Thin Filament Function Research Articles

Cardiac Myosin and Thin Filament as Targets for Lead and Cadmium Divalent Cations.

Lead and cadmium are heavy metals widely distributed in the environment and contribute significantly to cardiovascular morbidity and mortality. Using Leadmium Green dye, we have shown that lead and cadmium enter cardiomyocytes, distributing throughout the cell. Using an invitro motility assay, we have shown that sliding velocity of actin and native thin filaments over myosin decreases with increasing concentrations of Pb2+ and Cd2+. Significantly lower concentrations of Pb2+ and Cd2+ (0.6mM) were required to stop sliding of thin filaments over myosin compared to the stopping actin sliding over the same myosin (1.1-1.6mM). Lower concentration of Cd2+ (1.1mM) needed to stop actin sliding over myosin compared to the Pb2++Cd2+ combination (1.3mM) and lead alone (1.6mM). There were no differences found in the effects of lead and cadmium cations on relative force developed by myosin heads or number of actin filaments bound to myosin. Sliding velocity of actin over myosin in the left atrium, right and left ventricles changed equally when exposed to the same dose of the same metal. Thus, we have demonstrated for the first time that Pb2+ and Cd2+ can directly affect myosin and thin filament function, with Cd2+ exerting a more toxic influence on myosin function compared to Pb2+.

Troponin-I-induced tropomyosin pivoting defines thin-filament function in relaxed and active muscle.

Regulation of the crossbridge cycle that drives muscle contraction involves a reconfiguration of the troponin-tropomyosin complex on actin filaments. By comparing atomic models of troponin-tropomyosin fitted to cryo-EM structures of inhibited and Ca2+-activated thin filaments, we find that tropomyosin pivots rather than rolls or slides across actin as generally thought. We propose that pivoting can account for the Ca2+ activation that initiates muscle contraction and then relaxation influenced by troponin-I (TnI). Tropomyosin is well-known to occupy either of three meta-stable configurations on actin, regulating access of myosin motorheads to their actin-binding sites and thus the crossbridge cycle. At low Ca2+ concentrations, tropomyosin is trapped by TnI in an inhibitory B-state that sterically blocks myosin binding to actin, leading to muscle relaxation. Ca2+ binding to TnC draws TnI away from tropomyosin, while tropomyosin moves to a C-state location over actin. This partially relieves the steric inhibition and allows weak binding of myosin heads to actin, which then transition to strong actin-bound configurations, fully activating the thin filament. Nevertheless, the reconfiguration that accompanies the initial Ca2+-sensitive B-state/C-state shift in troponin-tropomyosin on actin remains uncertain and at best is described by moderate-resolution cryo-EM reconstructions. Our recent computational studies indicate that intermolecular residue-to-residue salt-bridge linkage between actin and tropomyosin is indistinguishable in B- and C-state thin filament configurations. We show here that tropomyosin can pivot about relatively fixed points on actin to accompany B-state/C-state structural transitions. We argue that at low Ca2+ concentrations C-terminal TnI domains attract tropomyosin, causing it to bend and then pivot toward the TnI, thus blocking myosin binding and contraction.

Functional assays reveal the pathogenic mechanism of a de novo tropomyosin variant identified in patient with dilated cardiomyopathy

Dilated cardiomyopathy (DCM) is a leading cause of heart failure and a major indicator for heart transplant. Human genetic studies have identified over a thousand causal mutations for DCM in genes involved in a variety of cellular processes, including sarcomeric contraction. A substantial clinical challenge is determining the pathogenicity of novel variants in disease-associated genes. This challenge of connecting genotype and phenotype has frustrated attempts to develop effective, mechanism-based treatments for patients. Here, we identified a de novo mutation (T237S) in TPM1, the gene that encodes the thin filament protein tropomyosin, in a patient with DCM and conducted in vitro experiments to characterize the pathogenicity of this novel variant. We expressed recombinant mutant protein, reconstituted it into thin filaments, and examined the effects of the mutation on thin filament function. We show that the mutation reduces the calcium sensitivity of thin filament activation, as previously seen for known pathogenic mutations. Mechanistically, this shift is due to mutation-induced changes in tropomyosin positioning along the thin filament. We demonstrate that the thin filament activator omecamtiv mecarbil restores the calcium sensitivity of thin filaments regulated by the mutant tropomyosin, which lays the foundation for additional experiments to explore the therapeutic potential of this drug for patients harboring the T237S mutation. Taken together, our results suggest that the TPM1 T237S mutation is likely pathogenic and demonstrate how functional in vitro characterization of pathogenic protein variants in the lab might guide precision medicine in the clinic.

Removal of MuRF1 Increases Muscle Mass in Nemaline Myopathy Models, but Does Not Provide Functional Benefits.

Nemaline myopathy (NM) is characterized by skeletal muscle weakness and atrophy. No curative treatments exist for this debilitating disease. NM is caused by mutations in proteins involved in thin-filament function, turnover, and maintenance. Mutations in nebulin, encoded by NEB, are the most common cause. Skeletal muscle atrophy is tightly linked to upregulation of MuRF1, an E3 ligase, that targets proteins for proteasome degradation. Here, we report a large increase in MuRF1 protein levels in both patients with nebulin-based NM, also named NEM2, and in mouse models of the disease. We hypothesized that knocking out MuRF1 in animal models of NM with muscle atrophy would ameliorate the muscle deficits. To test this, we crossed MuRF1 KO mice with two NEM2 mouse models, one with the typical form and the other with the severe form. The crosses were viable, and muscles were studied in mice at 3 months of life. Ultrastructural examination of gastrocnemius muscle lacking MuRF1 and with severe NM revealed a small increase in vacuoles, but no significant change in the myofibrillar fractional area. MuRF1 deficiency led to increased weights of various muscle types in the NM models. However, this increase in muscle size was not associated with increased in vivo or in vitro force production. We conclude that knocking out MuRF1 in NEM2 mice increases muscle size, but does not improve muscle function.

Using novel small molecules to alter cardiac thin filament function

Inherited cardiomyopathies such as hypertrophic cardiomyopathy (HCM) and dilated cardiomyopathy (DCM) have a high population prevalence of 1 in 500 and 1 in 250 individuals, respectively, in the US and Europe. Disease-related HCM and DCM effects range enormously from being asymptomatic to leading to heart failure and a need for heart transplantation. HCM is characterized by left ventricular (LV) hypertrophy, typically with increased calcium sensitivity of thin filament activation, while DCM exhibits LV dilatation and reduced calcium sensitivity.

Tirasemtiv and dATP Synergistically Reverse the Acidosis-induced Depression of Myosin’s Force and Motion Generating Capacity

In response to repeated, intense contractions muscle reduces its ability to generate force and velocity, characterizing fatigue. This reduction is due, in part, to the depressive direct effects of acidosis on myosin and thin-filament function, particularly at sub-saturating calcium concentrations. We sought to reverse these effects in vitro by 1) replacing ATP with 2-deoxy ATP (dATP), and 2) the addition of the troponin activator tirasemtiv (TR). We used a mini-ensemble laser trap assay to measure the force generating capacity with a regulated thin filament at pCa 7. Acidosis (pH 6.8 vs 7.4) significantly decreased myosin's force-generating capacity (0.80 ± 0.23 vs. 0.93 ± 0.29 pN; p < 0.05, at 6.8 and 7.4 respectively) as measured in a mini-ensemble laser trap assay. However, replacing the ATP with dATP and adding TR at pH 6.8 caused force to not only recover but increase above the control value at pH 7.4 (0.93 ± 0.007 vs. 1.32 ± 0.001; p < 0.05, for 7.4 and 6.8 respectively). The increased force at low pH (6.8) appeared to be due to an increased rate of myosin's attachment to the thin filament because the frequency of binding events was significantly increased under acidic conditions with dATP and TR (2.34 ± 2.94) added compared to ATP (0.47 ± 1.16; p < 0.05). Overall, these findings suggest that acidosis directly inhibits myosin's ability to bind and translocate a regulated actin filament, and that these agents that increase myosin's rate of attachment to actin can reverse these effects in vitro. Thus, these results suggest that these agents might offer hope in mitigating the deleterious effects of muscle fatigue in clinical populations.

The Nebulin Family LIM and SH3 Proteins Regulate Postsynaptic Development and Function.

Neuronal dendrites have specialized actin-rich structures called dendritic spines that receive and integrate most excitatory synaptic inputs. The stabilization of dendrites and spines during neuronal maturation is essential for proper neural circuit formation. Changes in dendritic morphology and stability are largely mediated by regulation of the actin cytoskeleton; however, the underlying mechanisms remain to be fully elucidated. Here, we present evidence that the nebulin family members LASP1 and LASP2 play an important role in the postsynaptic development of rat hippocampal neurons from both sexes. We find that both LASP1 and LASP2 are enriched in dendritic spines, and their knockdown impairs spine development and synapse formation. Furthermore, LASP2 exerts a distinct role in dendritic arbor and dendritic spine stabilization. Importantly, the actin-binding N-terminal LIM domain and nebulin repeats of LASP2 are required for spine stability and dendritic arbor complexity. These findings identify LASP1 and LASP2 as novel regulators of neuronal circuitry.SIGNIFICANCE STATEMENT Proper regulation of the actin cytoskeleton is essential for the structural stability of dendrites and dendritic spines. Consequently, the malformation of dendritic structures accompanies numerous neurologic disorders, such as schizophrenia and autism. Nebulin family members are best known for their role in regulating the stabilization and function of actin thin filaments in muscle. The two smallest family members, LASP1 and LASP2, are more structurally diverse and are expressed in a broader array of tissues. While both LASP1 and LASP2 are highly expressed in the brain, little is currently known about their function in the nervous system. In this study, we demonstrate the first evidence that LASP1 and LASP2 are involved in the formation and long-term maintenance of dendrites and dendritic spines.

A new twist on tropomyosin binding to actin filaments: perspectives on thin filament function, assembly and biomechanics.

Tropomyosin, best known for its role in the steric regulation of muscle contraction, polymerizes head-to-tail to form cables localized along the length of both muscle and non-muscle actin-based thin filaments. In skeletal and cardiac muscles, tropomyosin, under the control of troponin and myosin, moves in a cooperative manner between blocked, closed and open positions on filaments, thereby masking and exposing actin-binding sites necessary for myosin crossbridge head interactions. While the coiled-coil signature of tropomyosin appears to be simple, closer inspection reveals surprising structural complexity required to perform its role in steric regulation. For example, component α-helices of coiled coils are typically zippered together along a continuous core hydrophobic stripe. Tropomyosin, however, contains a number of anomalous, functionally controversial, core amino acid residues. We argue that the atypical residues at this interface, including clusters of alanines and a charged aspartate, are required for preshaping tropomyosin to readily fit to the surface of the actin filament, but do so without compromising tropomyosin rigidity once the filament is assembled. Indeed, persistence length measurements of tropomyosin are characteristic of a semi-rigid cable, in this case conducive to cooperative movement on thin filaments. In addition, we also maintain that tropomyosin displays largely unrecognized and residue-specific torsional variance, which is involved in optimizing contacts between actin and tropomyosin on the assembled thin filament. Corresponding twist-induced stiffness may also enhance cooperative translocation of tropomyosin across actin filaments. We conclude that anomalous core residues of tropomyosin facilitate thin filament regulatory behavior in a multifaceted way.

Testing of therapies in a novel nebulin nemaline myopathy model demonstrate a lack of efficacy

Nemaline myopathies are heterogeneous congenital muscle disorders causing skeletal muscle weakness and, in some cases, death soon after birth. Mutations in nebulin, encoding a large sarcomeric protein required for thin filament function, are responsible for approximately 50% of nemaline myopathy cases. Despite the severity of the disease there is no effective treatment for nemaline myopathy with limited research to develop potential therapies. Several supplements, including L-tyrosine, have been suggested to be beneficial and consequently self-administered by nemaline myopathy patients without any knowledge of their efficacy. We have characterized a zebrafish model for nemaline myopathy caused by a mutation in nebulin. These fish form electron-dense nemaline bodies and display reduced muscle function akin to the phenotypes observed in nemaline myopathy patients. We have utilized our zebrafish model to test and evaluate four treatments currently self-administered by nemaline myopathy patients to determine their ability to increase skeletal muscle function. Analysis of muscle pathology and locomotion following treatment with L-tyrosine, L-carnitine, taurine, or creatine revealed no significant improvement in skeletal muscle function emphasizing the urgency to develop effective therapies for nemaline myopathy.

Tropomyosin Must Interact Weakly with Actin to Effectively Regulate Thin Filament Function

Elongated tropomyosin, associated with actin-subunits along the surface of thin filaments, makes electrostatic interactions with clusters of conserved residues, K326, K328, and R147, on actin. The association is weak, permitting low-energy cost regulatory movement of tropomyosin across the filament during muscle activation. Interestingly, acidic D292 on actin, also evolutionarily conserved, lies adjacent to the three-residue cluster of basic amino acids and thus may moderate the combined local positive charge, diminishing tropomyosin-actin interaction and facilitating regulatory-switching. Indeed, charge neutralization of D292 is connected to muscle hypotonia in individuals with D292V actin mutations and linked to congenital fiber-type disproportion. Here, the D292V mutation may predispose tropomyosin-actin positioning to a myosin-blocking state, aberrantly favoring muscle relaxation, thus mimicking the low-Ca2+ effect of troponin even in activated muscles. To test this hypothesis, interaction energetics and in vitro function of wild-type and D292V filaments were measured. Energy landscapes based on F-actin-tropomyosin models show the mutation localizes tropomyosin in a blocked-state position on actin defined by a deeper energy minimum, consistent with augmented steric-interference of actin-myosin binding. In addition, whereas myosin-dependent motility of troponin/tropomyosin-free D292V F-actin is normal, motility is dramatically inhibited after addition of tropomyosin to the mutant actin. Thus, D292V-induced blocked-state stabilization appears to disrupt the delicately poised energy balance governing thin filament regulation. Our results validate the premise that stereospecific but necessarily weak binding of tropomyosin to F-actin is required for effective thin filament function.

Atomic resolution probe for allostery in the regulatory thin filament

Calcium binding and dissociation within the cardiac thin filament (CTF) is a fundamental regulator of normal contraction and relaxation. Although the disruption of this complex, allosterically mediated process has long been implicated in human disease, the precise atomic-level mechanisms remain opaque, greatly hampering the development of novel targeted therapies. To address this question, we used a fully atomistic CTF model to test both Ca(2+) binding strength and the energy required to remove Ca(2+) from the N-lobe binding site in WT and mutant troponin complexes that have been linked to genetic cardiomyopathies. This computational approach is combined with measurements of in vitro Ca(2+) dissociation rates in fully reconstituted WT and cardiac troponin T R92L and R92W thin filaments. These human disease mutations represent known substitutions at the same residue, reside at a significant distance from the calcium binding site in cardiac troponin C, and do not affect either the binding pocket affinity or EF-hand structure of the binding domain. Both have been shown to have significantly different effects on cardiac function in vivo. We now show that these mutations independently alter the interaction between the Ca(2+) ion and cardiac troponin I subunit. This interaction is a previously unidentified mechanism, in which mutations in one protein of a complex indirectly affect a third via structural and dynamic changes in a second to yield a pathogenic change in thin filament function that results in mutation-specific disease states. We can now provide atom-level insight that is potentially highly actionable in drug design.

Localization of the binding interface between leiomodin-2 and α-tropomyosin

The development of some familial dilated cardiomyopathies (DCM) correlates with the presence of mutations in proteins that regulate the organization and function of thin filaments in cardiac muscle cells. Harmful effects of some mutations might be caused by disruption of yet uncharacterized protein–protein interactions. We used nuclear magnetic resonance spectroscopy to localize the region of striated muscle α-tropomyosin (Tpm1.1) that interacts with leiomodin-2 (Lmod2), a member of tropomodulin (Tmod) family of actin-binding proteins. We found that 21 N-terminal residues of Tpm1.1 are involved in interactions with residues 7–41 of Lmod2. The K15N mutation in Tpm1.1, known to be associated with familial DCM, is located within the newly identified Lmod2 binding site of Tpm1.1. We studied the effect of this mutation on binding Lmod2 and Tmod1. The mutation reduced binding affinity for both Lmod2 and Tmod1, which are responsible for correct lengths of thin filaments. The effect of the K15N mutation on Tpm1.1 binding to Lmod2 and Tmod1 provides a molecular rationale for the development of familial DCM.

Cardiomyopathy-Associated Mutation K15N in Tropomyosin Affects its Interaction with Leiomodin and Tropomodulin

The development of some familial dilated cardiomyopathies (DCM ) correlates with the presence of mutations in proteins that regulate the organization and function of thin filaments in cardiac muscle cells. Whereas the biological consequence of mutations affecting protein folding, stability and levels of intracellular expression is more apparent, understanding of harmful effects of mutations interfering with yet uncharacterized protein-protein interactions might be elusive. We used nuclear magnetic resonance spectroscopy to precisely localize the region of striated muscle α-tropomyosin that interacts with leiomodin-2 (Lmod2), a member of tropomodulin family of actin-binding proteins. The K15N mutation known to be associated with familial DCM is located within the newly identified Lmod2 binding site of tropomyosin. We studied the effect of this mutation on binding Lmod2 and Tmod1, cardiac isoforms responsible for correct lengths of the thin filaments. The mutation reduced binding affinity for both Lmod2 and Tmod1, however the decrease was more pronounced for binding Lmod2. The reduced affinity and the uneven effect of the K15N mutation on TM binding to Lmod2 and Tmod1 provide a molecular rationale for the development of familial DCM.

Structure of the F-Actin-Tropomyosin Complex Revealed by Electron Cryomicroscopy

Filamentous actin (F-actin) is the major protein of muscle thin filaments, and actin microfilaments are the main component of the eukaryotic cytoskeleton. Mutations in different actin isoforms lead to early-onset autosomal dominant non-syndromic hearing loss, familial thoracic aortic aneurysms and dissections, and multiple variations of myopathies. In striated muscle fibres, the binding of myosin motors to actin filaments is mainly regulated by tropomyosin and troponin. Tropomyosin also binds to F-actin in smooth muscle and in non-muscle cells and stabilizes and regulates the filaments there in the absence of troponin. Although crystal structures for monomeric actin (G-actin) are available, a high-resolution structure of F-actin is still missing, hampering our understanding of how disease-causing mutations affect the function of thin muscle filaments and microfilaments. Here we report the three-dimensional structure of F-actin at a resolution of 3.7 A in complex with tropomyosin at a resolution of 6.5 A, determined by electron cryomicroscopy. The structure reveals that the D-loop is ordered and acts as a central region for hydrophobic and electrostatic interactions that stabilize the F-actin filament. We clearly identify map density corresponding to ADP and Mg2+ and explain the possible effect of prominent disease-causing mutants. A comparison of F-actin with G-actin reveals the conformational changes during filament formation and identifies the D-loop as their key mediator. We also confirm that negatively charged tropomyosin interacts with a positively charged groove on F-actin. Comparison of the position of tropomyosin in F-actin-tropomyosin with its position in our previously determined F-actin-tropomyosin-myosin structure reveals a myosin-induced transition of tropomyosin. Our results allow us to understand the role of individual mutations in the genesis of actin- and tropomyosin-related diseases and will serve as a strong foundation for the targeted development of drugs.

The N-Terminal Hypervariable Region of Troponin T Differentially Modulates the Affinity of Tropomyosin-Binding Sites

The troponin complex plays a central role in the allosteric function of sarcomeric thin filaments by enacting conformational changes during the Ca2+-regulated contraction and relaxation of striated muscle. The troponin subunit T (TnT) has two binding sites for tropomyosin (Tm) and is responsible for anchoring the troponin complex to the thin filament. Although the C-terminal and middle regions of the TnT polypeptide chain are highly conserved among the three muscle type isoforms, the hypervariable N-terminal region has evolutionarily diverged significantly among isoforms. Previous studies have shown that the N-terminal variable region fine-tunes Ca2+ regulation of muscle contractility via modulation of the overall molecular conformation of TnT, and its interactions with Tm. In the present study, we engineered intact TnT and representative fragments of TnT, expressed them in E. coli, and prepared purified proteins for functional studies. Tropomyosin binding affinity was analyzed using affinity chromatography and solid phase protein binding assays to investigate the modulatory effects of the N-terminal variable region. The results demonstrated that in the absence of the N-terminal variable region, TnT's conserved middle region and C-terminal T2 region Tm-binding sites showed comparable Tm-binding affinities across isoforms. The data demonstrate that without the modulatory effect of the N-terminal variable region, the intrinsic Tm-binding affinities of the two sites are both high. In contrast, the presence of the isoform specific N-terminal variable region differentially reduces the binding affinity of TnT for Tm, primarily at the middle region binding site. These novel findings indicate that the N-terminal variable region plays a key role in the functional difference of muscle fiber type-specific, developmental, splice variant, and pathogenic TnT isoforms by modulating the interactions with Tm during the contraction and relaxation of cardiac and skeletal muscle.

Structure of the F-actin-tropomyosin complex.

Filamentous actin (F-actin) is the major protein of muscle thin filaments, and actin microfilaments are the main component of the eukaryotic cytoskeleton. Mutations in different actin isoforms lead to early-onset autosomal dominant non-syndromic hearing loss, familial thoracic aortic aneurysms and dissections, and multiple variations of myopathies. In striated muscle fibres, the binding of myosin motors to actin filaments is mainly regulated by tropomyosin and troponin. Tropomyosin also binds to F-actin in smooth muscle and in non-muscle cells and stabilizes and regulates the filaments there in the absence of troponin. Although crystal structures for monomeric actin (G-actin) are available, a high-resolution structure of F-actin is still missing, hampering our understanding of how disease-causing mutations affect the function of thin muscle filaments and microfilaments. Here we report the three-dimensional structure of F-actin at a resolution of 3.7Å in complex with tropomyosin at a resolution of 6.5Å, determined by electron cryomicroscopy. The structure reveals that the D-loop is ordered and acts as a central region for hydrophobic and electrostatic interactions that stabilize the F-actin filament. We clearly identify map density corresponding to ADP and Mg(2+) and explain the possible effect of prominent disease-causing mutants. A comparison of F-actin with G-actin reveals the conformational changes during filament formation and identifies the D-loop as their key mediator. We also confirm that negatively charged tropomyosin interacts with a positively charged groove on F-actin. Comparison of the position of tropomyosin in F-actin-tropomyosin with its position in our previously determined F-actin-tropomyosin-myosin structure reveals a myosin-induced transition of tropomyosin. Our results allow us to understand the role of individual mutations in the genesis of actin- and tropomyosin-related diseases and will serve as a strong foundation for the targeted development of drugs.

The energy regulating upstream kinase complex LKB1/MO25/STRAD is a potential novel regulator of thin filament function (1081.3)

AMP‐activated protein kinase (AMPK) is a cellular energetic regulator that is known to modify both metabolic and contractile function in the heart. Phosphorylation by an upstream kinase complex (LKB1/MO25/STRAD; LMS) is required for AMPK activation. Yet, the relationship between the LMS, AMPK activation, and myofilament function is unknown. Accordingly, we hypothesized that the upstream kinase complex can regulate myofilament function independently or in combination with AMPK. To do this, demembranated (skinned) rat cardiac trabeculae were incubated with either the LMS or with varying ratios of the LMS with AMPK and myofilament function was measured. Skinned rat trabeculae treated with the LMS alone were desensitized to Ca2+ and had lowered maximum tension. Adding increasing amounts of AMPK in combination with LMS reversed this effect increasing Ca2+‐sensitivity of tension and maximum tension. We furthered hypothesized that the mechanism underlying this relationship is mediated through direct interaction of the LMS with thin filament proteins. Western blot analysis showed an association of the LMS with myofibrils while immunofluorescence indicated that Mo25 co‐localizes with the phalloidin stained thin filament. In conclusion, the LMS has been identified as a potential novel regulator of myofilament function perhaps through a direct interaction with the thin filament.Grant Funding Source: T32HL07249

Kinetic and Structural Characterization of Calcium Sensitizer Action on Thin Filament Function using FRET

The use of novel calcium sensitizing agents in the treatment of heart disease offers therapeutic value for patients suffering from a particularly prevalent and recalcitrant condition, however the elusive mechanisms of action for these drugs prevent increased and improved utilization of such agents clinically. Ideally the calcium sensitizer impact on the thin filament could be monitored directly in a physiologically relevant and dynamic way while still capturing the molecular level mechanisms involved. With that aim a homo-FRET scheme was developed which monitors the N-domain opening of the calcium binding subunit of cardiac troponin (N-cTnC) by labeling with TAMRA at cTnC(cys-13) and cTnC(cys-51). Using this novel FRET design the calcium binding properties of reconstituted troponin and their deactivation kinetics were measured in the presence of calcium sensitizers Levosimendan, Bepridil, Pimobendan, and EMD-57033 at various levels of in vitro reconstitution. We hypothesized that depending on the mechanism for each calcium sensitizer the effects on cTnC calcium binding and deactivation kinetics would vary based on the level of reconstitution. We expect that new insight into which thin filament proteins are necessary for sensitizer action will yield a clearer picture of the molecular level mechanisms underlying cardiotonic action. Although the study is currently ongoing preliminary results show significant sensitization for all four drugs in reconstituted ternary troponin compared to control and that this effect is abrogated in samples containing only cTnC and cardiac troponin inhibitory subunit (cTnI). Deactivation kinetics show a decreased transition rate for Levosimendan, Bepridil and Pimobendan but not for EMD-57033 both at the ternary troponin level and at the cTnI-cTnC level. Results from measurements with reconstituted thin filament preparation will be also discussed.

The Accuracy of Cardiac Myofilament Simulations is Enhanced by Permitting Calcium-Independent Tropomyosin Transitions

Conceptual and computational models have generally assumed that each regulatory unit (RU) of the thin filament remains in the blocked state until Ca2+ binds to troponin C. This includes our previous model (Campbell et al., Biophys J 98:2254, 2010), which simultaneously recapitulated key attributes of myofilament activation. However, the model failed to fully reproduce exchange experiments in which some fraction of myofilament troponin C is replaced with a non-Ca2+ binding mutant (xTnC). In simulations, xTnC caused much greater reductions in tension than were shown experimentally (Gillis et al., J Physiol 580:561, 2007). This effect was caused by the assumption of strong cooperative inhibition among nearest-neighbor RUs, which was required to produce basic myofilament activation behavior. We hypothesized that permitting some Ca2+-independent RU activation while maintaining cooperative inhibition would reconcile this discrepancy. In a new model, blocked-to-closed RU transitions were allowed without bound Ca2+ but at greater energetic cost (ΔG). Monte Carlo simulations showed that reducing ΔG to a finite value of 4.7 kJ/mol maintained cooperative myofilament activation while reproducing xTnC experiments (Figure). These results suggest that thin filament function does not require perfect Ca2+ switch fidelity.View Large Image | View Hi-Res Image | Download PowerPoint Slide

Gestalt-Binding of tropomyosin on actin during thin filament activation

Our thesis is that thin filament function can only be fully understood and muscle regulation then elucidated if atomic structures of the thin filament are available to reveal the positions of tropomyosin on actin in all physiological states. After all, it is tropomyosin influenced by troponin that regulates myosin-crossbridge cycling on actin and therefore controls contraction in all muscles. In addition, we maintain that a complete appreciation of thin filament activation also requires that the mechanical properties of tropomyosin itself are recognized and then related to the effect of myosin-association on actin. Taking the Gestalt-binding of tropomyosin into account, coupled with our electron microscopy structures and computational chemistry, we propose a comprehensive mechanism for tropomyosin regulatory movement over the actin filament surface that explains the cooperative muscle activation process. In fact, well-known point mutations of critical amino acids on the actin-tropomyosin binding interface disrupt Gestalt-binding and are associated with a number of inherited myopathies. Moreover, dysregulation of tropomyosin may also be a factor that interferes with the gatekeeping operation of non-muscle tropomyosin in the controlling interactions of a wide variety of cellular actin-binding proteins. The clinical relevance of Gestalt-binding is discussed in articles by the Marston and the Gunning groups in this special journal issue devoted to the impact of tropomyosin on biological systems.