CI 0.454-0.680], n = 119) or subgroups (0.625 [0.387-0.863], n = 27; 0.506 [0.360-0.652], n = 86), nor APPLE scores (fast 0.568 [0.453-0.682], full 0.585 [0.470-0.699], n = 100), were significant prognosticators for death. CONCLUSIONS AND CLINICAL IMPORTANCE: Abnormally high serum cTnI concentration was common in critically ill cats. Unlike in dogs, cTnI did not confer prognostic information regarding death.

Molecular regulation of stretch activation.

Stretch activation is defined as a delayed increase in force after rapid stretches. Although there is considerable evidence for stretch activation in isolated cardiac myofibrillar preparations, few studies have measured mechanisms of stretch activation in mammalian skeletal muscle fibers. We measured stretch activation following rapid step stretches [∼1%-4% sarcomere length (SL)] during submaximal Ca2+ activations of rat permeabilized slow-twitch skeletal muscle fibers before and after protein kinase A (PKA), which phosphorylates slow myosin binding protein-C. PKA significantly increased stretch activation during low (∼25%) Ca2+ activation and accelerated rates of delayed force development (kef) during both low and half-maximal Ca2+ activation. Following the step stretches and subsequent force development, fibers were rapidly shortened to original sarcomere length, which often elicited a shortening-induced transient force overshoot. After PKA, step shortening-induced transient force overshoot increased ∼10-fold following an ∼4% SL shortening during low Ca2+ activation levels. kdf following step shortening also increased after PKA during low and half-maximal Ca2+ activations. We next investigated thin filament regulation of stretch activation. We tested the interplay between cardiac troponin I (cTnI) phosphorylation at the canonical PKA and novel tyrosine kinase sites on stretch activation. Native slow-skeletal Tn complexes were exchanged with recombinant human cTn complex with different human cTnI N-terminal pseudo-phosphorylation molecules: 1) nonphosphorylated wild type (WT), 2) the canonical S22/23D PKA sites, 3) the tyrosine kinase Y26E site, and 4) the combinatorial S22/23D + Y26E cTnI. All three pseudo-phosphorylated cTnIs elicited greater stretch activation than WT. Following stretch activation, a new, elevated stretch-induced steady-state force was reached with pseudo-phosphorylated cTnI. Combinatorial S22/23D + Y26E pseudo-phosphorylated cTnI increased kdf. These results suggest that slow-skeletal myosin binding protein-C (sMyBP-C) phosphorylation modulates stretch activation by a combination of cross-bridge recruitment and faster cycling kinetics, whereas cTnI phosphorylation regulates stretch activation by both redundant and synergistic mechanisms; and, taken together, these sarcomere phosphoproteins offer precision targets for enhanced contractility.

Long-term predictive value of cardiac biomarkers in patients with COVID-19 infection.

OBJECTIVE: Several studies have investigated the association between cardiac biomarkers and short-term prognosis in the COVID-19 infection. However, the data on the predictive value of cardiac biomarkers to predict long-term prognosis in COVID-19 infection are limited. We aimed at determining the relationship between N-terminal brain-type natriuretic peptide (NT-proBNP) and high-sensitivity cardiac troponin I (hs-TnI) as cardiac biomarkers and in-hospital/long-term outcomes in COVID-19 infection. PATIENTS AND METHODS: The study included a total of 916 patients with confirmed COVID-19 infection. The primary outcome was in-hospital and 1-year mortality. The secondary outcome was intensive care need at admission or the need to be transferred to the intensive care unit later on. RESULTS: The study included 498 (54.4%) males and 418 (45.6%) females with a mean age of 55.1±18.5 years. The patients with known heart failure (HF), COVID-19-related HF, acute renal failure (ARF), chronic kidney disease (CKD), diabetes mellitus, hypertension, coronary artery disease (CAD), chronic obstructive pulmonary disease (COPD)/asthma, high CO-RADS score (≥ 4), lower EF, higher hs-TnI, and NT-proBNP levels had increased in-hospital and 1-year mortality. After multivariate analysis, NT-proBNP, hs-TnI, CKD, ARF, diabetes mellitus, and CAD were independent predictors of in-hospital and 1-year mortality. After ROC analysis, NT-proBNP cut-off levels of 1022.50 (sensitivity 87.5%, specificity 87.1%) and 1008 (sensitivity 88.6%, specificity 88.0%) were found to predict in-hospital and 1-year mortality, respectively. Hs-TnI cut-off levels of 49.6 (sensitivity 88.6%, specificity 88.9%) and 34.10 (sensitivity 83.8%, specificity 84.1%) were found to predict in-hospital and 1-year mortality, respectively. CONCLUSIONS: The current study suggests that NT-proBNP and hs-TnI can be used as valuable cardiac biomarkers to predict short-term and long-term parameters in COVID-19 infection.

Drugging the Sarcomere, a Delicate Balance: Position of N-Terminal Charge of the Inhibitor W7.

W7 is a sarcomere inhibitor that decreases the calcium sensitivity of force development in cardiac muscle. W7 binds to the interface of the regulatory domain of cardiac troponin C (cNTnC) and the switch region of troponin I (cTnI), decreasing the binding of cTnI to cNTnC, presumably by electrostatic repulsion between the -NH3+ group of W7 and basic amino acids in cTnI. W7 analogs with a -CO2- tail are inactive. To evaluate the importance of the location of the charged -NH3+, we used a series of compounds W4, W6, W8, and W9, which have three less, one less, one more, and two more methylene groups in the tail region than W7. W6, W8, and W9 all bind tighter to cNTnC-cTnI chimera (cChimera) than W7, while W4 binds weaker. W4 and, strikingly, W6 have no effect on calcium sensitivity of force generation, while W8 and W9 decrease calcium sensitivity, but less than W7. The structures of the cChimera-W6 and cChimera-W8 complexes reveal that W6 and W8 bind to the same hydrophobic cleft as W7, with the aliphatic tail taking a similar route to the surface. NMR relaxation data show that internal flexibility in the tail of W7 is very limited. Alignment of the cChimera-W7 structure with the recent cryoEM structures of the cardiac sarcomere in the diastolic and systolic states reveals the critical location of the amino group. Small molecule induced structural changes can therefore affect the tightly balanced equilibrium between tethered components required for rapid contraction.

C-Terminal Basic Region of Troponin T Alters the Ca2+-Dependent Changes in Troponin I Interactions.

The C-terminal 14-16 residues of human troponin T are required for full inactivation, and they prevent full activation at saturating Ca2+. Basic residues within that C-terminal region of TnT are essential for its function, but the mechanism of action is unknown. That region of TnT is natively disordered and does not appear in reconstructions of the troponin structure. We used Förster resonance energy transfer to determine if the C-terminal basic region of TnT alters transitions of TnI or if it operates independently. We also examined Ca2+-dependent changes in the C-terminal region of TnT itself. Probes on TnI-143 (inhibitory region) and TnI-159 (switch region) moved away from sites on actin and tropomyosin and toward TnC-84 at high Ca2+. Ca2+ also displaced C-terminal TnT from actin-tropomyosin but without movement toward TnC. Deletion of C-terminal TnT produced changes in TnI-143 like those effected by Ca2+, but effects on TnI-159 were muted; there was no effect on the distance of the switch region to TnC-84. Substituting Ala for basic residues within C-terminal TnT displaced C-terminal TnT from actin-tropomyosin. The results suggest that C-terminal TnT stabilizes tropomyosin in the inactive position on actin. Removal of basic residues from C-terminal TnT produced a Ca2+-like state except that the switch region of TnI was not bound to TnC. Addition of Ca2+ caused more extreme displacement from actin-tropomyosin as the active state became more fully occupied as in the case of wild-type TnT in the presence of both Ca2+ and bound rigor myosin S1.

Modulation of cardiac thin filament structure by phosphorylated troponin-I analyzed by protein-protein docking and molecular dynamics simulation.

Tropomyosin, controlled by troponin-linked Ca2+-binding, regulates muscle contraction by a macromolecular scale steric-mechanism that governs myosin-crossbridge-actin interactions. At low-Ca2+, C-terminal domains of troponin-I (TnI) trap tropomyosin in a position on thin filaments that interferes with myosin-binding, thus causing muscle relaxation. Steric inhibition is reversed at high-Ca2+ when TnI releases from F-actin-tropomyosin as Ca2+ and the TnI switch-peptide bind to the N-lobe of troponin-C (TnC). The opposite end of cardiac TnI contains a phosphorylation-sensitive ∼30 residue-long N-terminal peptide that is absent in skeletal muscle, and likely modifies these interactions in hearts. Here, PKA-dependent phosphorylation of serine 23 and 24 modulates Ca2+ and possibly switch-peptide binding to TnC, causing faster relaxation during the cardiac-cycle (lusitropy). The cardiac-specific N-terminal TnI domain is not captured in crystal structures of troponin or in cryo-EM reconstructions of thin filaments; thus, its global impact on thin filament structure and function is uncertain. Here, we used protein-protein docking and molecular dynamics simulation-based protocols to build a troponin model that was guided by and hence consistent with the recent seminal Yamada structure of Ca2+-activated thin filaments. We find that when present on thin filaments, phosphorylated Ser23/24 along with adjacent polar TnI residues interact closely with both tropomyosin and the N-lobe of TnC during our simulations. These interactions would likely bias tropomyosin to an off-state positioning on actin. In situ, such enhanced relaxation kinetics would promote cardiac lusitropy.

Truncation of the N-terminus of cardiac troponin I initiates adaptive remodeling of the myocardial proteosome via phosphorylation of mechano-sensitive signaling pathways.

The cardiac isoform of troponin I has a unique N-terminal extension (~ 1-30 amino acids), which contributes to the modulation of cardiac contraction and relaxation. Hearts of various species including humans produce a truncated variant of cardiac troponin I (cTnI-ND) deleting the first ~ 30 amino acids as an adaption in pathophysiological conditions. In this study, we investigated the impact of cTnI-ND chronic expression in transgenic mouse hearts compared to wildtype (WT) controls (biological n = 8 in each group). We aimed to determine the global phosphorylation effects of cTnI-ND on the cardiac proteome, thereby determining the signaling pathways that have an impact on cardiac function. The samples were digested and isobarically labeled and equally mixed for relative quantification via nanoLC-MS/MS. The peptides were then enriched for phospho-peptides and bioinformatic analysis was done with Ingenuity Pathway Analysis (IPA). We found approximately 77% replacement of the endogenous intact cTnI with cTnI-ND in the transgenic mouse hearts with 1674 phospho-proteins and 2971 non-modified proteins. There were 73 significantly altered phospho-proteins; bioinformatic analysis identified the top canonical pathways as associated with integrin, protein kinase A, RhoA, and actin cytoskeleton signaling. Among the 73 phospho-proteins compared to controls cTnI-ND hearts demonstrated a significant decrease in paxillin and YAP1, which are known to play a role in cell mechano-sensing pathways. Our data indicate that cTnI-ND modifications in the sarcomere are sufficient to initiate changes in the phospho-signaling profile that may underly the chronic-adaptive response associated with cTnI cleavage in response to stressors by modifying mechano-sensitive signaling pathways.

X-ray structure of a human cardiac muscle troponin C/troponin I chimera in two crystal forms.

The X-ray crystal structure of a human cardiac muscle troponin C/troponin I chimera has been determined in two different crystal forms and shows a conformation of the complex that differs from that previously observed by NMR. The chimera consists of the N-terminal domain of troponin C (cTnC; residues 1-80) fused to the switch region of troponin I (cTnI; residues 138-162). In both crystal forms, the cTnI residues form a six-turn α-helix that lays across the hydrophobic groove of an adjacent cTnC molecule in the crystal structure. In contrast to previous models, the cTnI helix runs in a parallel direction relative to the cTnC groove and completely blocks the calcium desensitizer binding site of the cTnC-cTnI interface.

Evolution of the N-Terminal Regulation of Cardiac Troponin I for Heart Function of Tetrapods: Lungfish Presents an Example of the Emergence of Novel Submolecular Structure to Lead the Capacity of Adaptation.

Troponin-based Ca2+ regulation of striated muscle contraction emerged approximately 700 million years ago with largely conserved functions during evolution. Troponin I (TnI) is the inhibitory subunit of troponin and has evolved into three muscle type-specific isoforms in vertebrates. Cardiac TnI is specifically expressed in the adult heart and has a unique N-terminal extension implicating a specific value during natural selection. The N-terminal extension of cardiac TnI in higher vertebrates contains ß-adrenergic-regulated protein kinase A (PKA) phosphorylation sites as a mechanism to enhance cardiac muscle relaxation and facilitate ventricular filling. Phylogenic studies showed that the N-terminal extension of cardiac TnI first emerged in the genomes of early tetrapods as well as primordial lobe-finned fishes such as the coelacanth whereas it is absent in ray-finned fish. This apparently rapid evolution of ß-adrenergic regulation of cardiac function suggests a high selection value for the heart of vertebrate animals on land to work under higher metabolic demands. Sequencing and PKA phosphorylation data showed that lungfish cardiac TnI has evolved with an amphibian-like N-terminal extension with prototype PKA phosphorylation sites while its overall structure remained fish like. The data demonstrate that the submolecular structure of TnI may evolve ahead of the whole protein for cardiac muscle contractility to adapt to new environmental conditions. Understanding the evolution of the ß-adrenergic regulation of TnI and cardiac adaptation to the increased energetic demands of life on land adds knowledge for the treatment of human heart diseases and failure.

Aptamer-modified magnetic SERS substrate for label-based determination of cardiac troponin I.

A sensitive label-based SERS strategy composed of magnetic bimetallic nanoparticles Fe3O4@Ag@Au, specific aptamer, and Bradford method was developed for the quantitative determination of cardiac troponin I (cTnI) in human serum. The prepared substrate with high magnetic character, signal enhancement, and uniformity exhibited significant Raman response. After the substrate was bound to the aptamer, the target protein cTnI was specifically captured, and it showed the Raman signal when the signal reporter Coomassie Brilliant Blue G-250 (CBBG) was supplied. The Raman signal intensity at 1621 cm-1 showed a wide linear relationship with the log value of the cTnI concentration in the range 0.01 to 100 ng·mL-1, and the estimated limit of detection (LOD) was 5.50 pg·mL-1. The recovery and relative standard deviation (RSD) of the spike experiment in human serum samples were 92-115% and 7.4-12.7%, respectively.

Interference-free photoelectrochemical immunoassays using carboxymethylated dextran-coated and gold-modified TiO2 nanotube arrays.

An interference-free photoelectrochemical (PEC) immunoassay was developed for cardiac troponin I (cTnI) detection. Covalent linkage of cTnI antibody to carboxymethylated (CM-) dextran pre-immobilized onto a gold nanoparticles (AuNPs)-modified TiO2 nanotube array (NTA) affords five consecutive analyte captures with surface regenerations in between. Changes in the photocurrents at this photoanode before and after cTnI captures can be well fitted with the Langmuir isotherm from 0.220 pM to 2.20 nM cTnI. Owing to the inherently high sensitivity of the PEC detection, the detection limit (2.20 pg/mL) is lower than the range attainable with the enzyme-linked immunosorbent assay (ELISA) (6.00-40.0 pg/mL). Furthermore, CM-dextran prevents species in complex biological matrices from nonspecifically adsorbing onto the sensor surface, a feature not attainable with uncoated semiconductor electrodes or those coated with non-hydrogel-based chemical modifiers. The excellent anti-fouling property of dextran hydrogel allowed us to validate the accuracy of our regenerable sensors through a comparison of PEC immunoassays of patient sera to those of ELISA.

Dilated Cardiomyopathy Mutations and Phosphorylation disrupt the Active Orientation of Cardiac Troponin C.

Cardiac troponin (cTn) is made up of three subunits, cTnC, cTnI, and cTnT. The regulatory N-terminal domain of cTnC (cNTnC) controls cardiac muscle contraction in a calcium-dependent manner. We show that calcium-saturated cNTnC can adopt two different orientations, with the "active" orientation consistent with the 2020 cryo-EM structure of the activated cardiac thin filament by Yamada et al. Using solution NMR 15N R2 relaxation analysis, we demonstrate that the two domains of cTnC tumble independently (average R2 10 s-1), being connected by a flexible linker. However, upon addition of cTnI1-77, the complex tumbles as a rigid unit (R2 30 s-1). cTnI phosphomimetic mutants S22D/S23D, S41D/S43D and dilated cardiomyopathy- (DCM-)associated mutations cTnI K35Q, cTnC D75Y, and cTnC G159D destabilize the active orientation of cNTnC, with intermediate 15N R2 rates (R2 17-23 s-1). The active orientation of cNTnC is stabilized by the flexible tails of cTnI, cTnI1-37 and cTnI135-209. Surprisingly, when cTnC is incorporated into complexes lacking these tails (cTnC-cTnI38-134, cTnC-cTnT223-288, or cTnC-cTnI38-134-cTnT223-288), the cNTnC domain is still immobilized, revealing a new interaction between cNTnC and the IT-arm that stabilizes a "dormant" orientation. We propose that the calcium sensitivity of the cardiac troponin complex is regulated by an equilibrium between active and dormant orientations, which can be shifted through post-translational modifications or DCM-associated mutations.

The muscle-relaxing C-terminal peptide from troponin I populates a nascent helix, facilitating binding to tropomyosin with a potent therapeutic effect.

The conserved C-terminal end segment of troponin I (TnI) plays a critical role in regulating muscle relaxation. This function is retained in the isolated C-terminal 27 amino acid peptide (residues 184-210) of human cardiac TnI (HcTnI-C27): When added to skinned muscle fibers, HcTnI-C27 reduces the Ca2+-sensitivity of activated myofibrils and facilitates relaxation without decreasing the maximum force production. However, the underlying mechanism of HcTnI-C27 function is unknown. We studied the conformational preferences of HcTnI-C27 and a myopathic mutant, Arg192His, (HcTnI-C27-H). Both peptides were mainly disordered in aqueous solution with a nascent helix involving residues from Trp191 to Ile195, as shown by NMR analysis and molecular dynamics simulations. The population of nascent helix was smaller in HcTnI-C27-H than in HcTnI-C27, as shown by circular dichroism (CD) titrations. Fluorescence and isothermal titration calorimetry (ITC) showed that both peptides bound tropomyosin (αTm), with a detectably higher affinity (∼10 µM) of HcTnI-C27 than that of HcTnI-C27-H (∼15 µM), consistent with an impaired Ca2+-desensitization effect of the mutant peptide on skinned muscle strips. Upon binding to αTm, HcTnI-C27 acquired a weakly stable helix-like conformation involving residues near Trp191, as shown by transferred nuclear Overhauser effect spectroscopy and hydrogen/deuterium exchange experiments. With the potent Ca2+-desensitization effect of HcTnI-C27 on skinned cardiac muscle from a mouse model of hypertrophic cardiomyopathy, the data support that the C-terminal end domain of TnI can function as an isolated peptide with the intrinsic capacity of binding tropomyosin, providing a promising therapeutic approach to selectively improve diastolic function of the heart.

Structural Basis of Tirasemtiv Activation of Fast Skeletal Muscle.

Troponin regulates the calcium-mediated activation of skeletal muscle. Muscle weakness in diseases such as amyotrophic lateral sclerosis and spinal muscular atrophy occurs from diminished neuromuscular output. The first direct fast skeletal troponin activator, tirasemtiv, amplifies the response of muscle to neuromuscular input. Tirasemtiv binds selectively and strongly to fast skeletal troponin, slowing the rate of calcium release and sensitizing muscle to calcium. We report the solution NMR structure of tirasemtiv bound to a fast skeletal troponin C-troponin I chimera. The structure reveals that tirasemtiv binds in a hydrophobic pocket between the regulatory domain of troponin C and the switch region of troponin I, which overlaps with that of Anapoe in the X-ray structure of skeletal troponin. Multiple interactions stabilize the troponin C-troponin I interface, increase the affinity of troponin C for the switch region of fast skeletal troponin I, and drive the equilibrium toward the active state.

C-terminal troponin-I residues trap tropomyosin in the muscle thin filament blocked-state.

Tropomyosin and troponin regulate muscle contraction by participating in a macromolecular scale steric-mechanism to control myosin-crossbridge - actin interactions and consequently contraction. At low-Ca2+, the C-terminal 30% of troponin subunit-I (TnI) is proposed to trap tropomyosin in a position on thin filaments that sterically interferes with myosin-binding, thus causing muscle relaxation. In contrast, at high-Ca2+, inhibition is released after the C-terminal domains dissociate from F-actin-tropomyosin as its component switch-peptide domain binds to the N-lobe of troponin-C (TnC). Recent, paradigm-shifting, cryo-EM reconstructions by the Namba group have revealed density attributed to TnI along cardiac muscle thin filaments at both low- and high-Ca2+ concentration. Modeling the reconstructions showed expected high-Ca2+ hydrophobic interactions of the TnI switch-peptide and TnC. However, under low-Ca2+ conditions, sparse interactions of TnI and tropomyosin, and in particular juxtaposition of non-polar switch-peptide residues and charged tropomyosin amino acids in the published model seem difficult to reconcile with an expected steric-blocking conformation. This anomaly is likely due to inaccurate fitting of tropomyosin into the cryo-EM volume. In the current study, the low-Ca2+ cryo-EM volume was fitted with a more accurate tropomyosin model and representation of cardiac TnI. Our results show that at low-Ca2+ a cluster of hydrophobic residues at the TnI switch-peptide and adjacent H4 helix (Ala149, Ala151, Met 154, Leu159, Gly160, Ala161, Ala163, Leu167, Leu169, Ala171, Leu173) draw-in tropomyosin surface residues (Ile143, Ile146, Ala151, Ile154), presumably attracting the entire tropomyosin cable to its myosin-blocking position on actin. The modeling confirms that neighboring TnI "inhibitory domain" residues (Arg145, Arg148) bind to thin filaments at actin residue Asp25, as previously suggested. ClusPro docking of TnI residues 137-184 to actin-tropomyosin, including the TnI inhibitory-domain, switch-peptide and Helix H4, verified the modeled configuration. Our residue-to-residue contact-mapping of the TnI-tropomyosin association lends itself to experimental validation and functional localization of disease-bearing mutations.

Potential impacts of the cardiac troponin I mobile domain on myofilament activation and relaxation.

The cardiac thin filament is regulated in a Ca2+-dependent manner through conformational changes of troponin and tropomyosin (Tm). It has been generally understood that under conditions of low Ca2+ the inhibitory peptide domain (IP) of troponin I (TnI) binds to actin and holds Tm over the myosin binding sites on actin to prevent crossbridge formation. More recently, evidence that the C-terminal mobile domain (MD) of TnI also binds actin has made for a more complex scenario. This study uses a computational model to investigate the consequences of assuming that TnI regulates Tm movement via two actin-binding domains rather than one. First, a 16-state model of the cardiac thin filament regulatory unit was created with TnI-IP as the sole regulatory domain. Expansion of this to include TnI-MD formed a 24-state model. Comparison of these models showed that assumption of a second actin-binding site allows the individual domains to have a lower affinity for actin than would be required for IP acting alone. Indeed, setting actin affinities of the IP and MD to 25% of that assumed for the IP in the single-site model was sufficient to achieve precisely the same degree of Ca2+ regulation. We also tested the 24-state model's ability to represent steady-state experimental data in the case of disruption of either the IP or MD. We were able to capture qualitative changes in several properties that matched what was seen in the experimental data. Lastly, simulations were run to examine the effect of disruption of the IP or MD on twitch dynamics. Our results suggest that both domains are required to keep diastolic cross-bridge activity to a minimum and accelerate myofilament relaxation. Overall, our analyses support a paradigm in which two domains of TnI bind with moderate affinity to actin, working in tandem to complete Ca2+-dependent regulation of the thin filament.