Myosin Research Articles

Calcium has a direct effect on thick filament activation in porcine myocardium.

Sarcomere activation in striated muscle requires both thin filament-based and thick filament-based activation mechanisms. Recent studies have shown that myosin heads on the thick filaments undergo OFF to ON structural transitions in response to calcium (Ca2+) in permeabilized porcine myocardium in the presence of a small molecule inhibitor that eliminated active force. The changes in X-ray diffraction signatures of OFF to ON transitions were interpreted as Ca2+ acting to activate the thick filaments. Alternatively, Ca2+ binding to troponin could initiate a Ca2+-dependent crosstalk from the thin filament to the thick filament via interfilament connections such as the myosin binding protein-C. Here, we exchanged native troponin in permeabilized porcine myocardium for troponin containing the cTnC D65A mutation, which disallows the activation of troponin through Ca2+ binding to determine if Ca2+-dependent thick filament activation persists in the absence of thin filament activation. After the exchange protocol, over 95% of the Ca2+-activated force was eliminated. Equatorial intensity ratio increased significantly in both WT and D65A exchanged myocardium with increasing Ca2+ concentration. The degree of helical ordering of the myosin heads decreased by the same amount in WT and D65A myocardium when Ca2+ concentration increased. These results are consistent with a direct effect of Ca2+ in activating the thick filament rather than an indirect effect due to Ca2+-mediated crosstalk between the thick and thin filaments.

Two Forms of Thick Filament in the Flight Muscle of Drosophila melanogaster.

Invertebrate striated muscle myosin filaments are highly variable in structure. The best characterized myosin filaments are those found in insect indirect flight muscle (IFM) in which the flight-powering muscles are not attached directly to the wings. Four insect orders, Hemiptera, Diptera, Hymenoptera, and Coleoptera, have evolved IFM. IFM thick filaments from the first three orders have highly similar myosin arrangements but differ significantly among their non-myosin proteins. The cryo-electron microscopy of isolated IFM myosin filaments from the Dipteran Drosophila melanogaster described here revealed the coexistence of two distinct filament types, one presenting a tubular backbone like in previous work and the other a solid backbone. Inside an annulus of myosin tails, tubular filaments show no noticeable densities; solid filaments show four paired paramyosin densities. Both myosin heads of the tubular filaments are disordered; solid filaments have one completely and one partially immobilized head. Tubular filaments have the protein stretchin-klp on their surface; solid filaments do not. Two proteins, flightin and myofilin, are identifiable in all the IFM filaments previously determined. In Drosophila, flightin assumes two conformations, being compact in solid filaments and extended in tubular filaments. Nearly identical solid filaments occur in the large water bug Lethocerus indicus, which flies infrequently. The Drosophila tubular filaments occur in younger flies, and the solid filaments appear in older flies, which fly less frequently if at all, suggesting that the solid filament form is correlated with infrequent muscle use. We suggest that the solid form is designed to conserve ATP when the muscle is not in active use.

The D75N and P161S Mutations in the C0-C2 Fragment of cMyBP-C Associated with Hypertrophic Cardiomyopathy Disturb the Thin Filament Activation, Nucleotide Exchange in Myosin, and Actin-Myosin Interaction.

About half of the mutations that lead to hypertrophic cardiomyopathy (HCM) occur in the MYBPC3 gene. However, the molecular mechanisms of pathogenicity of point mutations in cardiac myosin-binding protein C (cMyBP-C) remain poorly understood. In this study, we examined the effects of the D75N and P161S substitutions in the C0 and C1 domains of cMyBP-C on the structural and functional properties of the C0-C1-m-C2 fragment (C0-C2). Differential scanning calorimetry revealed that these mutations disorder the tertiary structure of the C0-C2 molecule. Functionally, the D75N mutation reduced the maximum sliding velocity of regulated thin filaments in an in vitro motility assay, while the P161S mutation increased it. Both mutations significantly reduced the calcium sensitivity of the actin-myosin interaction and impaired thin filament activation by cross-bridges. D75N and P161S C0-C2 fragments substantially decreased the sliding velocity of the F-actin-tropomyosin filament. ADP dose-dependently reduced filament sliding velocity in the presence of WT and P161S fragments, but the velocity remained unchanged with the D75N fragment. We suppose that the D75N mutation alters nucleotide exchange kinetics by decreasing ADP affinity to the ATPase pocket and slowing the myosin cycle. Our molecular dynamics simulations mean that the D75N mutation affects myosin S1 function. Both mutations impair cardiac contractility by disrupting thin filament activation. The results offer new insights into the HCM pathogenesis caused by missense mutations in N-terminal domains of cMyBP-C, highlighting the distinct effects of D75N and P161S mutations on cardiac contractile function.

Functional role of myosin-binding protein H in thick filaments of developing vertebrate fast-twitch skeletal muscle.

Myosin-binding protein H (MyBP-H) is a component of the vertebrate skeletal muscle sarcomere with sequence and domain homology to myosin-binding protein C (MyBP-C). Whereas skeletal muscle isoforms of MyBP-C (fMyBP-C, sMyBP-C) modulate muscle contractility via interactions with actin thin filaments and myosin motors within the muscle sarcomere "C-zone," MyBP-H has no known function. This is in part due to MyBP-H having limited expression in adult fast-twitch muscle and no known involvement in muscle disease. Quantitative proteomics reported here reveal that MyBP-H is highly expressed in prenatal rat fast-twitch muscles and larval zebrafish, suggesting a conserved role in muscle development and prompting studies to define its function. We take advantage of the genetic control of the zebrafish model and a combination of structural, functional, and biophysical techniques to interrogate the role of MyBP-H. Transgenic, FLAG-tagged MyBP-H or fMyBP-C both localize to the C-zones in larval myofibers, whereas genetic depletion of endogenous MyBP-H or fMyBP-C leads to increased accumulation of the other, suggesting competition for C-zone binding sites. Does MyBP-H modulate contractility in the C-zone? Globular domains critical to MyBP-C's modulatory functions are absent from MyBP-H, suggesting that MyBP-H may be functionally silent. However, our results suggest an active role. In vitro motility experiments indicate MyBP-H shares MyBP-C's capacity as a molecular "brake." These results provide new insights and raise questions about the role of the C-zone during muscle development.

Cryo-EM structures of cardiac muscle α-actin mutants M305L and A331P give insights into the structural mechanisms of hypertrophic cardiomyopathy

Cardiac muscle α-actin is a key protein of the thin filament in the muscle sarcomere that, together with myosin thick filaments, produce force and contraction important for normal heart function. Missense mutations in cardiac muscle α-actin can cause hypertrophic cardiomyopathy, a complex disorder of the heart characterized by hypercontractility at the molecular scale that leads to diverse clinical phenotypes. While the clinical aspects of hypertrophic cardiomyopathy have been extensively studied, the molecular mechanisms of missense mutations in cardiac muscle α-actin that cause the disease remain largely elusive. Here we used cryo-electron microscopy to reveal the structures of hypertrophic cardiomyopathy-associated actin mutations M305L and A331P in the filamentous state. We show that the mutations have subtle impacts on the overall architecture of the actin filament with mutation-specific changes in the nucleotide binding cleft active site, interprotomer interfaces, and local changes around the mutation site. This suggests that structural changes induced by M305L and A331P have implications for the positioning of the thin filament protein tropomyosin and the interaction with myosin motors. Overall, this study supports a structural model whereby altered interactions between thick and thin filament proteins contribute to disease mechanisms in hypertrophic cardiomyopathy.

Interacting myosin head dynamics and their modification by 2’-deoxy-ADP

The contraction of striated muscle is driven by cycling myosin motor proteins embedded within the thick filaments of sarcomeres. In addition to cross-bridge cycling with actin, these myosin proteins can enter an inactive, sequestered state in which the globular S1 heads rest along the thick filament surface and are inhibited from performing motor activities. Structurally, this state is called the interacting heads motif (IHM) and is a critical conformational state of myosin that regulates muscle contractility and energy expenditure. Structural perturbation of the sequestered state can pathologically disrupt IHM structure and the mechanical performance of muscle tissue. Thus, the IHM state has become a target for therapeutic intervention. An ATP analogue called 2’-deoxy-ATP (dATP) is a potent myosin activator that destabilizes the IHM. Here, we use molecular dynamics simulations to study the molecular mechanisms by which dATP modifies the structure and dynamics of myosin in a sequestered state. Simulations of the IHM state containing ADP.Pi in both nucleotide binding pockets revealed dynamic motions of the blocked head – free head interface, light chain binding domain, and S2 in this ‘inactive’ state of myosin. Replacement of ADP.Pi by dADP.Pi triggered a series of structural changes that increased heterogeneity among residue contact pairs at the blocked head – free head interface and a 14% decrease in the interaction energy at the interface. Dynamic changes to this interface were accompanied by dynamics in the light chain binding region. A comparative analysis of these dynamics predicted new structural sites that may affect IHM stability.

Phase change-mediated core-sheath 3D printing of hollow microlattice pseudocapacitive aerogel electrode with favorable electrochemical properties

Channel-interconnected regulation within 3D-printed pseudocapacitive electrode architecture to generate high active material loading/utilization and fast ion transport is pivotal but challenging for implementing high-performance pseudocapacitance system. Herein, we demonstrated an innovative phase change-mediated core-sheath direct ink writing (DIW) 3D printing strategy for controllably constructing pseudocapacitive hollow-microlattice graphene/NiCo2O4 aerogel (HGNA) electrode, with regular hollow channels in printed filaments and abundant well-interconnected hierarchical pores built by jointing tortuous pseudocapacitive graphene nanosheets. The unique architectural features facilitated highly efficient diffusion and infiltration of substance throughout the entire thick filaments from both “interior-exterior” and “exterior-interior” directions, thereby enabling high-level yet effective loading of active materials as well as unimpeded ion transport across the printed bulk-structured electrode. Kinetics analysis revealed that the capacitance of pseudocapacitive HGNA electrode was primarily contributed from fast kinetic process. An asymmetric device assembled with DIW-printed HGNA electrode boasted a high energy storage performance with capacitance of 3.43 F cm−2 and energy density of 1.01 mWh cm−2, while featuring remarkable cycling stability (87% after 8000 cycles) and superior capacity even at large electrode thickness. This work opens a promising route for processing rational pseudocapacitive electrode architectures toward high-capacity, fast-kinetics devices.

Myosin Isoform-Dependent Effect of Omecamtiv Mecarbil on the Regulation of Force Generation in Human Cardiac Muscle.

Omecamtiv mecarbil (OM) is a small molecule that has been shown to improve the function of the slow human ventricular myosin (MyHC) motor through a complex perturbation of the thin/thick filament regulatory state of the sarcomere mediated by binding to myosin allosteric sites coupled to inorganic phosphate (Pi) release. Here, myofibrils from samples of human left ventricle (β-slow MyHC-7) and left atrium (α-fast MyHC-6) from healthy donors were used to study the differential effects of μmolar [OM] on isometric force in relaxing conditions (pCa 9.0) and at maximal (pCa 4.5) or half-maximal (pCa 5.75) calcium activation, both under control conditions (15 °C; equimolar DMSO; contaminant inorganic phosphate [Pi] ~170 μM) and in the presence of 5 mM [Pi]. The activation state and OM concentration within the contractile lattice were rapidly altered by fast solution switching, demonstrating that the effect of OM was rapid and fully reversible with dose-dependent and myosin isoform-dependent features. In MyHC-7 ventricular myofibrils, OM increased submaximal and maximal Ca2+-activated isometric force with a complex dose-dependent effect peaking (40% increase) at 0.5 μM, whereas in MyHC-6 atrial myofibrils, it had no effect or-at concentrations above 5 µM-decreased the maximum Ca2+-activated force. In both ventricular and atrial myofibrils, OM strongly depressed the kinetics of force development and relaxation up to 90% at 10 μM [OM] and reduced the inhibition of force by inorganic phosphate. Interestingly, in the ventricle, but not in the atrium, OM induced a large dose-dependent Ca2+-independent force development and an increase in basal ATPase that were abolished by the presence of millimolar inorganic phosphate, consistent with the hypothesis that the widely reported Ca2+-sensitising effect of OM may be coupled to a change in the state of the thick filaments that resembles the on-off regulation of thin filaments by Ca2+. The complexity of this scenario may help to understand the disappointing results of clinical trials testing OM as inotropic support in systolic heart failure compared with currently available inotropic drugs that alter the calcium signalling cascade.

Abstract Mo116: Evaluating MYBPC3 and MYBPHL missense mutations on sarcomere incorporation

Hypertrophic cardiomyopathy (HCM) is one of the common genetic heart diseases and causes hypercontractility and pathological thickening of the myocardium. Cardiac myosin binding protein-C (cMyBP-C) is an important regulator of cardiac contractility and MYBPC3 mutations accounts for 40% of HCM cases, resulting in the truncation and loss of cMyBP-C. While cMyBP-C is expressed in both atria and ventricle, its recently identified homologous partner, myosin binding protein-HL (MyBP-HL) is solely expressed in the atria. Loss of function mutations in MYBPHL cause dilated cardiomyopathy in humans and mice. MyBP-HL and cMyBP-C bind to specific positions on the thick filament within the C-zone of sarcomere. In the atria, there is a 1:1 ratio between MyBP-HL and cMyBP-C, whereas cMyBP-C is solely expressed in the ventricle at double the cMyBP-C content of the the atria, establishing a stoichiometric relationship between these two proteins in their ability to bind to the thick filament within the sarcomere. Within the ventricle, heterozygous missense mutations within the necessary myosin binding domains of cMyBP-C may prevent proper myosin binding and sarcomere incorporation, but function may still be preserved due to the other unaffected allele. Since the atria expresses MyBP-HL and cMyBP-C, we hypothesize that MYBPC3 and MYBPHL missense mutations are more severe due to the competition for binding sites within the sarcomere between these proteins. We have identified MYBPC3 and MYBPHL missense mutations and variants to evaluate their pathogenicity and sarcomere incorporation via immunofluorescence microscopy within neonatal rat ventricular cardiomyocytes. To test these mutations, we created a “Mini-C” construct consisting of the thick filament binding domains of cMyBP-C. We validated our Mini-C construct via transfection and co-localization with endogenous cMyBP-C in neonatal rat ventricular cardiomyocytes (NRVMs). Our data demonstrate that known pathogenic MYBPC3 missense mutations have improper sarcomere incorporation within NRVMs. Disease associated MYBPHL missense mutations also show improper sarcomere incorporation. To test the effects of missense mutations on myosin binding protein stoichiometry within the sarcomere, we have created and validated a T2A/P2A construct, expressing our Mini-C construct, a fluorescent reporter, Td-Tomato, and our hMyBP-HL construct at consistent and reproducible expression levels via western blot and mass spectrometry.

Abstract Or107: MyBP-HL nonsense mutations fail in sarcomere incorporation leads to modifications in myocyte contraction trough calcium modifications.

Introduction: Heart function depends on the cardiomyocyte contractile apparatus and proper sarcomere protein expression. Mutations in sarcomere genes cause inherited forms of cardiomyopathy and arrhythmias, including atrial fibrillation. AF is the most common cardiac arrhythmia and is associated with ischemic stroke, heart failure, and substantial morbidity and mortality. Recently, our group described a novel sarcomere component, myosin binding protein-H like (MyBP-HL), that is mainly expressed in the cardiac atria. MyBP-HL is composed of two immunoglobulin domains and one fibronectin domain, with homology to last three C-terminal domains of cardiac myosin binding protein-C (cMyBP-C). cMyBP-C nonsense mutations prevent his incorporation into the sarcomere. C-MyBP-C truncated peptides are identified by cells as poisoning peptides, and they are rapidly degraded. cMyBP-C nonsense mutations are causative of hypertrophic cardiomyopathy (HCM). The MyBP-HL Arg255stop variant has been described in humans and has been linked to ventricular conduction system abnormalities, atrial enlargement, dilated cardiomyopathy, and atrial and ventricular arrhythmias. The gnomAD database reports 9 nonsense mutations for MYBPHL in humans (Gln29, Trp54, Arg113, Tyr123, Trp158, Trp192, Lys250, Arg255, and Tyr307). Hypothesis: MYBPHL nonsense mutations fail to incorporate into the sarcomere will lead to contractile disfunction. Results: We demonstrated that MyBP-HL overexpression showed to be able to incorporate in a similar pattern to cMyBP-C C-zone doublets. Nevertheless, nonsense mutations disrupt normal sarcomere localization. MyBP-HL nonsense mutations promote an increase in contraction amplitude and slower relaxation kinetics. Also, we observed an increase in sarcomere length. Similarly, MyBP-HL nonsense mutations lead to increase in amplitude peak of calcium transients, and reduction in velocity to maximal calcium peak. In accordance with calcium modifications, we observed through mass spectrometry an increase in calpain 6 abundance promoted by MyBP-HL nonsense mutations. Conclusion: Based on these data, we demonstrated that MYBPHL nonsense mutations prevent MyBP-HL incorporation into the thick filament. The failure of MyBP-HL truncating peptides to incorporate into the sarcomere leads to a deficiencies in myocytes contraction patterns, changes in calcium patterns and increase in degradation proteins abundance, which are related to AF prevalence.

Abstract Tu092: Role of Z-disc as a Driver of Hypertrophy and Sarcomere Assembly in Hypertrophic Cardiomyopathy

The Z-disc is an electron-dense structure demarcating the lateral boundaries of the sarcomere and postulated to play a key role in both cell signaling and sarcomere assembly. We performed EM on both HCM patient septal myectomies and human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) with 3 different HCM MYH7 mutations, finding marked Z-disc widening along with a hallmark feature of HCM, myofibrillar disarray. Mutations in MYH7 that cause HCM do so by increasing force at the sarcomere level, leading to the proposal that the Z-disc may act as a mechanosensor that converts myosin-generated force into the intracellular signaling that drives hypertrophy. We focused on understanding the mechanisms of Z-disc mechanotransduction of both hypertrophy and sarcomere disarray. RNA-seq from HCM myectomies demonstrated 52 Z-disc differentially expressed genes (DEGs). scRNA-seq from HCM hiPSC-CMs identified 24 Z-disc DEGs. 14 Z-disc DEGs were shared across both datasets, most notably 5 upstream regulators of calcineurin signalling. To correlate changes in gene expression with altered force we transfected hiPSC-CMs with a FRET-based tension sensor in the Z-disc protein vinculin allowing the direct measurement of force experienced by the Z-disc. Live-cell confocal imaging of hiPSC-CMs expressing EGFP-α-actinin-2 showed that indistinct α-actinin-2 foci coalesced into sarcomeres over the course of 24-48 h after replating. Imaging at nm scale using cryo-electron tomography revealed marked disorder in individual thick filaments in HCM hiPSC-CMs, distinct from the classical disarray at the level of whole myofibrils and which would not be visible on conventional EM. Our data suggest that early alterations in sarcomeric micro-structure and signaling induced by altered myosin force may precede hypertrophy and could occur decades before clinical signs (hypertrophy on ECG or echo) are present, with potential implications for early preventive intervention.

Abstract Mo122: Investigating the Role of the Lysine Methyltransferase SMYD1 in Striated Muscle Motor Domain Folding

Properly coordinated folding and assembly of myosin into the sarcomere thick filament is critical for cardiac and skeletal muscle development, function, growth, and maintenance. The motor domain of myosin heavy chain (MHC) requires muscle-specific chaperones for proper folding, making it currently impossible to fold in non-muscle cells. Previous studies found that HSP90 and UNC45B are necessary, but not sufficient, for folding the motor domain, indicating other factors are involved. Defining the minimal set of factors for motor domain folding would allow for development of an improved expression system and accelerate the study of myosin mutations and myosin-targeted drug development. We hypothesize that the muscle-specific lysine methyltransferase SMYD1 contributes to MHC folding and stability. We found SMYD1 associates with UNC45B, HSP90, and MHC in the adult human heart. To study motor domain folding we generated a human MYH7 motor domain fused to GFP (MD::GFP). Transfection into COS-7 cells resulted in unfolded aggregates while adenoviral infection of C 2 C 12 myotubes leads to folded and functional MD::GFP. Interestingly, the folded MD::GFP contains lysine trimethylation, which may arise from the methyltransferase activity of SMYD1. When COS-7 cells were co-transfected with UNC45B and SMYD1, we observed increased total protein levels of non-functional MD::GFP without affecting RNA levels as assessed by RT-qPCR. This effect was not observed when MD::GFP was co-transfected with UNC45B or SMYD1 alone and is dependent on HSP90. We found that UNC45B and SMYD1 stabilized MD::GFP as determined by cycloheximide chase assays. We also found SMYD1 associates with MD::GFP, suggesting this stabilizing effect is due to a direct interaction. Interestingly, a catalytically inactive SMYD1 also increased MD::GFP protein levels in COS-7 cells, consistent with our observation that no significant changes in global protein methylation were observed in the presence of WT SMYD1. Our data suggests that SMYD1 stabilizes the motor domain of striated muscle myosin in conjunction with UNC45B and HSP90, in a manner that is independent of its methyltransferase activity. However, UNC45B and SMYD1 are insufficient for motor domain folding in a non-muscle cell line, suggesting that additional folding factors or proper stoichiometric ratios of chaperones/co-chaperones may be required.

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.

<i>N</i>-terminal fragment of cardiac myosin binding protein C modulates cooperative mechanisms of the thin filament activation in the atria and ventricles

Cardiac myosin binding protein C (cMyBP-C) is one of the essential control components of the myosin cross-bridge cycle. The C-terminal part of cMyBP-C lies on the surface of the thick filament, and its N-terminal part interacts with actin, myosin, and tropomyosin, affecting both the kinetics of the ATP hydrolysis cycle and the lifetime of the cross bridge, as well as the calcium regulation of actin-myosin interaction, thereby modulating the contractile function of the myocardium. The role of cMyBP-C in atrial contraction is poorly studied. We examined the effect of the N-terminal C0-m-C2 (C0-C2) fragment of cMyBP-C on actin-myosin interaction using ventricular and atrial myosin in anin vitromotility assay. The C0-C2 fragment of cMyBP-C significantly reduced the maximum sliding velocity of thin filaments on both myosin isoforms and increased the calcium sensitivity of actin-myosin interaction. The C0-C2 fragment had different effects on the kinetics of nucleotide, ATP, and ADP exchange, increasing the affinity of ventricular myosin for ADP and decreasing the affinity of atrial myosin. The effect of the C0-C2 fragment on the activation of the thin filament depended on the myosin isoforms. Atrial myosin activates the thin filament less strongly than ventricular myosin, and the C0-C2 fragment makes these differences in myosin isoforms more pronounced.

Mechanisms of a novel regulatory light chain-dependent cardiac myosin inhibitor.

Hypertrophic cardiomyopathy (HCM) is a genetic disease of the heart characterized by thickening of the left ventricle (LV), hypercontractility, and impaired relaxation. HCM is caused primarily by heritable mutations in sarcomeric proteins, such as β myosin heavy chain. Until recently, medications in clinical use for HCM did not directly target the underlying contractile changes in the sarcomere. Here, we investigate a novel small molecule, RLC-1, identified in a bovine cardiac myofibril high-throughput screen. RLC-1 is highly dependent on the presence of a regulatory light chain to bind to cardiac myosin and modulate its ATPase activity. In demembranated rat LV trabeculae, RLC-1 decreased maximal Ca2+-activated force and Ca2+ sensitivity of force, while it increased the submaximal rate constant for tension redevelopment. In myofibrils isolated from rat LV, both maximal and submaximal Ca2+-activated force are reduced by nearly 50%. Additionally, the fast and slow phases of relaxation were approximately twice as fast as DMSO controls, and the duration of the slow phase was shorter. Structurally, x-ray diffraction studies showed that RLC-1 moved myosin heads away from the thick filament backbone and decreased the order of myosin heads, which is different from other myosin inhibitors. In intact trabeculae and isolated cardiomyocytes, RLC-1 treatment resulted in decreased peak twitch magnitude and faster activation and relaxation kinetics. In conclusion, RLC-1 accelerated kinetics and decreased force production in the demembranated tissue, intact tissue, and intact whole cells, resulting in a smaller cardiac twitch, which could improve the underlying contractile changes associated with HCM.

An atomistic model of myosin interacting heads motif dynamics and their modification by 2'-deoxy-ADP.

The contraction of striated muscle is driven by cycling myosin motor proteins embedded within the thick filaments of sarcomeres. In addition to cross-bridge cycling with actin, these myosin proteins can enter an inactive, sequestered state in which the globular S1 heads rest along the thick filament surface and are unable to perform motor activities. Structurally, this state is called the interacting heads motif (IHM) and is a critical conformational state of myosin that regulates muscle contractility and energy expenditure. Structural perturbation of the sequestered state via missense mutations can pathologically disrupt the mechanical performance of muscle tissue. Thus, the IHM state has become a target for therapeutic intervention. An ATP analogue called 2'-deoxy-ATP (dATP) is a potent myosin activator which destabilizes the IHM. Here we use molecular dynamics simulations to study the molecular mechanisms by which dATP modifies the structure and dynamics of myosin in a sequestered state. Simulations with IHM containing ADP.Pi in both nucleotide binding pockets revealed residual dynamics in an otherwise 'inactive' and 'sequestered' state of a motor protein. Replacement of ADP.Pi by dADP.Pi triggered a series of structural changes that modify the protein-protein interface that stabilizes the sequestered state, and changes to this interface were accompanied by allosteric changes in remote regions of the protein complex. A comparative analysis of these dynamics predicted new structural sites that may affect IHM stability.

Fast myosin binding protein C knockout in skeletal muscle alters length-dependent activation and myofilament structure.

In striated muscle, the sarcomeric protein myosin-binding protein-C (MyBP-C) is bound to the myosin thick filament and is predicted to stabilize myosin heads in a docked position against the thick filament, which limits crossbridge formation. Here, we use the homozygous Mybpc2 knockout (C2-/-) mouse line to remove the fast-isoform MyBP-C from fast skeletal muscle and then conduct mechanical functional studies in parallel with small-angle X-ray diffraction to evaluate the myofilament structure. We report that C2-/- fibers present deficits in force production and calcium sensitivity. Structurally, passive C2-/- fibers present altered sarcomere length-independent and -dependent regulation of myosin head conformations, with a shift of myosin heads towards actin. At shorter sarcomere lengths, the thin filament is axially extended in C2-/-, which we hypothesize is due to increased numbers of low-level crossbridges. These findings provide testable mechanisms to explain the etiology of debilitating diseases associated with MyBP-C.

Proteomic reference map for sarcopenia research: mass spectrometric identification of key muscle proteins located in the sarcomere, cytoskeleton and the extracellular matrix.

Sarcopenia of old age is characterized by the progressive loss of skeletal muscle mass and concomitant decrease in contractile strength. Age-related skeletal muscle dysfunctions play a key pathophysiological role in the frailty syndrome and can result in a drastically diminished quality of life in the elderly. Here we have used mass spectrometric analysis of the mouse hindlimb musculature to establish the muscle protein constellation at advanced age of a widely used sarcopenic animal model. Proteomic results were further analyzed by systems bioinformatics of voluntary muscles. In this report, the proteomic survey of aged muscles has focused on the expression patterns of proteins involved in the contraction-relaxation cycle, membrane cytoskeletal maintenance and the formation of the extracellular matrix. This includes proteomic markers of the fast versus slow phenotypes of myosin-containing thick filaments and actin-containing thin filaments, as well as proteins that are associated with the non-sarcomeric cytoskeleton and various matrisomal layers. The bioanalytical usefulness of the newly established reference map was demonstrated by the comparative screening of normal versus dystrophic muscles of old age, and findings were verified by immunoblot analysis.

Natural history and clinical outcomes of patients with hypertrophic cardiomyopathy from thin filament mutations.

Hypertrophic cardiomyopathy (HCM) due to thick filament variants is more common; however, HCM due to thin filament variants (HCM-Thin) may be associated with a more malignant phenotype with an increased risk of sudden cardiac death. The aim of this study was to review all the published cases of HCM-Thin to better understand the natural history and clinical outcomes of this disease. A literature review of HCM-Thin identified 21 studies with a total of 177 patients that were suitable for analysis. There were three outcomes of interest, which included a heart failure composite, a ventricular arrhythmia composite and a heart failure and arrhythmia composite outcome. Kaplan-Meier (KM) survival analyses for freedom from each of the abovementioned composite outcomes were completed for the entire cohort and stratified by age of onset and sarcomeric variant. The heart failure composite occurred in 24 (13.6%) patients, the ventricular arrhythmia composite occurred in 30 patients (16.9%) and the combined heart failure and arrhythmia composite occurred in 50 patients (28.2%). In regard to left ventricular ejection fraction (LVEF), the majority of patients were preserved (LVEF>50%) compared with mildly reduced (LVEF 41%-50%) and reduced (LVEF≤40%) (respectively 26.6% vs. 0.6% vs. 3.4%). The median maximal left ventricular wall thickness (LVWT) was 19.0mm [interquartile range (IQR) 5.3]. Only 10.7% of the cohort had evidence of left ventricular outflow tract (LVOT) obstruction. Those with paediatric-onset HCM had earlier onset and were at higher risk for each endpoint than their adult counterparts. When stratified by genetic variant, patients with TNNI3 and TPM1 were at a higher risk of the heart failure composite endpoint and the combined heart failure and arrhythmia composite endpoint in comparison with those with the other genetic variants. HCM-Thin is associated with significant morbidity and mortality, with a high arrhythmia burden despite low rates of cardiac obstruction and mild hypertrophy. The paediatric onset of disease and certain sarcomeric variants appear to be associated with a worse prognosis than their adult-onset and other sarcomeric variant counterparts. HCM-Thin seems to have a distinct phenotype, which may require a different management approach.

Constructing myosin/high-density lipoprotein composite emulsions: Roles of pH on emulsification stability, rheological and structural properties

The emulsification activity of myosin plays a significant role in affecting quality of emulsified meat products. High-density lipoprotein (HDL) possesses strong emulsification activity and stability due to its structural characteristics, suggesting potential for its utilization in developing functional emulsified meat products. In order to explore the effect of HDL addition on emulsification stability, rheological properties and structural features of myosin (MS) emulsions, HDL-MS emulsion was prepared by mixing soybean oil with isolated HDL and MS, with pH adjustments ranging from 3.0 to 11.0. The results found that emulsification activity and stability in two emulsion groups consistently improved as pH increased. Under identical pH, HDL-MS emulsion exhibited superior emulsification behavior as compared to MS emulsion. The HDL-MS emulsion under pH of 7.0–11.0 formed a viscoelastic protein layer at the interface, adsorbing more proteins and retarding oil droplet diffusion, leading to enhanced oxidative stability, compared to the MS emulsion. Raman spectroscopy analysis showed more flexible conformational changes in the HDL-MS emulsion. Microstructural observations corroborated these findings, showing a more uniform distribution of droplet sizes in the HDL-MS emulsion with smaller particle sizes. Overall, these determinations suggested that the addition of HDL enhanced the emulsification behavior of MS emulsions, and the composite emulsions demonstrated heightened responsiveness under alkaline conditions. This establishes a theoretical basis for the practical utilization of HDL in emulsified meat products.