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

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

Drug discovery for heart failure targeting myosin-binding protein C.

Cardiac MyBP-C (cMyBP-C) interacts with actin and myosin to fine-tune cardiac muscle contractility. Phosphorylation of cMyBP-C, which reduces the binding of cMyBP-C to actin and myosin, is often decreased in patients with heart failure (HF) and is cardioprotective in model systems of HF. Therefore, cMyBP-C is a potential target for HF drugs that mimic its phosphorylation and/or perturb its interactions with actin or myosin. We labeled actin with fluorescein-5-maleimide (FMAL) and the C0-C2 fragment of cMyBP-C (cC0-C2) with tetramethylrhodamine (TMR). We performed two complementary high-throughput screens (HTS) on an FDA-approved drug library, to discover small molecules that specifically bind to cMyBP-C and affect its interactions with actin or myosin, using fluorescence lifetime (FLT) detection. We first excited FMAL and detected its FLT, to measure changes in fluorescence resonance energy transfer (FRET) from FMAL (donor) to TMR (acceptor), indicating binding. Using the same samples, we then excited TMR directly, using a longer wavelength laser, to detect the effects of compounds on the environmentally sensitive FLT of TMR, to identify compounds that bind directly to cC0-C2. Secondary assays, performed on selected modulators with the most promising effects in the primary HTS assays, characterized the specificity of these compounds for phosphorylated versus unphosphorylated cC0-C2 and for cC0-C2 versus C1-C2 of fast skeletal muscle (fC1-C2). A subset of identified compounds modulated ATPase activity in cardiac and/or skeletal myofibrils. These assays establish the feasibility of the discovery of small-molecule modulators of the cMyBP-C-actin/myosin interaction, with the ultimate goal of developing therapies for HF.

Human cardiac myosin-binding protein C phosphorylation- and mutation-dependent structural dynamics monitored by time-resolved FRET.

Cardiac myosin-binding protein C (cMyBP-C) is a thick filament-associated protein of the sarcomere and a potential therapeutic target for treating contractile dysfunction in heart failure. Mimicking the structural dynamics of phosphorylated cMyBP-C by small-molecule drug binding could lead to therapies that modulate cMyBP-C conformational states, and thereby function, to improve contractility. We have developed a human cMyBP-C biosensor capable of detecting intramolecular structural changes due to phosphorylation and mutation. Using site-directed mutagenesis and time-resolved fluorescence resonance energy transfer (TR-FRET), we substituted cysteines in cMyBP-C N-terminal domains C0 through C2 (C0-C2) for thiol-reactive fluorescent probe labeling to examine C0-C2 structure. We identified a cysteine pair that upon donor-acceptor labeling reports phosphorylation-sensitive structural changes between the C1 domain and the tri-helix bundle of the M-domain that links C1 to C2. Phosphorylation reduced FRET efficiency by ~18%, corresponding to a ~11% increase in the distance between probes and a ~30% increase in disorder between them. The magnitude and precision of phosphorylation-mediated TR-FRET changes, as quantified by the Z'-factor, demonstrate the assay's potential for structure-based high-throughput screening of compounds for cMyBP-C-targeted therapies to improve cardiac performance in heart failure. Additionally, by probing C1's spatial positioning relative to the tri-helix bundle, these findings provide new molecular insight into the structural dynamics of phosphoregulation as well as mutations in cMyBP-C. Biosensor sensitivity to disease-relevant mutations in C0-C2 was demonstrated by examination of the hypertrophic cardiomyopathy mutation R282W. The results presented here support a screening platform to identify small molecules that regulate N-terminal cMyBP-C conformational states.

Cardiac myosin-binding protein C interaction with actin is inhibited by compounds identified in a high-throughput fluorescence lifetime screen.

Cardiac myosin-binding protein C (cMyBP-C) interacts with actin and myosin to modulate cardiac muscle contractility. These interactions are disfavored by cMyBP-C phosphorylation. Heart failure patients often display decreased cMyBP-C phosphorylation, and phosphorylation in model systems has been shown to be cardioprotective against heart failure. Therefore, cMyBP-C is a potential target for heart failure drugs that mimic phosphorylation or perturb its interactions with actin/myosin. Here we have used a novel fluorescence lifetime-based assay to identify small-molecule inhibitors of actin-cMyBP-C binding. Actin was labeled with a fluorescent dye (Alexa Fluor 568, AF568) near its cMyBP-C binding sites; when combined with the cMyBP-C N-terminal fragment, C0-C2, the fluorescence lifetime of AF568-actin decreases. Using this reduction in lifetime as a readout of actin binding, a high-throughput screen of a 1280-compound library identified three reproducible hit compounds (suramin, NF023, and aurintricarboxylic acid) that reduced C0-C2 binding to actin in the micromolar range. Binding of phosphorylated C0-C2 was also blocked by these compounds. That they specifically block binding was confirmed by an actin-C0-C2 time-resolved FRET (TR-FRET) binding assay. Isothermal titration calorimetry (ITC) and transient phosphorescence anisotropy (TPA) confirmed that these compounds bind to cMyBP-C, but not to actin. TPA results were also consistent with these compounds inhibiting C0-C2 binding to actin. We conclude that the actin-cMyBP-C fluorescence lifetime assay permits detection of pharmacologically active compounds that affect cMyBP-C-actin binding. We now have, for the first time, a validated high-throughput screen focused on cMyBP-C, a regulator of cardiac muscle contractility and known key factor in heart failure.

Human cardiac myosin-binding protein C restricts actin structural dynamics in a cooperative and phosphorylation-sensitive manner.

Cardiac myosin-binding protein C (cMyBP-C) is a thick filament-associated protein that influences actin-myosin interactions. cMyBP-C alters myofilament structure and contractile properties in a protein kinase A (PKA) phosphorylation-dependent manner. To determine the effects of cMyBP-C and its phosphorylation on the microsecond rotational dynamics of actin filaments, we attached a phosphorescent probe to F-actin at Cys-374 and performed transient phosphorescence anisotropy (TPA) experiments. Binding of cMyBP-C N-terminal domains (C0-C2) to labeled F-actin reduced rotational flexibility by 20-25°, indicated by increased final anisotropy of the TPA decay. The effects of C0-C2 on actin TPA were highly cooperative (n = ∼8), suggesting that the cMyBP-C N terminus impacts the rotational dynamics of actin spanning seven monomers (i.e. the length of tropomyosin). PKA-mediated phosphorylation of C0-C2 eliminated the cooperative effects on actin flexibility and modestly increased actin rotational rates. Effects of Ser to Asp phosphomimetic substitutions in the M-domain of C0-C2 on actin dynamics only partially recapitulated the phosphorylation effects. C0-C1 (lacking M-domain/C2) similarly exhibited reduced cooperativity, but not as reduced as by phosphorylated C0-C2. These results suggest an important regulatory role of the M-domain in cMyBP-C effects on actin structural dynamics. In contrast, phosphomimetic substitution of the glycogen synthase kinase (GSK3ß) site in the Pro/Ala-rich linker of C0-C2 did not significantly affect the TPA results. We conclude that cMyBP-C binding and PKA-mediated phosphorylation can modulate actin dynamics. We propose that these N-terminal cMyBP-C-induced changes in actin dynamics help explain the functional effects of cMyBP-C phosphorylation on actin-myosin interactions.

Site-directed spectroscopy of cardiac myosin-binding protein C reveals effects of phosphorylation on protein structural dynamics.

We have used the site-directed spectroscopies of time-resolved fluorescence resonance energy transfer (TR-FRET) and double electron-electron resonance (DEER), combined with complementary molecular dynamics (MD) simulations, to resolve the structure and dynamics of cardiac myosin-binding protein C (cMyBP-C), focusing on the N-terminal region. The results have implications for the role of this protein in myocardial contraction, with particular relevance to ß-adrenergic signaling, heart failure, and hypertrophic cardiomyopathy. N-terminal cMyBP-C domains C0-C2 (C0C2) contain binding regions for potential interactions with both thick and thin filaments. Phosphorylation by PKA in the MyBP-C motif regulates these binding interactions. Our spectroscopic assays detect distances between pairs of site-directed probes on cMyBP-C. We engineered intramolecular pairs of labeling sites within cMyBP-C to measure, with high resolution, the distance and disorder in the protein's flexible regions using TR-FRET and DEER. Phosphorylation reduced the level of molecular disorder and the distribution of C0C2 intramolecular distances became more compact, with probes flanking either the motif between C1 and C2 or the Pro/Ala-rich linker (PAL) between C0 and C1. Further insight was obtained from microsecond MD simulations, which revealed a large structural change in the disordered motif region in which phosphorylation unmasks the surface of a series of residues on a stable α-helix within the motif with high potential as a protein-protein interaction site. These experimental and computational findings elucidate structural transitions in the flexible and dynamic portions of cMyBP-C, providing previously unidentified molecular insight into the modulatory role of this protein in cardiac muscle contractility.

N-terminal extension in cardiac myosin-binding protein C regulates myofilament binding.

RATIONALE: Mutations in the gene encoding the sarcomeric protein cardiac myosin-binding protein C (cMyBP-C) are a leading cause of hypertrophic cardiomyopathy (HCM). Mouse models targeting cMyBP-C and use of recombinant proteins have been effective in studying its roles in contractile function and disease. Surprisingly, while the N-terminus of cMyBP-C is important to regulate myofilament binding and contains many HCM mutations, an incorrect sequence, lacking the N-terminal 8 amino acids has been used in many studies. OBJECTIVES: To determine the N-terminal cMyBP-C sequences in ventricles and investigate the roles of species-specific differences in cMyBP-C on myofilament binding. METHODS AND RESULTS: We determined cMyBP-C sequences in mouse and human by inspecting available sequence databases. N-terminal differences were confirmed using liquid chromatography-tandem mass spectrometry (LC-MS/MS). Cosedimentation assays with actin or myosin were used to examine binding in mouse, human and chimeric fusion proteins of cMyBP-C. Time-resolved FRET (TR-FRET) with site-directed probes on cMyBP-C was employed to measure structural dynamics. LC-MS/MS supported the sequencing data that mouse cMyBP-C contains an eight-residue N-terminal extension (NTE) not found in human. Cosedimentation assays revealed that cardiac myosin binding was strongly influenced by the presence of the NTE, which reduced binding by 60%. 75% more human C0-C2 than mouse bound to myosin. Actin binding of mouse C0-C2 was not affected by the NTE. 50% more human C0-C2 than mouse bound to actin. TR-FRET indicates that the NTE did not significantly affect structural dynamics across domains C0 and C1. CONCLUSIONS: Our functional results are consistent with the idea that cardiac myosin binding of N-terminal cMyBP-C is reduced in the mouse protein due to the presence of the NTE, which is proposed to interfere with myosin regulatory light chain (RLC) binding. The NTE is a critical component of mouse cMyBP-C, and should be considered in extrapolation of studies to cMyBP-C and HCM mechanisms in human.

Dysregulated Nox4 ubiquitination contributes to redox imbalance and age-related severity of acute lung injury.

Acute respiratory distress syndrome (ARDS) is a devastating critical illness disproportionately affecting the elderly population, with both higher incidence and mortality. The integrity of the lung endothelial cell (EC) monolayer is critical for preservation of lung function. However, mechanisms mediating EC barrier regulation in the context of aging remain unclear. We assessed the severity of acute lung injury (ALI) in young (2 mo) and aged (18 mo) mice using a two-hit preclinical model. Compared with young cohorts, aged mice exhibited increased ALI severity, with greater vascular permeability characterized by elevated albumin influx and levels of bronchoalveolar lavage (BAL) cells (neutrophils) and protein. Aged/injured mice also demonstrated elevated levels of reactive oxygen species (ROS) in the BAL, which was associated with upregulation of the ROS-generating enzyme, Nox4. We evaluated the role of aging in human lung EC barrier regulation utilizing a cellular model of replicative senescence. Senescent EC populations were defined by increases in ß-galactosidase activity and p16 levels. In response to lipopolysaccharide (LPS) challenge, senescent ECs demonstrate exacerbated permeability responses compared with control "young" ECs. LPS challenge led to a rapid induction of Nox4 expression in both control and senescent ECs, which was posttranslationally mediated via the proteasome/ubiquitin system. However, senescent ECs demonstrated deficient Nox4 ubiquitination, resulting in sustained expression of Nox4 and alterations in cellular redox homeostasis. Pharmacological inhibition of Nox4 in senescent ECs reduced LPS-induced alterations in permeability. These studies provide insight into the roles of Nox4/senescence in EC barrier responses and offer a mechanistic link to the increased incidence and mortality of ARDS associated with aging.

Estradiol modulates myosin regulatory light chain phosphorylation and contractility in skeletal muscle of female mice.

Impairment of skeletal muscle function has been associated with changes in ovarian hormones, especially estradiol. To elucidate mechanisms of estradiol on skeletal muscle strength, the hormone's effects on phosphorylation of the myosin regulatory light chain (pRLC) and muscle contractility were investigated, hypothesizing an estradiol-specific beneficial impact. In a skeletal muscle cell line, C2C12, pRLC was increased by 17ß-estradiol (E2) in a concentration-dependent manner. In skeletal muscles of C57BL/6 mice that were E2 deficient via ovariectomy (OVX), pRLC was lower than that from ovary-intact, sham-operated mice (Sham). The reduced pRLC in OVX muscle was reversed by in vivo E2 treatment. Posttetanic potentiation (PTP) of muscle from OVX mice was low compared with that from Sham mice, and this decrement was reversed by acute E2 treatment, demonstrating physiological consequence. Western blot of those muscles revealed that low PTP corresponded with low pRLC and higher PTP with greater pRLC. We aimed to elucidate signaling pathways affecting E2-mediated pRLC using a kinase inhibitor library and C2C12 cells as well as a specific myosin light chain kinase inhibitor in muscles. PI3K/Akt, MAPK, and CamKII were identified as candidate kinases sensitive to E2 in terms of phosphorylating RLC. Applying siRNA strategy in C2C12 cells, pRLC triggered by E2 was found to be mediated by estrogen receptor-ß and the G protein-coupled estrogen receptor. Together, these results provide evidence that E2 modulates myosin pRLC in skeletal muscle and is one mechanism by which this hormone can affect muscle contractility in females.

The myosin super-relaxed state is disrupted by estradiol deficiency.

We have used quantitative epifluorescence microscopy of fluorescent ATP to measure single-nucleotide turnover in skinned skeletal muscle fibers from mouse models of female aging and hormone treatment. Aging causes declines in muscle strength, often leading to frailty, disability, and loss of independence for the elderly. Female muscle is additionally affected by age due to reduction of ovarian hormone production with menopause. Estradiol (E2) is the key hormonal signal to skeletal muscle in females, and strength loss is attenuated by E2 treatment. To investigate E2 mechanisms on skeletal muscle, single fibers were isolated from sham-operated or ovariectomized (OVX) mice, with or without E2 treatment, and were incubated with 2'-(or-3')-O-(N-methylanthraniloyl) adenosine 5'-triphosphate (mantATP). We measured decay of mantATP fluorescence in an ATP-chase experiment, as pioneered by Cooke and coworkers, who unveiled a novel regulated state of muscle myosin characterized by slow nucleotide turnover on the order of minutes, termed the super-relaxed state (SRX). We detected a slow phase of nucleotide turnover in a portion of the myosin heads from sham fibers, consistent with SRX. Turnover was substantially faster in OVX fibers, with a turnover time constant for the slow phase of 65 ± 8s as compared to 102 ± 7s for sham fibers. 60-days E2 treatment in OVX mice substantially reversed this effect on SRX, while acute exposure of isolated muscles from OVX mice to E2 had no effect. We conclude that E2-mediated signaling reversibly regulates slow ATP turnover by myosin. Age- and hormone-related muscle functional losses may be targetable at the level of myosin structure/function for strategies to offset weakness and metabolic changes that occur with age.

Cardiac myosin binding protein-C restricts intrafilament torsional dynamics of actin in a phosphorylation-dependent manner.

We have determined the effects of myosin binding protein-C (MyBP-C) and its domains on the microsecond rotational dynamics of actin, detected by time-resolved phosphorescence anisotropy (TPA). MyBP-C is a multidomain modulator of striated muscle contraction, interacting with myosin, titin, and possibly actin. Cardiac and slow skeletal MyBP-C are known substrates for protein kinase-A (PKA), and phosphorylation of the cardiac isoform alters contractile properties and myofilament structure. To determine the effects of MyBP-C on actin structural dynamics, we labeled actin at C374 with a phosphorescent dye and performed TPA experiments. The interaction of all three MyBP-C isoforms with actin increased the final anisotropy of the TPA decay, indicating restriction of the amplitude of actin torsional flexibility by 15-20° at saturation of the TPA effect. PKA phosphorylation of slow skeletal and cardiac MyBP-C relieved the restriction of torsional amplitude but also decreased the rate of torsional motion. In the case of fast skeletal MyBP-C, its effect on actin dynamics was unchanged by phosphorylation. The isolated C-terminal half of cardiac MyBP-C (C5-C10) had effects similar to those of the full-length protein, and it bound actin more tightly than the N-terminal half (C0-C4), which had smaller effects on actin dynamics that were independent of PKA phosphorylation. We propose that these MyBP-C-induced changes in actin dynamics play a role in the functional effects of MyBP-C on the actin-myosin interaction.

Fluorescence lifetime-based assay reports structural changes in cardiac muscle mediated by effectors of contractile regulation.

Cardiac muscle contraction is regulated by Ca2+-induced structural changes of the thin filaments to permit myosin cross-bridge cycling driven by ATP hydrolysis in the sarcomere. In congestive heart failure, contraction is weakened, and thus targeting the contractile proteins of the sarcomere is a promising approach to therapy. However, development of novel therapeutic interventions has been challenging due to a lack of precise discovery tools. We have developed a fluorescence lifetime-based assay using an existing site-directed probe, N,N'-dimethyl-N-(iodoacetyl)-N'-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)ethylenediamine (IANBD) attached to human cardiac troponin C (cTnC) mutant cTnCT53C, exchanged into porcine cardiac myofibrils. We hypothesized that IANBD-cTnCT53C fluorescence lifetime measurements provide insight into the activation state of the thin filament. The sensitivity and precision of detecting structural changes in cTnC due to physiological and therapeutic modulators of thick and thin filament functions were determined. The effects of Ca2+ binding to cTnC and myosin binding to the thin filament were readily detected by this assay in mock high-throughput screen tests using a fluorescence lifetime plate reader. We then evaluated known effectors of altered cTnC-Ca2+ binding, W7 and pimobendan, and myosin-binding drugs, mavacamten and omecamtiv mecarbil, used to treat cardiac diseases. Screening assays were determined to be of high quality as indicated by the Z' factor. We conclude that cTnC lifetime-based probes allow for precise evaluation of the thin filament activation in functioning myofibrils that can be used in future high-throughput screens of small-molecule modulators of function of the thin and thick filaments.

Drug discovery for heart failure targeting myosin-binding protein C.

Cardiac MyBP-C (cMyBP-C) interacts with actin-myosin to fine-tune cardiac muscle contractility. Phosphorylation of cMyBP-C, which reduces binding of cMyBP-C to actin or myosin, is often decreased in heart failure (HF) patients, and is cardioprotective in model systems for HF. Therefore, cMyBP-C is a potential target for HF drugs that mimic phosphorylation and/or perturb its interactions with actin or myosin. We labeled actin with fluorescein-5-maleimide (FMAL), and the C0-C2 fragment of cMyBP-C (cC0-C2) with tetramethyl rhodamine (TMR). We performed two complementary high-throughput screens (HTS) on an FDA-approved drug library, to discover small molecules that specifically bind to cMyBP-C and affect its interactions with actin or myosin, using fluorescence lifetime (FLT) detection. We first excited FMAL and detected its FLT, to measure changes in fluorescence resonance energy transfer (FRET) from FMAL (donor) to TMR (acceptor), indicating binding and/or structural changes in the protein complex. Using the same samples, we then excited TMR directly, using a longer wavelength laser, to detect the effects of compounds on the environmentally sensitive FLT of TMR, to identify compounds that bind directly to cC0-C2. Secondary assays, performed on selected modulators with the most promising effects in the primary HTS assays, characterized specificity of these compounds for phosphorylated versus unphosphorylated cC0-C2 and for cC0-C2 versus C1-C2 of fast skeletal muscle (fskC1-C2). A subset of identified compounds modulated ATPase activity in cardiac and/or skeletal myofibrils. These assays establish feasibility for discovery of small-molecule modulators of the cMyBP-C-actin/myosin interaction, with the ultimate goal of developing therapies for HF.

Myosin binding protein-C phosphorylation is the principal mediator of protein kinase A effects on thick filament structure in myocardium.

Phosphorylation of cardiac myosin binding protein-C (cMyBP-C) is a regulator of pump function in healthy hearts. However, the mechanisms of regulation by cAMP-dependent protein kinase (PKA)-mediated cMyBP-C phosphorylation have not been completely dissociated from other myofilament substrates for PKA, especially cardiac troponin I (cTnI). We have used synchrotron X-ray diffraction in skinned trabeculae to elucidate the roles of cMyBP-C and cTnI phosphorylation in myocardial inotropy and lusitropy. Myocardium in this study was isolated from four transgenic mouse lines in which the phosphorylation state of either cMyBP-C or cTnI was constitutively altered by site-specific mutagenesis. Analysis of peak intensities in X-ray diffraction patterns from trabeculae showed that cross-bridges are displaced similarly from the thick filament and toward actin (1) when both cMyBP-C and cTnI are phosphorylated, (2) when only cMyBP-C is phosphorylated, and (3) when cMyBP-C phosphorylation is mimicked by replacement with negative charge in its PKA sites. These findings suggest that phosphorylation of cMyBP-C relieves a constraint on cross-bridges, thereby increasing the proximity of myosin to binding sites on actin. Measurements of Ca(2+)-activated force in myocardium defined distinct molecular effects due to phosphorylation of cMyBP-C or co-phosphorylation with cTnI. Echocardiography revealed that mimicking the charge of cMyBP-C phosphorylation protects hearts from hypertrophy and systolic dysfunction that develops with constitutive dephosphorylation or genetic ablation, underscoring the importance of cMyBP-C phosphorylation for proper pump function.

Structural dynamics of muscle protein phosphorylation.

We have used site-directed spectroscopic probes to detect structural changes, motions, and interactions due to phosphorylation of proteins involved in the regulation of muscle contraction and relaxation. Protein crystal structures provide static snapshots that provide clues to the conformations that are sampled dynamically by proteins in the cellular environment. Our site-directed spectroscopic experiments, combined with computational simulations, extend these studies into functional assemblies in solution, and reveal details of protein regions that are too dynamic or disordered for crystallographic approaches. Here, we discuss phosphorylation-mediated structural transitions in the smooth muscle myosin regulatory light chain, the striated muscle accessory protein myosin binding protein-C, and the cardiac membrane Ca(2+) pump modulator phospholamban. In each of these systems, phosphorylation near the N terminus of the regulatory protein relieves an inhibitory interaction between the phosphoprotein and its regulatory target. Several additional unifying themes emerge from our studies: (a) The effect of phosphorylation is not to change the affinity of the phosphoprotein for its regulated binding partner, but to change the structure of the bound complex without dissociation. (b) Phosphorylation induces transitions between order and dynamic disorder. (c) Structural states are only loosely coupled to phosphorylation; i.e., complete phosphorylation induces dramatic functional effects with only a partial shift in the equilibrium between ordered and disordered structural states. These studies, which offer atomic-resolution insight into the structural and functional dynamics of these phosphoproteins, were inspired in part by the ground-breaking work in this field by Michael and Kate Barany.

A high-throughput fluorescence lifetime-based assay to detect binding of myosin-binding protein C to F-actin.

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.

AZD2014, a dual mTOR inhibitor, attenuates cardiac hypertrophy in vitro and in vivo.

Cardiac hypertrophy is one of the most common genetic heart disorders and considered a risk factor for cardiac morbidity and mortality. The mammalian target of rapamycin (mTOR) pathway plays a key regulatory function in cardiovascular physiology and pathology in hypertrophy. AZD2014 is a small-molecule ATP competitive mTOR inhibitor working on both mTORC1 and mTORC2 complexes. Little is known about the therapeutic effects of AZD2014 in cardiac hypertrophy and its underlying mechanism. Here, AZD2014 is examined in in vitro model of phenylephrine (PE)-induced human cardiomyocyte hypertrophy and a myosin-binding protein-C (Mybpc3)-targeted knockout (KO) mouse model of cardiac hypertrophy. Our results demonstrate that cardiomyocytes treated with AZD2014 retain the normal phenotype and AZD2014 attenuates cardiac hypertrophy in the Mybpc3-KO mouse model through inhibition of dual mTORC1 and mTORC2, which in turn results in the down-regulation of the Akt/mTOR signaling pathway.

Differential roles of regulatory light chain and myosin binding protein-C phosphorylations in the modulation of cardiac force development.

Phosphorylation of myosin regulatory light chain (RLC) by myosin light chain kinase (MLCK) and myosin binding protein-C (cMyBP-C) by protein kinase A (PKA) independently accelerate the kinetics of force development in ventricular myocardium. However, while MLCK treatment has been shown to increase the Ca(2+) sensitivity of force (pCa(50)), PKA treatment has been shown to decrease pCa(50), presumably due to cardiac troponin I phosphorylation. Further, MLCK treatment increases Ca(2+)-independent force and maximum Ca(2+)-activated force, whereas PKA treatment has no effect on either force. To investigate the structural basis underlying the kinase-specific differential effects on steady-state force, we used synchrotron low-angle X-ray diffraction to compare equatorial intensity ratios (I(1,1)/I(1,0)) to assess the proximity of myosin cross-bridge mass relative to actin and to compare lattice spacings (d(1,0)) to assess the inter-thick filament spacing in skinned myocardium following treatment with either MLCK or PKA. As we showed previously, PKA phosphorylation of cMyBP-C increases I(1,1)/I(1,0) and, as hypothesized, treatment with MLCK also increased I(1,1)/I(1,0), which can explain the accelerated rates of force development during activation. Importantly, interfilament spacing was reduced by 2 nm (3.5%) with MLCK treatment, but did not change with PKA treatment. Thus, RLC or cMyBP-C phosphorylation increases the proximity of cross-bridges to actin, but only RLC phosphorylation affects lattice spacing, which suggests that RLC and cMyBP-C modulate the kinetics of force development by similar structural mechanisms; however, the effect of RLC phosphorylation to increase the Ca(2+) sensitivity of force is mediated by a distinct mechanism, most probably involving changes in interfilament spacing.

Protein kinase A-mediated phosphorylation of cMyBP-C increases proximity of myosin heads to actin in resting myocardium.

Protein kinase A-mediated (PKA) phosphorylation of cardiac myosin binding protein C (cMyBP-C) accelerates the kinetics of cross-bridge cycling and may relieve the tether-like constraint of myosin heads imposed by cMyBP-C. We favor a mechanism in which cMyBP-C modulates cross-bridge cycling kinetics by regulating the proximity and interaction of myosin and actin. To test this idea, we used synchrotron low-angle x-ray diffraction to measure interthick filament lattice spacing and the equatorial intensity ratio, I(11)/I(10), in skinned trabeculae isolated from wild-type and cMyBP-C null (cMyBP-C(-/-)) mice. In wild-type myocardium, PKA treatment appeared to result in radial or azimuthal displacement of cross-bridges away from the thick filaments as indicated by an increase (approximately 50%) in I(11)/I(10) (0.22+/-0.03 versus 0.33+/-0.03). Conversely, PKA treatment did not affect cross-bridge disposition in mice lacking cMyBP-C, because there was no difference in I(11)/I(10) between untreated and PKA-treated cMyBP-C(-/-) myocardium (0.40+/-0.06 versus 0.42+/-0.05). Although lattice spacing did not change after treatment in wild-type (45.68+/-0.84 nm versus 45.64+/-0.64 nm), treatment of cMyBP-C(-/-) myocardium increased lattice spacing (46.80+/-0.92 nm versus 49.61+/-0.59 nm). This result is consistent with the idea that the myofilament lattice expands after PKA phosphorylation of cardiac troponin I, and when present, cMyBP-C, may stabilize the lattice. These data support our hypothesis that tethering of cross-bridges by cMyBP-C is relieved by phosphorylation of PKA sites in cMyBP-C, thereby increasing the proximity of cross-bridges to actin and increasing the probability of interaction with actin on contraction.

Radial displacement of myosin cross-bridges in mouse myocardium due to ablation of myosin binding protein-C.

Myosin binding protein-C (cMyBP-C) is a thick filament accessory protein, which in cardiac muscle functions to regulate the kinetics of cross-bridge interaction with actin; however, the underlying mechanism is not yet understood. To explore the structural basis for cMyBP-C function, we used synchrotron low-angle X-ray diffraction to measure interfilament lattice spacing and the equatorial intensity ratio, I(11)/I(10), in skinned myocardial preparations isolated from wild-type (WT) and cMyBP-C null (cMyBP-C(-/-)). In relaxed myocardium, ablation of cMyBP-C appeared to result in radial displacement of cross-bridges away from the thick filaments, as there was a significant increase ( approximately 30%) in the I(11)/I(10) ratio for cMyBP-C(-/-) (0.37+/-0.03) myocardium as compared to WT (0.28+/-0.01). While lattice spacing tended to be greater in cMyBP-C(-/-) myocardium (44.18+/-0.68 nm) when compared to WT (42.95+/-0.43 nm), the difference was not statistically significant. Furthermore, liquid-like disorder in the myofilament lattice was significantly greater ( approximately 40% greater) in cMyBP-C(-/-) myocardium as compared to WT. These results are consistent with our working hypothesis that cMyBP-C normally acts to tether myosin cross-bridges nearer to the thick filament backbone, thereby reducing the likelihood of cross-bridge binding to actin and limiting cooperative activation of the thin filament.