- Assistant Professor, Cellular and Molecular Medicine
- Assistant Professor, Physiological Sciences - GIDP
- Assistant Professor, Biomedical Engineering
- Assistant Professor, BIO5 Institute
- Assistant Professor, Clinical Translational Sciences
My research interests include muscle physiology, muscle disease, and heart failure. The primary focus of my current research is cellular and molecular mechanisms underlying cardiac muscle dysfunction that occurs with genetic mutations in myosin binding protein-C (cMyBP-C), causing hypertrophic cardiomyopathy and leading to arrhythmias, heart failure, and sudden cardiac death. This work also involves development and application of site-directed spectroscopic probe methods for understanding structure, function, and dynamics of cardiac muscle proteins, which is needed to understand the basic mechanisms that are crucial to cardiac muscle physiology and malfunction in disease. The powerful combination of my doctoral training experience in muscle physiology and biophysics under Dr. Richard Moss and my postdoctoral training under Dr. David Thomas in biochemistry and spectroscopic analysis of muscle protein molecular dynamics, uniquely positioned me to undertake biophysical studies at the forefront of biomedicine and technology. I will now continue this line of study in my newly established lab’s research program and independent research career. At the University of Arizona, I aim to establish a strong program in striated muscle biology and cardiovascular sciences to study the molecular mechanisms of muscle proteins and their response to changing physiological demands in health and disease, combining several biophysical techniques from comprehensive analysis of contractile function at levels ranging from isolated muscles to actin-myosin molecular interactions, to high-resolution distance and disorder measurements of muscle protein structural dynamics in solution and in muscle cells, specially engineered with reporter probes. I expect my career development to continue in the presence of high-quality faculty colleagues in the research area of cardiovascular physiology and muscle biophysics in the Department of Cellular and Molecular Medicine, the Molecular Cardiovascular Research Group, and the Sarver Heart Center under direction of Drs. Carol Gregorio and Nancy Sweitzer. My career development will be further strengthened with Dr. Henk Granzier as my senior faculty mentor. I am confident my past training has rigorously prepared me to pursue very exciting medically-relevant spectroscopy studies, well-aligned for discovery of novel therapies for muscle dysfunction and heart failure that I have proposed to study as I start my independent investigator career, in order to understand and fix the molecular defects underlying rare and complex disease in skeletal and cardiac muscle. I will use these spectroscopic approaches to understand muscle structure and mechanical function and then apply these insights for the development of high-throughput assays for novel muscle disease therapies to improve muscle strength and cardiac performance.
- Ph.D. Physiology
- University of Wisconsin, Madison, Madison, Wisconsin, United States
- Ultrastructural basis for accelerated force development in myocardium due to phosphorylation of cMyBP-C
- M.S. Physiology
- University of Wisconsin, Madison, Madison, Wisconsin, United States
- B.S. Molecular Biology
- University of Wisconsin, Madison, Madison, Wisconsin, United States
- University of Minnesota, Minneapolis, Minnesota (2010 - 2015)
- University of Wisconsin, Madison, Wisconsin (2006)
- University of Wisconsin, Madison, Wisconsin (2002 - 2004)
- Precision Mouse Modeling Program
- UA GEMM Core, Fall 2017
- UA GEMM Core, Fall 2015
Licensure & Certification
- Graduate, National School on Neutron and X-ray Scattering, Argonne National Laboratories (2006)
Physiology, Complex Diseases, Biophysics, Biochemistry, Molecular Biology, Cardiovascular and Muscle Biology, Biomedical Engineering
Spectroscopy, Cardiovascular and Muscle Biology, Molecular Motors, Drug Discovery
Cardio Muscle Bio & DiseaseBME 484 (Spring 2019)
Cardio Muscle Bio & DiseaseCMM 584 (Spring 2019)
Cardio Muscle Bio & DiseaseMCB 484 (Spring 2019)
Cardio Muscle Bio & DiseasePSIO 484 (Spring 2019)
Cardio Muscle Bio & DiseasePSIO 584 (Spring 2019)
Scientific GrantsmanshipIMB 521 (Spring 2019)
Senior CapstoneBIOC 498 (Spring 2019)
Directed RsrchMCB 392 (Fall 2018)
Directed RsrchMCB 492 (Fall 2018)
Honors Independent StudyMCB 399H (Fall 2018)
Introduction to ResearchMCB 795A (Fall 2018)
ResearchCMM 900 (Fall 2018)
Senior CapstoneBIOC 498 (Fall 2018)
Cardio Muscle Bio & DiseasePSIO 484 (Spring 2018)
Cardio Muscle Bio & DiseasePSIO 584 (Spring 2018)
Crnt Tops in Translational MedCMM 604 (Spring 2018)
Directed ResearchBIOC 492 (Spring 2018)
Directed RsrchMCB 392 (Spring 2018)
Directed ResearchBIOC 392 (Fall 2017)
Directed ResearchCHEM 392 (Fall 2017)
Introduction to ResearchMCB 795A (Fall 2017)
Cardio Muscle Bio & DiseaseBME 484 (Spring 2017)
Cardio Muscle Bio & DiseaseBME 584 (Spring 2017)
Cardio Muscle Bio & DiseaseCMM 584 (Spring 2017)
Cardio Muscle Bio & DiseasePSIO 484 (Spring 2017)
Crnt Tops in Translational MedCMM 604 (Spring 2017)
Introduction to ResearchMCB 795A (Spring 2017)
Senior CapstoneBIOC 498 (Spring 2017)
Prin of Cell BiologyCMM 577 (Fall 2016)
Prin of Cell BiologyMCB 577 (Fall 2016)
Senior CapstoneBIOC 498 (Fall 2016)
Independent StudyBME 599 (Summer I 2016)
- 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.More infoMutations 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.
- 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.More infoThe 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.
- 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.More infoAcute 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.More infoWe 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.More infoImpairment 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.
- 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.More infoWe 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.
- 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.More infoWe 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.
- 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.More infoWe 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.More infoPhosphorylation 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.More infoWe 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.
- 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.More infoPhosphorylation 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.
- 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.More infoProtein 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.More infoMyosin 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.