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Velia M Fowler - One of the best experts on this subject based on the ideXlab platform.
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Tropomodulin 1 directly controls Thin Filament length in both wild-type and tropomodulin 4-deficient skeletal muscle.
Development, 2015Co-Authors: David S. Gokhin, Julien Ochala, Andrea A. Domenighetti, Velia M FowlerAbstract:The sarcomeric tropomodulin (Tmod) isoforms Tmod1 and Tmod4 cap Thin Filament pointed ends and functionally interact with the leiomodin (Lmod) isoforms Lmod2 and Lmod3 to control myofibril organization, Thin Filament lengths, and actomyosin crossbridge formation in skeletal muscle fibers. Here, we show that Tmod4 is more abundant than Tmod1 at both the transcript and protein level in a variety of muscle types, but the relative abundances of sarcomeric Tmods are muscle specific. We then generate Tmod4(-/-) mice, which exhibit normal Thin Filament lengths, myofibril organization, and skeletal muscle contractile function owing to compensatory upregulation of Tmod1, together with an Lmod isoform switch wherein Lmod3 is downregulated and Lmod2 is upregulated. However, RNAi depletion of Tmod1 from either wild-type or Tmod4(-/-) muscle fibers leads to Thin Filament elongation by ∼15%. Thus, Tmod1 per se, rather than total sarcomeric Tmod levels, controls Thin Filament lengths in mouse skeletal muscle, whereas Tmod4 appears to be dispensable for Thin Filament length regulation. These findings identify Tmod1 as the key direct regulator of Thin Filament length in skeletal muscle, in both adult muscle homeostasis and in developmentally compensated contexts.
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Alterations in Thin Filament length during postnatal skeletal muscle development and aging in mice
Frontiers in physiology, 2014Co-Authors: David S. Gokhin, Emily A. Dubuc, Kendra Q. Lian, Luanne L. Peters, Velia M FowlerAbstract:The lengths of the sarcomeric Thin Filaments vary in a skeletal muscle-specific manner and help specify the physiological properties of skeletal muscle. Since the extent of overlap between the Thin and thick Filaments determines the amount of contractile force that a sarcomere can actively produce, Thin Filament lengths are accurate predictors of muscle-specific sarcomere length-tension relationships and sarcomere operating length ranges. However, the striking uniformity of Thin Filament lengths wiThin sarcomeres, specified during myofibril assembly, has led to the widely held assumption that Thin Filament lengths remain constant throughout an organism's lifespan. Here, we rigorously tested this assumption by using computational super-resolution image analysis of confocal fluorescence images to explore the effects of postnatal development and aging on Thin Filament length in mice. We found that Thin Filaments shorten in postnatal tibialis anterior (TA) and gastrocnemius muscles between postnatal days 7 and 21, consistent with the developmental program of myosin heavy chain (MHC) gene expression in this interval. By contrast, Thin Filament lengths in TA and extensor digitorum longus (EDL) muscles remained constant between 2 mo and 2 yr of age, while Thin Filament lengths in soleus muscle became shorter, suggestive of a slow-muscle-specific mechanism of Thin Filament destabilization associated with aging. Collectively, these data are the first to show that Thin Filament lengths change as part of normal skeletal muscle development and aging, motivating future investigations into the cellular and molecular mechanisms underlying Thin Filament adaptation across the lifespan.
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congenital myopathy causing tropomyosin mutations induce Thin Filament dysfunction via distinct physiological mechanisms
Human Molecular Genetics, 2012Co-Authors: Julien Ochala, David S. Gokhin, I Penissonbesnier, Susana Quijanoroy, Nicole Monnier, Joel Lunardi, N Romero, Velia M FowlerAbstract:In humans, congenital myopathy-linked tropomyosin mutations lead to skeletal muscle dysfunction, but the cellular and molecular mechanisms underlying such dysfunction remain obscure. Recent studies have suggested a unifying mechanism by which tropomyosin mutations partially inhibit Thin Filament activation and prevent proper formation and cycling of myosin cross-bridges, inducing force deficits at the fiber and whole-muscle levels. Here, we aimed to verify this mechanism using single membrane-permeabilized fibers from patients with three tropomyosin mutations (TPM2-null, TPM3-R167H and TPM2-E181K) and measuring a broad range of parameters. Interestingly, we identified two divergent, mutation-specific pathophysiological mechanisms. (i) The TPM2-null and TPM3-R167H mutations both decreased cooperative Thin Filament activation in combination with reductions in the myosin cross-bridge number and force production. The TPM3-R167H mutation also induced a concomitant reduction in Thin Filament length. (ii) In contrast, the TPM2-E181K mutation increased Thin Filament activation, cross-bridge binding and force generation. In the former mechanism, modulating Thin Filament activation by administering troponin activators (CK-1909178 and EMD 57033) to single membrane-permeabilized fibers carrying tropomyosin mutations rescued the Thin Filament activation defect associated with the pathophysiology. Therefore, administration of troponin activators may constitute a promising therapeutic approach in the future.
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Thin-Filament length correlates with fiber type in human skeletal muscle
American journal of physiology. Cell physiology, 2011Co-Authors: David S. Gokhin, Nancy Kim, Sarah A. Lewis, Heinz R. Hoenecke, Darryl D. D'lima, Velia M FowlerAbstract:Force production in skeletal muscle is proportional to the amount of overlap between the Thin and thick Filaments, which, in turn, depends on their lengths. Both Thin- and thick-Filament lengths are precisely regulated and uniform wiThin a myofibril. While thick-Filament lengths are essentially constant across muscles and species (∼1.65 μm), Thin-Filament lengths are highly variable both across species and across muscles of a single species. Here, we used a high-resolution immunofluorescence and image analysis technique (distributed deconvolution) to directly test the hypothesis that Thin-Filament lengths vary across human muscles. Using deltoid and pectoralis major muscle biopsies, we identified Thin-Filament lengths that ranged from 1.19 ± 0.08 to 1.37 ± 0.04 μm, based on tropomodulin localization with respect to the Z-line. Tropomodulin localized from 0.28 to 0.47 μm further from the Z-line than the NH(2)-terminus of nebulin in the various biopsies, indicating that human Thin Filaments have nebulin-free, pointed-end extensions that comprise up to 34% of total Thin-Filament length. Furthermore, Thin-Filament length was negatively correlated with the percentage of type 2X myosin heavy chain wiThin the biopsy and shorter in type 2X myosin heavy chain-positive fibers, establishing the existence of a relationship between Thin-Filament lengths and fiber types in human muscle. Together, these data challenge the widely held assumption that human Thin-Filament lengths are constant. Our results also have broad relevance to musculoskeletal modeling, surgical reattachment of muscles, and orthopedic rehabilitation.
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tropomodulin isoforms regulate Thin Filament pointed end capping and skeletal muscle physiology
Journal of Cell Biology, 2010Co-Authors: Raymond A Lewis, Caroline R Mckeown, Roberta B Nowak, David Samuel Gokhin, Richard L Lieber, Ryan Littlefield, Velia M FowlerAbstract:During myofibril assembly, Thin Filament lengths are precisely specified to optimize skeletal muscle function. Tropomodulins (Tmods) are capping proteins that specify Thin Filament lengths by controlling actin dynamics at pointed ends. In this study, we use a genetic targeting approach to explore the effects of deleting Tmod1 from skeletal muscle. Myofibril assembly, skeletal muscle structure, and Thin Filament lengths are normal in the absence of Tmod1. Tmod4 localizes to Thin Filament pointed ends in Tmod1-null embryonic muscle, whereas both Tmod3 and -4 localize to pointed ends in Tmod1-null adult muscle. Substitution by Tmod3 and -4 occurs despite their weaker interactions with striated muscle tropomyosins. However, the absence of Tmod1 results in depressed isometric stress production during muscle contraction, systemic locomotor deficits, and a shift to a faster fiber type distribution. Thus, Tmod3 and -4 compensate for the absence of Tmod1 structurally but not functionally. We conclude that Tmod1 is a novel regulator of skeletal muscle physiology.
William Lehman - One of the best experts on this subject based on the ideXlab platform.
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a new twist on tropomyosin binding to actin Filaments perspectives on Thin Filament function assembly and biomechanics
Journal of Muscle Research and Cell Motility, 2020Co-Authors: William Lehman, Michael J Rynkiewicz, Jeffrey R MooreAbstract: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.
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The mechanism of Thin Filament regulation: Models in conflict?
The Journal of general physiology, 2019Co-Authors: Michael A. Geeves, Sherwin S. Lehrer, William LehmanAbstract:In a recent JGP article, Heeley et al. (2019. J. Gen. Physiol https://doi.org/10.1085/jgp.201812198) reopened the debate about two- versus three-state models of Thin Filament regulation. The authors review their work, which measures the rate constant of Pi release from myosin.ADP.Pi activated by actin or Thin Filaments under a variety of conditions. They conclude that their data can be described by a two-state model and raise doubts about the generally accepted three-state model as originally formulated by McKillop and Geeves (1993. Biophys. J. https://doi.org/10.1016/S0006-3495(93)81110-X). However, in the following article, we follow Plato's dictum that "twice and thrice over, as they say, good it is to repeat and review what is good." We have therefore reviewed the evidence for the three- and two-state models and present our view that the evidence is overwhelmingly in favor of three structural states of the Thin Filament, which regulate access of myosin to its binding sites on actin and, hence, muscle contractility.
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tropomyosin must interact weakly with actin to effectively regulate Thin Filament function
Biophysical Journal, 2017Co-Authors: Michael J Rynkiewicz, Steven B. Marston, Stefan Fischer, Jeffrey R Moore, Kathleen M Trybus, Thavanareth Prum, Stephen M Hollenberg, Farooq Ahmad Kiani, Patricia M Fagnant, William LehmanAbstract: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.
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structural determinants of muscle Thin Filament cooperativity
Archives of Biochemistry and Biophysics, 2016Co-Authors: Jeffrey R Moore, Stuart G Campbell, William LehmanAbstract:End-to-end connections between adjacent tropomyosin molecules along the muscle Thin Filament allow long-range conformational rearrangement of the multicomponent Filament structure. This process is influenced by Ca(2+) and the troponin regulatory complexes, as well as by myosin crossbridge heads that bind to and activate the Filament. Access of myosin crossbridges onto actin is gated by tropomyosin, and in the case of striated muscle Filaments, troponin acts as a gatekeeper. The resulting tropomyosin-troponin-myosin on-off switching mechanism that controls muscle contractility is a complex cooperative and dynamic system with highly nonlinear behavior. Here, we review key information that leads us to view tropomyosin as central to the communication pathway that coordinates the multifaceted effectors that modulate and tune striated muscle contraction. We posit that an understanding of this communication pathway provides a framework for more in-depth mechanistic characterization of myopathy-associated mutational perturbations currently under investigation by many research groups.
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Thin Filament structure and the steric blocking model
Comprehensive Physiology, 2016Co-Authors: William LehmanAbstract:By interacting with the troponin-tropomyosin complex on myofibrillar Thin Filaments, Ca2+ and myosin govern the regulatory switching processes influencing contractile activity of mammalian cardiac and skeletal muscles. A possible explanation of the roles played by Ca2+ and myosin emerged in the early 1970s when a compelling "steric model" began to gain traction as a likely mechanism accounting for muscle regulation. In its most simple form, the model holds that, under the control of Ca2+ binding to troponin and myosin binding to actin, tropomyosin strands running along Thin Filaments either block myosin-binding sites on actin when muscles are relaxed or move away from them when muscles are activated. Evidence for the steric model was initially based on interpretation of subtle changes observed in X-ray fiber diffraction patterns of intact skeletal muscle preparations. Over the past 25 years, electron microscopy coupled with three-dimensional reconstruction directly resolved Thin Filament organization under many experimental conditions and at increasingly higher resolution. At low-Ca2+, tropomyosin was shown to occupy a "blocked-state" position on the Filament, and switched-on in a two-step process, involving first a movement of tropomyosin away from the majority of the myosin-binding site as Ca2+ binds to troponin and then a further movement to fully expose the site when small numbers of myosin heads bind to actin. In this contribution, basic information on Ca2+-regulation of muscle contraction is provided. A description is then given relating the voyage of discovery taken to arrive at the present understanding of the steric regulatory model.
Jonathan P. Davis - One of the best experts on this subject based on the ideXlab platform.
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tri modal regulation of cardiac muscle relaxation intracellular calcium decline Thin Filament deactivation and cross bridge cycling kinetics
Biophysical Reviews, 2014Co-Authors: Brandon J. Biesiadecki, Mark T Ziolo, Jonathan P. Davis, Paul M.l. JanssenAbstract:Cardiac muscle relaxation is an essential step in the cardiac cycle. Even when the contraction of the heart is normal and forceful, a relaxation phase that is too slow will limit proper filling of the ventricles. Relaxation is too often thought of as a mere passive process that follows contraction. However, many decades of advancements in our understanding of cardiac muscle relaxation have shown it is a highly complex and well-regulated process. In this review, we will discuss three distinct events that can limit the rate of cardiac muscle relaxation: the rate of intracellular calcium decline, the rate of Thin-Filament de-activation, and the rate of cross-bridge cycling. Each of these processes are directly impacted by a plethora of molecular events. In addition, these three processes interact with each other, further complicating our understanding of relaxation. Each of these processes is continuously modulated by the need to couple bodily oxygen demand to cardiac output by the major cardiac physiological regulators. Length-dependent activation, frequency-dependent activation, and beta-adrenergic regulation all directly and indirectly modulate calcium decline, Thin-Filament deactivation, and cross-bridge kinetics. We hope to convey our conclusion that cardiac muscle relaxation is a process of intricate checks and balances, and should not be thought of as a single rate-limiting step that is regulated at a single protein level. Cardiac muscle relaxation is a system level property that requires fundamental integration of three governing systems: intracellular calcium decline, Thin Filament deactivation, and cross-bridge cycling kinetics.
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combined troponin i ser 150 and ser 23 24 phosphorylation sustains Thin Filament ca2 sensitivity and accelerates deactivation in an acidic environment
Journal of Molecular and Cellular Cardiology, 2014Co-Authors: Benjamin R Nixon, Mark T Ziolo, Jonathan P. Davis, Sean C. Little, Shane D Walton, Bo Zhang, Elizabeth A Brundage, Brandon J. BiesiadeckiAbstract:Abstract The binding of Ca 2 + to troponin C (TnC) in the troponin complex is a critical step regulating the Thin Filament, the actin–myosin interaction and cardiac contraction. Phosphorylation of the troponin complex is a key regulatory mechanism to match cardiac contraction to demand. Here we demonstrate that phosphorylation of the troponin I (TnI) subunit is simultaneously increased at Ser-150 and Ser-23/24 during in vivo myocardial ischemia. Myocardial ischemia decreases intracellular pH resulting in depressed binding of Ca 2 + to TnC and impaired contraction. To determine the pathological relevance of these simultaneous TnI phosphorylations we measured individual TnI Ser-150 (S150D), Ser-23/24 (S23/24D) and combined (S23/24/150D) pseudo-phosphorylation effects on Thin Filament regulation at acidic pH similar to that in myocardial ischemia. Results demonstrate that while acidic pH decreased Thin Filament Ca 2 + binding to TnC regardless of TnI composition, TnI S150D attenuated this decrease rendering it similar to non-phosphorylated TnI at normal pH. The dissociation of Ca 2 + from TnC was unaltered by pH such that TnI S150D remained slow, S23/24D remained accelerated and the combined S23/24/150D remained accelerated. This effect of the combined TnI Ser-150 and Ser-23/24 pseudo-phosphorylations to maintain Ca 2 + binding while accelerating Ca 2 + dissociation represents the first post-translational modification of troponin by phosphorylation to both accelerate Thin Filament deactivation and maintain Ca 2 + sensitive activation. These data suggest that TnI Ser-150 phosphorylation induced attenuation of the pH-dependent decrease in Ca 2 + sensitivity and its combination with Ser-23/24 phosphorylation to maintain accelerated Thin Filament deactivation may impart an adaptive role to preserve contraction during acidic ischemia pH without slowing relaxation.
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Protein kinase C phosphomimetics alter Thin Filament Ca2+ binding properties.
PloS one, 2014Co-Authors: Bin Liu, Brandon J. Biesiadecki, Joseph J. Lopez, Jonathan P. DavisAbstract:Adrenergic stimulation modulates cardiac function by altering the phosphorylation status of several cardiac proteins. The Troponin complex, which is the Ca2+ sensor for cardiac contraction, is a hot spot for adrenergic phosphorylation. While the effect of β-adrenergic related PKA phosphorylation of troponin I at Ser23/24 is well established, the effects of α-adrenergic induced PKC phosphorylation on multiple sites of TnI (Ser43/45, Thr144) and TnT (Thr194, Ser198, Thr203 and Thr284) are much less clear. By utilizing an IAANS labeled fluorescent troponin C, , we systematically examined the site specific effects of PKC phosphomimetic mutants of TnI and TnT on TnC’s Ca2+ binding properties in the Tn complex and reconstituted Thin Filament. The majority of the phosphomemetics had little effect on the Ca2+ binding properties of the isolated Tn complex. However, when incorporated into the Thin Filament, the phosphomimetics typically altered Thin Filament Ca2+ sensitivity in a way consistent with their respective effects on Ca2+ sensitivity of skinned muscle preparations. The altered Ca2+ sensitivity could be generally explained by a change in Ca2+ dissociation rates. WiThin TnI, phosphomimetic Asp and Glu did not always behave similar, nor were Ala mutations (used to mimic non-phosphorylatable states) benign to Ca2+ binding. Our results suggest that Troponin may act as a hub on the Thin Filament, sensing physiological stimuli to modulate the contractile performance of the heart.
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Disease-related cardiac troponins alter Thin Filament Ca2+ association and dissociation rates.
PloS one, 2012Co-Authors: Bin Liu, Svetlana B. Tikunova, Kristopher P. Kline, Jalal K. Siddiqui, Jonathan P. DavisAbstract:The contractile response of the heart can be altered by disease-related protein modifications to numerous contractile proteins. By utilizing an IAANS labeled fluorescent troponin C, , we examined the effects of ten disease-related troponin modifications on the Ca2+ binding properties of the troponin complex and the reconstituted Thin Filament. The selected modifications are associated with a broad range of cardiac diseases: three subtypes of familial cardiomyopathies (dilated, hypertrophic and restrictive) and ischemia-reperfusion injury. Consistent with previous studies, the majority of the protein modifications had no effect on the Ca2+ binding properties of the isolated troponin complex. However, when incorporated into the Thin Filament, dilated cardiomyopathy mutations desensitized (up to 3.3-fold), while hypertrophic and restrictive cardiomyopathy mutations, and ischemia-induced truncation of troponin I, sensitized the Thin Filament to Ca2+ (up to 6.3-fold). Kinetically, the dilated cardiomyopathy mutations increased the rate of Ca2+ dissociation from the Thin Filament (up to 2.5-fold), while the hypertrophic and restrictive cardiomyopathy mutations, and the ischemia-induced truncation of troponin I decreased the rate (up to 2-fold). The protein modifications also increased (up to 5.4-fold) or decreased (up to 2.5-fold) the apparent rate of Ca2+ association to the Thin Filament. Thus, the disease-related protein modifications alter Ca2+ binding by influencing both the association and dissociation rates of Thin Filament Ca2+ exchange. These alterations in Ca2+ exchange kinetics influenced the response of the Thin Filament to artificial Ca2+ transients generated in a stopped-flow apparatus. Troponin C may act as a hub, sensing physiological and pathological stimuli to modulate the Ca2+-binding properties of the Thin Filament and influence the contractile performance of the heart.
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disease related cardiac troponins alter Thin Filament ca2 association and dissociation rates
PLOS ONE, 2012Co-Authors: Bin Liu, Svetlana B. Tikunova, Kristopher P. Kline, Jalal K. Siddiqui, Jonathan P. DavisAbstract:The contractile response of the heart can be altered by disease-related protein modifications to numerous contractile proteins. By utilizing an IAANS labeled fluorescent troponin C, [Formula: see text], we examined the effects of ten disease-related troponin modifications on the Ca(2+) binding properties of the troponin complex and the reconstituted Thin Filament. The selected modifications are associated with a broad range of cardiac diseases: three subtypes of familial cardiomyopathies (dilated, hypertrophic and restrictive) and ischemia-reperfusion injury. Consistent with previous studies, the majority of the protein modifications had no effect on the Ca(2+) binding properties of the isolated troponin complex. However, when incorporated into the Thin Filament, dilated cardiomyopathy mutations desensitized (up to 3.3-fold), while hypertrophic and restrictive cardiomyopathy mutations, and ischemia-induced truncation of troponin I, sensitized the Thin Filament to Ca(2+) (up to 6.3-fold). Kinetically, the dilated cardiomyopathy mutations increased the rate of Ca(2+) dissociation from the Thin Filament (up to 2.5-fold), while the hypertrophic and restrictive cardiomyopathy mutations, and the ischemia-induced truncation of troponin I decreased the rate (up to 2-fold). The protein modifications also increased (up to 5.4-fold) or decreased (up to 2.5-fold) the apparent rate of Ca(2+) association to the Thin Filament. Thus, the disease-related protein modifications alter Ca(2+) binding by influencing both the association and dissociation rates of Thin Filament Ca(2+) exchange. These alterations in Ca(2+) exchange kinetics influenced the response of the Thin Filament to artificial Ca(2+) transients generated in a stopped-flow apparatus. Troponin C may act as a hub, sensing physiological and pathological stimuli to modulate the Ca(2+)-binding properties of the Thin Filament and influence the contractile performance of the heart.
Larry S. Tobacman - One of the best experts on this subject based on the ideXlab platform.
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dual regulatory functions of the Thin Filament revealed by replacement of the troponin i inhibitory peptide with a linker
Journal of Biological Chemistry, 2010Co-Authors: Julie Mouannes Kozaili, Daniel Leek, Larry S. TobacmanAbstract:Striated muscles are relaxed under low Ca2+ concentration conditions due to actions of the Thin Filament protein troponin. To investigate this regulatory mechanism, an 11-residue segment of cardiac troponin I previously termed the inhibitory peptide region was studied by mutagenesis. Several mutant troponin complexes were characterized in which specific effects of the inhibitory peptide region were abrogated by replacements of 4–10 residues with Gly-Ala linkers. The mutations greatly impaired two of troponin's actions under low Ca2+ concentration conditions: inhibition of myosin subfragment 1 (S1)-Thin Filament MgATPase activity and cooperative suppression of myosin S1-ADP binding to Thin Filaments with low myosin saturation. Inhibitory peptide replacement diminished but did not abolish the Ca2+ dependence of the ATPase rate; ATPase rates were at least 2-fold greater when Ca2+ rather than EGTA was present. This residual regulation was highly cooperative as a function of Ca2+ concentration, similar to the degree of cooperativity observed with WT troponin present. Other effects of the mutations included 2-fold or less increases in the apparent affinity of the Thin Filament regulatory Ca2+ sites, similar decreases in the affinity of troponin for actin-tropomyosin regardless of Ca2+, and increases in myosin S1-Thin Filament ATPase rates in the presence of saturating Ca2+. The overall results indicate that cooperative myosin binding to Ca2+-free Thin Filaments depends upon the inhibitory peptide region but that a cooperatively activating effect of Ca2+ binding does not. The findings suggest that these two processes are separable and involve different conformational changes in the Thin Filament.
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an atomic model of the Thin Filament in the relaxed and ca2 activated states
Journal of Molecular Biology, 2006Co-Authors: Alnoor Pirani, Larry S. Tobacman, Roger Craig, Victoria Hatch, Maia V Vinogradova, Paul M G Curmi, William A King, Robert J Fletterick, William LehmanAbstract:Contraction of striated muscles is regulated by tropomyosin strands that run continuously along actin-containing Thin Filaments. Tropomyosin blocks myosin-binding sites on actin in resting muscle and unblocks them during Ca2+-activation. This steric effect controls myosin-crossbridge cycling on actin that drives contraction. Troponin, bound to the Thin Filaments, couples Ca2+-concentration changes to the movement of tropomyosin. Ca2+-free troponin is thought to trap tropomyosin in the myosin-blocking position, while this constraint is released after Ca2+-binding. Although the location and movements of tropomyosin are well known, the structural organization of troponin on Thin Filaments is not. Its mechanism of action therefore remains uncertain. To determine the organization of troponin on the Thin Filament, we have constructed atomic models of low and high-Ca2+ states based on crystal structures of actin, tropomyosin and the “core domain” of troponin, and constrained by distances between Filament components and by their location in electron microscopy (EM) reconstructions. Alternative models were also built where troponin was systematically repositioned or reoriented on actin. The accuracy of the different models was evaluated by determining how well they corresponded to EM images. While the initial low and high-Ca2+ models fitted the data precisely, the alternatives did not, suggesting that the starting models best represented the correct structures. Thin Filament reconstructions were generated from the EM data using these starting models as references. In addition to showing the core domain of troponin, the reconstructions showed additional detail not present in the starting models. We attribute this to an extension of TnI linking the troponin core domain to actin at low (but not at high) Ca2+, thereby trapping tropomyosin in the OFF-state. The bulk of the core domain of troponin appears not to move significantly on actin, regardless of Ca2+ level. Our observations suggest a simple model for muscle regulation in which troponin affects the charge balance on actin and hence tropomyosin position.
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cardiomyopathic tropomyosin mutations that increase Thin Filament ca2 sensitivity and tropomyosin n domain flexibility
Journal of Biological Chemistry, 2003Co-Authors: Mark J Heller, Mahta Nili, Earl Homsher, Larry S. TobacmanAbstract:Abstract The relationship between tropomyosin thermal stability and Thin Filament activation was explored using two N-domain mutants of α-striated muscle tropomyosin, A63V and K70T, each previously implicated in familial hypertrophic cardiomyopathy. Both mutations had prominent effects on tropomyosin thermal stability as monitored by circular dichroism. Wild type tropomyosin unfolded in two transitions, separated by 10 °C. The A63V and K70T mutations decreased the melting temperature of the more stable of these transitions by 4 and 10 °C, respectively, indicating destabilization of the N-domain in both cases. Global analysis of all three proteins indicated that the tropomyosin N-domain and C-domain fold with a cooperative free energy of 1.0–1.5 kcal/mol. The two mutations increased the apparent affinity of the regulatory Ca2+ binding sites of Thin Filament in two settings: Ca2+-dependent sliding speed of unloaded Thin Filaments in vitro (at both pH 7.4 and 6.3), and Ca2+ activation of the Thin Filament-myosin S1 ATPase rate. Neither mutation had more than small effects on the maximal ATPase rate in the presence of saturating Ca2+ or on the maximal sliding speed. Despite the increased tropomyosin flexibility implied by destabilization of the N-domain, neither the cooperativity of Thin Filament activation by Ca2+ nor the cooperative binding of myosin S1-ADP to the Thin Filament was altered by the mutations. The combined results suggest that a more dynamic tropomyosin N-domain influences interactions with actin and/or troponin that modulate Ca2+ sensitivity, but has an unexpectedly small effect on cooperative changes in tropomyosin position on actin.
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the troponin tail domain promotes a conformational state of the Thin Filament that suppresses myosin activity
Journal of Biological Chemistry, 2002Co-Authors: Larry S. Tobacman, Roger Craig, William Lehman, Mahta Nihli, Carol Butters, Mark Heller, Victoria Hatch, Earl HomsherAbstract:Abstract In cardiac and skeletal muscles tropomyosin binds to the actin outer domain in the absence of Ca2+, and in this position tropomyosin inhibits muscle contraction by interfering sterically with myosin-actin binding. The globular domain of troponin is believed to produce this B-state of the Thin Filament (Lehman, W., Hatch, V., Korman, V. L., Rosol, M., Thomas, L. T., Maytum, R., Geeves, M. A., Van Eyk, J. E., Tobacman, L. S., and Craig, R. (2000) J. Mol. Biol. 302, 593–606) via troponin I-actin interactions that constrain the tropomyosin. The present study shows that the B-state can be promoted independently by the elongated tail region of troponin (the NH2 terminus (TnT-(1–153)) of cardiac troponin T). In the absence of the troponin globular domain, TnT-(1–153) markedly inhibited both myosin S1-actin-tropomyosin MgATPase activity and (at low S1 concentrations) myosin S1-ADP binding to the Thin Filament. Similarly, TnT-(1–153) increased the concentration of heavy meromyosin required to support in vitro sliding of Thin Filaments. Electron microscopy and three-dimensional reconstruction of Thin Filaments containing TnT-(1–153) and either cardiac or skeletal muscle tropomyosin showed that tropomyosin was in the B-state in the complete absence of troponin I. All of these results indicate that portions of the troponin tail domain, and not only troponin I, contribute to the positioning of tropomyosin on the actin outer domain, thereby inhibiting muscle contraction in the absence of Ca2+.
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Effects of a Cardiomyopathy-causing Troponin T Mutation on Thin Filament Function and Structure
The Journal of biological chemistry, 2001Co-Authors: James Burhop, Larry S. Tobacman, Michael Rosol, Roger Craig, William LehmanAbstract:Familial hypertrophic cardiomyopathy (FHC) is caused by missense or premature truncation mutations in proteins of the cardiac contractile apparatus. Mutant proteins are incorporated into the Thin Filament or thick Filament and eventually produce cardiomyopathy. However, it has been unclear how the several, genetically identified defects in protein structure translate into impaired protein and muscle function. We have studied the basis of FHC caused by premature truncation of the most frequently implicated Thin Filament target, troponin T. Electron microscope observations showed that the Thin Filament undergoes normal structural changes in response to Ca(2+) binding. On the other hand, solution studies showed that the mutation alters and destabilizes troponin binding to the Thin Filament to different extents in different regulatory states, thereby affecting the transitions among states that regulate myosin binding and muscle contraction. Development of hypertrophic cardiomyopathy can thus be traced to a defect in the primary mechanism controlling cardiac contraction, switching between different conformations of the Thin Filament.
Ryan Littlefield - One of the best experts on this subject based on the ideXlab platform.
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Thin Filament Pointed Ends Redistribute in Response to Altered Thick Filaments
Biophysical Journal, 2017Co-Authors: Ryan LittlefieldAbstract:Striated muscle generates contractile force and shortens as polar actin (Thin) Filaments slide past bipolar myosin (thick) Filaments. Maximal force is able to be produced when Thin Filaments completely overlap the motor domains (heads) which occurs when the Thin Filament slow-growing (pointed) ends extend to the central (∼0.2 micrometer) thick Filament bare zone devoid of myosin heads. The assembly of Thin and thick Filaments into regular contractile units (sarcomeres) is complex and highly-regulated, yet also robust; allowing diverse vertebrate and invertebrate animals to produce a variety of sarcomeric architectures with specific physiological contractile properties. Maximal Thin-thick Filament overlap in vivo is highly conserved among all types of striated muscle examined and highlights its importance. How Thin-thick Filament overlap is specified and maintained in different muscles is uncertain, but it manifests as the uniform and precise specification of Thin Filament lengths and involves the exchange of actin subunits at pointed ends and dynamic pointed-end capping by tropomodulin (Tmod). Here, we use CRISPR-Cas9 gene editing and fluorescence microscopy to investigate whether interactions between myosin heads and pointed ends are necessary for specifying Thin Filament lengths in C. elegans obliquely striated body wall muscle. To determine the position of Thin Filament pointed ends, we inserted the green fluorescent protein (GFP) coding sequence in-frame with the C. elegans Tmd1 gene to generate a GFP-Tmod fusion protein. Homozygous GFP-Tmod worms are indistinguishable from wildtype worms demonstrating that the GFP fusion is completely functional. In body wall muscle, GFP-Tmod appears as regular striations that co-localize with myosin thick Filaments. GFP-Tmod striations appear as single bands (singlets) or closely spaced double bands (doublets) in living worms confirming that Thin-thick Filament overlap is complete. In contrast, GFP-Tmod striations appear as more broadly-spaced doublets in headless-myoA transgenic worms that were designed to have an expanded (∼2 micrometer) thick Filament bare zone. Our results show that Thin Filament pointed ends redistribute their location in response to changing the size of the thick Filament bare zone. The response of pointed ends suggests that interactions with myosin heads stabilize pointed ends, increasing Thin Filament lengths until they fully overlap with thick Filaments and permit optimal force generation.
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tropomodulin isoforms regulate Thin Filament pointed end capping and skeletal muscle physiology
Journal of Cell Biology, 2010Co-Authors: Raymond A Lewis, Caroline R Mckeown, Roberta B Nowak, David Samuel Gokhin, Richard L Lieber, Ryan Littlefield, Velia M FowlerAbstract:During myofibril assembly, Thin Filament lengths are precisely specified to optimize skeletal muscle function. Tropomodulins (Tmods) are capping proteins that specify Thin Filament lengths by controlling actin dynamics at pointed ends. In this study, we use a genetic targeting approach to explore the effects of deleting Tmod1 from skeletal muscle. Myofibril assembly, skeletal muscle structure, and Thin Filament lengths are normal in the absence of Tmod1. Tmod4 localizes to Thin Filament pointed ends in Tmod1-null embryonic muscle, whereas both Tmod3 and -4 localize to pointed ends in Tmod1-null adult muscle. Substitution by Tmod3 and -4 occurs despite their weaker interactions with striated muscle tropomyosins. However, the absence of Tmod1 results in depressed isometric stress production during muscle contraction, systemic locomotor deficits, and a shift to a faster fiber type distribution. Thus, Tmod3 and -4 compensate for the absence of Tmod1 structurally but not functionally. We conclude that Tmod1 is a novel regulator of skeletal muscle physiology.
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a nebulin ruler does not dictate Thin Filament lengths
Biophysical Journal, 2009Co-Authors: Angelica Castillo, Kimberly P Littlefield, Roberta B Nowak, Velia M Fowler, Ryan LittlefieldAbstract:To generate force, striated muscle requires overlap between uniform-length actin and myosin Filaments. The hypothesis that a nebulin ruler mechanism specifies Thin Filament lengths by targeting where tropomodulin (Tmod) caps the slow-growing, pointed end has not been rigorously tested. Using fluorescent microscopy and quantitative image analysis, we found that nebulin extended 1.01–1.03 μm from the Z-line, but Tmod localized 1.13–1.31 μm from the Z-line, in seven different rabbit skeletal muscles. Because nebulin does not extend to the Thin Filament pointed ends, it can neither target Tmod capping nor specify Thin Filament lengths. We found instead a strong correspondence between Thin Filament lengths and titin isoform sizes for each muscle. Our results suggest the existence of a mechanism whereby nebulin specifies the minimum Thin Filament length and sarcomere length regulates and coordinates pointed-end dynamics to maintain the relative overlap of the Thin and thick Filaments during myofibril assembly.
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Thin Filament length regulation in striated muscle sarcomeres : Pointed-end dynamics go beyond a nebulin ruler
Seminars in Cell & Developmental Biology, 2008Co-Authors: Ryan Littlefield, Velia M FowlerAbstract:The actin (Thin) Filaments in striated muscle are highly regulated and precisely specified in length to optimally overlap with the myosin (thick) Filaments for efficient myofibril contraction. Here, we review and critically discuss recent evidence for how Thin Filament lengths are controlled in vertebrate skeletal, vertebrate cardiac, and invertebrate (arthropod) sarcomeres. Regulation of actin polymerization dynamics at the slow-growing (pointed) ends by the capping protein tropomodulin provides a unified explanation for how Thin Filament lengths are physiologically optimized in all three muscle types. Nebulin, a large protein thought to specify Thin Filament lengths in vertebrate skeletal muscle through a ruler mechanism, may not control pointed-end actin dynamics directly, but instead may stabilize a large core region of the Thin Filament. We suggest that this stabilizing function for nebulin modifies the lengths primarily specified by pointed-end actin dynamics to generate uniform Filament lengths in vertebrate skeletal muscle. We suggest that nebulette, a small homolog of nebulin, may stabilize a correspondingly shorter core region and allow individual Thin Filament lengths to vary according to working sarcomere lengths in vertebrate cardiac muscle. We present a unified model for Thin Filament length regulation where these two mechanisms cooperate to tailor Thin Filament lengths for specific contractile environments in diverse muscles.
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nebulin deficient mice exhibit shorter Thin Filament lengths and reduced contractile function in skeletal muscle
Journal of Cell Biology, 2006Co-Authors: Marie Louise Bang, Shannon N Bremner, Andrea Thor, Kirk U Knowlton, Xiaodong Li, Richard L Lieber, Ryan Littlefield, Ju ChenAbstract:Nebulin is a giant modular sarcomeric protein that has been proposed to play critical roles in myofibrillogenesis, Thin Filament length regulation, and muscle contraction. To investigate the functional role of nebulin in vivo, we generated nebulin-deficient mice by using a Cre knock-in strategy. Lineage studies utilizing this mouse model demonstrated that nebulin is expressed uniformly in all skeletal muscles. Nebulin-deficient mice die wiThin 8–11 d after birth, with symptoms including decreased milk intake and muscle weakness. Although myofibrillogenesis had occurred, skeletal muscle Thin Filament lengths were up to 25% shorter compared with wild type, and Thin Filaments were uniform in length both wiThin and between muscle types. Ultrastructural studies also demonstrated a critical role for nebulin in the maintenance of sarcomeric structure in skeletal muscle. The functional importance of nebulin in skeletal muscle function was revealed by isometric contractility assays, which demonstrated a dramatic reduction in force production in nebulin-deficient skeletal muscle.
