Troponin C Research Articles

Relationship between calcium circulation-related factors and muscle strength in rat sciatic nerve injury model.

Objective(s):The purpose of this study is to investigate the indication function of the calcium circulation-related factors on the damage to muscle strength and contraction function after nerve injury. The target factors include ryanodine receptor (RyR), inositol-1,4,5-triphosphate receptor (IP3R), phospholamban (PLN), cryptocalcitonin (CASQ), ATPase and troponin C (TNNC).Materials and Methods:Sprague-Dawley (SD) rats were randomly divided into sham-operated group (SO), sciatic nerve injury group (SNI) and sciatic nerve disconnection group (SNT). Sciatic nerve function index and stretching test were used to examine the changes to muscle strength; bilateral gastrocnemius muscles were extracted after execution for gastrocnemius wet weight ratio test. HE staining slides and average cross-sectional area of muscle fibers were acquired to analyze the muscle atrophy. The transcription level of the factors was also measured.Results:Sciatic nerve damage in SNI group was significantly higher than that in SO group in the 6 weeks, but there was no significant difference between SNT and SO groups fallowing sciatic nerve damage. Sciatic nerve function in SNT group was worse than that in SNI group. The average cross-sectional area of gastrocnemius muscle fibers in SNI and SNT groups was significantly reduced compared to that in SO group. The transcriptional levels of RyR, PLN, CASQ, ATPase and TNNC in SNI and SNT groups were significantly different from those in SO group.Conclusion:Calcium circulation-related factors could be used as potential indicators for assessment of damages to muscle strength.

Molecular characterization of troponin C (TnC) in Scylla paramamosain and its role in white spot syndrome virus and Vibrio alginolyticus infection

Troponin C (TnC) is one member of the EF-hand superfamily. In many species, this gene had been identified and related functions had been elucidated. The TnC gene was still blank in the Scylla paramamosain. We obtained the TnC gene for the first time in the S. paramamosain. And we systematically analyzed the possible role of this gene in the innate immunity of S. paramamosain while infected with white spot syndrome virus (WSSV) or Vibrio alginolyticus. The full-length 1427 bp sequence of TnC contains a 453 bp open reading frame (ORF) for encoding a 151 amino acid protein. Detection of tissue specificity of gene expression showed that the TnC was primarily expressed in muscle tissue. The expression of TnC was successfully inhibited by RNA interference technology, and several immune genes were affected. The activity of phenoloxidase and superoxide dismutase increased, and the total hemocytes counts increased after RNAi of TnC. It was found that after infection with V. alginolyticus and WSSV, the expression of TnC in hemocytes decreased. Infected with V. alginolyticus and WSSV, the cumulative mortality and apoptotic rate of hemocytes increased after silencing the TnC gene. Our results indicate that TnC takes participate in the innate immunity of S. paramamosain and may plays a different role in the antiviral and antibacterial immune response.

Computational Studies of Cardiac and Skeletal Troponin.

Troponin is a key regulatory protein in muscle contraction, consisting of three subunits troponin C (TnC), troponin I (TnI), and troponin T (TnT). Calcium association to TnC initiates contraction by causing a series of dynamic and conformational changes that allow the switch peptide of TnI to bind and subsequently cross bridges to form between the thin and thick filament of the sarcomere. Owing to its pivotal role in contraction regulation, troponin has been the focus of numerous computational studies over the last decade. These studies elegantly supplemented a large volume of experimental work and focused on the structure, dynamics and function of the whole troponin complex, individual subunits, and even on segments of the thin filament. Molecular dynamics, Brownian dynamics, and free energy simulations have been used to elucidate the conformational dynamics and underlying free energy landscape of troponin, calcium, and switch peptide binding, as well as the effect of disease mutations, small molecules and post-translational modifications such as phosphorylation. Frequently, simulations have been used to confirm or explain experimental observations. Computer-aided drug discovery tools have been employed to identify novel potential calcium sensitizing agents binding to the TnC-TnI interface. Finally, Markov modeling has contributed to simulating contraction within the sarcomere on the mesoscale. Here we are reviewing and classifying the existing computational work on troponin and its subunits, outline current gaps in simulations elucidating troponin's role in contraction and suggest potential future developments in the field.

Characterization of the L29Q Hypertrophic Cardiomyopathy Mutation in Cardiac Troponin C by Paramagnetic Relaxation Enhancement Nuclear Magnetic Resonance.

The key events in regulating muscle contraction involve the troponin (Tn) heterotrimeric protein complex in which the binding to and release of Ca2+ from the highly conserved troponin C (TnC) subunit trigger a series of structural changes within Tn, and the other thin filament proteins, to result in contraction. In the heart, the control of contraction and relaxation events can be altered by many single-point mutations that may result in cardiomyopathy and sometimes sudden cardiac death. Here we have examined the structural effects of one hypertrophic cardiomyopathy mutation, L29Q, on Ca2+-induced structural transitions within whole TnC. This mutation is of particular interest as several physiological and structural studies have indicated that the response of TnC to Ca2+ binding is altered in the presence of the L29Q mutation, but the structural nature of these changes continues to be debated. In addition, little is known about the effect of this mutation in the Ca2+ free state. Here we have used paramagnetic relaxation enhancement nuclear magnetic resonance (PRE-NMR) to assess the structural effects arising from the L29Q mutation. PRE-NMR distances obtained from a nitroxide spin-label at Cys84 showed that the L29Q mutation perturbs the structure of the TnC N-domain in the presence and absence of Ca2+, with a more "open" TnC N-domain observed in the apo form. In addition, binding of Ca2+ to the TnC-L29Q construct triggers a change in the orientation between the two domains of TnC. Together, these structural perturbations, revealed by PRE-NMR, provide insight into the pathogenesis of this mutation.

Isolation and characterization of three skeletal troponin genes and association with growth-related traits in Exopalaemon carinicauda.

Growth is among the most important traits for animal breeding. Muscle growth is controlled by different cellular and molecular pathways and environments, and it also relies heavily on high-quality muscle contractions. The troponin complex, composed of troponin T (TnT), troponin C (TnC) and troponin I (TnI), plays a vital role in the regulation of muscle contraction. In this study, the cDNA of EcTnT, EcTnC and EcTnI of the ridgetail white prawn Exopalaemon carinicauda were cloned and characterized. The full length cDNA of EcTnT, EcTnC and EcTnI were 1 373bp, 692bp, and 1 475bp, encoding a protein of 385, 150 and 193 amino acid residues, respectively. The expression of all genes was predominantly detected in abdominal muscle, while extremely lesser expressed in gill and hepatopancreas. Higher expression level of EcTnI was observed in heavier shrimp of the same age during different developmental stages, excepted for 120 days. Eleven single nucleotide polymorphisms (SNPs) were revealed in the three skeletal troponin genes, and only c.TnI66 A>G from EcTnI was significantly associated with both body weight and body length (P < 0.05). In summary, the result of this study suggested that EcTnI is growth-related gene of the troponin complex gene and the presence of SNP suggests that it could be a candidate gene for shrimp genetic improvement research.

Troponin Structure and Function in Health and Disease

<h3>ABSTRACT</h3> Troponin (Tn) is a heterotrimeric protein containing 3 subunits (C, T, and I) with different molecular weights and distinctive functions. The subunits of Tn work cohesively to regulate the contraction and relaxation activities of striated muscles: troponin-C (TnC) binds calcium (Ca²⁺); troponin-T (TnT) interacts with tropomyosin (Tm) and anchors Tn to actin; and troponin-I (TnI) inhibits the adenosine triphosphatase (ATPase) activity of the actomyosin cross-bridge and effectively blocks the myosin-binding site on actin subunits. At a genetic level, there are 8 distinctive Tn genes (isoforms) that code for tissue-specific heart and skeletal muscle protein subunits: TNNI1, TNNI2, TNNI3, TNNT1, TNNT2, TNNT3, TNNC1, and TNNC2. The gene isoforms are regulated throughout development via posttranscriptional and posttranslational modifications. Genetic mutations in any of the 3 protein subunits could be linked to hypertrophic, dilated, and restrictive cardiomyopathies. Tn release by damaged cardiomyocytes is clinically used as a biomarker for myocardial infarction (MI), and its release into the serum is measured at specific times postinjury for diagnostic or prognostic purposes. Current tests that measure serum Tn are fifth generation assays, which have improved sensitivity and specificity compared with previous assays. However, increased serum Tn levels have been seen in chronic diseases such as Fabry disease and chronic renal disease. It is important to remember, when clinically examining a patient, that Tn levels are only one piece of the puzzle. A patient’s history or symptoms are essential for making an accurate diagnosis.

Jack-of-many-trades: discovering new roles for troponin C.

Jack-of-many-trades: discovering new roles for troponin C.

Molecular Dynamics and Umbrella Sampling Simulations Elucidate Differences in Troponin C Isoform and Mutant Hydrophobic Patch Exposure.

Troponin C (TnC) facilitates muscle contraction through calcium-binding within its N-terminal region (NTnC). As previously observed using molecular dynamics (MD) simulations, this calcium-binding event leads to an increase in the dynamics of helices lining a hydrophobic patch on TnC. Simulation times of multiple microseconds were required to even see a partial opening of the hydrophobic patch, limiting the ability to thoroughly and quantitatively investigate these rare events. Here we describe the application of umbrella sampling to probe the TnC hydrophobic patch opening in a more targeted and quantitative fashion. Umbrella sampling was utilized to investigate the differences in the free energy of opening between cardiac (cTnC) and fast skeletal TnC (sTnC). We found that, in agreement with previous reports, holo (calcium-bound) sTnC had a lower free energy of opening compared with holo cTnC. Additionally, differences in the free energy of opening of hypertrophic (HCM) and dilated cardiomyopathy (DCM) cTnC systems were investigated. MD simulations and umbrella sampling revealed a lower free energy of opening for the HCM mutations A8V and A31S, as well as the calcium-sensitizing mutation L48Q. The DCM mutations, Y5H, Q50R, and E59D/D75Y, all exhibited a higher free energy of opening. An umbrella sampling simulation of cTnI-bound holo cTnC exhibited the lowest free energy in the open configuration, in agreement with experimental data. In conclusion, this study presents a novel and successful protocol for applying umbrella sampling simulations to quantitatively study the molecular basis of muscle contraction and proposes a mechanism by which HCM and DCM-associated mutations influence contraction.

Double Quantum Coherence EPR Reveals the Structure–Function Relationships of the Cardiac Troponin C–Troponin I Complex Regulated by Ca2+ Ions and a Phosphomimetic

Troponin (Tn) is a protein that consists of three subunits, troponin C (TnC), troponin I (TnI), and troponin T (TnT), and Tn controls cardiac muscle contraction by calcium ion binding and phosphorylation. The Ca2+-binding site is the E–F hand motif (C helix–loop–D helix) in the N-terminal domain of TnC, and the structural transition induced by Ca2+ is the opening of these helices and the interaction with TnI, probably at the A and B helices. In this paper, we studied structural changes in the TnC–TnI binary complex on Ca2+ binding by double quantum coherence (DQC) distance measurements. We used a binary complex of the cardiac troponin C and I (cTnC and cTnI) complexes, chose four positions of nitroxide spin label at helices A, B, C, and D in the N-terminal domain and chose the E helix in the C-terminal domain as the reference position to study the structural changes on Ca2+ addition. The label positions were (A22C/S98C), (M47C/S98C), (Q58C/S98C), and (C84/S98C) for the A, B, C, and D helices, respectively. The effects of phosphorylation of the cardiac-specific N-terminal region of cTnI were studied using a phosphomimetic cTnI mutant. Analysis of the modulation of the DQC echo signals provided the distribution of the spin–spin distance. The distances averaged over the distribution showed that the labels on the A, B, and C helices decreased, i.e., moved to the E helix, on Ca2+ binding, while the distance of the label on the D helix showed almost no change. Shoulders and/or small separate peaks were observed in the shape of the distribution and were analyzed as the sum of a few Gaussian functions. The Gaussian functions were grouped into two components, components 1 and 2, at the longer and shorter distances, respectively, separated by 0.7–1.5 nm. The fractions of component 2 were ca. 0.1–0.2 in the Ca2+-free state and increased by 0.2–0.3 on Ca2+ addition, suggesting that the increase in component 2 is related to physiological control of cardiac muscle contraction. The phosphomimetic-modification effects on the Ca2+-induced changes of the fraction of components and the distances of the C- and D-helix labels are small. On the other hand, in the A and B helices, there are significant effects on the Ca2+-induced changes in the distances of the components. The different behaviors of A/B and C/D helices support the current model of the phosphorylation effects in which both N-terminal region and regulatory domain of cTnI interact with the A and B helices of cTnC.

Genetically encoded calcium indicator with NTnC-like design and enhanced fluorescence contrast and kinetics

BackgroundThe recently developed genetically encoded calcium indicator (GECI), called NTnC, has a novel design with reduced size due to utilization of the troponin C (TnC) as a Ca2+-binding moiety inserted into the mNeonGreen fluorescent protein. NTnC binds two times less Ca2+ ions while maintaining a higher fluorescence brightness at the basal level of Ca2+ in neurons as compared with the calmodulin-based GECIs, such as GCaMPs. In spite of NTnC’s high brightness, pH-stability, and high sensitivity to single action potentials, it has a limited fluorescence contrast (F-Ca2+/F+Ca2+) and slow Ca2+ dissociation kinetics.ResultsHerein, we developed a new NTnC-like GECI with enhanced fluorescence contrast and kinetics by replacing the mNeonGreen fluorescent subunit of the NTnC indicator with EYFP. Similar to NTnC, the developed indicator, named iYTnC2, has an inverted fluorescence response to Ca2+ (i.e. becoming dimmer with an increase of Ca2+ concentration). In the presence of Mg2+ ions, iYTnC2 demonstrated a 2.8-fold improved fluorescence contrast in vitro as compared with NTnC. The iYTnC2 indicator has lower brightness and pH-stability, but similar photostability as compared with NTnC in vitro. Stopped-flow fluorimetry studies revealed that iYTnC2 has 5-fold faster Ca2+ dissociation kinetics than NTnC. When compared with GCaMP6f GECI, iYTnC2 has up to 5.6-fold faster Ca2+ association kinetics and 1.7-fold slower dissociation kinetics. During calcium transients in cultured mammalian cells, iYTnC2 demonstrated a 2.7-fold higher fluorescence contrast as compared with that for the NTnC. iYTnC2 demonstrated a 4-fold larger response to Ca2+ transients in neuronal cultures than responses of NTnC. iYTnC2 response in neurons was additionally characterized using whole-cell patch clamp. Finally, we demonstrated that iYTnC2 can visualize neuronal activity in vivo in the hippocampus of freely moving mice using a nVista miniscope.ConclusionsWe demonstrate that expanding the family of NTnC-like calcium indicators is a promising strategy for the development of the next generation of GECIs with smaller molecule size and lower Ca2+ ions buffering capacity as compared with commonly used GECIs.

Cardiomyopathy-Linked Mutation K15N in Tropomyosin Alters Calcium-Dependent Regulation of Reconstituted Cardiac Thin Filaments

In striated muscle, actin-based thin filaments and myosin-based thick filaments are organized into basic contractile units called sarcomeres. Tropomyosin (Tpm), a coiled-coil dimer, polymerizes head-to-tail along the thin filament, stabilizes the filament and, together with the troponin complex, regulates Ca2+-dependent actomyosin interactions which are molecular prerequisites for muscle contraction. Troponin, consisting of troponin I (TnI), troponin C (TnC), and troponin T (TnT), initiates muscle contraction through a series of structural changes within the complex, which is triggered by the binding of Ca2+ to TnC. The structural changes spread through TnT which in turn move Tpm's position on the thin filament, enabling actomyosin interactions. In this work, we studied two missense mutations in striated muscle tropomyosin (Tpm1.1), K15N and R21H, which are associated with dilated cardiomyopathy and hypertrophic cardiomyopathy, respectively. We investigated the mutations’ effects on Tpm1.1's affinity to F-actin and on the Ca2+-dependent regulation of reconstituted cardiac thin filaments using Fluorescence Resonance Energy Transfer (FRET). We labeled residues 151 and 167 of cardiac TnI with IAEDANS as FRET donors, and residue 89 of cardiac TnC with DDPM as FRET acceptor. We found that while both mutations reduced Tpm1.1's affinity to F-actin, the K15N mutation decreased Ca2+-sensitivity and Ca2+-dissociation-induced kinetics of reconstituted cardiac thin filaments. In the presence of the K15N mutant, the interprobe distances were greater than in the presence of wild type Tpm1.1, indicating that the mutation affected cation-induced conformational changes within troponin. Our data demonstrated that the K15N mutation in Tpm1.1 affected Ca2+-dependent thin filament regulation while the R21H mutation had almost no effect.

Regulated Model of Steady-State Cardiac Length-Dependent Activation

Recent data suggests length-dependent activation derives from titin interacting with super-relaxed myosin crossbridges on the thick filament. This feature was incorporated into an existing model of crossbridge dynamics (Caremani et al, 2015) by replacing the detached post-ATP-hydrolysis state with an equilibrium mixture of rapidly interacting super-relaxed, detached, and weakly bound crossbridges. The model also included: (1) cooperative activation of the thin filament by both Ca++ and strongly bound crossbridges, (2) loosely coupled interaction that allows Ca++ to dissociate from troponin C (TnC) before strong crossbridges detach, (3) Ca++ bound more strongly to TnC when a strong crossbridge is also bound nearby. As suggested by data (Sun & Irving, 2015), the equilibrium between super-relaxed and detached crossbridges equaled 3:1 for a long sarcomere length. We supposed that the reduced forces exerted by titin on the thick filament at low sarcomere length made it more likely to form a super-relaxed crossbridge. As the force exerted by titin increases, the super-relaxed myosin transitions to an unfettered detached state and becomes available to form weak crossbridges. We altered the Gibbs free energy to form a super-relaxed crossbridge by less than 1 kT as titin's force decreased over sarcomere lengths from 2.25 to 1.9 µm. This caused the maximal steady-state force for isometric contraction at saturating Ca++ to reduce by ∼ 25%, as data suggests (Dobesh et al, 2002). The model thus unites existing data and provides a more quantitative understanding of how titin interacts with the thick filament at different sarcomere lengths. We anticipate that this model insight and more extensive data will lead to deeper understanding of cardiac length-dependent activation. (Supported by the Center for Engineering MechanoBiology through a grant from NSF's STC program (CMMI):15-48571).

RNA interference-mediated knockdown and virus-induced suppression of Troponin C gene adversely affect the behavior or fitness of the green rice leafhopper, Nephotettix cincticeps.

The green rice leafhopper, Nephotettix cincticeps, is a major rice pest in Southeast Asia and Southern China. Novel control strategies must be explored to control the rice pest. Behavior or fitness regulation of insect by modulating the Troponin C (TnC) may be a novel strategy in the comprehensive management of the insect pest. However, characterizations and functions of TnC, especially regarding effect of its RNA interference-mediated gene knockdown on the behavior or fitness of N. cincticeps remain unknown. Here, we successfully cloned and characterized TnC gene from N. cincticeps (Nc-TnC). We demonstrated that Nc-TnC ubiquitously transcribed at all development stages and special tissues in adult insects, with relative higher levels at the adult stage and in the intestinal canal. Microinjection- or oral membrane feeding-based transient knockdown of Nc-TnC adversely affected the performance or fitness, such as the decreased survival, feeding capacity, weight, and fecundity of N. cincticeps. Furthermore, we revealed that the expression of Nc-TnC was suppressed by its interaction with rice dwarf virus-encoded nonstructural protein 10, which ultimately affected detrimentally the corresponding parameters of the performance or fitness of N. cincticeps. In conclusion, our data deepen understanding of Nc-TnC functions during the development of and viral infection in N. cincticeps. It imply Nc-TnC may serve as a potential target for N. cincticeps control in future.

Abstract 63: A Novel Inotropic Gene Therapy Approach for Aging Induced Cardiac Dysfunction

We have developed a novel approach to increase cardiac contractility without the deleterious effects of existing inotropes (beta agonists, Ca2+ sensitizers). Using the strengths of protein engineering, we have shown that cardiac function can be directly controlled and strongly influenced by manipulating the Ca2+ dependent switch in cardiac muscle, troponin C (TnC). Our smartly formulated construct, L48Q TnC, is a strong Ca2+ sensitizer. Although effective acutely, long-term inotropic support is detrimental. We transduced mice with AAV9 L48Q TnC at 3 months of age and serially performed echocardiography for 2 years. The current dogma would suggest that chronic Ca2+ sensitization with L48Q TnC should result in a hypertrophic cardiomyopathy and compromised function. Astonishingly, the L48Q TnC mice maintained ejection fraction unlike the progressive decline observed in the controlled, age-matched mice. Furthermore, L48Q TnC mice had an increased rate of survival. Considering cardiovascular disease is an elderly issue, we reasoned L48Q TnC would be beneficial in other age-related cardiovascular problems (i.e., hypertension and pressure-overload). We tested this in two models, transverse aortic constriction (TAC, pressure-overload) and angiotensin II infusion (AngII, hypertension). Consistent with the aging results, L48Q TnC mice displayed enhanced cardiac function (ejection fraction, contractility, relaxation), performance (VO 2max , distance run before exhaustion), less adverse remodeling, and enhanced survival. These studies provide a promising approach (i.e., inotropic support via L48Q TnC) that can be used therapeutically to enhance heart function in a variety of cardiomyopathies associated with aging.

Myosin Rod Hypophosphorylation and CB Kinetics in Papillary Muscles from a TnC-A8V KI Mouse Model

The cardiac troponin C (TnC)-A8V mutation is associated with hypertrophic and restrictive cardiomyopathy (HCM and RCM) in human and mice. The residue affected lies in the N-helix, a region known to affect Ca2+-binding affinity to the N-terminal domain. Here we report on the functional effects of this mutation in skinned papillary muscle fibers from homozygous knock-in TnC-A8V mice. Muscle fibers from left ventricle were activated at 25°C under the ionic conditions of working cardiomyocytes. The pCa-tension relationship showed a 3× increase in Ca2+-sensitivity and a decrease (0.8×) in cooperativity (nH) in mutant fibers. The elementary steps of the cross-bridge (CB) cycle were investigated by sinusoidal analysis. The ATP study revealed that there is no significant change in the affinity of ATP (K1) for the myosin head. In TnC-A8V mutant fibers, the CB detachment rate (k2) and its equilibrium constant (K2) increased (1.5×). The phosphate study revealed that rate constant of the force-generation step (k4) decreased (0.5×), reversal step (k−4) increased (2×), and the phosphate-release step (1/K5) increased (2×). Pro-Q Diamond staining of the skinned fibers samples revealed no significant changes in total phosphorylation of multiple sarcomeric proteins. Further investigation using liquid chromatography-tandem mass spectrometry revealed hypophosphorylation of the rod domain of myosin heavy chain in TnC-A8V mutant fibers compared to wild-type. Immunoblotting confirmed the results observed in the mass spectrometry analysis. The results suggest perturbed CB kinetics—possibly caused by changes in the α-myosin heavy chain phosphorylation profile—as a novel mechanism, to our knowledge, by which a mutation in TnC can have rippling effects in the myofilament and contribute to the pathogenesis of HCM/RCM.

Doublet stimulation increases Ca2+ binding to troponin C to ensure rapid force development in skeletal muscle.

Fast-twitch skeletal muscle fibers are often exposed to motor neuron double discharges (≥200 Hz), which markedly increase both the rate of contraction and the magnitude of the resulting force responses. However, the mechanism responsible for these effects is poorly understood, likely because of technical limitations in previous studies. In this study, we measured cytosolic Ca2+ during doublet activation using the low-affinity indicator Mag-Fluo-4 at high temporal resolution and modeled the effects of doublet stimulation on sarcoplasmic reticulum (SR) Ca2+ release, binding of Ca2+ to cytosolic buffers, and force enhancement in fast-twitch fibers. Single isolated fibers respond to doublet pulses with two clear Ca2+ spikes, at doublet frequencies up to 1 KHz. A 200-Hz doublet at the start of a tetanic stimulation train (70 Hz) decreases the drop in free Ca2+ between the first three Ca2+ spikes of the transient, maintaining a higher overall free Ca2+ level during first 20-30 ms of the response. Doublet stimulation also increased the rate of force development in isolated fast-twitch muscles. We also modeled SR Ca2+ release rates during doublet stimulation and showed that Ca2+-dependent inactivation of ryanodine receptor activity is rapid, occurring ≤1ms after initial release. Furthermore, we modeled Ca2+ binding to the main intracellular Ca2+ buffers of troponin C (TnC), parvalbumin, and the SR Ca2+ pump during Ca2+ release and found that the main effect of the second response in the doublet is to more rapidly increase the occupation of the second Ca2+-binding site on TnC (TnC2), resulting in earlier activation of force. We conclude that doublet stimulation maintains high cytosolic Ca2+ levels for longer in the early phase of the Ca2+ response, resulting in faster saturation of TnC2 with Ca2+, faster initiation of cross-bridge cycling, and more rapid force development.

Will you still need me (Ca2+, TnT, and DHPR), will you still cleave me (calpain), when I'm 64?

Will you still need me (Ca²⁺, TnT, and DHPR), will you still cleave me (calpain), when I'm 64?

Myofilament Calcium Sensitivity: Consequences of the Effective Concentration of Troponin I.

Control of calcium binding to and dissociation from cardiac troponin C (TnC) is essential to healthy cardiac muscle contraction/relaxation. There are numerous aberrant post-translational modifications and mutations within a plethora of contractile, and even non-contractile, proteins that appear to imbalance this delicate relationship. The direction and extent of the resulting change in calcium sensitivity is thought to drive the heart toward one type of disease or another. There are a number of molecular mechanisms that may be responsible for the altered calcium binding properties of TnC, potentially the most significant being the ability of the regulatory domain of TnC to bind the switch peptide region of TnI. Considering TnI is essentially tethered to TnC and cannot diffuse away in the absence of calcium, we suggest that the apparent calcium binding properties of TnC are highly dependent upon an “effective concentration” of TnI available to bind TnC. Based on our previous work, TnI peptide binding studies and the calcium binding properties of chimeric TnC-TnI fusion constructs, and building upon the concept of effective concentration, we have developed a mathematical model that can simulate the steady-state and kinetic calcium binding properties of a wide assortment of disease-related and post-translational protein modifications in the isolated troponin complex and reconstituted thin filament. We predict that several TnI and TnT modifications do not alter any of the intrinsic calcium or TnI binding constants of TnC, but rather alter the ability of TnC to “find” TnI in the presence of calcium. These studies demonstrate the apparent consequences of the effective TnI concentration in modulating the calcium binding properties of TnC.

A new design for a green calcium indicator with a smaller size and a reduced number of calcium-binding sites.

Genetically encoded calcium indicators (GECIs) are mainly represented by two- or one-fluorophore-based sensors. One type of two-fluorophore-based sensor, carrying Opsanus troponin C (TnC) as the Ca2+-binding moiety, has two binding sites for calcium ions, providing a linear response to calcium ions. One-fluorophore-based sensors have four Ca2+-binding sites but are better suited for in vivo experiments. Herein, we describe a novel design for a one-fluorophore-based GECI with two Ca2+-binding sites. The engineered sensor, called NTnC, uses TnC as the Ca2+-binding moiety, inserted in the mNeonGreen fluorescent protein. Monomeric NTnC has higher brightness and pH-stability in vitro compared with the standard GECI GCaMP6s. In addition, NTnC shows an inverted fluorescence response to Ca2+. Using NTnC, we have visualized Ca2+ dynamics during spontaneous activity of neuronal cultures as confirmed by control NTnC and its mutant, in which the affinity to Ca2+ is eliminated. Using whole-cell patch clamp, we have demonstrated that NTnC dynamics in neurons are similar to those of GCaMP6s and allow robust detection of single action potentials. Finally, we have used NTnC to visualize Ca2+ neuronal activity in vivo in the V1 cortical area in awake and freely moving mice using two-photon microscopy or an nVista miniaturized microscope.

Molecular evolution of troponin I and a role of its N‐terminal extension in nematode locomotion

The troponin complex, composed of troponin T (TnT), troponin I (TnI), and troponin C (TnC), is the major calcium‐dependent regulator of muscle contraction, which is present widely in both vertebrates and invertebrates. Little is known about evolutionary aspects of troponin in the animal kingdom. Using a combination of data mining and functional analysis of TnI, we report evidence that an N‐terminal extension of TnI is present in most of bilaterian animals as a functionally important domain. Troponin components have been reported in species in most of representative bilaterian phyla. Comparison of TnI sequences shows that the core domains are conserved in all examined TnIs, and that N‐ and C‐terminal extensions are variable among isoforms and species. In particular, N‐terminal extensions are present in all protostome TnIs and chordate cardiac TnIs but lost in a subset of chordate TnIs including vertebrate skeletal‐muscle isoforms. Transgenic rescue experiments in Caenorhabditis elegans striated muscle show that the N‐terminal extension of TnI (UNC‐27) is required for coordinated worm locomotion but not in sarcomere assembly and single muscle‐contractility kinetics. These results suggest that N‐terminal extensions of TnIs are retained from a TnI ancestor as a functional domain. © 2016 Wiley Periodicals, Inc.