Thick Myosin Research Articles

Murf1 alters myosin replacement rates in cultured myotubes in a myosin isoform-dependent manner.

Skeletal muscle tissue increases or decreases its volume by synthesizing or degrading myofibrillar proteins. The ubiquitin-proteasome system plays a pivotal role during muscle atrophy, where muscle ring finger proteins (Murf) function as E3 ubiquitin ligases responsible for identifying and targeting substrates for degradation. Our previous study demonstrated that overexpression of Ozz, an E3 specific to embryonic myosin heavy chain (Myh3), precisely reduced the Myh3 replacement rate in the thick filaments of myotubes (E. Ichimura et al., Physiol Rep. 9:e15003, 2021). These findings strongly suggest that E3 plays a critical role in regulating myosin replacement. Here, we hypothesized that the Murf isoforms, which recognize Myhs as substrates, reduced the myosin replacement rates through the enhanced Myh degradation by Murfs. First, fluorescence recovery after a photobleaching experiment was conducted to assess whether Murf isoforms affected the GFP-Myh3 replacement. In contrast to Murf2 or Murf3 overexpression, Murf1 overexpression selectively facilitated the GFP-Myh3 myosin replacement. Next, to examine the effects of Murf1 overexpression on the replacement of myosin isoforms, Cherry-Murf1 was coexpressed with GFP-Myh1, GFP-Myh4, or GFP-Myh7 in myotubes. Intriguingly, Murf1 overexpression enhanced the myosin replacement of GFP-Myh4 but did not affect those of GFP-Myh1 or GFP-Myh7. Surprisingly, overexpression of Murf1 did not enhance the ubiquitination of proteins. These results indicate that Murf1 selectively regulated myosin replacement in a Myh isoform-dependent fashion, independent of enhanced ubiquitination. This suggests that Murf1 may have a role beyond functioning as a ubiquitin ligase E3 in thick filament myosin replacement.

Cardiac myosin-binding protein C N-terminal interactions with myosin and actin filaments: Opposite effects of phosphorylation and M-domain mutations

N-terminal cardiac myosin-binding protein C (cMyBP-C) domains (C0-C2) bind to thick (myosin) and thin (actin) filaments to coordinate contraction and relaxation of the heart. These interactions are regulated by phosphorylation of the M-domain situated between domains C1 and C2. In cardiomyopathies and heart failure, phosphorylation of cMyBP-C is significantly altered. We aimed to investigate how cMyBP-C interacts with myosin and actin. We developed complementary, high-throughput, C0-C2 FRET-based binding assays for myosin and actin to characterize the effects due to 5 HCM-linked variants or functional mutations in unphosphorylated and phosphorylated C0-C2. The assays indicated that phosphorylation decreases binding to both myosin and actin, whereas the HCM mutations in M-domain generally increase binding. The effects of mutations were greatest in phosphorylated C0-C2, and some mutations had a larger effect on actin than myosin binding. Phosphorylation also altered the spatial relationship of the probes on C0-C2 and actin. The magnitude of these structural changes was dependent on C0-C2 probe location (C0, C1, or M-domain). We conclude that binding can differ between myosin and actin due to phosphorylation or mutations. Additionally, these variables can change the mode of binding, affecting which of the interactions in cMyBP-C N-terminal domains with myosin or actin take place. The opposite effects of phosphorylation and M-domain mutations is consistent with the idea that cMyBP-C phosphorylation is critical for normal cardiac function. The precision of these assays is indicative of their usefulness in high-throughput screening of drug libraries for targeting cMyBP-C as therapy.

Dependence of myosin filament structure on intracellular calcium concentration in skeletal muscle.

Contraction of skeletal muscle is triggered by an increase in intracellular calcium concentration that relieves the structural block on actin-binding sites in resting muscle, potentially allowing myosin motors to bind and generate force. However, most myosin motors are not available for actin binding because they are stabilized in folded helical tracks on the surface of myosin-containing thick filaments. High-force contraction depends on the release of the folded motors, which can be triggered by stress in the thick filament backbone, but additional mechanisms may link the activation of the thick filaments to that of the thin filaments or to intracellular calcium concentration. Here, we used x-ray diffraction in combination with temperature-jump activation to determine the steady-state calcium dependence of thick filament structure and myosin motor conformation in near-physiological conditions. We found that x-ray signals associated with the perpendicular motors characteristic of isometric force generation had almost the same calcium sensitivity as force, but x-ray signals associated with perturbations in the folded myosin helix had a much higher calcium sensitivity. Moreover, a new population of myosin motors with a longer axial periodicity became prominent at low levels of calcium activation and may represent an intermediate regulatory state of the myosin motors in the physiological pathway of filament activation.

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.

The organisation of titin at the centre of the vertebrate striated muscle thick filament.

Striated muscle sarcomeres operate by sliding of the regular arrays of interdigitating thin (actin) and thick (myosin) filaments. Thick filaments are held together at their centre by M-band bridges, maintaining sarcomere structure during contraction and relaxation. The giant sarcomeric protein titin is instrumental in maintaining order, since C-terminal truncations of titin lead to loss of A-band alignment and sarcomere failure. The C-terminal sequence of titin encodes a kinase domain, 10 Ig domains and linker sequences, and is associated with the bare zone at the centre of the sarcomere. Unlike the cross-bridge associated sequence, it does not show any correlation with the organisation of the thick filament bare zone and associated M-band bridges. The precise disposition of titin remains unclear, e.g. whether anti-parallel molecules overlap at centre of the filament. We used superresolution light microscopy with new titin antibodies to establish the position of several titin epitopes in the bare zone, together with key markers for the M-band proteins myomesin and M-protein. We show that the titin kinase domain is at the edge of the bare zone, about 70 nm from the M-band centre at the first myosin motor domains and may therefore account for the first of the accessory protein stripes in the A-band. SAXS data of two titin constructs covering several domains of the bare-zone associated sequence, one from the beginning including the kinase and the other from the end, suggest that they are extended structures but with compact linkers. Conversely, the long insert sequence between M-band Ig-domains 3 and 4 (Ig-162 and Ig-163) appears to be more extensive. Our combined data suggests that in the bare zone, titin molecules from each side of the thick filament overlap each other by the width of the M-band.

Fitting a model to multiscale data suggests thick filament activation can produce force depression in muscle fibers.

Multiscale muscle models aim to connect muscle's molecular and organ-scale function to, e.g., inform the treatment of muscle diseases. Current muscle models have not quantitatively fit data spanning the molecular to cellular scales due, in part, to a lack of self-consistent multiscale data. To address this gap, we measured the force response from single skinned rabbit psoas muscle fibers to ramp shortenings (5% amplitude) and step stretches (0.25%, 0.5%, 0.75% amplitude), performed on the plateau region of the force-length relationship. We isolated myosin from the same muscle source and, under similar conditions, performed single molecule and mini ensemble measurements of event duration and displacement using optical trapping and in vitro motility assays. We fit the fiber data by developing a partial differential equation model that includes thick filament activation, whereby an increase in force on the thick filament pulls myosin out of the superrelaxed state, a series elastic element and a parallel elastic element. This parallel elastic element models a titin-actin interaction proposed to account for the increase in isometric force following stretch (residual force enhancement). By optimizing the model fit to fiber measurements, we specified seven unknown parameters. The model then successfully predicted our molecular measurements from the optical trap and in vitro motility at different ATP concentrations. The success of the model suggests that our multiscale data are self-consistent and can serve as a testbed for other multiscale modeling efforts. Moreover, the model captures the decrease in isometric force observed in our muscle fibers after active shortening (force depression), suggesting a molecular mechanism for force depression, whereby a parallel elastic element combined with thick filament activation serve to decrease the number of cycling cross-bridges.

Cryo-ET reveals sarcomere structures at molecular resolution.

Muscles underpin movement and heart function. Individual muscle fibers can be very large syncytia of up to several centimeters in length and are comprised of the force-generating and load-bearing devices of muscles called sarcomeres. Contraction and relaxation of sarcomeres relies on the sliding between two types of filaments—the thin filament (comprising mainly F-actin, tropomyosin, and troponin) and the thick myosin filament. In addition, several other proteins are involved in the contraction mechanism and their mutational malfunction can lead to debilitating and even life-threatening diseases. In my presentation, I will explain how we managed to obtain high-resolution structures of native sarcomeres using cryo-electron tomography (cryo-ET). Our cryo-ET reconstructions reveal molecular details of the three-dimensional organization and interaction of actin and myosin in the A-band, I-band and Z-disc and demonstrate that α-actinin cross-links antiparallel actin filaments by forming doublets with 6 nm spacing. Structures of myosin, tropomyosin, actin, and nebulin at up to 4.5 Å further reveal two conformations of “double-headed” myosin, where the flexible orientation of the lever arm and light chains enable myosin not only to interact with the same actin filament, but also to split between two actin filaments. The structures provide high-resolution details of the interaction between nebulin and actin, demonstrating the stabilising role of nebulin. Unexpectedly, nebulin does not interact with myosin or tropomyosin, but with a troponin-T linker through two potential binding motifs on nebulin, explaining its regulatory role. Our results provide unexpected insights into the fundamental organization of vertebrate skeletal muscle and serve as a strong foundation for future investigations of muscle diseases.

Cryo-Electron Microscopy Reveals Cardiac Myosin Binding Protein-C M-Domain Interactions with the Thin Filament

Cardiac myosin binding protein C (cMyBP-C) modulates cardiac contraction via direct interactions with cardiac thick (myosin) and thin (actin) filaments (cTFs). While its C-terminal domains (e.g. C8-C10) anchor cMyBP-C to the backbone of the thick filament, its N-terminal domains (NTDs) (e.g. C0, C1, M, and C2) bind to both myosin and actin to accomplish its dual roles of inhibiting thick filaments and activating cTFs. While the positions of C0, C1 and C2 on cTF have been reported, the binding site of the M-domain on the surface of the cTF is unknown. Here, we used cryo-EM to reveal that the M-domain interacts with actin via helix 3 of its ordered tri-helix bundle region, while the unstructured part of the M-domain does not maintain extensive interactions with actin. We combined the recently obtained structure of the cTF with the positions of all the four NTDs on its surface to propose a complete model of the NTD binding to the cTF. The model predicts that the interactions of the NTDs with the cTF depend on the activation state of the cTF. At the peak of systole, when bound to the extensively activated cTF, NTDs would inhibit actomyosin interactions. In contrast, at falling Ca2+ levels, NTDs would not compete with the myosin heads for binding to the cTF, but would rather promote formation of active cross-bridges at the adjacent regulatory units located at the opposite cTF strand. Our structural data provides a testable model of the cTF regulation by the cMyBP-C.

Interaction of the M-domain of cardiac myosin binding protein-C with the native cardiac thin filament

Cardiac muscle contraction depends on precise interactions between thin (actin) and thick (myosin) filaments (TFs). TFs are regulated by intracellular Ca2+ levels as well as other sarcomeric proteins, such as cardiac myosin binding protein C (cMyBP-C) and myosin. Recent studies have shown the first four N-terminal domains (NTDs) of cMyBP-C (i.e. C0, C1, M and C2), are effective modulators of the TF activity. Their collective activation of the TF is regulated by the phosphorylation of the M domain but the mechanism is not fully understood.

Interaction of the C2 Ig-like Domain of Cardiac Myosin Binding Protein-C with F-actin

Cardiac muscle contraction depends on interactions between thick (myosin) and thin (actin) filaments (TFs). TFs are regulated by intracellular Ca2+ levels. Under activating conditions Ca2+ binds to the troponin complex and displaces tropomyosin from myosin binding sites on the TF surface to allow actomyosin interactions. Recent studies have shown that in addition to Ca2+, the first four N-terminal domains (NTDs) of cardiac myosin binding protein C (cMyBP-C) (e.g. C0, C1, M and C2), are potent modulators of the TF activity, but the mechanism of their collective action is poorly understood. Previously, we showed that C1 activates the TF at low Ca2+ and C0 stabilizes binding of C1 to the TF, but the ability of C2 to bind and/or affect the TF remains unknown. Here we obtained 7.5 Å resolution cryo-EM reconstruction of C2-decorated actin filaments to demonstrate that C2 binds to actin in a single structural mode that does not activate the TF unlike the polymorphic binding of C0 and C1 to actin. Comparison of amino acid sequences of C2 with either C0 or C1 shows low levels of identity between the residues involved in interactions with the TF but high levels of conservation for residues involved in Ig fold stabilization. This provides a structural basis for strikingly different interactions of structurally homologous C0, C1 and C2 with the TF. Our detailed analysis of the interaction of C2 with the actin filament provides crucial information required to model the collective action of cMyBP-C NTDs on the cardiac TF.

Contiguity and Structural Impacts of a Non-Myosin Protein within the Thick Filament Myosin Layers.

Simple SummaryHexapods and crustaceans (Pancrustacea) represent nearly 80% of known living animals. Species within this clade exhibit exquisite muscle types propelling ingenious means of locomotion, likely contributing to their evolutionary success. Flightin, a myosin-binding protein, first identified in the flight muscle of Drosophila, is defined by WYR, a protein domain exclusive to Pancrustacea. In Drosophila, flightin imparts stiffness to the thick filament and is essential for their length determination and structural integrity. Here, we build on results from the three-dimensional reconstruction of the Lethocerus flight muscle thick filament to advance the hypothesis that flightin influences thick filament mechanics, and by extension muscle function, by acting as a cinch in the filament core.Myosin dimers arranged in layers and interspersed with non-myosin densities have been described by cryo-EM 3D reconstruction of the thick filament in Lethocerus at 5.5 Å resolution. One of the non-myosin densities, denoted the ‘red density’, is hypothesized to be flightin, an LMM-binding protein essential to the structure and function of Drosophila indirect flight muscle (IFM). Here, we build upon the 3D reconstruction results specific to the red density and its engagement with the myosin coiled-coil rods that form the backbone of the thick filament. Each independent red density winds its way through the myosin dimers, such that it links four dimers in a layer and one dimer in a neighboring layer. This area in which three distinct interfaces within the myosin rod are contacted at once and the red density extends to the thick filament core is designated the “multiface”. Present within the multiface is a contact area inclusive of E1563 and R1568. Mutations in the corresponding Drosophila residues (E1554K and R1559H) are known to interfere with flightin accumulation and phosphorylation in Drosophila. We further examine the LMM area in direct apposition to the red density and identified potential binding residues spanning up to ten helical turns. We find that the red density is associated within an expanse of the myosin coiled-coil that is unwound by the third skip residue and the coiled-coil is re-oriented while in contact with the red density. These findings suggest a mechanism by which flightin induces ordered assembly of myosin dimers through its contacts with multiple myosin dimers and brings about reinforcement on the level of a single myosin dimer by stabilization of the myosin coiled-coil.

Molecular basis of F-actin regulation and sarcomere assembly via myotilin.

Sarcomeres, the basic contractile units of striated muscle cells, contain arrays of thin (actin) and thick (myosin) filaments that slide past each other during contraction. The Ig-like domain-containing protein myotilin provides structural integrity to Z-discs-the boundaries between adjacent sarcomeres. Myotilin binds to Z-disc components, including F-actin and α-actinin-2, but the molecular mechanism of binding and implications of these interactions on Z-disc integrity are still elusive. To illuminate them, we used a combination of small-angle X-ray scattering, cross-linking mass spectrometry, and biochemical and molecular biophysics approaches. We discovered that myotilin displays conformational ensembles in solution. We generated a structural model of the F-actin:myotilin complex that revealed how myotilin interacts with and stabilizes F-actin via its Ig-like domains and flanking regions. Mutant myotilin designed with impaired F-actin binding showed increased dynamics in cells. Structural analyses and competition assays uncovered that myotilin displaces tropomyosin from F-actin. Our findings suggest a novel role of myotilin as a co-organizer of Z-disc assembly and advance our mechanistic understanding of myotilin's structural role in Z-discs.

MICAL2 is essential for myogenic lineage commitment

Contractile myofiber units are mainly composed of thick myosin and thin actin (F-actin) filaments. F-Actin interacts with Microtubule Associated Monooxygenase, Calponin And LIM Domain Containing 2 (MICAL2). Indeed, MICAL2 modifies actin subunits and promotes actin filament turnover by severing them and preventing repolymerization. In this study, we found that MICAL2 increases during myogenic differentiation of adult and pluripotent stem cells (PSCs) towards skeletal, smooth and cardiac muscle cells and localizes in the nucleus of acute and chronic regenerating muscle fibers. In vivo delivery of Cas9–Mical2 guide RNA complexes results in muscle actin defects and demonstrates that MICAL2 is essential for skeletal muscle homeostasis and functionality. Conversely, MICAL2 upregulation shows a positive impact on skeletal and cardiac muscle commitments. Taken together these data demonstrate that modulations of MICAL2 have an impact on muscle filament dynamics and its fine-tuned balance is essential for the regeneration of muscle tissues.

Tropomyosin as a Stretch Sensor in the Troponin Bridges of Insect Flight Muscle

The flight muscles of many insects contract at high frequency and nearly constant concentrations of calcium. The fibres are activated by rapid stretches, and bridges between thick and thin filaments have been identified as possible stretch sensors. The bridges are spaced at 38.7 nm at the position of troponin on the thin filament, alternating with force-producing crossbridges at target sites in actin. The tropomyosin-troponin complex (Tm-Tn) in Lethocerus flight muscle has two isoforms of tropomyosin (Tm1 and Tm2). Tm-Tn and Tm1 bind to thick filaments or myosin, whereas Tm2 does not. The Tm isoforms exist as homodimers in situ and could act independently: Tm1 forming part of a Tn bridge and Tm2 acting as conventional Tm. Electron micrographs of Tm-Tn incubated with isolated thick filaments showed the complex following the helical arrangement of myosin molecules, with projections every ∼40 nm. Thick filaments were sometimes crosslinked by Tm-Tn or by Tm1 alone. Atomic force microscopy (AFM) of thick filaments in relaxing buffer showed crossbridge crowns every ∼43 nm that were ∼30 nm in height above the mica surface, and intermediate crowns ∼19 nm above the surface. The higher crowns would be more likely to interact with Tm-Tn on thin filaments. Thick filaments crosslinked by Tm1 were also observed by tapping mode AFM. Direct binding of Tm1 to myosin confirms the suggestion that non-force producing crossbridges could activate the thin filament directly when fibres are stretched, pulling Tm from an inhibitory position on actin every cycle of oscillatory contraction, without changes in calcium concentration. We will describe the differences in biophysical properties of Tm1 and Tm2.

Septins, a cytoskeletal protein family, with emerging role in striated muscle

Appropriate organization of cytoskeletal components are required for normal distribution and intracellular localization of different ion channels and proteins involved in calcium homeostasis, signal transduction, and contractile function of striated muscle. Proteins of the contractile system are in direct or indirect connection with the extrasarcomeric cytoskeleton. A number of other molecules which have essential role in regulating stretch-, voltage-, and chemical signal transduction from the surface into the cytoplasm or other intracellular compartments are already well characterized. Sarcomere, the basic contractile unit, is comprised of a precisely organized system of thin (actin), and thick (myosin) filaments. Intermediate filaments connect the sarcomeres and other organelles (mitochondria and nucleus), and are responsible for the cellular integrity. Interacting proteins have a very diverse function in coupling of the intracellular assembly components and regulating the normal physiological function. Despite the more and more intense investigations of a new cytoskeletal protein family, the septins, only limited information is available regarding their expression and role in striated, especially in skeletal muscles. In this review we collected basic and specified knowledge regarding this protein group and emphasize the importance of this emerging field in skeletal muscle biology.

Atomically Detailed Simulation of the Powerstroke in Myosin II by Milestoning

The interaction between actin and myosin II plays an important role in a variety of cellular functions. In particular, myosin II is involved in muscle contraction, which is attributed to the sliding of thin filament actin past the thick myosin II filaments. Past studies on the structure of myosin have linked severe pathologies to defects in myosin, making it important to understand the mechanism of the system. In this study, we focus our analysis on the powerstroke of the myosin II cross bridge cycle, which is the active process of muscle contraction. To do this, we use an algorithm called Milestoning which partitions the dynamics into a sequence of trajectories between “milestones” along the reaction pathway. The structure of myosin II bound to actin in the rigor state was used as a starting point, and a structure for the bound pre-powerstroke state was developed using existing published structures for the unbound pre-powerstroke state as well as experimental data gathered about the movement of myosin II during the powerstroke. With both the beginning and final states of the powerstroke, we can interpolate between these structures to build intermediate states along the reaction pathway. A total of 97 all-atom structures along the pathway of the powerstroke were developed to serve as guides and “anchors” along the reaction pathway. Milestoning short trajectories between cells defined by the anchors will allow for the computation thermodynamics and kinetics of the myosin II powerstroke. This work will lead to a significant improvement in our understanding of the complete powerstroke mechanism, which will in turn facilitate future research on the effects of structural defects in myosin II on powerstroke function and muscle contraction.

Thick Filament Protein Network, Functions, and Disease Association.

Sarcomeres consist of highly ordered arrays of thick myosin and thin actin filaments along with accessory proteins. Thick filaments occupy the center of sarcomeres where they partially overlap with thin filaments. The sliding of thick filaments past thin filaments is a highly regulated process that occurs in an ATP-dependent manner driving muscle contraction. In addition to myosin that makes up the backbone of the thick filament, four other proteins which are intimately bound to the thick filament, myosin binding protein-C, titin, myomesin, and obscurin play important structural and regulatory roles. Consistent with this, mutations in the respective genes have been associated with idiopathic and congenital forms of skeletal and cardiac myopathies. In this review, we aim to summarize our current knowledge on the molecular structure, subcellular localization, interacting partners, function, modulation via posttranslational modifications, and disease involvement of these five major proteins that comprise the thick filament of striated muscle cells. © 2018 American Physiological Society. Compr Physiol 8:631-709, 2018.

Lessons from a tarantula: new insights into muscle thick filament and myosin interacting-heads motif structure and function.

The tarantula skeletal muscle X-ray diffraction pattern suggested that the myosin heads were helically arranged on the thick filaments. Electron microscopy (EM) of negatively stained relaxed tarantula thick filaments revealed four helices of heads allowing a helical 3D reconstruction. Due to its low resolution (5.0nm), the unambiguous interpretation of densities of both heads was not possible. A resolution increase up to 2.5nm, achieved by cryo-EM of frozen-hydrated relaxed thick filaments and an iterative helical real space reconstruction, allowed the resolving of both heads. The two heads, "free" and "blocked", formed an asymmetric structure named the "interacting-heads motif" (IHM) which explained relaxation by self-inhibition of both heads ATPases. This finding made tarantula an exemplar system for thick filament structure and function studies. Heads were shown to be released and disordered by Ca2+-activation through myosin regulatory light chain phosphorylation, leading to EM, small angle X-ray diffraction and scattering, and spectroscopic and biochemical studies of the IHM structure and function. The results from these studies have consequent implications for understanding and explaining myosin super-relaxed state and thick filament activation and regulation. A cooperative phosphorylation mechanism for activation in tarantula skeletal muscle, involving swaying constitutively Ser35 mono-phosphorylated free heads, explains super-relaxation, force potentiation and post-tetanic potentiation through Ser45 mono-phosphorylated blocked heads. Based on this mechanism, we propose a swaying-swinging, tilting crossbridge-sliding filament for tarantula muscle contraction.

Overview of the Muscle Cytoskeleton.

Cardiac and skeletal striated muscles are intricately designed machines responsible for muscle contraction. Coordination of the basic contractile unit, the sarcomere, and the complex cytoskeletal networks are critical for contractile activity. The sarcomere is comprised of precisely organized individual filament systems that include thin (actin), thick (myosin), titin, and nebulin. Connecting the sarcomere to other organelles (e.g., mitochondria and nucleus) and serving as the scaffold to maintain cellular integrity are the intermediate filaments. The costamere, on the other hand, tethers the sarcomere to the cell membrane. Unique structures like the intercalated disc in cardiac muscle and the myotendinous junction in skeletal muscle help synchronize and transmit force. Intense investigation has been done on many of the proteins that make up these cytoskeletal assemblies. Yet the details of their function and how they interconnect have just started to be elucidated. A vast number of human myopathies are contributed to mutations in muscle proteins; thus understanding their basic function provides a mechanistic understanding of muscle disorders. In this review, we highlight the components of striated muscle with respect to their interactions, signaling pathways, functions, and connections to disease. © 2017 American Physiological Society. Compr Physiol 7:891-944, 2017.

Comparison of the Regulation of Contraction in Insect Flight Muscle and Vertebrate Cardiac Muscle

The indirect flight muscle of insects (IFM), such as Drosophila and Lethocerus, is activated by periodic stretches at a priming concentration of calcium. The alternate activation of opposing muscles produces the rapid wing movements needed for flight. Cardiac muscle develops more force at longer sarcomere lengths (length-dependent activation), and force is enhanced by a rapid stretch at each beat. Troponin bridges between thick and thin filaments have been identified in Lethocerus IFM (Perz-Edwards et al, 2014) and these may be stretch sensors. Cardiac TnC and the TnC-F2 isoform in IFM both bind one regulatory calcium in the N-lobe. The force-pCa curve for IFM with only the TnC-F2 isoform (pCa50 5.8 and co-operativity nH 3.2) is similar to that of cardiac trabeculae, suggesting the co-operative behaviour is produced in the same way. TnC-F1, the IFM isoform needed for stretch activation, binds one calcium in the C-lobe, which is not regulatory. The force-pCa curve for IFM fibres, in which TnC-F1 is the main isoform, shows greater calcium-sensitivity and less co-operativity (pCa50 6.2, nH 1.3). The force-pCa curve of IFM fibres is similar to that of cardiac trabeculae with bound C1-C2 fragment of MyBP-C, in which calcium-sensitivity is increased and co-operativity greatly reduced. In this case, cardiac and IFM fibres achieve the same mechanical properties by different mechanisms. In order to determine if troponin forms part of a link between thick and thin filaments in IFM, we have tested binding between a tropomyosin-troponin complex and isolated thick filaments or myosin. The subunits of tropomyosin-troponin and the conditions needed for binding will be described.