Troponin Structural Dynamics in the Native Cardiac Thin Filament Revealed by Cryo Electron Microscopy.

Cardiac muscle contraction occurs due to repetitive interactions between myosin thick and actin thin filaments (TF) regulated by Ca2+ levels, active cross-bridges, and cardiac myosin-binding protein C (cMyBP-C). The cardiac TF (cTF) has two nonequivalent strands, each comprised of actin, tropomyosin (Tm), and troponin (Tn). Tn shifts Tm away from myosin-binding sites on actin at elevated Ca2+ levels to allow formation of force-producing actomyosin cross-bridges. The Tn complex is comprised of three distinct polypeptides - Ca2+-binding TnC, inhibitory TnI, and Tm-binding TnT. The molecular mechanism of their collective action is unresolved due to lack of comprehensive structural information on Tn region of cTF. C1 domain of cMyBP-C activates cTF in the absence of Ca2+ to the same extent as rigor myosin. Here we used cryo-EM of native cTFs to show that cTF Tn core adopts multiple structural conformations at high and low Ca2+ levels and that the two strands are structurally distinct. At high Ca2+ levels, cTF is not entirely activated by Ca2+ but exists in either partially or fully activated state. Complete dissociation of TnI C-terminus is required for full activation. In presence of cMyBP-C C1 domain, Tn core adopts a fully activated conformation, even in absence of Ca2+. Our data provide a structural description for the requirement of myosin to fully activate cTFs and explain increased affinity of TnC to Ca2+ in presence of active cross-bridges. We suggest that allosteric coupling between Tn subunits and Tm is required to control actomyosin interactions.

Structural Biochemistry of Muscle Contraction.

Muscles are essential for movement and heart function. Contraction and relaxation of muscles rely on the sliding of two types of filaments-the thin filament and the thick myosin filament. The thin filament is composed mainly of filamentous actin (F-actin), tropomyosin, and troponin. Additionally, several other proteins are involved in the contraction mechanism, and their malfunction can lead to diverse muscle diseases, such as cardiomyopathies. We review recent high-resolution structural data that explain the mechanism of action of muscle proteins at an unprecedented level of molecular detail. We focus on the molecular structures of the components of the thin and thick filaments and highlight the mechanisms underlying force generation through actin-myosin interactions, as well as Ca2+-dependent regulation via the dihydropyridine receptor, the ryanodine receptor, and troponin. We particularly emphasize the impact of cryo-electron microscopy and cryo-electron tomography in leading muscle research into a new era.

50 Years of the steric-blocking mechanism in vertebrate skeletal muscle: a retrospective.

Fifty years have now passed since Parry and Squire proposed a detailed structural model that explained how tropomyosin, mediated by troponin, played a steric-blocking role in the regulation of vertebrate skeletal muscle. In this Special Issue dedicated to the memory of John Squire it is an opportune time to look back on this research and to appreciate John's key contributions. A review is also presented of a selection of the developments and insights into muscle regulation that have occurred in the years since this proposal was formulated.

Structural and Thermodynamic Model for the Activation of Cardiac Troponin.

Cardiac troponin is a regulatory protein complex located on the sarcomere that regulates the engagement of myosin on actin filaments. Low-molecular weight modulators of troponin that bind allosterically with the calcium ion have the potential to improve cardiac contractility in patients with reduced cardiac function. Here we propose an approach to the rational design of troponin modulators through the combined use of solution nuclear magnetic resonance and isothermal titration calorimetry methods. In contrast to traditional approaches limited to calcium and activator-bound troponin structures, here we analyzed the structural and thermodynamic impact of an activator in the context of the troponin functional cycle. This led us to propose a rationale for developing an efficacious troponin activator.

Locusta migratoria flight muscle troponin partially activates thin filament in a calcium-dependent manner.

The troponin (Tn) complex, the sensor for Ca2+ to regulate contraction of striated muscle, is composed of three subunits, that is, TnT, TnI and TnC. Different isoforms of TnI and TnC are expressed in the thorax dorsal longitudinal muscle (flight muscle) and the hind leg extensor tibiae muscle (jump muscle) of the migratory locust, Locusta migratoria. The major Tn complexes in the flight muscle and the jump muscle are Tn-171 (TnT1/TnI7/TnC1) and Tn-153 (TnT1/TnI5/TnC3), respectively. Here, we prepared a number of recombinant Tn complexes and the reconstituted thin filaments, and investigated their regulation on thin filament. Although both Tn-171 and Tn-153 regulate thin filament in a Ca2+ -dependent manner, the extent of Ca2+ activation mediated by Tn-171 was significantly lower than that by Tn-153. We demonstrated that TnC1 and TnC3, rather than TnI5 and TnI7, are responsible for the different levels of the thin filament activation. Mutagenesis of TnC1 and TnC3 shows that the low level of TnC1-mediated thin filament activation can be attributed to the noncanonical residue Leu60 in the EF-hand-II of TnC1. We therefore propose that, in addition to Ca2+ , other regulatory mechanism(s) is required for the full activation of locust flight muscle.

Troponin Variants in Congenital Myopathies: How They Affect Skeletal Muscle Mechanics.

The troponin complex is a key regulator of muscle contraction. Multiple variants in skeletal troponin encoding genes result in congenital myopathies. TNNC2 has been implicated in a novel congenital myopathy, TNNI2 and TNNT3 in distal arthrogryposis (DA), and TNNT1 and TNNT3 in nemaline myopathy (NEM). Variants in skeletal troponin encoding genes compromise sarcomere function, e.g., by altering the Ca2+ sensitivity of force or by inducing atrophy. Several potential therapeutic strategies are available to counter the effects of variants, such as troponin activators, introduction of wild-type protein through AAV gene therapy, and myosin modulation to improve muscle contraction. The mechanisms underlying the pathophysiological effects of the variants in skeletal troponin encoding genes are incompletely understood. Furthermore, limited knowledge is available on the structure of skeletal troponin. This review focusses on the physiology of slow and fast skeletal troponin and the pathophysiology of reported variants in skeletal troponin encoding genes. A better understanding of the pathophysiological effects of these variants, together with enhanced knowledge regarding the structure of slow and fast skeletal troponin, will direct the development of treatment strategies.

Does high sensitivity troponin add prognostic value to validated risk scores to predict in-hospital mortality in patients with acute heart failure?

Troponin elevation correlates with an increased short and long-term mortality in patients with acute decompensated heart failure (AHF). However, it has not been included in the development of multiple validated predictive models of mortality. We aim to  determine whether the addition of high-sensitivity troponin T (hs-TnT) to clinical risk scores improves the prediction of in-hospital mortality in patients with AHF. A retrospective analysis of a prospective and consecutive cohort was performed. Adult patients hospitalized between 2015 and 2019 with a primary diagnosis of AHF were included. Hs-TnT was measured on admission. OPTIMIZE-HF, GWTG-HF, and ADHERE risks score were calculated for each patient. The primary endpoint was all-cause in-hospital mortality. Discrimination of isolated hs-TnT and the risk scores with and without the addition of hs-TnT were evaluated using the area under the ROC curve (AUC-ROC). A subanalysis was performed according to left ventricular ejection fraction (LVEF). Of 712 patients, 562 (79%) had hs-TnT measurement upon admission, and was elevated in 91%. In-hospital mortality was 8.7% (n = 49). The AUC-ROC was 0.70 (95% CI 0.63-0.77) for isolated hs-TnT, and 0.80 (0.74-0.87), 0.79 (0.72 -0.86) and 0.79 (0.71-0.86) for the OPTIMIZE-HF, GWTG-HF and ADHERE scores, respectively. The addition of hs-TnT to the models did not increase the AUC: 0.72 (0.66-0.79) for the OPTIMIZE-HF + hs-TnT score (difference between AUC - 0.08 p = 0.04), 0.74 (0.68-0.80) for GWTG-HF (difference between AUC-0.04, p = 0.2) and 0.7 (0.63-0.77) for ADHERE (difference between AUC - 0.085 p = 0.07). The models presented good calibration (p > 0.05). In the sub-analysis, no differences were found between risk scores with the addition of hs-TnT in the population with LVEF < 40% and ≥ 40%. Elevated hs-TnT on admission was frequent and its incorporation into the validated risk scores did not prove an incremental prognostic benefit in patients hospitalized for AHF, regardless of LVEF. Isolated hs-TnT had a modest ability to predict hospital mortality. Additional prospective studies are needed to validate these findings.

The structure of the native cardiac thin filament at systolic Ca2+ levels.

Every heartbeat relies on cyclical interactions between myosin thick and actin thin filaments orchestrated by rising and falling Ca2+ levels. Thin filaments are comprised of two actin strands, each harboring equally separated troponin complexes, which bind Ca2+ to move tropomyosin cables away from the myosin binding sites and, thus, activate systolic contraction. Recently, structures of thin filaments obtained at low (pCa ∼9) or high (pCa ∼3) Ca2+ levels revealed the transition between the Ca2+-free and Ca2+-bound states. However, in working cardiac muscle, Ca2+ levels fluctuate at intermediate values between pCa ∼6 and pCa ∼7. The structure of the thin filament at physiological Ca2+ levels is unknown. We used cryoelectron microscopy and statistical analysis to reveal the structure of the cardiac thin filament at systolic pCa = 5.8. We show that the two strands of the thin filament consist of a mixture of regulatory units, which are composed of Ca2+-free, Ca2+-bound, or mixed (e.g., Ca2+ free on one side and Ca2+ bound on the other side) troponin complexes. We traced troponin complex conformations along and across individual thin filaments to directly determine the structural composition of the cardiac native thin filament at systolic Ca2+ levels. We demonstrate that the two thin filament strands are activated stochastically with short-range cooperativity evident only on one of the two strands. Our findings suggest a mechanism by which cardiac muscle is regulated by narrow range Ca2+ fluctuations.

Further Investigation into the Biochemical Effects of Phosphorylation of Tropomyosin Tpm1.1(α). Serine-283 Is in Communication with the Midregion.

The phosphorylated and unphosphorylated forms of tropomyosin Tpm1.1(α) are prepared from adult rabbit heart and compared biochemically. Electrophoresis confirms the high level of enrichment of the chromatography fractions and is consistent with a single site of phosphorylation. Covalently bound phosphate groups at position 283 of Tpm1.1(α) increase the rate of digestion at Leu-169, suggestive of a conformational rearrangement that extends to the midregion. Such a rearrangement, which is supported by ellipticity measurements between 25 and 42 °C, is consistent with a phosphorylation-mediated tightening of the interaction between various myofilament components. In a nonradioactive, co-sedimentation assay [30 mM KCl, 1 mM Mg(II), and 4 °C], phosphorylated Tpm1.1(α) displays a higher affinity for F-actin compared to that of the unphosphorylated control (Kd, 0.16 µM vs 0.26 µM). Phosphorylation decreases the concentration of thin filaments (pCa 4 plus ATP) required to attain a half-maximal rate of release of product from a pre-power stroke complex [myosin-S1-2-deoxy-3-O-(N-methylanthraniloyl)ADP-Pi], as investigated by double-mixing stopped-flow fluorescence, suggestive of a change in the proportion of active (turned on) and inactive (turned off) conformers, but similar maximum rates of product release are observed with either type of reconstituted thin filament. Phosphorylated thin filaments (pCa 4 and 8) display a higher affinity for myosin-S1(ADP) versus the control scenario without affecting isotherm steepness. Specific activities of ATP and Tpm1.1(α) are determined during an in vitro incubation of rat cardiac tissue [12 day-old, 50% phosphorylated Tpm1.1(α)] with [32P]orthophosphate. The incorporation of an isotope into tropomyosin lags behind that of ATP by a factor of approximately 10, indicating that transfer is a comparatively slow process.

Looking for Targets to Restore the Contractile Function in Congenital Myopathy Caused by Gln147Pro Tropomyosin.

We have used the technique of polarized microfluorimetry to obtain new insight into the pathogenesis of skeletal muscle disease caused by the Gln147Pro substitution in ß-tropomyosin (Tpm2.2). The spatial rearrangements of actin, myosin and tropomyosin in the single muscle fiber containing reconstituted thin filaments were studied during simulation of several stages of ATP hydrolysis cycle. The angular orientation of the fluorescence probes bound to tropomyosin was found to be changed by the substitution and was characteristic for a shift of tropomyosin strands closer to the inner actin domains. It was observed both in the absence and in the presence of troponin, Ca2+ and myosin heads at all simulated stages of the ATPase cycle. The mutant showed higher flexibility. Moreover, the Gln147Pro substitution disrupted the myosin-induced displacement of tropomyosin over actin. The irregular positioning of the mutant tropomyosin caused premature activation of actin monomers and a tendency to increase the number of myosin cross-bridges in a state of strong binding with actin at low Ca2+.

The Dark Side of Actin: Cardiac actin variants highlight the role of allostery in disease development.

Mutations in the α-cardiac actin ACTC1 gene cause dilated or hypertrophic cardiomyopathy. These diseases are the result of changes in protein interactions between ACTC protein and force-generating ß-myosin or the calcium-dependent cardiac-tropomyosin (cTm) and cardiac troponin (cTn) regulatory complex, altering the overall contractile force. The T126I and S271F ACTC variants possess amino acid substitutions on the other side of actin relative to the myosin or regulatory protein binding sites on what we call the "dark side" of actin. The T126I change results in hyposensitivity to calcium, in accordance with the calcium sensitivity pathway of cardiomyopathy development while the S271F change alters the maximum in vitro motility sliding speed, reflecting a change in maximum force. These results demonstrate the role of actin allostery in the cardiac disease development.

Triggering typical nemaline myopathy with compound heterozygous nebulin mutations reveals myofilament structural changes as pathomechanism.

Nebulin is a giant protein that winds around the actin filaments in the skeletal muscle sarcomere. Compound-heterozygous mutations in the nebulin gene (NEB) cause typical nemaline myopathy (NM), a muscle disorder characterized by muscle weakness with limited treatment options. We created a mouse model with a missense mutation p.Ser6366Ile and a deletion of NEB exon 55, the Compound-Het model that resembles typical NM. We show that Compound-Het mice are growth-retarded and have muscle weakness. Muscles have a reduced myofibrillar fractional-area and sarcomeres are disorganized, contain rod bodies, and have longer thin filaments. In contrast to nebulin-based severe NM where haplo-insufficiency is the disease driver, Compound-Het mice express normal amounts of nebulin. X-ray diffraction revealed that the actin filament is twisted with a larger radius, that tropomyosin and troponin behavior is altered, and that the myofilament spacing is increased. The unique disease mechanism of nebulin-based typical NM reveals novel therapeutic targets.

Cardiac troponin and tropomyosin bind to F-actin cooperatively, as revealed by fluorescence microscopy.

In cardiac muscle, binding of troponin (Tn) and tropomyosin (Tpm) to filamentous (F)-actin forms thin filaments capable of Ca2+ -dependent regulation of contraction. Tpm binds to F-actin in a head-to-tail fashion, while Tn stabilizes these linkages. Valuable structural and functional information has come from biochemical, X-ray, and electron microscopy data. However, the use of fluorescence microscopy to study thin filament assembly remains relatively underdeveloped. Here, triple fluorescent labeling of Tn, Tpm, and F-actin allowed us to track thin filament assembly by fluorescence microscopy. It is shown here that Tn and Tpm molecules self-organize on actin filaments and give rise to decorated and undecorated regions. Binding curves based on colocalization of Tn and Tpm on F-actin exhibit cooperative binding with a dissociation constant Kd of ~ 0.5 µm that is independent of the Ca2+ concentration. Binding isotherms based on the intensity profile of fluorescently labeled Tn and Tpm on F-actin show that binding of Tn is less cooperative relative to Tpm. Computational modeling of Tn-Tpm binding to F-actin suggests two equilibrium steps involving the binding of an initial Tn-Tpm unit (nucleation) and subsequent recruitment of adjacent Tn-Tpm units (elongation) that stabilize the assembly. The results presented here highlight the utility of employing fluorescence microscopy to study supramolecular protein assemblies.

Identification and characterization of troponin genes in Locusta migratoria.

Troponin complex comprises three subunits, namely troponin C (TpnC), troponin I (TpnI) and troponin T (TpnT), and regulates the contraction of striated muscle. We found that the locust Locusta migratoria genome has one TpnT gene (LmTpnT), one TpnI gene (LmTpnI) and three TpnC genes (LmTpnC1, LmTpnC2 and LmTpnC3). Through alternative splicing, LmTpnT and LmTpnI potentially encode two and eight isoforms, respectively. The flight muscle and the jump muscle of L. migratoria express an identical LmTpnT isoform, but different LmTpnC isoforms and LmTpnI isoforms. LmTpnC2 and LmTpnC3 both contain highly conserved residues essential for calcium binding in the EF-hand II and IV, thus belonging two-site isoform. LmTpnC1 contains non-conserved substitutions in the EF-hand II and all highly conserved residues for calcium binding in the EF-hand IV. Mutagenesis and tyrosine fluorescence spectroscopic analysis show that both the EF-hand II and IV of LmTpnC1 can serve as calcium-binding site. Therefore, all three LmTpnC isoforms belong to two-site isoform. This is in contrast to the situation in the insect with asynchronous flight muscle, which expresses both one-site isoform and two-site isoform of TpnC. Those results suggest that the origination of insect asynchronous flight muscle is associated with the emergence of one-site isoform of TpnC.

Skin wound healing with composite biomembranes loaded by tiopronin or captopril.

Novel wound dressings composed of chitosan (Ch) and hyaluronan (HA) loaded with tiopronin or captopril as antiinflammatory drugs were prepared. Composite biomembranes were examined in skin wounds of ischemic rabbits with the aim to accelerate the process of healing. The results proved that the biomembranes composed of Ch/HA/tiopronin or Ch/HA/captopril facilitated healing of skin wounds compared to untreated animals and animals treated with Ch/HA membranes. These results were confirmed by histology. Cu(II) ions and ascorbate-induced high-molar-mass HA degradation by means of rotational viscometry was performed and the ability of the both drugs to scavenge reactive oxygen species was evaluated. The results showed that captopril as well as tiopronin decreased the rate of HA degradation exclusively at higher concentrations.

Myosin and Other Energy-Transducing ATPases: Structural Dynamics Studied by Electron Paramagnetic Resonance.

The objective of this article was to document the energy-transducing and regulatory interactions in supramolecular complexes such as motor, pump, and clock ATPases. The dynamics and structural features were characterized by motion and distance measurements using spin-labeling electron paramagnetic resonance (EPR) spectroscopy. In particular, we focused on myosin ATPase with actin-troponin-tropomyosin, neural kinesin ATPase with microtubule, P-type ion-motive ATPase, and cyanobacterial clock ATPase. Finally, we have described the relationships or common principles among the molecular mechanisms of various energy-transducing systems and how the large-scale thermal structural transition of flexible elements from one state to the other precedes the subsequent irreversible chemical reactions.

Troponin destabilization impairs sarcomere-cytoskeleton interactions in iPSC-derived cardiomyocytes from dilated cardiomyopathy patients.

The sarcomeric troponin-tropomyosin complex is a critical mediator of excitation-contraction coupling, sarcomeric stability and force generation. We previously reported that induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) from patients with a dilated cardiomyopathy (DCM) mutation, troponin T (TnT)-R173W, display sarcomere protein misalignment and impaired contractility. Yet it is not known how TnT mutation causes dysfunction of sarcomere microdomains and how these events contribute to misalignment of sarcomeric proteins in presence of DCM TnT-R173W. Using a human iPSC-CM model combined with CRISPR/Cas9-engineered isogenic controls, we uncovered that TnT-R173W destabilizes molecular interactions of troponin with tropomyosin, and limits binding of PKA to local sarcomere microdomains. This attenuates troponin phosphorylation and dysregulates local sarcomeric microdomains in DCM iPSC-CMs. Disrupted microdomain signaling impairs MYH7-mediated, AMPK-dependent sarcomere-cytoskeleton filament interactions and plasma membrane attachment. Small molecule-based activation of AMPK can restore TnT microdomain interactions, and partially recovers sarcomere protein misalignment as well as impaired contractility in DCM TnT-R173W iPSC-CMs. Our findings suggest a novel therapeutic direction targeting sarcomere- cytoskeleton interactions to induce sarcomere re-organization and contractile recovery in DCM.

Cryo-EM structures of cardiac thin filaments reveal the 3D architecture of troponin.

Troponin is an essential component of striated muscle and it regulates the sliding of actomyosin system in a calcium-dependent manner. Despite its importance, the structure of troponin has been elusive due to its high structural heterogeneity. In this study, we analyzed the 3D structures of murine cardiac thin filaments using a cryo-electron microscope equipped with a Volta phase plate (VPP). Contrast enhancement by a VPP enabled us to reconstruct the entire repeat of the thin filament. We determined the orientation of troponin relative to F-actin and tropomyosin, and characterized the interactions between troponin and tropomyosin. This study provides a structural basis for understanding the molecular mechanism of actomyosin system.

High-speed AFM reveals subsecond dynamics of cardiac thin filaments upon Ca2+ activation and heavy meromyosin binding.

High-speed atomic force microscopy (HS-AFM) can be used to study dynamic processes with real-time imaging of molecules within 1- to 5-nm spatial resolution. In the current study, we evaluated the 3-state model of activation of cardiac thin filaments (cTFs) isolated as a complex and deposited on a mica-supported lipid bilayer. We studied this complex for dynamic conformational changes 1) at low and high [Ca2+] (pCa 9.0 and 4.5), and 2) upon myosin binding to the cTF in the nucleotide-free state or in the presence of ATP. HS-AFM was used to directly visualize the tropomyosin-troponin complex and Ca2+-induced tropomyosin movements accompanied by structural transitions of actin monomers within cTFs. Our data show that cTFs at relaxing or activating conditions are not ultimately in a blocked or activated state, respectively, but rather the combination of states with a prevalence that is dependent on the [Ca2+] and the presence of weakly or strongly bound myosin. The weakly and strongly bound myosin induce similar changes in the structure of cTFs as confirmed by the local dynamical displacement of individual tropomyosin strands in the center of a regulatory unit of cTF at the relaxed and activation conditions. The displacement of tropomyosin at the relaxed conditions had never been visualized directly and explains the ability of myosin binding to TF at the relaxed conditions. Based on the ratios of nonactivated and activated segments within cTFs, we proposed a mechanism of tropomyosin switching from different states that includes both weakly and strongly bound myosin.