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.

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.

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.

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.

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.

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.

Age-specific treatment effects of orexin/hypocretin-receptor antagonism on methamphetamine-seeking behavior.

BACKGROUND: Worldwide methamphetamine (METH) use has increased significantly over the last 10 years, and in the US, METH dependence has sky-rocketed among individuals with opioid use disorder. Of significant concern, METH use is gaining popularity among groups with susceptibility to developing severe substance use disorders, such as women and adolescents. Nevertheless, there is no established pharmacotherapy for METH addiction. Emerging evidence has identified the orexin/hypocretin system as an important modulator of reward-driven behavior and a potential target for the treatment of drug addiction and relapse. However, to date, there have been no investigations into the therapeutic efficacy of orexin/hypocretin receptor antagonists for METH-motivated behavior in adolescents or adults. In the present study, we examined the effects of selective antagonists of the orexin-1 (SB-334867, 20 mg/kg) and orexin-2 (TCS-OX2-29, 20 mg/kg) receptors on the reinstatement of METH seeking in both adolescent and adult male and female rats. METHODS: Rats were trained to self-administer METH (0.05 mg/kg/inf, iv) during two 2-h sessions/day for 5 days. Following 20 sessions of extinction over 10 days, a within-subjects design was used to test for METH seeking precipitated by METH (1 mg/kg, ip) or METH cues after systemic pretreatment with SB-334867 or TCS-OX2-29. RESULTS: SB-334867 reduced cue-induced reinstatement in males and females, regardless of age. Additionally, METH-induced METH seeking was attenuated by SB-334867 in adolescents and by TCS-OX2-29 in adults. CONCLUSION: Selective orexin/hypocretin receptor antagonists have significant therapeutic potential for diminishing METH-seeking behavior, although their treatment efficacy may be influenced by age.

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.

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.

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.

Oxygen consumption deficit in Huntington disease mouse brain under metabolic stress.

In vivo evidence for brain mitochondrial dysfunction in animal models of Huntington disease (HD) is scarce. We applied the novel 17O magnetic resonance spectroscopy (MRS) technique on R6/2 mice to directly determine rates of oxygen consumption (CMRO2) and assess mitochondrial function in vivo Basal respiration and maximal CMRO2 in the presence of the mitochondrial uncoupler dinitrophenol (DNP) were compared using 16.4 T in isoflurane anesthetized wild type (WT) and HD mice at 9 weeks. At rest, striatal CMRO2 of R6/2 mice was equivalent to that of WT, indicating comparable mitochondrial output despite onset of motor symptoms in R6/2. After DNP injection, the maximal CMRO2 in both striatum and cortex of R6/2 mice was significantly lower than that of WT, indicating less spare energy generating capacity. In a separate set of mice, oligomycin injection to block ATP generation decreased CMRO2 equally in brains of R6/2 and WT mice, suggesting oxidative phosphorylation capacity and respiratory coupling were equivalent at rest. Expression levels of representative mitochondrial proteins were compared from harvested tissue samples. Significant differences between R6/2 and WT included: in striatum, lower VDAC and the mitochondrially encoded cytochrome oxidase subunit I relative to actin; in cortex, lower tricarboxylic acid cycle enzyme aconitase and higher protein carbonyls; in both, lower glycolytic enzyme enolase. Therefore in R6/2 striatum, lowered CMRO2 may be attributed to a decrease in mitochondria while the cortical CMRO2 decrease may result from constraints upstream in energetic pathways, suggesting regionally specific changes and possibly rates of metabolic impairment.