bioRxiv Science⌕ Search

Biology subjects

Irving, T.

Publications and source records attributed to Irving, T..

15 recordsLinked to original sources

Symmetry-Based Center and Rotation Refinement for Fiber Diffraction Patterns

X-ray fiber diffraction patterns exhibit four-fold symmetry that can be exploited, through folding and averaging, to improve signal-to-noise ratio. Accurate folding requires a precise sub-pixel estimate of the symmetry center and precise orientation of the meridional pattern axis to the fiber axis: small center or angular errors blur diffraction features, reduce layer-line sharpness, and introduce errors in spacing measurements. A pixel-level estimate is often too imprecise for this purpose, and detector gaps further complicate the alignment objective. We formulate the masked quadrant-folding problem, define a four-quadrant symmetry loss that consistently excludes invalid pixels, and evaluate several refinement strategies: hierarchical coarse-to-fine grid search; ECC-based rigid registration with global center/orientation correction fitting; ECC registration followed by local gradient refinement; and a hybrid that appends a local grid search on a cropped pattern. Direct gradient optimization from the rough QF alignment was found to be unreliable. Grid search provides a robust, interpretable baseline that directly minimizes the folding objective but is substantially slower than registration; ECC gives a fast near-correct alignment, and the hybrid closes the accuracy gap to brute-force search at a fraction of its runtime. On real datasets with calibration data, applying a calibration center with optimized rotation is effectively optimal. The hybrid center-refinement method has been integrated into the MuscleX package.

biophysics↗

LysM Receptor Proteins are Required for Ectomycorrhizal Symbiosis in Poplar

Successful symbiotic associations often rely on the perception of specific microbial signals by host plants through pattern recognition receptors. LysM receptor-like kinases are key receptors in both rhizobial and arbuscular mycorrhizal symbioses. In this study, we provide the first evidence that such LysM receptor-like kinases also control the establishment of ectomycorrhizal associations in poplar. Using CRISPR/Cas9-mediated genome editing, we knocked out three NFP-like candidate genes in various combinations. We examined their roles in the perception of the ectomycorrhizal fungus Laccaria bicolor, as well as chitin oligomers and lipo-chitooligosaccharides. We demonstrate that all three receptors are necessary for the recognition of symbiosis-related signals, calcium spiking, symbiotic gene expression, and ectomycorrhizal root phenotypes.

plant biology↗

Annotating the X-ray diffraction pattern of vertebrate striated muscle

Low-angle X-ray diffraction is a powerful technique for analyzing the molecular structure of the myofilaments of striated muscle in situ. It has contributed greatly to our understanding of the relaxed, 430-[A]-repeating organization of myosin heads in thick filaments in skeletal and cardiac muscle. Using X-ray diffraction, changes in filament structure can be detected on the [A] length scale and millisecond time scale, leading to models that are the foundation of our understanding of the structural basis of contraction. As with all X-ray fiber diffraction studies, interpretation requires modeling, which has previously been based on low-resolution knowledge of thick filament structure and is complicated by the contributions of multiple filament components to most X-ray reflections. Here, we use an atomic model of the human cardiac thick filament C-zone, derived from cryo-EM, to compute objectively the contributions of myosin heads, tails, titin, and cMyBP-C to the diffraction pattern, by including/excluding these components in the calculations. Our results support some previous interpretations but contradict others. We confirm that the myosin heads are responsible for most of the intensity on the myosin layer-lines, including the M3 meridional. Contrary to expectation, we find that myosin tails contribute little to the pattern, including the M6 meridional; this reflection arises mainly from heads and other components. The M11 layer line (39 [A] spacing) arises mostly from the curved and kinked structure of titin, which allows eleven [~]42-[A]-long domains to fit into the 430 [A] repeat. The M11 spacing can be used as a measure of strain in the myosin filament backbone as there is negligible head contribution. These insights should aid future understanding of the X-ray pattern of intact muscle in different conditions such as contraction and drug treatment. Significance statementX-ray diffraction is widely used to study the structure of striated muscle, revealing the molecular organization of the thick and thin filaments in situ. Changes in the X-ray pattern during contraction provide insights into contractile mechanisms on the [A] length scale and millisecond timescale. Interpretation of X-ray patterns is based on modeling, which is complicated by contributions of multiple filament components to different reflections and the lack of a reliable thick filament model. Here, we use a cryo-EM-based atomic model of the thick filament to compute contributions of different filament components to the diffraction pattern, by including/excluding these components in the calculations. The insights gained will aid interpretation of the X-ray pattern in relaxation and contraction and following drug treatment.

biophysics↗

Myosin modulator Aficamten inhibits force in cardiac muscle by altering myosin's biochemical activity without changing thick filament structure

BackgroundInhibiting contractility by targeting cardiac myosin is an effective treatment for patients with hypertrophic cardiomyopathy (HCM). Aficamten is a second in class myosin inhibitor with promising clinical data showing improvements in hemodynamics and symptoms in patients with HCM. While it is known that Aficamten inhibits force and cardiomyocyte contractility by stabilizing the weak pre-powerstroke conformation, effects on myosin structure and kinetics during loaded contraction are lacking. MethodsPermeabilized porcine cardiac tissue and myofibrils were used for single-molecule imaging of ATP turn over, X-ray diffraction, and mechanical measurements. Engineered heart tissues from human induced pluripotent stem cell cardiomyocytes were used to evaluate effects on force and contraction kinetics. ResultsIn contrast to Mavacamten, Aficamten does not structurally sequester myosin heads along the thick filament. Aficamten inhibits ATPase activity by shifting myosin heads from higher to slower ATPase state, with the emergence of a super slow biochemical nucleotide turnover state. This results in decreased force and calcium sensitivity without altering cross-bridge cycling. These contractile mechanical changes are comparable to Mavacamten. Our myofibril mechanical assay showed inhibition of force with accelerated relaxation. In EHTs, while Mavacamten and Aficamten inhibit cardiac twitch forces, Mavacamten reduces the activation kinetics while both result in faster relaxation. ConclusionsWe used a combination of biochemical and biomechanical assays to show that Aficamten inhibits myosin ATPase without appreciably altering myosin structure. This is different from Mavacamten that strongly affects both. While both compounds inhibit contractility, differences in mechanisms of action and kinetics of force activation and relaxation could allow use in different patient populations.

biophysics↗

Probing relaxed myosin states in hypertrophic cardiomyopathy by second harmonic-generation microscopy

This study explores the use of polarized second-harmonic generation (pSHG) to investigate myosin conformation in the relaxed state, differentiating between the actin-available, disordered (ON) state and the energy-conserving, ordered (OFF) state. By shifting the ON/OFF equilibrium using both physical and chemical manipulations, we demonstrate the sensitivity of pSHG in quantifying the ON/OFF ratio in skeletal and cardiac tissues. Comparisons with X-ray diffraction measurements further validate our findings. Applying this approach to a sarcomeric mutation associated with hypertrophic cardiomyopathy, we show that R403Q/MYH7-mutated minipig ventricle tissue exhibits a higher ON fraction compared to controls. This difference is abolished under high concentrations of a myosin activator (2-deoxyATP) and an inhibitor (Mavacamten), indicating structural similarity between R403Q and controls in these two states. ATPase assays reveal increased resting ATPase activity in R403Q samples, which persists even in the presence of 2-deoxyATP, suggesting that the elevated energy consumption in the R403Q mutation is driven by both a population shift toward the ON state and enhanced myosin ATPase activity per motor head.

physiology↗

Mavacamten facilitates myosin head ON-to-OFF transitions and shortens thin filament length in relaxed skeletal muscle

The first-in-its-class cardiac drug mavacamten reduces the proportion of so-called ON-state myosin heads in relaxed sarcomeres, altering contraction performance. However, mavacamten is not completely specific to cardiac myosin and can also affect skeletal muscle myosin, an important consideration since mavacamten is administered orally and so will also be present in skeletal tissue. Here, we studied the effect of mavacamten on skeletal muscle structure using small-angle X-ray diffraction. Mavacamten treatment reduced the proportion of ON myosin heads but did not eliminate the molecular underpinnings of length-dependent activation, demonstrating similar effects to those observed in cardiac muscle. These findings provide valuable insights for the potential use of mavacamten as a tool to study muscle contraction across striated muscle.

physiology↗

Unlocking the Role of sMyBP-C: A Key Player in Skeletal Muscle Development and Growth

Skeletal muscle is the largest organ in the body, responsible for gross movement and metabolic regulation. Recently, variants in the MYBPC1 gene have been implicated in a variety of developmental muscle diseases, such as distal arthrogryposis. How MYBPC1 variants cause disease is not well understood. Here, through a collection of novel gene-edited mouse models, we define a critical role for slow myosin binding protein-C (sMyBP-C), encoded by MYBPC1, across muscle development, growth, and maintenance during prenatal, perinatal, postnatal and adult stages. Specifically, Mybpc1 knockout mice exhibited early postnatal lethality and impaired skeletal muscle formation and structure, skeletal deformity, and respiratory failure. Moreover, a conditional knockout of Mybpc1 in perinatal, postnatal and adult stages demonstrates impaired postnatal muscle growth and function secondary to disrupted actomyosin interaction and sarcomere structural integrity. These findings confirm the essential role of sMyBP-C in skeletal muscle and reveal specific functions in both prenatal embryonic musculoskeletal development and postnatal muscle growth and function.

physiology↗

The structural OFF and ON states of myosin can be decoupled from the biochemical super-relaxed and disordered-relaxed states.

There is a growing awareness that both thick filament and classical thin filament regulation play central roles in modulating muscle contraction. Myosin ATPase assays have demonstrated that under relaxed conditions, myosin may reside in either a high energy-consuming disordered-relaxed (DRX) state available for binding actin to generate force, or in an energy-sparing super-relaxed (SRX) state unavailable for actin binding. X-ray diffraction studies have shown the majority of myosin heads are in a quasi-helically ordered OFF state in a resting muscle and that this helical ordering is lost when myosin heads are turned ON for contraction. It has been assumed that myosin heads in SRX and DRX states are equivalent to the OFF and ON state respectively and the terms have been used interchangeably. Here, we use X-ray diffraction and ATP turnover assays to track the structural and biochemical transitions of myosin heads respectively induced with either omecamtiv mecarbil (OM) or piperine in relaxed porcine myocardium. We find that while OM and piperine induce dramatic shifts of myosin heads from the OFF to ON states, there are no appreciable changes in the population of myosin heads in the SRX and DRX states in both unloaded and loaded preparations. Our results show that biochemically defined SRX and DRX can be decoupled from structurally-defined OFF and ON states. In summary, while SRX/DRX and OFF/ON transitions can be correlated in some cases, these two phenomena are measured using different approaches, do not necessarily reflect the same properties of the thick filament and should be investigated and interpreted separately. SignificanceMyosin based thick filament regulation is now known to be critical for muscle contraction with myosin dysregulation found in hypertrophic and dilated cardiomyopathies. While previously thought to be synonymous, this study finds that biochemical and structural thick filament disengagement are distinct properties and should be investigated as independent phenomena. Understanding the details of thick filament regulation will be of great relevance to defining sarcomere-level dysfunction in myopathies and understanding and better designing and testing sarcomere therapies aimed at reversing them for treatment of cardiomyopathy.

biophysics↗

Myosin in autoinhibited off state(s), stabilized by mavacamten, can be recruited via inotropic effectors

Mavacamten is a novel, FDA-approved, small molecule therapeutic designed to regulate cardiac function by selectively but reversibly inhibiting the enzymatic activity of myosin. It shifts myosin towards ordered off states close to the thick filament backbone. It remains unresolved whether mavacamten permanently sequesters these myosin heads in the off state(s) or whether these heads can be recruited in response to physiological stimuli when required to boost cardiac output. We show that cardiac myosins stabilized in these off state(s) by mavacamten are recruitable by Ca2+, increased heart rate, stretch, and {beta}-adrenergic ({beta}-AR) stimulation, all known physiological inotropic effectors. At the molecular level, we show that, in presence of mavacamten, Ca2+ increases myosin ATPase activity by shifting myosin heads from the reserve super-relaxed (SRX) state to the active disordered relaxed (DRX) state. At the myofilament level, both Ca2+ and passive lengthening can shift ordered off myosin heads from positions close to the thick filament backbone to disordered on states closer to the thin filaments in the presence of mavacamten. In isolated rat cardiomyocytes, increased stimulation rates enhanced shortening fraction in mavacamten-treated cells. This observation was confirmed in vivo in telemetered rats, where left-ventricular dP/dtmax, an index of inotropy, increased with heart rate in mavacamten treated animals. Finally, we show that {beta}-AR stimulation in vivo increases left-ventricular function and stroke volume in the setting of mavacamten. Our data demonstrate that the mavacamten-promoted off states of myosin in the thick filament are activable, at least partially, thus leading to preservation of cardiac reserve mechanisms. Significance statementMavacamten is the first myosin-targeted small molecule inhibitor approved by the FDA to treat obstructive hypertrophic cardiomyopathy by attenuating myocardial hyperdynamic contraction. The recruitment of cardiac contractility is, however, vital to ensure sufficient cardiac output during increased physiological demand. Here we show that major inotropic effectors are at least partially preserved in the setting of mavacamten, resulting in maintenance of cardiac reserve mechanisms. These results not only suggest an alternative mechanistic explanation, beyond mere LV outflow tract obstruction removal, for the clinically observed increase in peak oxygen uptake with exercise in HCM patients receiving mavacamten, but also lay the groundwork for a potential methodology to investigate the sarcomeric basis of chronotropic incompetence in disease states to motivate new therapeutic interventions.

physiology↗

The distinctive mechanical and structural signatures of residual force enhancement in myofibers

In muscle, titin proteins connect myofilaments together and are thought to be critical for contraction, especially during residual force enhancement (RFE) when force is elevated after an active stretch. We investigated titins function during contraction using small-angle X-ray diffraction to track structural changes before and after 50% titin cleavage and in the RFE-deficient, mdm titin mutant. We report that the RFE state is structurally distinct from pure isometric contractions, with increased thick filament strain and decreased lattice spacing, most likely caused by elevated titin-based forces. Furthermore, no RFE structural state was detected in mdm muscle. We posit that decreased lattice spacing, increased thick filament stiffness, and increased non-crossbridge forces are the major contributors to RFE. We conclude that titin directly contributes to RFE. One-Sentence SummaryTitin contributes to active force production and residual force enhancement in skeletal muscles.

physiology↗

Thick Filament Activation Is Different in Fast and Slow-Twitch Skeletal Muscle

The contractile properties of fast-twitch and slow-twitch skeletal muscles are primarily determined by the myosin isoform content and modulated by a variety of sarcomere proteins. X-ray diffraction studies of regulatory mechanisms in muscle contraction have focused predominately on fast-or mixed-fiber muscle with slow muscle being much less studied. Here, we used time-resolved x-ray diffraction to investigate the dynamic behavior of the myofilament proteins in relatively pure slow fiber rat soleus (SOL) and pure fast fiber rat extensor digitorum longus (EDL) muscle during twitch and tetanic contractions at optimal length. During twitch contractions the diffraction signatures indicating a transition in the myosin heads from ordered OFF states, where heads are held close to the thick filament backbone, to disordered ON states, where heads are free to bind to thin filaments, were found in EDL and not in SOL muscle. During tetanic contraction, changes in the disposition of myosin heads as active tension develops is a cooperative process in EDL muscle whereas in SOL muscle this relationship is less cooperative. The observed reduced extensibility of the thick filaments in SOL muscle as compared to EDL muscles indicate a molecular basis for this behavior. These data indicate that for the EDL thick filament activation is a cooperative strain-induced mechano-sensing mechanism, whereas for the SOL thick filament activation has a more graded response. These different approaches to thick filament regulation in fast- and slow-twitch muscles may be adaptations for short duration, strong contractions versus sustained, finely controlled contractions, respectively. Key PointsO_LIFast twitch muscle and slow-twitch muscle are optimized for strong, short duration contractions and sustained smaller movements respectively. C_LIO_LIStructural events (OFF to ON transitions) in the myosin containing thick filaments in fast muscle help determine the timing and strength of contractions but these have not been studied in slow-twitch muscle. C_LIO_LIThe X-ray diffraction signatures of structural OFF to ON transitions are different in fast extensor digitorum longus (EDL) and slow soleus (SOL) muscle, being completely absent during twitches in soleus muscle and blunted during tetanic contractions SOL as compared to EDL C_LIO_LICooperative thick filament structural OFF to ON transitions in fast twitch muscle may be an adaptation for rapid and ballistic movements whereas more graded OFF to ON structural transitions in slow-twitch muscle may be an adaptation for slower, finer motions. C_LI

physiology↗

Cardiac myosin filaments are directly regulated by calcium

Contraction of cardiac and skeletal muscle is initiated by calcium (Ca2+) binding to regulatory proteins on actin-containing thin filaments. During muscle contraction, structural changes in Ca2+-dependent thin filament allow the binding of myosin motors to drive muscle contraction1. The dynamic switching between the resting off states and the active on states of myosin is also critical in regulating muscle contractility2-4. However, the molecular switch on the myosin-containing thick filament that drives this process is not understood. Here we show that cardiac thick filaments are directly Ca2+-regulated. We find that Ca2+ progressively moves the myosin heads from ordered off states close to the thick filament backbone to disordered on states closer to the thin filaments. This Ca2+-dependent structural shift of myosin is accompanied by a biochemically defined transition from the inactive super-relaxed state(s) to the active disordered relaxed state(s)3. Furthermore, we find that this Ca2+-mediated molecular switching is an intrinsic property of cardiac myosin but only when assembled into thick filaments. This novel concept of Ca2+ as a regulatory modulator of the thick filament provides a fresh perspective on cardiac muscle regulation, which may be particularly valuable for devising restorative treatments for pathologies altering the Ca2+ sensitivity of the sarcomere.

biophysics↗

Myofibril orientation as a metric for characterizing heart disease

Myocyte disarray is a hallmark of many cardiac disorders. However, the relationship between alterations in the orientation of individual myofibrils and myofilaments to disease progression has been largely underexplored. This oversight has predominantly been due to a paucity of methods for objective and quantitative analysis. Here we introduce a novel, less-biased approach to quantify myofibrillar and myofilament orientation in cardiac muscle under near physiological conditions and demonstrate its superiority as compared to conventional histological assessments. Using small-angle X-ray diffraction, we first investigated changes in myofibrillar orientation at increasing sarcomere lengths in permeabilized, relaxed, wildtype mouse myocardium by assessing the angular spread of the 1,0 equatorial reflection (angle {sigma}). At a sarcomere length (SL) of 1.9 m, the angle {sigma} was 0.23{+/-}0.01 rad, decreased to 0.19{+/-}0.01 rad at a SL of 2.1 m, and further decreased to 0.15{+/-}0.01 rad at a SL of 2.3 m (p<0.0001). Angle {sigma} was significantly larger in R403Q, a MYH7 hypertrophic cardiomyopathy (HCM) model, porcine myocardium (0.24{+/-}0.01 rad) compared to WT myocardium (0.14{+/-}0.005 rad, p<0.0001) as well as in human heart failure tissue (0.19{+/-}0.006 rad) when compared to non-failing samples (0.17{+/-}0.007 rad, p=0.01). These data indicate that diseased myocardium suffers from greater myofibrillar disorientation compared to healthy controls. Finally, we showed that conventional, histology-based analysis of disarray can be subject to user bias and/or sampling error and lead to false positives. Our method for directly assessing myofibrillar orientation avoids the artifacts introduced by conventional histological approaches that assess myocyte orientation and only indirectly evaluate myofibrillar orientation, and provides a precise and objective metric for phenotypically characterizing myocardium. The ability to obtain excellent X-ray diffraction patterns from frozen human myocardium provides a new tool for investigating structural anomalies associated with cardiac diseases. Statement of SignificanceWe introduce a precise and quantitative approach to directly measure myofibrillar and myofilament orientation in cardiac muscle under near physiological conditions as a novel tool for phenotypically characterizing striated muscle systems. We use this technique to demonstrate that myocardium from disease model organisms and failing human myocardium suffers from greater myofibrillar disorientation compared to healthy controls. We also demonstrate that excellent diffraction patterns can be obtained from frozen and thawed human myocardium. Given the ready availability of frozen human heart tissue in tissue banks, this capability opens up a large space of potential experiments relating sarcomere structure to dysfunction in cardiac disorders.

biophysics↗

Lattice arrangement of myosin filaments correlates with fiber type in rat skeletal muscle

The thick (myosin-containing) filaments of vertebrate skeletal muscle are arranged in a hexagonal lattice, interleaved with an array of thin (actin-containing) filaments with which they interact to produce contraction. X-ray diffraction and electron microscopy have shown that there are two types of thick filament lattice. In the simple lattice, all filaments have the same orientation about their long axis, while in the super lattice, nearest neighbors have rotations differing by 60{degrees}. Tetrapods (amphibians, reptiles, birds, mammals) typically have only a super lattice, while the simple lattice is confined to fish. We have carried out X-ray diffraction and electron microscopy of the soleus (SOL) and extensor digitorum longus (EDL) muscles of the rat and found that while the EDL has a super-lattice, as expected, the SOL has a simple lattice. The EDL and SOL of the rat are unusual in being essentially pure fast and slow muscles respectively. The mixed fiber content of most tetrapod muscles and/or lattice disorder may explain why the simple lattice has not been apparent in these vertebrates before. This is supported by only weak simple lattice diffraction in the X-ray pattern of mouse SOL, which has a greater mix of fiber types than rat. We conclude that the simple lattice might be common in tetrapods. The correlation between fiber type and filament lattice arrangement suggests that the lattice arrangement may contribute to the functional properties of a muscle.\n\nSummaryThe three-dimensional arrangement of thick filaments in skeletal muscle is studied by X-ray diffraction and electron microscopy. A correlation is found between thick filament lattice type (simple or super lattice) and fiber type (fast/slow). This suggests that lattice organization contributes to muscle functional properties

biophysics↗

A nanometer difference in myofilament lattice spacing of two cockroach leg muscles explains their different functions

Muscle is highly organized across scales. Consequently, small changes in arrangement of myofilaments can influence macroscopic function. Two leg muscles of a cockroach, have identical innervation, mass, twitch responses, length-tension curves, and force-velocity relationships. However, during running, one muscle is dissipative, while the other produces significant positive mechanical work. Using time resolved x-ray diffraction in intact, contracting muscle, we simultaneously measured the myofilament lattice spacing, packing structure, and macroscopic force production of these muscle to test if nanoscale differences could account for this conundrum. While the packing patterns are the same, one muscle has 1 nm smaller lattice spacing at rest. Under isometric activation, the difference in lattice spacing disappeared explaining the two muscles identical steady state behavior. During periodic contractions, one muscle undergoes a 1 nm greater change in lattice spacing, which correlates with force. This is the first identified feature that can account for the muscles different functions.

biophysics↗