Myofilaments and their associated proteins, which together constitute the sarcomeres, provide the molecular-level basis for contractile function in all muscle types. In intact muscle, sarcomere-level contraction is strongly coupled to other cellular subsystems, in particular the sarcolemmal membrane. Skinned muscle preparations (where the sarcolemma has been removed or permeabilized) are an experimental system designed to probe contractile mechanisms independently of the sarcolemma. Over the last few decades, experiments performed using permeabilized preparations have been invaluable for clarifying the understanding of contractile mechanisms in both skeletal and cardiac muscle. Today, the technique is increasingly harnessed for preclinical and/or pharmacological studies that seek to understand how interventions will impact intact muscle contraction. In this context, intrinsic functional and structural differences between skinned and intact muscle pose a major interpretational challenge. This review first surveys measurements that highlight these differences in terms of the sarcomere structure, passive and active tension generation, and calcium dependence. We then highlight the main practical challenges and caveats faced by experimentalists seeking to emulate the physiological conditions of intact muscle. Gaining an awareness of these complexities is essential for putting experiments in due perspective.

Super-relaxation 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 (relaxed) state, is ubiquitous and 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, which switches the heads off. However, recent studies show that even isolated myosin heads can exhibit this slow rate. Here, we review the role of head interactions in creating the super-relaxed state and show how increased numbers of interactions in thick filaments underlie the high levels of super-relaxation found in intact muscle. We suggest how a third, even more inhibited, state of myosin (a hyper-relaxed state) seen in certain species results from additional interactions involving the heads. We speculate on the relationship between animal lifestyle and level of super-relaxation in different species and on the mechanism of formation of the super-relaxed state. We also review how super-relaxed thick filaments are activated and how the super-relaxed state is modulated in healthy and diseased muscles.

Denervation leads to muscle atrophy, which is described as muscle mass and force loss, the latter exceeding expectation from mass loss. The objective of this study was to determine the efficiency of angiotensin (Ang) 1–7 at reducing muscle atrophy in mouse extensor digitorum longus (EDL) and soleus following 14- and 28-d denervation periods. Some denervated mice were treated with Ang 1–7 or diminazene aceturate (DIZE), an ACE2 activator, to increase Ang 1–7 levels. Ang 1–7/DIZE treatment had little effect on muscle mass loss and fiber cross-sectional area reduction. Ang 1–7 and DIZE fully prevented the loss of tetanic force normalized to cross-sectional area and accentuated the increase in twitch force in denervated muscle. However, they did not prevent the shift of the force–frequency relationship toward lower stimulation frequencies. The Ang 1–7/DIZE effects on twitch and tetanic force were completely blocked by A779, a MasR antagonist, and were not observed in MasR^−/− muscles. Ang 1–7 reduced the extent of membrane depolarization, fully prevented the loss of membrane excitability, and maintained the action potential overshoot in denervated muscles. Ang 1–7 had no effect on the changes in α-actin, myosin, or MuRF-1, atrogin-1 protein content or the content of total or phosphorylated Akt, S6, and 4EPB. This is the first study that provides evidence that Ang 1–7 maintains normal muscle function in terms of maximum force and membrane excitability during 14- and 28-d periods after denervation.

The myosin motors in resting skeletal muscle are folded back against their tails in the thick filament in a conformation that makes them unavailable for binding to actin. When muscles are activated, calcium binding to troponin leads to a rapid change in the structure of the actin-containing thin filaments that uncovers the myosin binding sites on actin. Almost as quickly, myosin motors leave the folded state and move away from the surface of the thick filament. To test whether motor unfolding is triggered by the availability of nearby actin binding sites, we measured changes in the x-ray reflections that report motor conformation when muscles are activated at longer sarcomere length, so that part of the thick filaments no longer overlaps with thin filaments. We found that the intensity of the M3 reflection from the axial repeat of the motors along the thick filaments declines almost linearly with increasing sarcomere length up to 2.8 µm, as expected if motors in the nonoverlap zone had left the folded state and become relatively disordered. In a recent article in JGP, Squire and Knupp challenged this interpretation of the data. We show here that their analysis is based on an incorrect assumption about how the interference subpeaks of the M3 reflection were reported in our previous paper. We extend previous models of mass distribution along the filaments to show that the sarcomere length dependence of the M3 reflection is consistent with <10% of no-overlap motors remaining in the folded conformation during active contraction, confirming our previous conclusion that unfolding of myosin motors on muscle activation is not due to the availability of local actin binding sites.

Sarcomeric contraction in cardiomyocytes serves as the basis for the heart’s pump functions. It has generally been considered that in cardiac muscle as well as in skeletal muscle, sarcomeres equally contribute to myofibrillar dynamics in myocytes at varying loads by producing similar levels of active and passive force. In the present study, we expressed α-actinin–AcGFP in Z-disks to analyze dynamic behaviors of sequentially connected individual sarcomeres along a myofibril in a left ventricular (LV) myocyte of the in vivo beating mouse heart. To quantify the magnitude of the contribution of individual sarcomeres to myofibrillar dynamics, we introduced the novel parameter “contribution index” (CI) to measure the synchrony in movements between a sarcomere and a myofibril (from −1 [complete asynchrony] to 1 [complete synchrony]). First, CI varied markedly between sarcomeres, with an average value of ∼0.3 during normal systole. Second, when the movements between adjacent sarcomeres were asynchronous (CI < 0), a sarcomere and the ones next to the adjacent sarcomeres and farther away moved in synchrony (CI > 0) along a myofibril. Third, when difference in LV pressure in diastole and systole (ΔLVP) was lowered to <10 mm Hg, diastolic sarcomere length increased. Under depressed conditions, the movements between adjacent sarcomeres were in marked asynchrony (CI, −0.3 to −0.4), and, as a result, average CI was linearly decreased in association with a decrease in ΔLVP. These findings suggest that in the left ventricle of the in vivo beating mouse heart, (1) sarcomeres heterogeneously contribute to myofibrillar dynamics due to an imbalance of active and passive force between neighboring sarcomeres, (2) the force imbalance is pronounced under depressed conditions coupled with a marked increase in passive force and the ensuing tug-of-war between sarcomeres, and (3) sarcomere synchrony via the distal intersarcomere interaction regulates the heart's pump function in coordination with myofibrillar contractility.

X-ray diffraction studies of muscle have provided a wealth of information on muscle structure and physiology, and the meridian of the diffraction pattern is particularly informative. Reconditi et al. (2014. J. Physiol. https://doi.org/10.1113/jphysiol.2013.267849 ) performed superb experiments on changes to the M3 meridional peak as a function of sarcomere length (SL). They found that the M3 intensity dropped almost linearly as sarcomere length increased at least to about SL = 3.0 µm, and that it followed the same track as tension, pointing toward zero at the end of overlap at ∼3.6 µm. They concluded that, just as tension could only be generated by overlapped myosin heads, so ordered myosin heads contributing to the M3 intensity could only occur in the overlap region of the A-band, and that nonoverlapped heads must be highly disordered. Here we show that this conclusion is not consistent with x-ray diffraction theory; it would not explain their observations. We discuss one possible reason for the change in M3 intensity with increasing sarcomere length in terms of increasing axial misalignment of the myosin filaments that at longer sarcomere lengths is limited by the elastic stretching of the M-band and titin.

Hypertrophic cardiomyopathy (HCM) is an inherited disorder caused primarily by mutations to thick and thinfilament proteins. Although thin filament mutations are less prevalent than their oft-studied thick filament counterparts, they are frequently associated with severe patient phenotypes and can offer important insight into fundamental disease mechanisms. We have performed a detailed study of tropomyosin (TPM1) E192K, a variant of uncertain significance associated with HCM. Molecular dynamics revealed that E192K results in a more flexible TPM1 molecule, which could affect its ability to regulate crossbridges. In vitro motility assays of regulated actin filaments containing TPM1 E192K showed an overall loss of Ca²⁺ sensitivity. To understand these effects, we used multiscale computational models that suggested a subtle phenotype in which E192K leads to an inability to completely inhibit actin–myosin crossbridge activity at low Ca²⁺. To assess the physiological impact of the mutation, we generated patient-derived engineered heart tissues expressing E192K. These tissues showed disease features similar to those of the patients, including cellular hypertrophy, hypercontractility, and diastolic dysfunction. We hypothesized that excess residual crossbridge activity could be triggering cellular hypertrophy, even if the overall Ca²⁺ sensitivity was reduced by E192K. To test this hypothesis, the cardiac myosin–specific inhibitor mavacamten was applied to patient-derived engineered heart tissues for 4 d followed by 24 h of washout. Chronic mavacamten treatment abolished contractile differences between control and TPM1 E192K engineered heart tissues and reversed hypertrophy in cardiomyocytes. These results suggest that the TPM1 E192K mutation triggers cardiomyocyte hypertrophy by permitting excess residual crossbridge activity. These studies also provide direct evidence that myosin inhibition by mavacamten can counteract the hypertrophic effects of mutant tropomyosin.

Muscle ankyrin repeat protein 1 (MARP1) is frequently up-regulated in stressed muscle, but its effect on skeletal muscle function is poorly understood. Here, we focused on its interaction with the titin–N2A element, found in titin’s molecular spring region. We show that MARP1 binds to F-actin, and that this interaction is stronger when MARP1 forms a complex with titin–N2A. Mechanics and super-resolution microscopy revealed that MARP1 “locks” titin–N2A to the sarcomeric thin filament, causing increased extension of titin’s elastic PEVK element and, importantly, increased passive force. In support of this mechanism, removal of thin filaments abolished the effect of MARP1 on passive force. The clinical relevance of this mechanism was established in diaphragm myofibers of mechanically ventilated rats and of critically ill patients. Thus, MARP1 regulates passive force by locking titin to the thin filament. We propose that in stressed muscle, this mechanism protects the sarcomere from mechanical damage.

The actin-, myosin-, and calmodulin-binding protein caldesmon (CaD) is expressed in two splice isoforms: h-CaD, which is an integral part of the actomyosin domain of smooth muscle cells, and l-CaD, which is widely expressed and is involved in many cellular functions. Despite extensive research for many years, CaD's in vivo function has remained elusive. To explore the role of CaD in smooth muscle contraction in vivo, we generated a mutant allele that ablates both isoforms. Heterozygous animals were viable and had a normal life span, but homozygous mutants died perinatally, likely because of a persistent umbilical hernia. The herniation was associated with hypoplastic and dysmorphic abdominal wall muscles. We assessed mechanical parameters in isometrically mounted longitudinal strips of E18.5 urinary bladders and in ring preparations from abdominal aorta using wire myography. Ca²⁺ sensitivity was higher and relaxation rate was slower in Cald1^−/− compared with Cald1^+/+ skinned bladder strips. However, we observed no change in the content and phosphorylation of regulatory proteins of the contractile apparatus and myosin isoforms known to affect these contractile parameters. Intact fibers showed no difference in actin and myosin content, regardless of genotype, although KCl-induced force tended to be lower in homozygous and higher in heterozygous mutants than in WTs. Conversely, in skinned fibers, myosin content and maximal force were significantly lower in Cald1^−/− than in WTs. In KO abdominal aortas, resting and U46619 elicited force were lower than in WTs. Our results are consistent with the notion that CaD impacts smooth muscle function dually by (1) acting as a molecular brake on contraction and (2) maintaining the structural integrity of the contractile machinery. Most importantly, CaD is essential for resolution of the physiological umbilical hernia and ventral body wall closure.

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.

Mavacamten (MYK-461) is a small-molecule allosteric inhibitor of sarcomeric myosins being used in preclinical/clinical trials for hypertrophic cardiomyopathy treatment. A better understanding of its impact on force generation in intact or skinned striated muscle preparations, especially for human cardiac muscle, has been hindered by diffusional barriers. These limitations have been overcome by mechanical experiments using myofibrils subject to perturbations of the contractile environment by sudden solution changes. Here, we characterize the action of mavacamten in human ventricular myofibrils compared with fast skeletal myofibrils from rabbit psoas. Mavacamten had a fast, fully reversible, and dose-dependent negative effect on maximal Ca²⁺-activated isometric force at 15°C, which can be explained by a sudden decrease in the number of heads functionally available for interaction with actin. It also decreased the kinetics of force development in fast skeletal myofibrils, while it had no effect in human ventricular myofibrils. For both myofibril types, the effects of mavacamten were independent from phosphate in the low-concentration range. Mavacamten did not alter force relaxation of fast skeletal myofibrils, but it significantly accelerated the relaxation of human ventricular myofibrils. Lastly, mavacamten had no effect on resting tension but inhibited the ADP-stimulated force in the absence of Ca²⁺. Altogether, these effects outline a motor isoform–specific dependence of the inhibitory effect of mavacamten on force generation, which is mediated by a reduction in the availability of strongly actin-binding heads. Mavacamten may thus alter the interplay between thick and thin filament regulation mechanisms of contraction in association with the widely documented drug effect of stabilizing myosin motor heads into autoinhibited states.

Mutations in the cardiac myosin regulatory light chain (RLC, MYL2 gene) are known to cause inherited cardiomyopathies with variable phenotypes. In this study, we investigated the impact of a mutation in the RLC (K104E) that is associated with hypertrophic cardiomyopathy (HCM). Previously in a mouse model of K104E, older animals were found to develop cardiac hypertrophy, fibrosis, and diastolic dysfunction, suggesting a slow development of HCM. However, variable penetrance of the mutation in human populations suggests that the impact of K104E may be subtle. Therefore, we generated human cardiac myosin subfragment-1 (M2β-S1) and exchanged on either the wild type (WT) or K104E human ventricular RLC in order to assess the impact of the mutation on the mechanochemical properties of cardiac myosin. The maximum actin-activated ATPase activity and actin sliding velocities in the in vitro motility assay were similar in M2β-S1 WT and K104E, as were the detachment kinetic parameters, including the rate of ATP-induced dissociation and the ADP release rate constant. We also examined the mechanical performance of α-cardiac myosin extracted from transgenic (Tg) mice expressing human wild type RLC (Tg WT) or mutant RLC (Tg K104E). We found that α-cardiac myosin from Tg K104E animals demonstrated enhanced actin sliding velocities in the motility assay compared with its Tg WT counterpart. Furthermore, the degree of incorporation of the mutant RLC into α-cardiac myosin in the transgenic animals was significantly reduced compared with wild type. Therefore, we conclude that the impact of the K104E mutation depends on either the length or the isoform of the myosin heavy chain backbone and that the mutation may disrupt RLC interactions with the myosin lever arm domain.

Familial hypertrophic cardiomyopathy (HCM), a leading cause of sudden cardiac death, is primarily caused by mutations in sarcomeric proteins. The pathogenesis of HCM is complex, with functional changes that span scales, from molecules to tissues. This makes it challenging to deconvolve the biophysical molecular defect that drives the disease pathogenesis from downstream changes in cellular function. In this study, we examine an HCM mutation in troponin T, R92Q, for which several models explaining its effects in disease have been put forward. We demonstrate that the primary molecular insult driving disease pathogenesis is mutation-induced alterations in tropomyosin positioning, which causes increased molecular and cellular force generation during calcium-based activation. Computational modeling shows that the increased cellular force is consistent with the molecular mechanism. These changes in cellular contractility cause downstream alterations in gene expression, calcium handling, and electrophysiology. Taken together, our results demonstrate that molecularly driven changes in mechanical tension drive the early disease pathogenesis of familial HCM, leading to activation of adaptive mechanobiological signaling pathways.

The two main phases of the mammalian cardiac cycle are contraction and relaxation; however, whether there is a connection between them in humans is not well understood. Routine exercise has been shown to improve cardiac function, morphology, and molecular signatures. Likewise, the acute and chronic changes that occur in the heart in response to injury, disease, and stress are well characterized, albeit not fully understood. In this study, we investigated how exercise and myocardial injury affect contraction–relaxation coupling. We retrospectively analyzed the correlation between the maximal speed of contraction and the maximal speed of relaxation of canine myocardium after receiving surgically induced myocardial infarction, followed by either sedentary recovery or exercise training for 10–12 wk. We used isolated right ventricular trabeculae, which were electrically paced at different lengths, frequencies, and with increasing β-adrenoceptor stimulation. In all conditions, contraction and relaxation were linearly correlated, irrespective of injury or training history. Based on these results and the available literature, we posit that contraction–relaxation coupling is a fundamental myocardial property that resides in the structural arrangement of proteins at the level of the sarcomere and that this may be regulated by the actions of cardiac myosin binding protein C (cMyBP-C) on actin and myosin.

Our review focuses on sarcomere regulatory mechanisms with a discussion of cardiac-specific modifications to the three-state model of thin filament activation from a blocked to closed to open state. We discuss modulation of these thin filament transitions by Ca²⁺, by crossbridge interactions, and by thick filament–associated proteins, cardiac myosin–binding protein C (cMyBP-C), cardiac regulatory light chain (cRLC), and titin. Emerging evidence supports the idea that the cooperative activation of the thin filaments despite a single Ca²⁺ triggering regulatory site on troponin C (cTnC) cannot be considered in isolation of other functional domains of the sarcomere. We discuss long- and short-range interactions among these domains with the regulatory units of thin filaments, including proteins at the barbed end at the Z-disc and the pointed end near the M-band. Important to these discussions is the ever-increasing understanding of the role of cMyBP-C, cRLC, and titin filaments. Detailed knowledge of these control processes is critical to the understanding of mechanisms sustaining physiological cardiac state with varying hemodynamic load, to better defining genetic and acquired cardiac disorders, and to developing targets for therapies at the level of the sarcomeres.

Microtubules tune cytoskeletal stiffness, which affects cytoskeletal mechanics and mechanotransduction of striated muscle. While recent evidence suggests that microtubules enriched in detyrosinated α-tubulin regulate these processes in healthy muscle and increase them in disease, the possible contribution from several other α-tubulin modifications has not been investigated. Here, we used genetic and pharmacologic strategies in isolated cardiomyocytes and skeletal myofibers to increase the level of acetylated α-tubulin without altering the level of detyrosinated α-tubulin. We show that microtubules enriched in acetylated α-tubulin increase cytoskeletal stiffness and viscoelastic resistance. These changes slow rates of contraction and relaxation during unloaded contraction and increased activation of NADPH oxidase 2 (Nox2) by mechanotransduction. Together, these findings add to growing evidence that microtubules contribute to the mechanobiology of striated muscle in health and disease.

The Frank–Starling relationship establishes that elevated end-diastolic volume progressively increases ventricular pressure and stroke volume in healthy hearts. The relationship is modulated by a number of physiological inputs and is often depressed in human heart failure. Emerging evidence suggests that cardiac myosin-binding protein-C (cMyBP-C) contributes to the Frank–Starling relationship. We measured contractile properties at multiple levels of structural organization to determine the role of cMyBP-C and its phosphorylation in regulating (1) the sarcomere length dependence of power in cardiac myofilaments and (2) the Frank–Starling relationship in vivo. We compared transgenic mice expressing wild-type cMyBP-C on the null background, which have ∼50% phosphorylated cMyBP-C (Controls), to transgenic mice lacking cMyBP-C (KO) and to mice expressing cMyBP-C that have serine-273, -282, and -302 mutated to aspartate (cMyBP-C t3SD) or alanine (cMyBP-C t3SA) on the null background to mimic either constitutive PKA phosphorylation or nonphosphorylated cMyBP-C, respectively. We observed a continuum of length dependence of power output in myocyte preparations. Sarcomere length dependence of power progressively increased with a rank ordering of cMyBP-C KO = cMyBP-C t3SA < Control < cMyBP-C t3SD. Length dependence of myofilament power translated, at least in part, to hearts, whereby Frank–Starling relationships were steepest in cMyBP-C t3SD mice. The results support the hypothesis that cMyBP-C and its phosphorylation state tune sarcomere length dependence of myofibrillar power, and these regulatory processes translate across spatial levels of myocardial organization to control beat-to-beat ventricular performance.

Binding properties of actin-binding proteins are typically evaluated by cosedimentation assays. However, this method is time-consuming, involves multiple steps, and has a limited throughput. These shortcomings preclude its use in screening for drugs that modulate actin-binding proteins relevant to human disease. To develop a simple, quantitative, and scalable F-actin–binding assay, we attached fluorescent probes to actin's Cys-374 and assessed changes in fluorescence lifetime upon binding to the N-terminal region (domains C0–C2) of human cardiac myosin-binding protein C (cMyBP-C). The lifetime of all five probes tested decreased upon incubation with cMyBP-C C0–C2, as measured by time-resolved fluorescence (TR-F), with IAEDANS being the most sensitive probe that yielded the smallest errors. The TR-F assay was compared with cosedimentation to evaluate in vitro changes in binding to actin and actin–tropomyosin arising from cMyBP-C mutations associated with hypertrophic cardiomyopathy (HCM) and tropomyosin binding. Lifetime changes of labeled actin with added C0–C2 were consistent with cosedimentation results. The HCM mutation L352P was confirmed to enhance actin binding, whereas PKA phosphorylation reduced binding. The HCM mutation R282W, predicted to disrupt a PKA recognition sequence, led to deficits in C0–C2 phosphorylation and altered binding. Lastly, C0–C2 binding was found to be enhanced by tropomyosin and binding capacity to be altered by mutations in a tropomyosin-binding region. These findings suggest that the TR-F assay is suitable for rapidly and accurately determining quantitative binding and for screening physiological conditions and compounds that affect cMyBP-C binding to F-actin for therapeutic discovery.

In myocardium, phosphorylation of cardiac myosin-binding protein-C (cMyBP-C) is thought to modulate the cooperative activation of the thin filament by binding to myosin and/or actin, thereby regulating the probability of cross-bridge binding to actin. At low levels of Ca²⁺ activation, unloaded shortening velocity (V_o) in permeabilized cardiac muscle is comprised of an initial high-velocity phase and a subsequent low-velocity phase. The velocities in these phases scale with the level of activation, culminating in a single high-velocity phase (V_max) at saturating Ca²⁺. To test the idea that cMyBP-C phosphorylation contributes to the activation dependence of V_o, we measured V_o before and following treatment with protein kinase A (PKA) in skinned trabecula isolated from mice expressing either wild-type cMyBP-C (tWT), nonphosphorylatable cMyBP-C (t3SA), or phosphomimetic cMyBP-C (t3SD). During maximal Ca²⁺ activation, V_max was monophasic and not significantly different between the three groups. Although biphasic shortening was observed in all three groups at half-maximal activation under control conditions, the high- and low-velocity phases were faster in the t3SD myocardium compared with values obtained in either tWT or t3SA myocardium. Treatment with PKA significantly accelerated both the high- and low-velocity phases in tWT myocardium but had no effect on V_o in either the t3SD or t3SA myocardium. These results can be explained in terms of a model in which the level of cMyBP-C phosphorylation modulates the extent and rate of cooperative spread of myosin binding to actin.

Myosin and actin filaments are highly organized within muscle sarcomeres. Myosin-binding protein C (MyBP-C) is a flexible, rod-like protein located within the C-zone of the sarcomere. The C-terminal domain of MyBP-C is tethered to the myosin filament backbone, and the N-terminal domains are postulated to interact with actin and/or the myosin head to modulate filament sliding. To define where the N-terminal domains of MyBP-C are localized in the sarcomere of active and relaxed mouse myocardium, the relative positions of the N terminus of MyBP-C and actin were imaged in fixed muscle samples using super-resolution fluorescence microscopy. The resolution of the imaging was enhanced by particle averaging. The images demonstrate that the position of the N terminus of MyBP-C is biased toward the actin filaments in both active and relaxed muscle preparations. Comparison of the experimental images with images generated in silico, accounting for known binding partner interactions, suggests that the N-terminal domains of MyBP-C may bind to actin and possibly the myosin head but only when the myosin head is in the proximity of an actin filament. These physiologically relevant images help define the molecular mechanism by which the N-terminal domains of MyBP-C may search for, and capture, molecular binding partners to tune cardiac contractility.