Super-relaxed Research Articles

Abstract 9592: The Super Relaxed State of Myosin Contributes to the Pathogenesis of Right Ventricular Dysfunction in Pulmonary Hypertension

Introduction: Patients with pulmonary hypertension (PH) often succumb to right ventricular (RV) failure yet little is known regarding the underlying pathological mechanisms. Recently, the transition of myosin from the disordered relaxed (DRX) to the super relaxed (SRX) state during diastole has been identified as a pathological feature contributing to myocardial dysfunction in heart failure. Using synchrotron radiation (SR) x-ray diffraction (SAXS), the proximity of myosin heads relative to actin in the in situ beating rat heart can be evaluated in different layers of the myocardial wall. We performed SR SAXS at different timepoints post-PH induction with simultaneous PV-loop acquisition to assess if SRX transition contributes to progressive global RV dysfunction in a rat model of PH. Methods: PH was induced in rats using the Sugen5416/3-week 10% hypoxia method, with rats being returned to normoxia for 3- (SuHx3wk) or 6-weeks (SuHx6wk) after the hypoxic period. SR SAXS was performed at BL40XU, SPring-8 Synchrotron, Japan. Global and site-specific RV myofilament phosphorylation status was also examined. Results: Compared to control rats (n=7), SuHx3wk (n=7) and SuHx6wk (n=7) rats exhibited progressive increases in RV size, RV end systolic pressure (ESP), RV dP/dt max and dP/dt min (all P<0.05 vs. Control). In the deeper subendocardial layer of the RV free wall, there were virtually no myosin heads within the proximity of actin at end diastole in SuHx6wk rats, whereas in the control and SuHx3wk rats, around 30-50% of myosin heads were in proximity to actin (P<0.05, SuHx6wk vs. Control and SuHx3wk). No significant differences in diastolic sarcomere length were found, suggesting a transition of myosin from DRX to SRX in the SuHx6wk rats. Importantly, indices for global RV function (ESP, dP/dt max, dP/dt min ) significantly correlated with diastolic myosin mass transfer values. Analysis of RV myofilament phosphorylation revealed a progressive and significant reduction in MLC-2v phosphorylation over the time course of PH in rats. Conclusion: PH is associated with progressive changes in global RV function, which may be attributed to a transition from DRX to SRX state of myosin in the RV. Reduced MLC-2v phosphorylation may underlie the DRX to SRX transition in PH.

Variants of the myosin interacting-heads motif.

Under relaxing conditions, the two heads of myosin II interact with each other and with the proximal part (S2) of the myosin tail, establishing the interacting-heads motif (IHM), found in myosin molecules and thick filaments of muscle and nonmuscle cells. The IHM is normally thought of as a single, unique structure, but there are several variants. In the simplest ("canonical") IHM, occurring in most relaxed thick filaments and in heavy meromyosin, the interacting heads bend back and interact with S2, and the motif lies parallel to the filament surface. In one variant, occurring in insect indirect flight muscle, there is no S2-head interaction and the motif is perpendicular to the filament. In a second variant, found in smooth and nonmuscle single myosin molecules in their inhibited (10S) conformation, S2 is shifted ∼20 Å from the canonical form and the tail folds twice and wraps around the interacting heads. These molecule and filament IHM variants have important energetic and pathophysiological consequences. (1) The canonical motif, with S2-head interaction, correlates with the super-relaxed (SRX) state of myosin. The absence of S2-head interaction in insects may account for the lower stability of this IHM and apparent absence of SRX in indirect flight muscle, contributing to the quick initiation of flight in insects. (2) The ∼20 Å shift of S2 in 10S myosin molecules means that S2-head interactions are different from those in the canonical IHM. This variant therefore cannot be used to analyze the impact of myosin mutations on S2-head interactions that occur in filaments, as has been proposed. It can be used, instead, to analyze the structural impact of mutations in smooth and nonmuscle myosin.

Cardiac myosin filaments are directly regulated by calcium.

Classically, striated muscle contraction is initiated by calcium (Ca2+)-dependent structural changes in regulatory proteins on actin-containing thin filaments, which allow the binding of myosin motors to generate force. Additionally, dynamic switching between resting off and active on myosin states has been shown to regulate muscle contractility, a recently validated mechanism by novel myosin-targeted therapeutics. The molecular nature of this switching, however, is not understood. Here, using a combination of small-angle x-ray fiber diffraction and biochemical assays with reconstituted systems, we show that cardiac thick filaments are directly Ca2+-regulated. We find that Ca2+ induces a structural transition of myosin heads from ordered off states close to the thick filament to disordered on states closer to the thin filaments. Biochemical assays show a Ca2+-induced transition from an inactive super-relaxed (SRX) state(s) to an active disordered-relaxed (DRX) state(s) in synthetic thick filaments. We show that these transitions are an intrinsic property of cardiac myosin only when assembled into thick filaments and provide a fresh perspective on nature's two orthogonal mechanisms to regulate muscle contraction through the thin and the thick filaments.

Functional comparison of phosphomimetic S15D and T160D mutants of myosin regulatory light chain exchanged in cardiac muscle preparations of HCM and WT mice.

In this study, we investigated the rescue potential of two phosphomimetic mutants of the myosin regulatory light chain (RLC, MYL2 gene), S15D, and T160D RLCs. S15D-RLC mimics phosphorylation of the established serine-15 site of the human cardiac RLC. T160D-RLC mimics the phosphorylation of threonine-160, identified by computational analysis as a high-score phosphorylation site of myosin RLC. Cardiac myosin and left ventricular papillary muscle (LVPM) fibers were isolated from a previously generated model of hypertrophic cardiomyopathy (HCM), Tg-R58Q, and Tg-wild-type (WT) mice. Muscle specimens were first depleted of endogenous RLC and then reconstituted with recombinant human cardiac S15D and T160D phosphomimetic RLCs. Preparations reconstituted with recombinant human cardiac WT-RLC and R58Q-RLC served as controls. Mouse myosins were then tested for the actin-activated myosin ATPase activity and LVPM fibers for the steady-state force development and Ca2+-sensitivity of force. The data showed that S15D-RLC significantly increased myosin ATPase activity compared with T160D-RLC or WT-RLC reconstituted preparations. The two S15D and T160D phosphomimetic RLCs were able to rescue Vmax of Tg-R58Q myosin reconstituted with recombinant R58Q-RLC, but the effect of S15D-RLC was more pronounced than T160D-RLC. Low tension observed for R58Q-RLC reconstituted LVPM from Tg-R58Q mice was equally rescued by both phosphomimetic RLCs. In the HCM Tg-R58Q myocardium, the S15D-RLC caused a shift from the super-relaxed (SRX) state to the disordered relaxed (DRX) state, and the number of heads readily available to interact with actin and produce force was increased. At the same time, T160D-RLC stabilized the SRX state at a level similar to R58Q-RLC reconstituted fibers. We report here on the functional superiority of the established S15 phospho-site of the human cardiac RLC vs. C-terminus T160-RLC, with S15D-RLC showing therapeutic potential in mitigating a non-canonical HCM behavior underlined by hypocontractile behavior of Tg-R58Q myocardium.

Molecular basis of force-pCa relation in MYL2 cardiomyopathy mice: Role of the super-relaxed state of myosin

In this study, we investigated the role of the super-relaxed (SRX) state of myosin in the structure-function relationship of sarcomeres in the hearts of mouse models of cardiomyopathy-bearing mutations in the human ventricular regulatory light chain (RLC, MYL2 gene). Skinned papillary muscles from hypertrophic (HCM-D166V) and dilated (DCM-D94A) cardiomyopathy models were subjected to small-angle X-ray diffraction simultaneously with isometric force measurements to obtain the interfilament lattice spacing and equatorial intensity ratios (I11/I10) together with the force-pCa relationship over a full range of [Ca2+] and at a sarcomere length of 2.1 μm. In parallel, we studied the effect of mutations on the ATP-dependent myosin energetic states. Compared with wild-type (WT) and DCM-D94A mice, HCM-D166V significantly increased the Ca2+ sensitivity of force and left shifted the I11/I10-pCa relationship, indicating an apparent movement of HCM-D166V cross-bridges closer to actin-containing thin filaments, thereby allowing for their premature Ca2+ activation. The HCM-D166V model also disrupted the SRX state and promoted an SRX-to-DRX (super-relaxed to disordered relaxed) transition that correlated with an HCM-linked phenotype of hypercontractility. While this dysregulation of SRX ↔ DRX equilibrium was consistent with repositioning of myosin motors closer to the thin filaments and with increased force-pCa dependence for HCM-D166V, the DCM-D94A model favored the energy-conserving SRX state, but the structure/function-pCa data were similar to WT. Our results suggest that the mutation-induced redistribution of myosin energetic states is one of the key mechanisms contributing to the development of complex clinical phenotypes associated with human HCM-D166V and DCM-D94A mutations.

Dilated cardiomyopathy mutation E525K in human beta-cardiac myosin stabilizes the interacting heads motif and super-relaxed state of myosin

Formation of the auto-inhibited super-relaxed (SRX) state of cardiac myosin is thought to be crucial for regulating contraction, relaxation, and energy conservation in the heart. We found that a single point mutation, E525K, associated with dilated cardiomyopathy (DCM) dramatically enhances the stability of the SRX state. We expressed human cardiac myosin containing the subfragment 1 domain, 15 heptads of the subfragment 2 coiled-coil region and a leucine zipper that facilitates dimerization, as well as a C-terminal GFP tag (M2β 15HPZ.GFP).

Structural basis of the super-relaxed and hyper-relaxed state of myosin II

Super-relaxation (SRX) is a state of muscle thick filaments in which ATP turnover by myosin is much slower than that of myosin II in solution. This inhibited state, in equilibrium with a faster, disordered-relaxed state (DRX), is ubiquitous across muscle types and species. It is thought to be fundamental to muscle function, acting as a mechanism for switching off energy-consuming myosin motors when they are not being used. The structural basis of super-relaxation is usually taken to be a motif formed by myosin in which the two heads interact with each other and with the proximal tail, forming an interacting-heads motif (IHM), which switches the heads off.

Structure and strength of human cardiac myosin S1-S2 interaction

The super-relaxed (SRX) state was recently discovered in cardiac muscle fibers as an OFF state of myosin where the myosin S1 heads have reduced ATPase activity. The SRX state is thought to be a regulatory state of myosin heads that modulates the availability of functionally accessible myosin heads. A hypothesis is that the protein-protein interactions which form the SRX state are related to the interacting-head motif (IHM) state of myosin in which the S1 heads are folded back onto their coiled-coil S2 tails due to head-to-head interactions.

Two Classes of Myosin Inhibitors, Para-nitroblebbistatin and Mavacamten, Stabilize β-Cardiac Myosin in Different Structural and Functional States

In addition to a conventional relaxed state, a fraction of myosins in the cardiac muscle exists in a low-energy consuming super-relaxed (SRX) state, which is kept as a reserve pool that may be engaged under sustained increased cardiac demand. The conventional relaxed and the super-relaxed states are widely assumed to correspond to a structure where myosin heads are in an open configuration, free to interact with actin, and a closed configuration, inhibiting binding to actin, respectively. Disruption of the myosin SRX population is an emerging model in different heart diseases, such as hypertrophic cardiomyopathy, which results in excessive muscle contraction, and stabilizing them using myosin inhibitors is budding as an attractive therapeutic strategy. Here we examined the structure–function relationships of two myosin ATPase inhibitors, mavacamten and para-nitroblebbistatin, and found that binding of mavacamten at a site different than para-nitroblebbistatin populates myosin into the SRX state. Para-nitroblebbistatin, binding to a distal pocket to the myosin lever arm near the nucleotide-binding site, does not affect the usual myosin SRX state but instead appears to render myosin into a new, perhaps much more inhibited, ‘ultra-relaxed’ state. X-ray scattering-based rigid body modeling shows that both mavacamten and para-nitroblebbistatin induce novel conformations in human β-cardiac heavy meromyosin that diverge significantly from the hypothetical open and closed states, and furthermore, mavacamten treatment causes greater compaction than para-nitroblebbistatin. Taken together, we conclude that mavacamten and para-nitroblebbistatin stabilize myosin in different structural states, and such states may give rise to different functional energy-sparing states.

Cardiomyopathic mutations in essential light chain reveal mechanisms regulating the super relaxed state of myosin.

In this study, we assessed the super relaxed (SRX) state of myosin and sarcomeric protein phosphorylation in two pathological models of cardiomyopathy and in a near-physiological model of cardiac hypertrophy. The cardiomyopathy models differ in disease progression and severity and express the hypertrophic (HCM-A57G) or restrictive (RCM-E143K) mutations in the human ventricular myosin essential light chain (ELC), which is encoded by the MYL3 gene. Their effects were compared with near-physiological heart remodeling, represented by the N-terminally truncated ELC (Δ43 ELC mice), and with nonmutated human ventricular WT-ELC mice. The HCM-A57G and RCM-E143K mutations had antagonistic effects on the ATP-dependent myosin energetic states, with HCM-A57G cross-bridges fostering the disordered relaxed (DRX) state and the RCM-E143K model favoring the energy-conserving SRX state. The HCM-A57G model promoted the switch from the SRX to DRX state and showed an ∼40% increase in myosin regulatory light chain (RLC) phosphorylation compared with the RLC of normal WT-ELC myocardium. On the contrary, the RCM-E143K-associated stabilization of the SRX state was accompanied by an approximately twofold lower level of myosin RLC phosphorylation compared with the RLC of WT-ELC. Upregulation of RLC phosphorylation was also observed in Δ43 versus WT-ELC hearts, and the Δ43 myosin favored the energy-saving SRX conformation. The two disease variants also differently affected the duration of force transients, with shorter (HCM-A57G) or longer (RCM-E143K) transients measured in electrically stimulated papillary muscles from these pathological models, while no changes were displayed by Δ43 fibers. We propose that the N terminus of ELC (N-ELC), which is missing in the hearts of Δ43 mice, works as an energetic switch promoting the SRX-to-DRX transition and contributing to the regulation of myosin RLC phosphorylation in full-length ELC mice by facilitating or sterically blocking RLC phosphorylation in HCM-A57G and RCM-E143K hearts, respectively.

Cardiac myosin super relaxation (SRX): a perspective on fundamental biology, human disease and therapeutics.

ABSTRACTThe fundamental basis of muscle contraction ‘the sliding filament model’ (Huxley and Niedergerke, 1954; Huxley and Hanson, 1954) and the ‘swinging, tilting crossbridge-sliding filament mechanism’ (Huxley, 1969; Huxley and Brown, 1967) nucleated a field of research that has unearthed the complex and fascinating role of myosin structure in the regulation of contraction. A recently discovered energy conserving state of myosin termed the super relaxed state (SRX) has been observed in filamentous myosins and is central to modulating force production and energy use within the sarcomere. Modulation of myosin function through SRX is a rapidly developing theme in therapeutic development for both cardiovascular disease and infectious disease. Some 70 years after the first discoveries concerning muscular function, modulation of myosin SRX may bring the first myosin targeted small molecule to the clinic, for treating hypertrophic cardiomyopathy (Olivotto et al., 2020). An often monogenic disease HCM afflicts 1 in 500 individuals, and can cause heart failure and sudden cardiac death. Even as we near therapeutic translation, there remain many questions about the governance of muscle function in human health and disease. With this review, we provide a broad overview of contemporary understanding of myosin SRX, and explore the complexities of targeting this myosin state in human disease.This article has an associated Future Leaders to Watch interview with the authors of the paper.

Future Leaders to Watch – Christopher Toepfer and Manuel Schmid

ABSTRACTFuture Leaders to Watch is a series of interviews with the first authors of a selection of papers published in Biology Open, helping early-career researchers promote themselves alongside their papers. Christopher Toepfer and Manuel Schmid are co-first authors on ‘Myosin super relaxation (SRX) a perspective on fundamental biology, human disease, and cardiac therapeutics’, published in BiO. Christopher is a Sir Henry Wellcome Postdoctoral Fellow and British Heart Foundation Centre of Research Excellence (CRE) Intermediate Transition Fellow in the department of Cardiovascular Medicine at the Radcliffe Department of Medicine, University of Oxford, UK. Manuel is a Research Assistant in the department of Cardiovascular Medicine at the Radcliffe Department of Medicine, University of Oxford, UK. They are investigating the mechanisms that control heart function and impact acquired and inherited cardiovascular conditions.

To lie or not to lie: Super-relaxing with myosins.

Since the discovery of muscle in the 19th century, myosins as molecular motors have been extensively studied. However, in the last decade, a new functional super-relaxed (SRX) state of myosin has been discovered, which has a 10-fold slower ATP turnover rate than the already-known non-actin-bound, disordered relaxed (DRX) state. These two states are in dynamic equilibrium under resting muscle conditions and are thought to be significant contributors to adaptive thermogenesis in skeletal muscle and can act as a reserve pool that may be recruited when there is a sustained demand for increased cardiac muscle power. This report provides an evolutionary perspective of how striated muscle contraction is regulated by modulating this myosin DRX↔SRX state equilibrium. We further discuss this equilibrium with respect to different physiological and pathophysiological perturbations, including insults causing hypertrophic cardiomyopathy, and small-molecule effectors that modulate muscle contractility in diseased pathology.

Synthetic thick filaments: A new avenue for better understanding the myosin super-relaxed state in healthy, diseased, and mavacamten-treated cardiac systems

A hallmark feature of myosin-II is that it can spontaneously self-assemble into bipolar synthetic thick filaments (STFs) in low-ionic-strength buffers, thereby serving as a reconstituted in vitro model for muscle thick filaments. Although these STFs have been extensively used for structural characterization, their functional evaluation has been limited. In this report, we show that myosins in STFs mirror the more electrostatic and cooperative interactions that underlie the energy-sparing super-relaxed (SRX) state, which are not seen using shorter myosin subfragments, heavy meromyosin (HMM) and myosin subfragment 1 (S1). Using these STFs, we show several pathophysiological insults in hypertrophic cardiomyopathy, including the R403Q myosin mutation, phosphorylation of myosin light chains, and an increased ADP:ATP ratio, destabilize the SRX population. Furthermore, WT myosin containing STFs, but not S1, HMM, or STFs-containing R403Q myosin, recapitulated the ADP-induced destabilization of the SRX state. Studies involving a clinical-stage small-molecule inhibitor, mavacamten, showed that it is more effective in not only increasing myosin SRX population in STFs than in S1 or HMM but also in increasing myosin SRX population equally well in STFs made of healthy and disease-causing R403Q myosin. Importantly, we also found that pathophysiological perturbations such as elevated ADP concentration weakens mavacamten’s ability to increase the myosin SRX population, suggesting that mavacamten-bound myosin heads are not permanently protected in the SRX state but can be recruited into action. These findings collectively emphasize that STFs serve as a valuable tool to provide novel insights into the myosin SRX state in healthy, diseased, and therapeutic conditions.

Ablation of the N terminus of cardiac essential light chain promotes the super-relaxed state of myosin and counteracts hypercontractility in hypertrophic cardiomyopathy mutant mice.

In this study, we focus on the molecular mechanisms associated with the A57G (Ala57-to-Gly57) mutation in myosin essential light chains (ELCs), found to cause hypertrophic cardiomyopathy (HCM) in humans and in mice. Specifically, we studied the effects of A57G on the super-relaxed (SRX) state of myosin that may contribute to the hypercontractile cross-bridge behavior and ultimately lead to pathological cardiac remodeling in transgenic Tg-A57G mice. The disease model was compared to Tg-WT mice, expressing the wild-type human ventricular ELC, and analyzed against Tg-Δ43 mice, expressing the N-terminally truncated ELC, whose hearts hypertrophy with time but do not show any abnormalities in cardiac morphology or function. Our data suggest a new role for the N terminus of cardiac ELC (N-ELC) in modulation of myosin cross-bridge function in the healthy as well as in HCM myocardium. The lack of N-ELC in Tg-Δ43 mice was found to significantly stabilize the SRX state of myosin and increase the number of myosin heads occupying a low-energy state. In agreement, Δ43 hearts showed significantly decreased ATP utilization and low actin-activated myosin ATPase compared with A57G and WT hearts. The hypercontractile activity of A57G-ELC cross-bridges was manifested by the inhibition of the SRX state, increased number of myosin heads available for interaction with actin, and higher ATPase activity. Fiber mechanics studies, echocardiography examination, and assessment of fibrosis confirmed the development of two distinct forms of cardiac remodeling in these two ELC mouse models, with pathological cardiac hypertrophy in Tg-A57G, and near physiologic cardiac growth in Tg-Δ43 animals.

Observing the Myosin Super-Relaxed State (SRX) in Cardiac Thick Filaments

We aim to develop time-resolved fluorescence resonance energy transfer (FRET) sensors to detect the structural state of myosin in cardiac thick filaments. In relaxation, these filaments are hypothesized to isomerize between the disordered relaxed state (DRX), which has the potential to interact with actin and generate force in the sarcomere, and an inactive state termed super-relaxed (SRX), which has much lower ATPase activity than DRX, apparently due to inhibitory interactions between the two heads of myosin (Hooijman et al, 2011). It has been proposed that (a) regulation of the transition between these states underlies the Frank-Starling law of the heart, and (b) dysregulation of the transition (destabilization of SRX) can lead to a form of heart failure known as hypertrophic cardiomyopathy (HCM). The structural basis of the SRX remains incompletely understood, but it has been proposed that it corresponds to the interacting heads motif (IHM) that has been observed by electron microscopy. To investigate directly the structural transition between DRX and SRX, we have developed a time-resolved FRET assay that measures the intermolecular distance between regulatory light chains on the two heads of myosin. The TRFRET data is best fit to two populations of Gaussian-distributed distances, suggesting that this FRET pair observes both the shorter distance predicted for the IHM (SRX) state and the longer distance predicted for the DRX state. This assay provides a structure-based assay for quantitating these states in functional muscle fibers, and it has the potential to create a high-throughput, high-precision assay for therapeutic development.

Deletion of the N-Terminus of Myosin Essential Light Chain (N-ELC) in the Background of HCM-A57G Mutation in Double Mutant Mice Rescues Hypercontractile Myosin Phenotype

Cardiac pathologic remodeling including fibrosis, diastolic dysfunction and increased Ca2+ sensitivity of force has been observed in mice expressing the A57G mutation in myosin ventricular ELC (MYL3 gene) with clinical phenotype of hypertrophic cardiomyopathy (HCM). Our previous investigation showed that the lack of the unique 43 amino acid-long N-terminus ELC (N-ELC) in >7-month-old Δ43 mice results in non-pathological cardiac hypertrophy. Papillary muscles (PM) from ∼8 mo-old Δ43 hearts were subjected to mantATP-chase assay and compared to those from age-matched WT-ELC and A57G mice. The data showed that Δ43 fibers favor the super-relaxed (SRX) state of myosin with increased population of slowly cycling heads compared to PM from age-matched WT-ELC mice. The opposite was true for A57G mice whose PM demonstrated a pathologic hypercontractile behavior with significantly inhibited SRX. We then tested the effect of N-ELC deletion in A57G mice on the SRX in offspring resulting from breeding Δ43 with A57G mice (Δ43xA57G) and expressing 0-22% of Δ43 and 0-84% of A57G. Single nucleotide turnover assays on PM from all Δ43xA57G animals showed stabilization of the SRX state that was reminiscent of Δ43 alone, and different from all other groups of mice. Interestingly, echocardiographic evaluation of ∼8-month-old Δ43xA57G mice revealed a decrease in cardiac hypertrophy (decrease in LV mass) compared to A57G and Δ43 animals. Myocardial performance index was also improved in Δ43xA57G crosses compared to HCM-A57G mice, and not different compared to WT or Δ43 mice. Our studies suggest that the deletion of N-ELC in the background of HCM-ELC mutation may be beneficial in reversing some of the pathological myosin motor phenotypes associated with HCM. Supported by NIH R01HL123255 and R56HL146133 (DSC).

Decoupling the Interacting Head Motif and the Super Relaxed State of Myosin in Reconstituted Cardiac Bipolar Thick Filaments

Like many other classes of myosins, vertebrate cardiac myosin is suggested to exist in possibly two different structural states: an open head configuration in which myosin heads are readily accessible to bind and interact with actin and a closed head configuration where the heads are sequestered by the proximal part of the myosin tail, traditionally known as the Interacting Head Motif (IHM). At the biochemical level, this sequestered state is loosely correlated to the Super Relaxed (SRX) state of myosin, which has ∼10-fold prolonged ATP turnover rate (∼ 0.003 s−1) as compared to that of the open head configuration.

Factors that Modulate the Stability of the Super Relaxed State of Myosin in Skeletal Muscle Fibers

The Super Relaxed State of myosin (SRX) has an ATPase activity that is 10 times slower than the Disordered Relaxed State (DRX). Factors that destabilize the SRX could be effective in treating obesity and type 2 diabetes. We have explored two such possible factors, binding of calcium and addition of covalently attached glucosamine. The regulatory light chain of myosin contains a site for binding divalent cations, either magnesium or calcium. We found that addition of calcium increased the basal myosin ATPase activity by 60% with a half maximum at pCa = 6.1 ± 0.1 and a Hill coefficient of 1.9 ± 0.6. We conclude that during periods of heavy activation magnesium is replaced by calcium, which destabilizes the SRX. Another control system that is present in all eukaryotic cells involves the reaction of glucosamine (GlcNac) with serines and threonines. Both myosin heavy chain and RLC have been shown to be labeled with GlcNac in skeletal muscle. We found that addition of free GlcNac to rabbit skeletal fibers inhibits the population of the SRX by 29 ± 8%. This inhibition presumably occurs because the free GlcNac competes off endogenous GlcNac thus reversing its effect. This would suggest that attachment of GlcNac to proteins in the fiber stabilizes the SRX. Attempts to add more GlcNac moieties to fibers by incubation in the transferase involved, along with its substrates, had no effect on the population of the SRX but increased the lifetime by 60 ± 8%, again suggesting greater stability of the SRX when GlcNac is attached. These very preliminary results suggest GlcNac increases the stability of the SRX. We suggest that such a system modulates the SRX in skeletal muscle.

The Skeletal Muscle Super Relaxed State (SRX) is Localized to the C-Zone

Under relaxing (Low/No Calcium) conditions, ATP hydrolysis by myosin in striated muscle shows two distinct subpopulations: a faster “Disordered Relaxed” (DRX) population, and a strongly inhibited “Super Relaxed” (SRX) population. This SRX state contributes to the low metabolic rate of resting muscle and SRX dysregulation may underlie some myopathies. Presently, the spatial arrangement of these two hydrolytic states at the sarcomere level is unclear, although it has been reported that Myosin Binding Protein C (MyBP-C) may stabilize the SRX state, which implies possible localization to the C-zone where MyBP-C exists. Here, we imaged single molecules of fluorescently labeled ATP being hydrolyzed by myosin in relaxed myofibrils from rat soleus skeletal muscle. First, we showed that BODIPY-ATP supports actomyosin activity comparable to non-fluorescent ATP in an in vitro motility assay. Then, we estimated the myosin BODIPY-ATP hydrolysis rate by measuring the lifetimes of individual BODIPY-ATP molecules binding to myofibrils followed presumably by the release of BODIPY-ADP. Lifetimes were well fit as two populations with lifetimes of 33.5±4.1s and 181±42s; consistent with reported kinetic rates for DRX and SRX myosin in skeletal muscle. Each individual ATP binding event was then mapped to its distance from the center of the sarcomere (M-line), as imaged by fluorescent antibody labeling of myomesin. Interestingly, ATP hydrolysis lifetimes within the C-zone (200 - 500nm from the M-line) were significantly longer than those of the flanking non-MyBP-C containing regions. Within the C-zone, hydrolysis lifetimes were well fit as two equal sized populations, with lifetimes of 34.4±14.2s and 178±54s, while in the non-MyBP-C containing regions, 95% of the events were well fit as a single population with a lifetime of 34.6±5.2s. Our data suggest that the strongly inhibited SRX myosins exist predominantly within the C-zone.