Brett Colson

Interests

Research

Spectroscopy, Cardiovascular and Muscle Biology, Molecular Motors, Drug Discovery

Teaching

Physiology, Complex Diseases, Biophysics, Biochemistry, Molecular Biology, Cardiovascular and Muscle Biology, Biomedical Engineering

Courses

Scholarly Contributions

Chapters

• Thomas, D. D., Muretta, J. M., Colson, B. A., Mello, R., & Kast, D. J. (2012). Spectroscopic probes of muscle proteins. In Muscle Biophysics. Elsevier Inc. doi:10.1016/B978-0-12-374920-8.00415-X
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  The use of site-directed spectroscopic probes in elucidating the molecular mechanism of force generation in muscle is reviewed, focusing on recent technical and conceptual advances. High-resolution crystal structures of actin and myosin suggest mechanistic models for force generation, but spectroscopic probe experiments under functional conditions are required to test and refine these models. An array of complementary spectroscopic techniques has been developed that can detect disorder and dynamics directly, resolve multiple conformational states, and can be applied to complex protein assemblies, taking advantage of the beautiful symmetry of the myofibrillar lattice. Recently developed instrumentation has the capacity to resolve simultaneously both biochemical and structural kinetics of myosin and/or actin. These new insights support a model in which structural and biochemical transitions are not tightly coupled, and disorder-to-order transitions play central roles in both force generation and its regulation.

Journals/Publications

• Wong, F. L., Bunch, T. A., Lepak, V. C., Steedman, A. L., & Colson, B. A. (2024). Cardiac myosin-binding protein C N-terminal interactions with myosin and actin filaments: Opposite effects of phosphorylation and M-domain mutations. Journal of molecular and cellular cardiology, 186, 125-137.
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  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.
• Bunch, T. A., Guhathakurta, P., Thompson, A. R., Lepak, V. C., Carter, A. L., Thomas, J. J., Thomas, D. D., & Colson, B. A. (2023). Drug discovery for heart failure targeting myosin-binding protein C. The Journal of biological chemistry, 299(12), 105369.
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  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.
• Dvornikov, A. V., Bunch, T. A., Lepak, V. C., & Colson, B. A. (2023). Fluorescence lifetime-based assay reports structural changes in cardiac muscle mediated by effectors of contractile regulation. The Journal of general physiology, 155(3).
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  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.
• Kanassatega, R. S., Bunch, T. A., Lepak, V. C., Wang, C., & Colson, B. A. (2022). Human cardiac myosin-binding protein C phosphorylation- and mutation-dependent structural dynamics monitored by time-resolved FRET. Journal of molecular and cellular cardiology, 166, 116-126.
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  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.
• Wong, F., Bunch, T., Lepak, V., & Colson, B. (2022). N-terminal cardiac myosin-binding protein C interactions with myosin and actin filaments using time-resolved FRET. bioRxiv. doi:https://doi.org/10.1101/2022.09.07.507024
• Bunch, T. A., Guhathakurta, P., Lepak, V. C., Thompson, A. R., Kanassatega, R. S., Wilson, A., Thomas, D. D., & Colson, B. A. (2021). Cardiac myosin-binding protein C interaction with actin is inhibited by compounds identified in a high-throughput fluorescence lifetime screen. The Journal of biological chemistry, 297(1), 100840.
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  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.
• Bunch, T. A., Lepak, V. C., Bortz, K. M., & Colson, B. A. (2021). A high-throughput fluorescence lifetime-based assay to detect binding of myosin-binding protein C to F-actin. The Journal of general physiology, 153(3).
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  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.
• Cha, B. H., Jung, M., Kim, A. S., Lepak, V. C., Colson, B. A., Bull, D. A., & Won, Y. (2021). AZD2014, a dual mTOR inhibitor, attenuates cardiac hypertrophy in vitro and in vivo. Journal of biological engineering, 15(1), 24.
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  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.
• Colson, B. A. (2021). In the eye of the STORM: Tracking the myosin-binding protein C N terminus in heart muscle. The Journal of general physiology, 153(3).
• Tardiff, J. C., Lehman, S. J., Klass, M. M., Kanassatega, R., Davis, J. P., & Colson, B. A. (2021). Exploring the Effects of Mutations and Thick Filament Proteins on Myofilament Calcium Kinetics Via Stopped-Flow. Biophysical Journal, 120(3). doi:10.1016/j.bpj.2020.11.566
• Thompson, A. R., Thomas, D. D., Lepak, V. C., Guhathakurta, P., Colson, B. A., & Bunch, T. A. (2021). High-Throughput Fluorescence Lifetime-Based Screen Detects Compounds that Bind to Myosin-Binding Protein C and Modulate Interactions with Actin. Biophysical Journal, 120(3). doi:10.1016/j.bpj.2020.11.1631
• Wilson, A., Thompson, A. R., Thomas, D. D., Lepak, V. C., Kanassatega, R., Guhathakurta, P., Colson, B. A., & Bunch, T. A. (2021). Cardiac myosin-binding protein C interaction with actin is inhibited by compounds identified in a high-throughput fluorescence lifetime screen.. The Journal of biological chemistry, 297(1), 100840. doi:10.1016/j.jbc.2021.100840
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  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.
• Lepak, V. C., Colson, B. A., & Bunch, T. A. (2020). A High-Throughput Fluorescence Lifetime-Based Assay for Detecting Binding of Myosin Binding Protein-C to F-Actin-Tropomyosin. Biophysical Journal, 118(3), 423a. doi:10.1016/j.bpj.2019.11.2383
• Lepak, V. C., Dvornikov, A. V., Colson, B. A., & Bunch, T. A. (2020). Altered Thick and Thin Filament Structural Dynamics in Mouse Myocardium Due to Ablation and Phosphorylation of Myosin Binding Protein-C. Biophysical Journal, 118(3), 425a. doi:10.1016/j.bpj.2019.11.2390
• Wang, C., Lepak, V. C., Kanassatega, R., Colson, B. A., & Bunch, T. A. (2020). A FRET-Based Biosensor for Detecting Phosphorylation-dependent Structural Dynamics in Human Myosin Binding Protein-C. Biophysical Journal, 118(3), 8a. doi:10.1016/j.bpj.2019.11.235
• Bunch, T. A., Kanassatega, R. S., Lepak, V. C., & Colson, B. A. (2019). Human cardiac myosin-binding protein C restricts actin structural dynamics in a cooperative and phosphorylation-sensitive manner. The Journal of biological chemistry, 294(44), 16228-16240.
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  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 ( = ∼8), suggesting that the cMyBP-C N terminus impacts the rotational dynamics of actin spanning seven monomers ( 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 (GSK3β) 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.
• Colson, B. A. (2019). What a drag!: Skeletal myosin binding protein-C affects sarcomeric shortening. The Journal of general physiology, 151(5), 614-618.
• Lepak, V. C., Kanassatega, R., Colson, B. A., & Bunch, T. A. (2019). Human Cardiac Myosin-Binding Protein C N-Terminal Domains Cooperatively Impact Actin Structural Dynamics. Biophysical Journal, 116(3), 266a. doi:10.1016/j.bpj.2018.11.1442
• Bunch, T. A., Lepak, V. C., Kanassatega, R. S., & Colson, B. A. (2018). N-terminal extension in cardiac myosin-binding protein C regulates myofilament binding. Journal of molecular and cellular cardiology, 125, 140-148.
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  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.
• Lepak, V. C., Colson, B. A., & Bunch, T. C. (2018). Differences in Myofilament Interactions and Structural Dynamics between Mouse and Human Cardiac Myosin-Binding Protein C. Biophysical Journal, 114(3), 497a. doi:10.1016/j.bpj.2017.11.2722
• Phung, L. A., Karvinen, S. M., Colson, B. A., Thomas, D. D., & Lowe, D. A. (2018). Age affects myosin relaxation states in skeletal muscle fibers of female but not male mice. PloS one, 13(9), e0199062.
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  The recent discovery that myosin has two distinct states in relaxed muscle-disordered relaxed (DRX) and super-relaxed (SRX)-provides another factor to consider in our fundamental understanding of the aging mechanism in skeletal muscle, since myosin is thought to be a potential contributor to dynapenia (age-associated loss of muscle strength independent of atrophy). The primary goal of this study was to determine the effects of age on DRX and SRX states and to examine their sex specificity. We have used quantitative fluorescence microscopy of the fluorescent nucleotide analog 2'/3'-O-(N-methylanthraniloyl) ATP (mantATP) to measure single-nucleotide turnover kinetics of myosin in skinned skeletal muscle fibers under relaxing conditions. We examined changes in DRX and SRX in response to the natural aging process by measuring the turnover of mantATP in skinned fibers isolated from psoas muscle of adult young (3-4 months old) and aged (26-28 months old) C57BL/6 female and male mice. Fluorescence decays were fitted to a multi-exponential decay function to determine both the time constants and mole fractions of fast and slow turnover populations, and significance was analyzed by a t-test. We found that in females, both the DRX and SRX lifetimes of myosin ATP turnover at steady state were shorter in aged muscle fibers compared to young muscle fibers (p ≤ 0.033). However, there was no significant difference in relaxation lifetime of either DRX (p = 0.202) or SRX (p = 0.804) between young and aged male mice. No significant effects were measured on the mole fractions (populations) of these states, as a function of sex or age (females, p = 0.100; males, p = 0.929). The effect of age on the order of myosin heads at rest and their ATPase function is sex specific, affecting only females. These findings provide new insight into the molecular factors and mechanisms that contribute to aging muscle dysfunction in a sex-specific manner.
• Thomas, D. D., Phung, L. A., Lowe, D. A., Karvinen, S. M., & Colson, B. A. (2018). Myosin Super-relaxed State is Affected by Aging in Female But Not Male Skeletal Muscle: 117 Board #6 May 30 9 30 AM - 11 30 AM. Medicine and Science in Sports and Exercise, 50(5S), 9. doi:10.1249/01.mss.0000535114.38958.86
• Thomas, D. D., Phung, L. A., Petersen, K. J., Lowe, D. A., Karvinen, S., & Colson, B. A. (2018). Ovarian Hormone Affects the Regulation of Super-Relaxation in Skeletal Muscle. Biophysical Journal, 114(3), 212a. doi:10.1016/j.bpj.2017.11.1184
• Lepak, V. C., Colson, B. A., & Bunch, T. A. (2017). Structural Dynamics of Human Cardiac Myosin-Binding Protein C Revealed by Time-Resolved FRET. Biophysical Journal, 112(3), 557a. doi:10.1016/j.bpj.2016.11.3005
• Palumbo, S., Shin, Y. J., Ahmad, K., Desai, A. A., Quijada, H., Mohamed, M., Knox, A., Sammani, S., Colson, B. A., Wang, T., Garcia, J. G., & Hecker, L. (2017). Dysregulated Nox4 ubiquitination contributes to redox imbalance and age-related severity of acute lung injury. American journal of physiology. Lung cellular and molecular physiology, ajplung.00305.2016.
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  Acute respiratory distress syndrome (ARDS) is a devastating critical illness disproportionately affecting the elderly population(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 aging remain unclear. We assessed the severity of acute lung injury (ALI) in young (2 months) and aged (18 months) mice using a two-hit pre-clinical model. Compared to young cohorts, aged mice exhibited increased ALI severity, with greater vascular permeability characterized by elevated albumin influx, levels of bronchoalveolar lavage (BAL) cells (neutrophils) and protein. Aged/injured mice also demonstrated elevated levels of reactive oxygen species (ROS) in the BAL, 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 beta-galactosidase activity and p16 levels. In response to lipopolysaccharide (LPS) challenge, senescent ECs demonstrate exacerbated permeability responses compared to control "young" ECs. LPS challenge led to a rapid induction of Nox4 expression in both control and senescent ECs, which was post-translationally mediated via the proteasome/ubiquitin system. However, senescent ECs demonstrated deficient Nox4 ubiquitination, resulting in sustained expression of Nox4and alterations in cellular redox homeostasis. Pharmacologic 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.
• Colson, B. A., Thompson, A. R., Espinoza-Fonseca, L. M., & Thomas, D. D. (2016). Site-directed spectroscopy of cardiac myosin-binding protein C reveals effects of phosphorylation on protein structural dynamics. Proceedings of the National Academy of Sciences of the United States of America.
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  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 β-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.
• Lai, S., Collins, B. C., Colson, B. A., Kararigas, G., & Lowe, D. A. (2016). Estradiol modulates myosin regulatory light chain phosphorylation and contractility in skeletal muscle of female mice. American journal of physiology. Endocrinology and metabolism, 310(9), E724-33.
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  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 17β-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-β 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.
• Thomas, D. D., Espinoza-fonseca, L. M., & Colson, B. A. (2016). Structural Measurements within the M-Domain Reveal Unique Details of Cardiac Myosin Binding Protein-C Phosphorylation. Biophysical Journal, 110(3), 2-3. doi:10.1016/j.bpj.2015.11.1584
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  We have used molecular dynamics (MD) simulations in atomic-level detail to resolve the structural changes associated with cardiac myosin binding protein-C (cMyBP-C) phosphorylation by protein kinase A (PKA), focusing on the N-terminal phosphorylation motif (pm/M-domain). The results are supported by spectroscopic measurements, and have implications for cMyBP-C's role in myocardial contraction, with particular relevance to -adrenergic signaling, heart failure, and hypertrophic cardiomyopathy. PKA phosphorylation in the pm mediates the affinity of cMyBP-C interactions with actin and the myosin-neck that modulate cardiac muscle contractility. Our spectroscopic studies using time-resolved FRET (TR-FRET) had previously detected distance distributions between probe pairs engineered at sites of cMyBP-C domains C0 through C2. Phosphorylation reduces the level of molecular disorder and the distribution of N-terminal inter-domain distances become more compact (e.g., C1-to-C2). Here, we gain further insight from 20 independent microsecond-long MD simulations of the intrinsically disordered pm. We modeled the motif structure (residues His255-Lys356) and simulated the unphosphorylated and phosphorylated (Ser- 273, 282, and 302) characteristics of molecular disorder, secondary and tertiary structure, intra-motif distance measurements, surface accessibility, and predictions of binding probability. A large structural change in the disordered region of pm was revealed, whereby phosphorylation unmasks a surface of positively-charged residues on an α-helix within the ordered region of pm. Distance measurements between residues in the disordered N-terminal portion of the motif to the helix residues of the motif C-terminus changed proximity to one another by as much as 2 nm upon phosphorylation. These findings help to elucidate cMyBP-C structural dynamics, providing new molecular insight into the modulatory role of cMyBP-C in force development. This work was supported by NIH grants to DDT (R01 AR032961) and to BC (R00 HL122397) and to LME-F from the American Heart Association (12SDG12060656).
• Thomas, D. D., Petersen, K. J., Lowe, D. A., Colson, B. A., Collins, B. C., & Bunch, T. A. (2016). The Super-Relaxed State of Myosin is Altered by Estradiol in Skeletal Muscle of Aged Female Mice. Biophysical Journal, 110(3), 303a-304a. doi:10.1016/j.bpj.2015.11.1632
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  We have used quantitative epifluorescence microscopy of fluorescent ATP to measure single-nucleotide turnover in skinned skeletal muscle fibers from old male and female mice. Aging causes declines in muscle strength, often leading to weakness-related health problems for the elderly. Female muscle has additional functional decrements with age due to reduced ovarian hormone production. Estradiol (E2) is the major sex hormone signal to skeletal muscle in females, and strength loss is rescued by E2 treatment in ovariectomized (OVX) mice. We previously showed that E2-mediated signaling reversibly regulates slow ATP turnover by myosin in single fibers isolated from OVX mice. To investigate E2 mechanisms on aged skeletal muscle, single fibers were isolated from young (2 months) or old (28 months) male and female mice, and were incubated with mantATP. Old female mice receiving 60-day E2 treatment were also studied. We measured the decay of mantATP fluorescence in an ATP-chase experiment to characterize the slow nucleotide turnover, called the super-relaxed state (SRX), detected in approximately one-third of the myosin heads. The SRX turnover was faster in aged female fibers compared to young female fibers. In contrast, ATP turnover in old male fibers was not significantly different than in young males, and even trended toward slower rather than faster turnover with aging. We conclude that E2-mediated signaling reversibly regulates slow ATP turnover by myosin in aging female mice. Age- and hormone-related functional deficits may be targetable at the level of myosin and other contractile protein structure/function for strategies to offset muscle weakness and metabolic changes that occur with age. This work was supported by NIH grants to DDT (R37 AG026160 and R01 AR032961), to DL (R01 AG31743), to BAC (R00 HL122397), and to KJP and BCC (T32 AR7612).
• Thomas, D. D., Sadayappan, S., Lin, B., Gallegos, A., & Colson, B. A. (2016). The N-Terminal Domains of MyBPC3 Restrict Actin Dynamics and Increase Resilience in a Phosphorylation-Dependent Manner. Biophysical Journal, 110(3), 293a. doi:10.1016/j.bpj.2015.11.1582
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  We have determined the effects of myosin binding protein-C (MyBP-C) N-terminal domains on the microsecond rotational dynamics of actin, detected by time-resolved phosphorescence anisotropy (TPA). MyBP-C modulates muscle contractility and is capable of interacting with thin filaments. Protein kinase A (PKA) phosphorylates cardiac and slow skeletal MyBP-C, altering myofilament structure and function. We previously used TPA to show that full-length MyBP-C restricts actin torsional dynamics, and effects by cardiac and slow skeletal MyBP-C (cMyBP-C; ssMyBP-C) are relieved by PKA phosphorylation. To determine the effects of MyBP-C N-terminal domains 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 flexibility at saturation of the TPA effect. PKA phosphorylation of ssMyBP-C domains C1-C2 and cMyBP-C domains C0-C2 relieved the restriction of torsional amplitude but also increased the rate of torsional motion, thus increasing actin resilience (rate/amplitude; less brittle). Moreover, effects of cardiac C0-C2 on actin resilience were also PKA-dependent, whereas slow skeletal C1-C2 effects which promote actin resilience persisted independently of phosphorylation. In the case of fast skeletal C1-C2, actin resilience was unaffected and its effect to restrict actin dynamics was unchanged by phosphorylation. The N-terminal domains of cMyBP-C (C0-C2) and skeletal MyBP-C's (C1-C2) had effects on actin dynamics similar to those determined for the full-length MyBP-C isoforms. Effects of phosphomimetic mutations were also investigated. These unique isoform-dependent MyBP-C-induced changes in actin dynamics may play a role in the functional effects of MyBP-C on contraction. This work was supported by NIH grants to DDT (R01 AR032961), to SS (R01 AR067279), and to BC (R00 HL122397).
• Colson, B. A., Petersen, K. J., Collins, B. C., Lowe, D. A., & Thomas, D. D. (2015). The myosin super-relaxed state is disrupted by estradiol deficiency. Biochemical and biophysical research communications, 456(1), 151-5.
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  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.
• Thomas, D. D., Petersen, K. J., Lowe, D. A., Colson, B. A., & Collins, B. C. (2015). The Myosin Super-Relaxed State is Regulated by Estradiol. Biophysical Journal, 108(2), 339a. doi:10.1016/j.bpj.2014.11.1850
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  We have used quantitative epifluorescence microscopy of fluorescent ATP to measure single-nucleotide turnovers in skinned muscle fibers from mouse models of female aging and hormone treatment. The loss of muscle strength is an undesirable consequence of aging, 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. Single skeletal muscle fibers, isolated from sham-operated or ovariectomized (OVX) mice with or without E2 treatment, were incubated with mantATP. We measured decay of mantATP fluorescence intensity in an ATP chase experiment, as pioneered by Cooke and colleagues (Stewart et al., 2010; Cooke, 2011). These studies by Cooke 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 approximately one-third of sham-operated myosin heads, consistent with SRX. Turnover was substantially faster in OVX fibers. Strikingly, the chronic lack of E2 in OVX fibers did not alter the fraction of heads with slow turnover but rather decreased the turnover time, suggesting disordering of SRX. E2 treatment in OVX mice partially reversed this effect on SRX, while acute E2 treatment in the muscle bath had no effect. All experiments were conducted with uniformly low myosin light chain phosphorylation. We conclude that E2-mediated signaling regulates slow ATP turnover and ordering of heads, probably via pathways distinct from myosin phosphorylation status. Age- and hormone-related muscle functional losses may be targetable at the level of thick filament structure for strategies to offset weakness and metabolic changes that occur with age.
• Espinoza-Fonseca, L. M., Colson, B. A., & Thomas, D. D. (2014). Effects of pseudophosphorylation mutants on the structural dynamics of smooth muscle myosin regulatory light chain. Molecular bioSystems, 10(10), 2693-8.
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  We have performed 50 independent molecular dynamics (MD) simulations to determine the effect of pseudophosphorylation mutants on the structural dynamics of smooth muscle myosin (SMM) regulatory light chain (RLC). We previously showed that the N-terminal phosphorylation domain of RLC simultaneously populates two structural states in equilibrium, closed and open, and that phosphorylation at S19 induces a modest shift toward the open state, which is sufficient to activate smooth muscle. However, it remains unknown why pseudophosphorylation mutants poorly mimic phosphorylation-induced activation of SMM. We performed MD simulations of unphosphorylated, phosphorylated, and three pseudophosphorylated RLC mutants: S19E, T18D/S19D and T18E/S19E. We found that the S19E mutation does not shift the equilibrium toward the open state, indicating that simple charge replacement at position S19 does not mimic the activating effect of phosphorylation, providing a structural explanation for previously published functional data. In contrast, mutants T18D/S19D and T18E/S19E shift the equilibrium toward the open structure and partially activate in vitro motility, further supporting the model that an increase in the mol fraction of the open state is coupled to SMM motility. Structural analyses of the doubly-charged pseudophosphorylation mutants suggest that alterations in an interdomain salt bridge between residues R4 and D100 results in impaired signal transmission from RLC to the catalytic domain of SMM, which explains the low ATPase activity of these mutants. Our results demonstrate that phosphorylation produces a unique structural balance in the RLC. These observations have important implications for our understanding of the structural aspects of activation and force potentiation in smooth and striated muscle.
• Thomas, D. D., Klein, J. C., James, Z. M., & Colson, B. A. (2014). Structural Dynamics of Cardiac Myosin Binding Protein-C and its Myofilament Binding Partners, Detected by Site-Directed Spectroscopy. Biophysical Journal, 106(2), 162a. doi:10.1016/j.bpj.2013.11.923
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  We have used site-directed labeling with pulsed dipolar electron-electron paramagnetic resonance (DEER) and time-resolved FRET (TR-FRET) spectroscopies to resolve the structure and dynamics of myosin binding protein-C (MyBP-C)'s cardiac isoform, with implications for the pathophysiology of hypertrophic cardiomyopathy (HCM). N-terminal cMyBP-C domains C0 through C2 contain binding regions for several muscle protein interaction partners, including myosin heavy chain subfragment 2 (S2), the actin filament, the regulatory light chain (RLC), and Ca2+-calmodulin (CaM). We previously determined the cMyBP-C-induced changes in actin torsional dynamics (Colson et al., Proc Natl Acad Sci USA. 2012 Dec 11; 109(50): 20437-42), and now extend our spectroscopic assays for microsecond protein motion to include distance detection via probes on cMyBP-C itself. We engineered intramolecular pairs of labeling sites within either cMyBP-C or CaM to measure, with high resolution, distance and disorder between either protein's flexible and dynamic regions using DEER and TR-FRET. Changes in distance and disorder were assessed for labeled proteins free in solution and when bound to an interaction partner (e.g., labeled-cMyBP-C titrated with unlabeled S2 or actin, or labeled-CaM titrated with unlabeled cMyBP-C). Understanding conformational transitions in the flexible and dynamic portions of cMyBP-C and CaM provides new molecular insight into defining cMyBP-C's modulatory role in cardiac muscle force development. Acknowledgments: Spectroscopy was performed in the Biophysical Spectroscopy Center at the University of Minnesota, with assistance from Fluorescence Innovations, Inc. (Greg Gillispie, President). This work was funded by an American Heart Association postdoctoral fellowship to BAC and NIH grants to DDT (R01 AR32961, P30 AR0507220, T32 AR007612).
• Thomas, D. D., Roopnarine, O., Mauseth, M. A., Kast, D. J., Espinoza-fonseca, L. M., & Colson, B. A. (2014). Atomic-Level Visualization of Smooth Muscle Activation by Phosphorylation of the Myosin Regulatory Light Chain. Biophysical Journal, 106(2), 161a-162a. doi:10.1016/j.bpj.2013.11.922
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  We have engineered site-directed probe pairs in the regulatory light chain (RLC) for exchange into smooth muscle heavy meromyosin (HMM) and subfragment-1 (S1), in order to examine the phosphorylation-induced changes in RLC structural states using time-resolved FRET (TR-FRET). Phosphorylation of the RLC is required for activation of contraction in smooth muscle and modulates force in striated muscle. Force development in smooth muscle is triggered by phosphorylation of the RLC's N-terminal phosphorylation domain (PD) at Ser19, alleviating inhibitory interactions between the two heads. Since crystal structures of the RLC lack the PD, the mechanism by which RLC phosphorylation allosterically triggers disruption of HMM auto-inhibition remains unresolved. Our site-directed FRET probe pairs resolved intramolecular atomic distance measurements between the RLC core and the dynamic PD. Smooth muscle RLC assumes two structural states in both states of activation: phosphorylation shifts the equilibrium of these two structural states from primarily occupying the compact closed state to favoring the extended open state of the PD, relative to the RLC core. Compared to single-headed S1 (Kast et al., PNAS., 2010), the open state RLC conformation of unphosphorylated HMM is more compact, presumably due to head-head or RLC-RLC interactions. All-atom MD simulations corroborate our TR-FRET studies, and reveal specific salt-bridges that stabilize each structural state. The hypotheses generated from our MD simulations are being tested experimentally by TR-FRET via site-directed mutagenesis to manipulate charge. These studies offer the first atomic-resolution insight into the structural dynamics of RLC phosphorylation, and a similar approach should be applicable to striated muscle. Spectroscopy was performed in the Biophysical Spectroscopy Center at the University of Minnesota. This work was funded by NIH grants to DDT (R01 AR32961, P30 AR0507220, T32 AR007612).
• Thomas, D. D., James, Z. M., & Colson, B. A. (2013). Structural Dynamics of Actin-Myosin Bound and Unbound States of Cardiac Myosin Binding Protein-C Detected by Dipolar EPR. Biophysical Journal, 104(2), 1-7. doi:10.1016/j.bpj.2012.11.1054
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  We have used site-directed spin labeling and pulsed dipolar electron-electron paramagnetic resonance (DEER) to resolve the structure and dynamics of flexible and disordered regions of myosin binding protein-C (MyBP-C)'s cardiac isoform, with implications for the pathophysiology of hypertrophic cardiomyopathy (HCM). N-terminal domains of cMyBP-C contain binding domains for several interaction partners in the myofilament, including myosin heavy chain subfragment 2 (S2) and actin. We engineered pairs of labeling sites in protein fragments of mouse cMyBP-C to measure with high resolution distance and disorder between (1) domains C0 and C1, flanking the flexible Pro/Ala-rich linker, and between (2) domains C1 and C2, flanking the partially disordered phosphorylation motif, using DEER. Changes in distance and disorder were assessed for double-Cys mutant cMyBP-C's free in solution and when bound to myosin S2 or actin, with or without cMyBP-C phosphorylation by protein kinase A (PKA). Understanding conformational transitions in the flexible and dynamic portions of cMyBP-C upon actin-myosin binding and phosphorylation provide new molecular insight into defining its modulatory role in muscle force development. (NIH-F32 to BAC; NIH-R01 to DDT)
• Colson, B. A., Gruber, S. J., & Thomas, D. D. (2012). Structural dynamics of muscle protein phosphorylation. Journal of muscle research and cell motility, 33(6), 419-29.
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  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.
• Colson, B. A., Patel, J. R., Chen, P. P., Bekyarova, T., Abdalla, M. I., Tong, C. W., Fitzsimons, D. P., Irving, T. C., & Moss, R. L. (2012). Myosin binding protein-C phosphorylation is the principal mediator of protein kinase A effects on thick filament structure in myocardium. Journal of molecular and cellular cardiology, 53(5), 609-16.
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  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.
• Colson, B. A., Rybakova, I. N., Prochniewicz, E., Moss, R. L., & Thomas, D. D. (2012). Cardiac myosin binding protein-C restricts intrafilament torsional dynamics of actin in a phosphorylation-dependent manner. Proceedings of the National Academy of Sciences of the United States of America, 109(50), 20437-42.
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  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.
• Thomas, D. D., Rybakova, I. N., Prochniewicz, E., Moss, R. L., & Colson, B. A. (2012). Effects of Myosin Binding Protein-C Isoform, Phosphorylation, and Domains on the Rotational Dynamics of Actin Filaments. Biophysical Journal, 102(3), 435a. doi:10.1016/j.bpj.2011.11.2383
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  We have determined the effects of Myosin binding protein-C (MyBP-C) and its domains on the microsecond time-scale rotational dynamics of actin, using time-resolved phosphorescence anisotropy (TPA). MyBP-C is a multi-domain thick filament-associated modulator of striated muscle contraction, spanning the interfilament spacing to contact both myosin and actin. Cardiac (c-) and slow skeletal (ss-) MyBP-C are known substrates for Protein kinase-A (PKA), and phosphorylation of cMyBP-C alters contractile properties and myofilament structure. To determine the effects of MyBP-C on actin's microsecond structural dynamics, we labeled actin at C374 with erythrosine iodoacetamide and performed TPA experiments. The interaction of all three MyBP-C isoforms with actin increased the final anisotropy (r∞) of the TPA decay in a concentration-dependent manner, indicating restriction of the rotational amplitude of actin dynamics. While PKA phosphorylation had little effect on fast skeletal (fs-) MyBP-C, phosphorylation of cMyBP-C and ssMyBP-C nearly eliminated the effects of these proteins to restrict actin dynamics, despite no change in binding affinity with phosphorylation. Skeletal MyBP-C (C1-C10) affected actin anisotropy at lower concentrations than cMyBP-C (C0-C10), suggesting that skeletal and cardiac N-terminal MyBP-C interactions with actin have distinct properties. The effects of truncated cMyBP-C on actin anisotropy determined that C-terminal domains are important for restricting rotational dynamics, whereas N-terminal domains are important for regulating this effect. These MyBP-C-induced changes in actin dynamics may play a role in the known effects of MyBP-C on the functional actin-myosin interaction. This work was funded by grants from NIH (F32 HL107039-01 to BAC and T32 AR007612 to DDT).
• Thomas, D. D., Rybakova, I. N., Prochniewicz, E., Moss, R. L., & Colson, B. A. (2011). Effects of Cardiac Myosin Binding Protein-C and its Domains on the Rotational Dynamics of Actin Filaments. Biophysical Journal, 100(3), 585a. doi:10.1016/j.bpj.2010.12.3379
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  Cardiac myosin binding protein-C (cMyBP-C) is a multi-domain thick filament-associated modulator of contraction, but it remains unknown whether cMyBP-C alters myosin S1 access to the thin filament by its interaction with myosin S2 and/or by its interaction with actin. Recently, actin binding properties of baculovirus-expressed full-length mouse cMyBP-C and its domains (i.e., C0-C10), assessed by cosedimentation, showed that cMyBP-C interacts with F-actin via a single moderate-affinity site localized to the C-terminal half of cMyBP-C, with no effect on binding due to phosphorylation (Rybakova et al., 2010, J Biol Chem., in press). Here, we have determined the effects of cMyBP-C and its domains on the microsecond time-scale rotational dynamics of actin labeled at C374 with erythrosine iodoacetamide, using time-resolved phosphorescence anisotropy (TPA). The interaction of cMyBP-C with actin increased the final anisotropy (rinfinity) of the TPA decay in a concentration-dependent manner, indicating restriction of the rotational amplitude of actin dynamics. The N-terminal domains C0C1 had no detectable effect to on the final anisotropy of actin, probably due to its inability to bind actin, whereas C0C4 moderately increased final anisotropy. Fragments containing the C-terminal domains, such as delta-C0C1, increased final anisotropy to a similar extent as full-length cMyBP-C, suggesting that the C-terminal domains are important for restricting rotational dynamics of actin. Protein kinase A (PKA) phosphorylation of cMyBP-C or delta-C0C1 reduced, but did not eliminate, the effects of these proteins to increase the final anisotropy of the TPA decay. Increased anisotropy was not caused by actin bundling, as shown by electron microscopy observation. These cMyBP-C-induced changes in actin dynamics may play a role in the known effects of cMyBP-C on the functional actin-myosin interaction.
• Colson, B. A., Locher, M. R., Bekyarova, T., Patel, J. R., Fitzsimons, D. P., Irving, T. C., & Moss, R. L. (2010). Differential roles of regulatory light chain and myosin binding protein-C phosphorylations in the modulation of cardiac force development. The Journal of physiology, 588(Pt 6), 981-93.
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  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.
• Schemmel, P. P., Moss, R. L., Irving, T. C., Fitzsimons, D. P., Colson, B. A., Chen, P. P., & Bekyarova, T. (2010). Phosphorylation of Myosin Binding Protein-C Alters the Proximity of Cross-Bridges to Actin and Accelerates Myocardial Twitch Kinetics. Biophysical Journal, 98(3), 347a. doi:10.1016/j.bpj.2009.12.1877
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  The strength and kinetics of cardiac contraction vary on a beat-to-beat basis in efforts to match cardiac output in response to changing circulatory demands. In living myocardium, the beta-adrenoreceptor agonist dobutamine initiates protein kinase A (PKA)-mediated phosphorylations of Ca2+ handling proteins and contractile proteins including cardiac myosin binding protein-C (cMyBP-C) and cardiac troponin I (cTnI), which leads to potentiation of twitch force and faster kinetics of force development and relaxation. Our previous studies in skinned myocardium suggest that PKA phosphorylation of cMyBP-C disrupts its interaction with myosin subfragment 2 (S2), which relieves the tether-like constraint of myosin heads imposed by cMyBP-C, and thereby accelerates cross-bridge cycling kinetics. To examine the relative role of cMyBP-C phosphorylation in altering twitch kinetics, we recorded twitch force and low-angle x-ray diffraction patterns in between twitches and near maximum twitch force in intact trabeculae isolated from murine myocardium electrically stimulated at 0.5 Hz in the presence and absence of dobutamine. Our data suggest that phosphorylation of cMyBP-C caused a radial or azimuthal displacement of cross-bridges towards the thin filament in vivo prior to the twitch, which contributes to the accelerated contraction kinetics following twitch stimulation. These results suggest that interactions between cMyBP-C and the S2-domain of myosin heavy chain are dynamically regulated by phosphorylation of cMyBP-C and function to modulate the availability and cooperative binding of cross-bridges to actin during the myocardial twitch.
• Tong, C. W., Powers, P. A., Moss, R. L., Locher, M. R., Irving, T. C., Fitzsimons, D. P., Colson, B. A., & Bekyarova, T. (2009). Constitutive Phosphorylation of Cardiac Myosin Binding Protein-C Increases the Probability of Myosin Cross-bridge Interaction with Actin. Biophysical Journal, 96(3), 232a. doi:10.1016/j.bpj.2008.12.1142
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  Protein kinase A-mediated (PKA) phosphorylation of cardiac myosin binding protein-C (cMyBP-C) accelerates the kinetics of cross-bridge cycling and appears to relieve the tether-like constraint of myosin heads imposed by cMyBP-C (Colson et al., 2008, Circ Res., 103:244-251). We favor a mechanism in which phosphorylation of the 3 PKA sites in cMyBP-C modulates cross-bridge kinetics by regulating the proximity and interaction of myosin with actin. To test this idea, we used synchrotron low-angle x-ray diffraction and mechanical measurements in skinned myocardium isolated from a mouse model with phosphomimetic substitutions in cMyBP-C, i.e., the CTSD mouse. The substitutions were introduced by transgenic expression of cMyBP-C with Ser-to-Asp mutations on a cMyBP-C null background. Western blots showed that expression of CTSD cMyBP-C was 85% of wild-type (WT), and the heart weight to body weight ratio was similar (5.2 ± 0.2 mg/g) in CTSD and WT mice. Expression of WT cMyBP-C on the knockout background served as control (i.e., the CTWT mouse). Skinned myocardium from CTSD and CTWT mice exhibited similar maximum active forces (mN/mm2: 17.7 ± 3.7 vs 13.2 ± 2.9), Ca2+-sensitivities of force (pCa50: 5.55 ± 0.03 vs 5.58 ± 0.04), and maximum rates of force development (ktr, sec-1: 20.2 ± 1.7 vs 22.5 ± 1.9; kdf, sec-1: 37.6 ± 3.7 vs 43.2 ± 2.3). I11/I10 intensity ratios and d10 lattice spacings determined from equatorial reflections from CTSD and CTWT myocardium were used to determine the effect of constitutive cMyBP-C phosphorylation on the distribution of cross-bridge mass between the thick and thin filaments and on interfilament lattice spacing. The results suggest that interactions between cMyBP-C and the S2 domain of myosin heavy chain are dynamically regulated by phosphorylations in the cMyBP-C motif. (AHA-predoctoral fellowship (BAC); NIH-HL-R01-82900)
• Colson, B. A., Bekyarova, T., Locher, M. R., Fitzsimons, D. P., Irving, T. C., & Moss, R. L. (2008). Protein kinase A-mediated phosphorylation of cMyBP-C increases proximity of myosin heads to actin in resting myocardium. Circulation research, 103(3), 244-51.
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  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.
• Colson, B. A., Bekyarova, T., Fitzsimons, D. P., Irving, T. C., & Moss, R. L. (2007). Radial displacement of myosin cross-bridges in mouse myocardium due to ablation of myosin binding protein-C. Journal of molecular biology, 367(1), 36-41.
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  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.