David M Warshaw
University Of Vermont & St Agric College
Project start date: 2000-09-01
Project end date: 2012-03-31
Sponsored Links Excellgen http://Excellgen.com
Grants awarded to David M Warshaw
TRAINING PROGRAM IN THE MOLECULAR BASIS OF CARDIOVASCULAR DISEASE
David M Warshaw, Professor And Chair
University Of Vermont & St Agric College, 85 South Prospect Street, Burlington, Vt 05405
Grant 5T32HL007944-10 from National Heart, Lung, And Blood Institute
Abstract: This training program in the Molecular Basis of Cardiovascular Disease is the first competitive renewal of a program originally titled "Molecular Basis of Muscle Contraction". During the past 5 years we have added new mentors and thus re-titled the program to match its broader emphasis on cardiovascular function. The original program supported 3 pre- and 4 postdoctoral positions and matriculated 12 trainees (6 pre-, 6 postdoctoral), with the present request for one additional pre-doctoral position. The Training Program has been very successful in training under-represented minorities, including two African-Americans and one Hispanic out of the 11 total trainees. Our training program has a small but highly focused and collaborative faculty that share their science, resources, and students. The 20 program faculty span multiple departments but given their active collaboration, all 20 have primary or secondary appointments in the closely knit Department of Molecular Physiology & Biophysics and the Department of Pharmacology. These faculty provide the desired blend of junior and senior investigators in cardiovascular biology, with expertise ranging from single-molecule biophysics to whole-animal physiology. The core of our training program is hands-on experience, relentless mentoring, and an exciting scientific environment. The close proximity and the opportunities for day-to-day interaction among our faculty provide a "dream" environment for individuals wishing to enter or excel in this field. With the existing collaborations, trainees benefit by having multiple mentors and numerous laboratory colleagues. This proposal describes our successes in training both pre- and postdoctoral researchers, in attracting and recruiting a diverse applicant pool, as well as describing our approaches to future enhancements of the program. The program provides extensive opportunities for trainees to learn essential "survival skills" for a successful career in biomedical research. Yearly program retreats with external scientific review, seminar series, journal clubs, program courses in cardiovascular biology, and opportunities to experience the writing of NIH proposals equip trainees to present their science within manuscripts, grant proposals, and public forums. Cardiovascular disease is still the leading cause of death in the United States. Thus, the proposed continuation of a Training Program in the Molecular Basis of Cardiovascular Disease at the University of Vermont addresses the need to train scientists capable of contributing to future discoveries in this critical area. (End of )
Keywords: Cardiovascular Diseases; Molecular; Training Programs; base; cardiovascular disorder
Project start date: 2000-09-01
Project end date: 2011-03-31
Budget start date: 1-APR-2010
Budget end date: 31-MAR-2011
PFA/PA: PA-02-109
5T32HL007944-10 (2010): $231611
5T32HL007944-07 (2007): $381477
2T32HL007944-06 (2006): $258425
CARDIAC MYOSIN TRANSGENESIS:MOLECULAR DESIGN/PERFORMANCE
David M Warshaw, Professor And Chariman
University Of Vermont And St Agric College 85 South Prospect Street Burlington, Vt 05405
Grant 5R01HL066157-04 from National Heart, Lung, And Blood Institute IRG: CVA
Abstract: The power output of the heart is a key determinant that differentiates ventricular performance in patients with or without heart failure. Normal and abnormal cardiac function depends, in part, on the force and motion generating capacity of the myosin motor. We plan to study how myosin s molecular structure contributes to its enzymatic and mechanical function and how this then translates into the mechanical performance of the myocardium. We exploit the fact that there are two isoforms of myosin in heart muscle, V1 and V3, which are 95% homologous and exhibit marked differences in mechanical and enzymatic behavior (ATPase, shortening velocity, Average Force). The differing amino acids fall into clusters that are situated on the myosin molecule at functionally important sites (based on the S1 crystal structure). Two such sites of divergence are found in the surface loops that span the nucleotide binding pocket (loop 1) and the actin binding region (loop2). It has been hypothesized (Spudich, 1994) that loop 1 controls the rate of ATP binding and ADP release and thus shortening velocity while loop2 controls the rate of myosin binding to actin and thus the ATPase activity, while the combination of the two contributes to the average force. We will use transgenic techniques to produce mouse hearts with a series of chimeric myosins consisting of a V1 backbone with either a V3 loop 1, V3 loop2, or both V3 loops (1 + 2). The chimeric myosin will then be compared to myosin from wild type V1 hearts an transgenic V3 preparations. State-of-the-art in vitro motility and laser trap techniques will be used to determine the performance of these myosins at the level of a single myosin molecule (e.g. actin filament velocity, average force, unitary displacement, unitary force, attachment time). At a more organized level, skinned fiber and whole heart mechanics will provide detailed mechanical information regarding the translation of molecular mechanics to whole organ mechanics. These studies should provide important information about myosin s molecular structure and function and how alterations to myosin s molecular performance is mechanically expressed in the ventricular power output.
Keywords: biomechanics, cardiac output, enzyme mechanism, isozyme, myocardium, myosin, protein structure function, genetically modified animal, laboratory mouse
Project start date: 2001-02-01
Project end date: 2006-01-31
5R01HL066157-04 (2004): $405069
Molecular Mechanics Of FHC And DCM Mutant Actomyosin
David M Warshaw, Professor And Chariman
University Of Vermont And St Agric College 85 South Prospect Street Burlington, Vt 05405
Grant 5P01HL059408-080001 from National Heart, Lung, And Blood Institute IRG: HLBP
Abstract: The goal of this proposal is to provide a molecular basis for the clinical impact that point mutations to cardiac actin and myosin s molecular structure have on patients afflicted with familial hypertrophic (FHC) and dilated cardiomyopathy (DCM). FHC is characterized by a thick, hypercontractile ventricular wall, whereas DCM patients have ventricles that are thin and hypocontractile. To understand how point mutations within actin or myosin result in such drastically different pathologies, information about the molecular mechanics of the mutant actomyosin motor is required. Our approach will take advantage of both transgenic mice and the Baculovirus system to express mutant cardiac actin and myosin that have single amino acid substitutions found in either FHC or DCM. Using state-of-the-art laser trapping techniques in an in vitro motility assay in force clamp mode, we will measure the forcevelocity relationship of a small myosin ensemble (<50 molecules) as it interacts with a single actin or regulated thin filament. These data will provide an estimate of the maximum power that can be produced by a mutant actomyosin motor. Any alterations in power will be probed at the level of a single myosin molecule to determine if changes have occurred to the inherent motion generating capacity or to the rates of actomyosin transitions. Since the chosen mutations are localized throughout the myosin heavy chain (FHC R403Q, R453C, G741R; DCM S532P, F764L), we will directly pinpoint crucial intramolecular domains that are important to myosin s ability to generate force and motion. With actin being a key element in force production and thin filament regulation, mutations to actin that lead to FHC (E99K, A331P) and DCM (R312H, E361G) will be assessed for their impact on actin flexural rigidity, actomyosin power production, and thin filament regulation. These studies will help determine whether the impact of these point mutations on actomyosin s mechanical performance results in functional alterations that are common and distinct for a given form of hypertrophy so that one triggers a cascade of events resulting in either FHC or DCM.
Keywords: actin, asymmetric septal hypertrophy, gene mutation, hypertrophic myocardiopathy, myosin, aminoacid, binding site, biomechanics, intermolecular interaction, point mutation, protein structure function, clinical research, genetically modified animal, laboratory mouse, laser, transfection /expression vector
MOLECULAR MECHANICS OF FHC & DCM MUTANT ACTOMYOSIN
David M Warshaw, Professor And Chariman
University Of Vermont And St Agric College 85 South Prospect Street Burlington, Vt 05405
Grant 1R01HL075570-01 from National Heart, Lung, And Blood Institute IRG: CVA
Abstract: The goal of this proposal is to provide a molecular basis for the clinical impact that point mutations to cardiac actin and myosin s molecular structure have on patients afflicted with familial hypertrophic (FHC) and dilated cardiomyopathy (DCM). FHC is characterized by a thick, hypercontractile ventricular wall, whereas DCM patients have ventricles that are thin and hypocontractile. To understand how point mutations within actin or myosin result in such drastically different pathologies, information about the molecular mechanics of the mutant actomyosin motor is required. Our approach will take advantage of both transgenic mice and the Baculovirus system to express mutant cardiac actin and myosin that have single amino acid substitutions found in either FHC or DCM. Using state-of-the-art laser trapping techniques in an in vitro motility assay in force clamp mode, we will measure the forcevelocity relationship of a small myosin ensemble (<50 molecules) as it interacts with a single actin or regulated thin filament. These data will provide an estimate of the maximum power that can be produced by a mutant actomyosin motor. Any alterations in power will be probed at the level of a single myosin molecule to determine if changes have occurred to the inherent motion generating capacity or to the rates of actomyosin transitions. Since the chosen mutations are localized throughout the myosin heavy chain (FHC R403Q, R453C, G741R; DCM S532P, F764L), we will directly pinpoint crucial intramolecular domains that are important to myosin s ability to generate force and motion. With actin being a key element in force production and thin filament regulation, mutations to actin that lead to FHC (E99K, A331P) and DCM (R312H, E361G) will be assessed for their impact on actin flexural rigidity, actomyosin power production, and thin filament regulation. These studies will help determine whether the impact of these point mutations on actomyosin s mechanical performance results in functional alterations that are common and distinct for a given form of hypertrophy so that one triggers a cascade of events resulting in either FHC or DCM.
Keywords: actin, hypertrophic myocardiopathy, molecular genetics, myosin, point mutation, protein structure function, protein protein interaction, structural biology, tropomyosin, troponin, Baculoviridae, genetically modified animal, laboratory mouse
Project start date: 2003-12-15
Project end date: 2007-11-30
1R01HL075570-01 (2004): $378750
Molecular Mechanics Of FHC And DCM Mutant Actomyosin
David M Warshaw, Professor And Chariman
University Of Vermont & St Agric College
85 South Prospect Street
burlington, Vt 05405
Grant 5P01HL059408-090001 from National Heart, Lung, And Blood Institute IRG: HLBP
Abstract: The goal of this proposal is to provide a molecular basis for the clinical impact that point mutations to cardiac actin and myosin´s molecular structure have on patients afflicted with familial hypertrophic (FHC) and dilated cardiomyopathy (DCM). FHC is characterized by a thick, hypercontractile ventricular wall, whereas DCM patients have ventricles that are thin and hypocontractile. To understand how point mutations within actin or myosin result in such drastically different pathologies, information about the molecular mechanics of the mutant actomyosin motor is required. Our approach will take advantage of both transgenic mice and the Baculovirus system to express mutant cardiac actin and myosin that have single amino acid substitutions found in either FHC or DCM. Using state-of-the-art laser trapping techniques in an in vitro motility assay in force clamp mode, we will measure the forcevelocity relationship of a small myosin ensemble (<50 molecules) as it interacts with a single actin or regulated thin filament. These data will provide an estimate of the maximum power that can be produced by a mutant actomyosin motor. Any alterations in power will be probed at the level of a single myosin molecule to determine if changes have occurred to the inherent motion generating capacity or to the rates of actomyosin transitions. Since the chosen mutations are localized throughout the myosin heavy chain (FHC R403Q, R453C, G741R; DCM S532P, F764L), we will directly pinpoint crucial intramolecular domains that are important to myosin´s ability to generate force and motion. With actin being a key element in force production and thin filament regulation, mutations to actin that lead to FHC (E99K, A331P) and DCM (R312H, E361G) will be assessed for their impact on actin flexural rigidity, actomyosin power production, and thin filament regulation. These studies will help determine whether the impact of these point mutations on actomyosin´s mechanical performance results in functional alterations that are common and distinct for a given form of hypertrophy so that one triggers a cascade of events resulting in either FHC or DCM
Keywords: actin, asymmetric septal hypertrophy, gene mutation, hypertrophic myocardiopathy, myosin aminoacid, binding site, biomechanics, intermolecular interaction, point mutation, protein structure function clinical research, genetically modified animal, laboratory mouse, laser, transfection /expression vector
Sponsored Links Excellgen http://Excellgen.com
MOLECULAR MECHANICS OF FHC MUTANT MYSOSINS
David M Warshaw, Professor And Chariman
University Of Vermont And St Agric College 85 South Prospect Street Burlington, Vt 05405
Grant 5P01HL059408-050003 from National Heart, Lung, And Blood Institute
Abstract: The goal of this proposal is to provide a molecular basis for the clinical impact that mutations to cardiac myosin s molecular structure have on patients afflicted with familial hypertrophic cardiomyopathy (FHC). Our approach will take advantage of the transgenic mouse model to express mutant alpha-cardiac and the baculovirus system to express smooth muscle HMMs that have identical signal amino acid substitutions found in FHC patients. This comparative approach, where similar mutations have been genetically engineered into different myosin species, should provide far greater information about presumed key functional domains of the myosin molecule and the consequence of point mutations to these domains. Since the chosen mutations are localized throughout the myosin heavy chain and light chains, we will directly pinpoint crucial intramolecular atomic interactions that are important to myosin s ability to generate force and motion. Using state-of-the-art ultra-compliant microneedle and laser optical trapping techniques in an in vitro motility assay, we will directly measure the force and motion generating capacity of a small myosin ensemble (<50 molecules) and that of a single myosin molecule as it interacts with a single actin filament. These studies will provide the most direct assessment of how FHC point mutations might functionally impact myosin s mechanical performance at the molecular level.
Keywords: hypertrophic myocardiopathy, intermolecular interaction, muscle function, myosin, point mutation, protein engineering, protein isoform, protein structure function, biomechanics, microfilament, muscle strength, laboratory mouse, nanotechnology, transgenic animal
SMOOTH MUSCLE CROSSBRIDGE INTERACTIONS--MOTILITY ASSAY
David M Warshaw, Professor And Chariman
University Of Vermont & St Agric College
85 South Prospect Street
burlington, Vt 05405
Grant 5R01HL045161-04 from National Heart, Lung, And Blood Institute IRG: PHY
Abstract: Smooth muscle contraction is essential to the normal function of many organ systems within the body. Therefore, an understanding of its normal contractile process is required before studying disease states such as hypertension in which smooth muscle function may be abnormal. The ability of smooth muscle to sustain prolonged isometric contractions with very little energy consumption (i.e. ATP) may relate to the most basic contractile unit, the myosin crossbridge, and its cyclic interaction with actin. Although the smooth muscle crossbridge cycle may be qualitatively similar to that in skeletal muscle, its mode of regulation is quite different. Specifically, phosphorylation of smooth muscle´s 20kD myosin light chain initiates contraction and crossbridge cycling. However, the degree of light chain phosphorylation may also modulate the crossbridge cycling rate. To characterize the effect of light chain phosphorylation on the crossbridge cycling rate, single actin filament velocity will be measured as the actin interacts with synthetic smooth muscle myosin filaments containing known proportions of dephosphorylated and phosphorylated crossbridges. In addition, techniques will be developed for recording force sustained by a single actin filament as it interacts with a myosin coated glass coverslip. The motility assay provides a unique opportunity to probe the most basic contractile mechanism in muscle at the molecular level
Keywords: hormone regulation /control mechanism, hypertension, muscle contraction, smooth muscle actin, adenosine triphosphate, copolymer, myosin, phosphorylation laboratory rabbit, sulfhydryl group
Project start date: 1990-07-01
Project end date: 1995-06-30
5R01HL045161-04 (1993): $148709
5R01HL045161-03 (1992): $108466
SMOOTH MUSCLE CROSSBRIDGE INTERACTIONS--A MOTILITY ASSAY
David M Warshaw
University Of Vermont And St Agric College 85 South Prospect Street Burlington, Vt 05405
Grant 5P01HL028001-110008 from National Heart, Lung, And Blood Institute
Abstract: Smooth muscle contraction is essential to the normal function of many organ systems within the body. Therefore, an understanding of its normal contractile process is required before studying disease states such as hypertension in which smooth muscle function may be abnormal. The ability of smooth muscle to sustain prolonged isometric contractions with very little energy consumption (i.e. ATP) may relate to the most basic contractile unit, the myosin crossbridge, and its cyclic interaction with actin. Although the smooth muscle crossbridge cycle may be qualitatively similar to that in skeletal muscle, its mode of regulation is quite different. Specifically, phosphorylation of smooth muscle s 2OkD myosin light chain initiates contraction and crossbridge cycling. However, the degree of light chain phosphorylation may also modulate the crossbridge cycling rate. To characterize the effect of light chain phosphorylation on the crossbridge cycling rate, a motility assay has been developed in which the motion of single fluorescently labelled actin filaments will be measured as these filaments are propelled by synthetic smooth muscle myosin filaments containing known proportions of dephosphorylated and phosphorylated crossbridges. In addition, techniques will be developed for recording force sustained by a single actin filament as it interacts with a myosin coated glass coverslip. The motility assay provides a unique opportunity to probe the molecular regulation of smooth muscle contractile proteins.
Keywords: hypertension, molecular biology, muscle contraction, smooth muscle, actin, bioenergetics, crosslink, myosin, phosphorylation, fluorescent dye /probe, laboratory rabbit
SMOOTH MUSCLE CROSSBRIDGES--MOLECULAR MECHANICS
David M Warshaw, Professor And Chariman
University Of Vermont And St Agric College 85 South Prospect Street Burlington, Vt 05405
Grant 5R01AR042231-05 from National Institute Of Arthritis And Musculoskeletal And Skin Diseases IRG: PHY
Abstract: Smooth muscle contraction is essential to the normal function of many organ systems within the body. Therefore an understanding of its normal contractile process is required before studying disease states such as hypertension in which smooth muscle function may be abnormal. An intriguing mechanical property of smooth muscle is its ability to produce as much force per cross-sectional area of muscle as skeletal muscle with far less myosin (i.e. fewer cross-bridges). These data suggest that smooth muscle cross-bridges may generate more average force than skeletal muscle cross-bridges. Since force generation in these two muscle types is believed to result from the cyclic interaction of myosin cross-bridges with actin, smooth muscle cross-bridges may achieve a higher average force by spending a larger fraction of each cross-bridge cycle attached to actin in a high force producing state (i.e. higher duty cycle). To address this question, an in vitro motility assay will be used to directly measure both the velocity and force of a single fluorescently labeled actin filament as it slides over a myosin coated glass coverslip. This proposal begins to characterize smooth muscle force generation at the molecular level.
Keywords: biomechanics, muscle contraction, smooth muscle, actin, cytoskeletal protein, molecular biology, myosin ATPase, chicken, fluorescent dye /probe
Project start date: 1993-08-01
Project end date: 1999-07-31
5R01AR042231-05 (1997): $257886
5R01AR042231-03 (1995): $237646
Sponsored Links Excellgen http://Excellgen.com
5R01AR042231-02 (1994): $183686
1R01AR042231-01 (1993): $230568
CROSSBRIDGE CYCLE KINETICS IN SINGLE SMOOTH MUSCLE CELLS
David M Warshaw, Professor And Chariman
University Of Vermont And St Agric College 85 South Prospect Street Burlington, Vt 05405
Grant 5R01AR034872-08 from National Institute Of Arthritis And Musculoskeletal And Skin Diseases IRG: PHY
Abstract: Smooth muscle contraction is essential to normal function of many organ systems in the body. Therefore, an understanding of its normal contractile process is required before studying disease states such as hypertension in which smooth muscle function may be abnormal. Smooth muscle is characterized by its slow shortening velocity and economical useage of ATP during force production. These contractile properties may reflect both the mechanics of the basic force generating element, the myosin crossbridge, and its cyclic interaction with actin. To characterize crossbridge properties in smooth muscle, stare-of-the-art techniques will be used to measure mechanical responses from a single smooth muscle cell, isolated from the toad stomach. The single cell approach will avoid the heterogeneity of cellular responses that can occur in multicellular tissue preparations. To correlate a smooth muscle cell s contractile capabilities to its crossbridge properties, changes in cell length will be used as a means of probing the crossbridge cycle. In addition to mechanical perturbations, membrane permeabilized cells will provide the opportunity to modify the cell s interior chemical environment. Thus changes in the cell s free intracellular concentrations of specific ions (e.g. MgATP) and the effect these chemical perturbations have upon cell mechanics will help identify the specific steps in the crossbridge cycle. Finally, a mathematical model of the crossbridge cycle in smooth muscle, that incorporates the mechanical data obtained in this proposal, will be used to formulate a view of the crossbridge cycle in smooth muscle to help explain smooth muscle s slow, economical contraction.
Keywords: cell membrane, muscle cell, muscle contraction, smooth muscle, actin, adenosinetriphosphatase, biomechanics, hypertension, magnesium, mathematical model, mechanical pressure, muscle tension, myofibril, myosin, sarcomere, Anura, computer simulation
Project start date: 1984-12-01
Project end date: 1993-07-31
5R01AR034872-08 (1992): $152353
SMOOTH MUSCLE MYOSIN: MOLECULAR MECHANICS AND INTRAMOLECULAR COMMUNICATION
David M Warshaw, Professor And Chair
University Of Vermont & St Agric College, 85 South Prospect Street, Burlington, Vt 05405
Grant 5R01HL085489-04 from National Heart, Lung, And Blood Institute
Abstract: Smooth muscle cells line the walls of every blood vessel. It is their contractile function that is critical to the control of blood pressure and when altered leads to diseases such as hypertension. At the molecular level, smooth muscle contraction is the result of the myosin molecular motor and its cyclic interaction with actin, a process powered by myosin´s hydrolysis of ATP. Smooth muscle myosin is distinguished from the striated muscle myosins by its myosin phosphorylation-dependent regulation and force maintenance with little energy (i.e. ATP) expenditure. This proposal will investigate how smooth muscle myosin´s molecular structure defines its mechanical performance. We will combine the power of structural mutagenesis through the use of the Baculovirus expression system with state-of-the-art single molecule biophysical techniques such as the laser trap to assess how myosin´s double-headed structure contributes to phosphorylation-dependent regulation. In addition, mutant myosins will be designed that will help characterize the role of each of smooth muscle myosin´s two heads in generating maximal force and motion. All muscles respond to load by varying their speed of shortening. Therefore, we will identify the structural domains within the smooth muscle myosin molecule that sense load and how load modulates the various steps of myosin´s hydrolysis of ATP. Our initial focus will be on the myosin converter and lever arm domains. We will also take advantage of naturally occurring isoforms found in tonic (e.g. blood vessels) and phasic (e.g. intestine) smooth muscles, which have dramatically different contractile properties but with slight differences in their molecular structure. The differences are specifically a 7-amino acid insert in the myosin head and two essential light chain isoforms. These myosins will be characterized by applying load to single smooth muscle myosin molecules using a novel laser trap force clamp assay. The proposed experiments will provide insight to smooth muscle´s ability to maintain vascular tone with little energy expenditure. Since the myosin molecular motor is found in every smooth muscle cell and shares significant similarities to other muscle myosins, understanding smooth muscle myosins molecular structure and function will impact not only how we may treat diseases of the vasculature but cardiomyopathies as well
Keywords: ATP Hydrolysis; ATPase, Actin-Activated; ATPase, Actomyosin; Actins; Active Sites; Actomyosin Adenosinetriphosphatase; Adenosine Triphosphatase, Myosin; Adenosinetriphosphatase, Actomyosin; Affect; Affinity; Amino Acids; Arm; Arts; Assay; BP control; Baculovirus Expression System; Binding; Binding (Molecular Function); Bioassay; Biologic Assays; Biological Assay; Blood Pressure, High; Blood Vessels; Cardiomyopathies; Cell Line; Cell Lines, Strains; CellLine; Communication; Dependence; Disease; Disorder; Electromagnetic, Laser; Elements; Energy Expenditure; Energy Metabolism; Expenditure; Genetics-Mutagenesis; Head; Hypertension; Intestinal; Intestines; Investigators; Isoforms; Kinetic; Kinetics; Lasers; Leiomyocyte; Light; Macromolecular Structure; Maintenance; Maintenances; Measures; Mechanics; Methods and Techniques; Methods, Other; Molecular; Molecular Biology, Mutagenesis; Molecular Interaction; Molecular Motors; Molecular Structure; Motion; Muscle; Muscle Cell Contraction; Muscle Contraction; Muscle Tissue; Muscle, Involuntary; Muscle, Smooth; Muscular Contraction; Mutagenesis; Mutate; Mycocardium Disease; Myocardial Diseases; Myocardial Disorder; Myocardiopathies; Myocytes, Smooth Muscle; Myosin ATPase; Myosin Adenosinetriphosphatase; Myosins; Neck; Pathway interactions; Performance; Phosphorylation; Photoradiation; Point Mutation; Process; Programs (PT); Programs [Publication Type]; Property; Property, LOINC Axis 2; Protein Isoforms; Protein Phosphorylation; Radiation, Laser; Regulation; Research Personnel; Researchers; Role; Sites, Active; Skeletal Muscle Myosins; Smooth Muscle Cells; Smooth Muscle Myocytes; Smooth Muscle Myosins; Smooth Muscle Tissue Cell; Smooth muscle (tissue); Speed; Speed (motion); Striated Muscle Tissue; Striated Muscles; Structure; Techniques; Upper arm; Vascular Hypertensive Disease; Vascular Hypertensive Disorder; Work; aminoacid; blood pressure control; blood pressure homeostasis; blood pressure regulation; bowel; cultured cell line; design; designing; disease/disorder; experiment; experimental research; experimental study; hyperpiesia; hyperpiesis; hypertensive disease; insight; molecular mechanics; mutant; myocardium disorder; myosin ATP phosphohydrolase (actin translocating); novel; pathway; programs; research study; single molecule; social role; vascular
Project start date: 2006-08-15
Project end date: 2010-06-30
Budget start date: 1-JUL-2009
Budget end date: 30-JUN-2010
5R01HL085489-04 (2009): $368980
5R01HL085489-03 (2008): $368980
5R01HL085489-02 (2007): $368980
1R01HL085489-01 (2006): $380000
CARDIAC MYOSIN BINDING PROTEIN-C: STRUCTURE, FUNCTION, AND REGULATION
David M Warshaw, Professor And Chair
University Of Vermont & St Agric College, 85 South Prospect Street, Burlington, Vt 05405
Grant 2P01HL059408-11 from National Heart, Lung, And Blood Institute
Abstract: Myosin-binding protein C (MyBP-C) is a component of the thick filaments of vertebrate skeletal and cardiac muscle. Phosphorylafion of the cardiac isoform, cMyBP-C, plays a key role in modulating cardiac contractility in response to p-adrenergic stimulation. Mutations in cMyBP-C have been shown to be a prime cause of the cardiac disease, familial hypertrophic cardiomyopathy. Our long term goal is to understand the structural basis of cMyBP-C function. In this project electron microscopy and image processing will be used to elucidate the structure of the molecule, its organization in the sarcomere, its interaction with thin filaments, and the changes that occur when it is phosphorylated. Experiments will make use of expressed mutant and wild type cMyBP-C molecules and N-terminal fragments (produced in Core C), native thick filaments from wild type and transgenic hearts (Core C), and intact muscle. Three specific aims will be addressed. (1) How is cMyBPC organized at the molecular, thick filament and sarcomeric level? We will determine whether the MyBP-C molecule has specific structural features required to carry out its function, whether it wraps around, extends along, or projects away from the filament surface to interact with neighboring filaments, and how it influences myosin head organization. (2) What is the structural basis of cMyBP-C´s modulation of acfin-myosin interaction? We will determine whether cMyBP-C competes with myosin heads for actin binding, and whether it influences the position of tropomyosin on thin fliaments at high or low Ca^* levels. (3) What are the structural effects of cMyBP-C phosphorylation? We will determine whether phosphorylation alters cMyBP-C flexibility, thick filament structure (e.g. head conformation), and its interaction with thin filaments. These goals will be achieved using negative stain, cryo-EM, antibody labeling and muscle sectioning approaches, combined with single particle, helical and tomographic 3D reconstruction techniques. Results will be correlated with, and structurally underpin, parallel single molecule biophysics experiments (Project 2) and whole heart functional data (Project 3, Core B). The project will provide new insights into the structural mechanisms by which cMyBP-C functions in the heart. (End of ) INDIVIDUAL PROJECTS AND CORE UNITS PROJECT 1. cMyBP-C and Native Thick Filament Structure (Craig, Roger) RESUME AND SUMMARY OF DISCUSSION Mutations in cardiac cMYBPC are responsible for at least 40% of the cases of FHC, but the way in which normal cMYBPC modulates contractility and the mutant forms produce the cardiomyopathy is not understood. This project asks important questions about the structure and its relation to function of cMYBPC, and its influence on the structure of the thick filament and the sarcomere in cardiac muscle. Dr Craig has been a leader in the study of the functional implications of structure in striated muscle and is very well qualified to carry out these studies. The paper from his lab in PNAS 2008 is a land mark report, describing the difference in the structure of isolated thick filaments from WT and KO preparations. Some concern was expressed about the absence of data from studies of cardiac muscle demonstrating the existence of sufficient resolving power to rigorously answer the proposed questions. However it was the consensus that the group of investigators is well qualified to produce important new information about the structure-function relations of this protein. Myosin-binding protein C (MyBP-C) is a component of the thick filaments of vertebrate skeletal and cardiac muscle. Phosphorylafion of the cardiac isoform, cMyBP-C, plays a key role in modulating cardiac contractility in response to p-adrenergic stimulation. Mutations in cMyBP-C have been shown to be a prime cause of the cardiac disease, familial hypertrophic cardiomyopathy. Our long term goal is to understand the structural basis of cMyBP-C function. In this project electron microscopy and image processing will be used to elucidate the structure of the molecule, its organization in the sarcomere, its interaction with thin filaments, and the changes that occur when it is phosphorylated. Experiments will make use of expressed mutant and wild type cMyBP-C molecules and N-terminal fragments (produced in Core C), native thick filaments from wild type and transgenic hearts (Core C), and intact muscle. Three specific aims will be addressed. (1) How is cMyBPC organized at the molecular, thick filament and sarcomeric level? We will determine whether the MyBP-C molecule has specific structural features required to carry out its function, whether it wraps around, extends along, or projects away from the filament surface to interact with neighboring filaments, and how it influences myosin head organization. (2) What is the structural basis of cMyBP-C´s modulation of acfin-myosin interaction? We will determine whether cMyBP-C competes with myosin heads for actin binding, and whether it influences the position of tropomyosin on thin fliaments at high or low Ca^* levels. (3) What are the structural effects of cMyBP-C phosphorylation? We will determine whether phosphorylation alters cMyBP-C flexibility, thick filament structure (e.g. head conformation), and its interaction with thin filaments. These goals will be achieved using negative stain, cryo-EM, antibody labeling and muscle sectioning approaches, combined with single particle, helical and tomographic 3D reconstruction techniques. Results will be correlated with, and structurally underpin, parallel single molecule biophysics experiments (Project 2) and whole heart functional data (Project 3, Core B). The project will provide new insights into the structural mechanisms by which cMyBP-C functions in the heart. (End of )
Keywords: No Project Terms available
Project start date: 2000-02-01
Project end date: 2015-01-31
Budget start date: 1-FEB-2010
Budget end date: 31-JAN-2011
2P01HL059408-11 (2010): $2181684
FAMILIAL HYPERTROPHIC CARDIOMYOPATHY--SARCOMERIC DISEASE
David M Warshaw, Professor And Chariman
University Of Vermont And St Agric College 85 South Prospect Street Burlington, Vt 05405
Grant 5P01HL059408-05 from National Heart, Lung, And Blood Institute IRG: HLBP
Abstract: Familial hypertrophic cardiomyopathy (FHC), an inherited disease with a high incidence of premature death due to cardiac failure, has its genetic loci in the contractile proteins in the heart. Thus, FHC may be a disease of the sarcomere, muscle s most basic contractile unit. In the sarcomere, myosin, a molecular motor, interacts with actin to generate the power of the heart. This Program Project (3 projects and 3 cores) focuses on mutations to myosin and the actin regulatory proteins, troponinT, and tropomyosin. Using state-of-the-art techniques, we will characterize FHC from the mechanics of the whole heart down the molecular mechanics of a single contractile protein, to assess how structural alterations to these proteins affect the mechanical properties of the sarcomere, the muscle fiber, and the whole heart. Project #1 will study the mechanical properties of the whole heart and papillary muscles obtained from transgenic mice with mutations in either myosin, troponinT, or alpha- tropomyosin. Project #2 will genetically engineer FHC mutations into myosin using an in vitro protein expression system. Project #2 will biochemically characterize these proteins, while project #3 will use the laser optical trap to assess the force and motion generating capacity of these mutants at the single molecule level. All projects use the Analytical and Modeling Core (Unit B) for expertise in data collection, analysis, and modeling. In addition, the Mouse Production and Ventricular Function Core (Unit C) will generate mice with FHC mutant hearts that will be studied at all anatomical levels by the various projects. In addition, this Core will also characterize the in vivo ventricular performance of the hearts within the transgenic mice. The long-term goals are 1) to utilize FHC-related point mutations as a means of identifying key structural domains within the mutant sarcomeric proteins and to determine how these domains relate to the protein s molecular function; 2) to understand how point mutations in contractile proteins compromise sarcomere function, and how these mutations, in turn, may trigger cardiac hypertrophy.
Keywords: hypertrophic myocardiopathy, sarcomere
Project start date: 1999-02-18
Project end date: 2004-12-14
5P01HL059408-05 (2003): $1568713
5P01HL059408-04 (2002): $1538554
Sponsored Links Excellgen http://Excellgen.com
5P01HL059408-03 (2001): $1449031
5P01HL059408-02 (2000): $1241193
1P01HL059408-01A1 (1999): $1265002
Molecular Basis Of Dilated & Hypertrophic Cardiomyopathy
David M Warshaw, Professor And Chariman
Molecular Physiology And Biophysicsuniversity Of Vermont & St Agric College
Grant 5P01HL059408-10 from National Heart, Lung, And Blood Institute IRG: HLBP
Abstract: Familial hypertrophic (FHC) and dilated cardiomyopathy (DCM) are distinct forms of cardiac hypertrophy that progress to heart failure. FHC is characterized by a thick, hypercontractile left ventricule, whereas DCM patients have thin and hypocontractile ventricles. Both FHC and DCM can result from genetic mutations to either cardiac myosin or actin. Thus, both FHC and DCM can be classified as diseases of the sarcomere, the most basic contractile unit of muscle. In the sarcomere, myosin, a molecular motor, interacts with actin to generate the power of the heart. This Program Project (3 Projects and 3 cores) focuses on mutations to myosin and actin. Using state-of-the-art techniques, we will characterize both FHC and DCM from the mechanics of the whole heart down to the molecular mechanics of a single contractile protein, to assess how structural alterations to these proteins affect the mechanical properties of the sarcomere, the muscle fiber, and the whole heart. Project 1 will use the laser trap to assess the force and motion generation of these mutants at the single molecule level. Project 2 will biochemically and structurally characterize these proteins, while Project 3 will study the mechanical properties of skinned muscle fibers and myofibrils from trans-genic mouse hearts with mutations in either myosin or actin. Project 2 will genetically engineer FHC and DCM muta-tions into myosin and actin using in vitro expression systems. The Technologies for Experimentation, Modeling and Analysis Core (Core B) will support the projects in their data collection, analysis, modeling, and biological preparation development. The Mouse Production and Ventricular Function Core (Core C) will generate mice with FHC and DCM mutant hearts to be studied at all anatomical levels by the various projects. This Core will also characterize the in vivo and in vitro ventricular performance of the transgenic hearts over time as the disease phenotype develops. The long term goals are 1) to utilize FHC and DCM-related point mutations as a means of identifying key structural domains within the mutant sarcomeric proteins and to determine how these domains relate to the protein´s molecular function; 2) to understand how point mutations in contractile proteins alter sarcomere and cardiac function so that two drastically different cardiac hypertrophies, i.e. FHC and DCM, develop
Keywords: actin, asymmetric septal hypertrophy, gene mutation, hypertrophic myocardiopathy, idiopathic dilated cardiomyopathy, myosin
Project start date: 2000-02-01
Project end date: 2009-11-30
Molecular Basis Of Dilated And Hypertrophic Cardiomyopathy
David M Warshaw, Professor And Chariman
University Of Vermont And St Agric College 85 South Prospect Street Burlington, Vt 05405
Grant 5P01HL059408-08 from National Heart, Lung, And Blood Institute IRG: HLBP
Abstract: Familial hypertrophic (FHC) and dilated cardiomyopathy (DCM) are distinct forms of cardiac hypertrophy that progress to heart failure. FHC is characterized by a thick, hypercontractile left ventricule, whereas DCM patients have thin and hypocontractile ventricles. Both FHC and DCM can result from genetic mutations to either cardiac myosin or actin. Thus, both FHC and DCM can be classified as diseases of the sarcomere, the most basic contractile unit of muscle. In the sarcomere, myosin, a molecular motor, interacts with actin to generate the power of the heart. This Program Project (3 Projects and 3 cores) focuses on mutations to myosin and actin. Using state-of-the-art techniques, we will characterize both FHC and DCM from the mechanics of the whole heart down to the molecular mechanics of a single contractile protein, to assess how structural alterations to these proteins affect the mechanical properties of the sarcomere, the muscle fiber, and the whole heart. Project 1 will use the laser trap to assess the force and motion generation of these mutants at the single molecule level. Project 2 will biochemically and structurally characterize these proteins, while Project 3 will study the mechanical properties of skinned muscle fibers and myofibrils from trans-genic mouse hearts with mutations in either myosin or actin. Project 2 will genetically engineer FHC and DCM muta-tions into myosin and actin using in vitro expression systems. The Technologies for Experimentation, Modeling and Analysis Core (Core B) will support the projects in their data collection, analysis, modeling, and biological preparation development. The Mouse Production and Ventricular Function Core (Core C) will generate mice with FHC and DCM mutant hearts to be studied at all anatomical levels by the various projects. This Core will also characterize the in vivo and in vitro ventricular performance of the transgenic hearts over time as the disease phenotype develops. The long term goals are 1) to utilize FHC and DCM-related point mutations as a means of identifying key structural domains within the mutant sarcomeric proteins and to determine how these domains relate to the protein s molecular function; 2) to understand how point mutations in contractile proteins alter sarcomere and cardiac function so that two drastically different cardiac hypertrophies, i.e. FHC and DCM, develop.
Keywords: actin, asymmetric septal hypertrophy, gene mutation, hypertrophic myocardiopathy, idiopathic dilated cardiomyopathy, myosin
Project start date: 2000-02-01
Project end date: 2009-11-30
5P01HL059408-08 (2007): $2098335
5P01HL059408-07 (2006): $1971638
2P01HL059408-06A1 (2005): $1990786
Cardiac Myosin Binding Protein-C: Molecular Mechanisms Of Actomyosin Modulation
David M Warshaw, Professor And Chariman
Molecular Physiology And Biophysicsuniversity Of Vermont & St Agric College
Grant 5R01HL086728-02 from National Heart, Lung, And Blood Institute IRG: CCHF
Abstract: Cardiac Myosin Binding Protein-C (cMyBP-C) is critical to normal cardiac performance as evidenced by genetic mutations in cMyBP-C being one of the leading causes of familial hypertrophic cardiomyopathy. Despite its functional importance, the molecular mechanism by which cMyBP-C exerts its effect on the myosin molecular motor as it interacts with actin to generate force and motion remains largely undefined. A yet unanswered question is with its low ratio relative to myosin and it being located in distinct regions of the thick filament, we will determine how cMyBP-C´s modulates actomyosin´s power generation by interacting with only a limited population of crossbridges within the thick filament. To address this in Aim #1, we will use state-of- the-art single molecule biophysical techniques (e.g. laser trap assay) to probe the effect that cMyBP-C exerts on actomyosin function along the length a single native thick filament. In Aim #2, we will use expressed N-terminal fragments of cMyBP-C to probe the binding affinity of these fragments for actin, the regulated thin filament, and/or myosin. In combination with motility and laser trap assays, we will determine if the N- terminus of CMyBP-C limits myosin´s attachment rate to the thin filament or if it directly affects myosin´s inherent molecular mechanics and kinetics. Finally, in Aim #3 we will characterize how phosphorylation regulates cMyBP-C action. Using existing transgenic mouse models expressing cMyBP C mutants having alanine or aspartic acid substitutions for all three phosphorylatable serines, we will determine the functional importance of phosphorylation using native thick filaments containing mutant cMyBP-C as well as N-terminal fragments having the same mutations. Once the molecular mechanism of cMyBP-C is defined, the potential for novel therapeutics or clinical intervention may be possible in cases of heart failure associated with genetic mutations in cMyBP-C. PUBLIC HEALTH RELEVANCE Cardiac Myosin Binding Protein-C (cMyBP-C) is critical to normal cardiac performance as evidenced by genetic mutations in cMyBP-C being one of the leading causes of familial hypertrophic cardiomyopathy. Despite its functional importance, the molecular mechanism by which cMyBP-C exerts its effect on the myosin molecular motor as it interacts with actin to generate force and motion remains largely undefined
Project start date: 2008-08-01
Project end date: 2012-05-30
Mechanics And Enzymology Of A Single Myosin Motor
David M Warshaw, Professor And Chariman
University Of Vermont And St Agric College 85 South Prospect Street Burlington, Vt 05405
Grant 5P01AR047906-020002 from National Institute Of Arthritis And Musculoskeletal And Skin Diseases IRG: ZAR1
Abstract: Biological motility, ranging from muscle contraction to intracellular vesicular to transport, can be attributed to the myosin molecular motor. At present, there are 15 distinct classes of myosin motors. At present, there are distinct classes of myosin motors. All myosins have the ability to convert energy from the hydrolysis of ATP into mechanical work as they cyclically interact with actin. Although much is known about myosin s mechanical performance, two major questions remain 1) How is the chemistry of ATP hydrolysis coupled to force and motion generation at the molecular level 2) What is the structural basis for the mechanics of the myosin motor? To address these questions, we have developed a laser trap-total internal reflectance (TIRF) microscope? This instrument has the capacity to measure the force and generation generated by a single myosin molecule as it interacts with an actin filament, as well as simultaneously measuring fluorescence and polarization of a single fluorophore. Using the laser trap-TIRF microscope, we propose to determine 1) How force and motion are coupled to specific steps in the hydrolysis of MgATP by measuring single myosin molecular motor displacements simultaneously with fluorescence data that indicate the presence of fluorescently-labeled MgATP and its hydrolysis products in the myosin active site; 2) What is the structural basis for the mechanics of the myosin motor? To address these questions, we have developed a laser trap-total internal reflectance (TIRF) microscope. This instrument has the capacity to measure the force and motion generated by a single myosin molecule as it interacts with an actin filament, as well as simultaneously measuring fluorescence and polarization of a single fluorophore. Using laser trap-TIRF microscope, we propose to determine 1) How force and motion are coupled to specific steps in the hydrolysis of MgATP by measuring single myosin molecular motor displacements simultaneously with fluorescence data that indicate the presence of fluorescently-labeled MgATP and its hydrolysis products in the myosin active site; 2) If the myosin light chain binding domain acts a lever that rotates during the myosin powerstroke, then by fluorescently-labeling the regulatory light chain, we will be able to correlate fluorescence polarization measurements with unitary displacements as a means of determining whether or not the light chain binding domain rotates during the myosin powerstroke. In addition, the mechanics of Myosin V neck length mutants will be studied to test the lever arm hypothesis.
Keywords: actin, biomechanics, enzyme mechanism, myosin, physical chemical interaction, protein binding, protein protein interaction, structural biology, microfilament, fluorescence microscopy, fluorescent dye /probe, microscopy, polarimetry
Training In The Molecular Basis Of Muscle Contraction
David M Warshaw, Professor And Chariman
University Of Vermont And St Agric College 85 South Prospect Street Burlington, Vt 05405
Grant 5T32HL007944-05 from National Heart, Lung, And Blood Institute IRG: ZHL1
Abstract: Adapted from applicant s ) Within the Department of Molecular Physiology and Biophysics at the University of Vermont, a core group of researchers has long focused their work on the mechanisms of muscle contraction. New approaches, including molecular genetics, protein crystallography, single molecule mechanics, and myosin fluorescence studies have expanding both the depth and the breadth of this research. Additional perspectives are brought by investigators dedicating their work to the mechanisms of motor-protein based disease (for example Familial Hypertrophic Cardiomyopathy). This multidisciplinary and multidepartmental cluster of laboratories therefore both spans and integrates investigations of muscle mechanisms from individual molecules through whole organisms. This group forms a "dream" environment for training at either the pre- or postdoctoral level, with "down-the-hall" access to an unprecedented spectrum of state-of-the-art methodologies and world-class expertise. Ongoing activities with the Department of Physiology, and in collaboration with other departments, provide an environment for continued research career development. Besides the regular graduate courses, s have several ongoing study groups that meet regularly in specifically focusses areas. Although this group has also been active for many years in training new investigators, the size of the program has always been small. They currently have 5 graduate students and 4 post-doctoral fellows working within the 18 fully established laboratories that constitute this program. It is proposed here to increase the participation of young researchers by developing a specifically targeted training program in the molecular basis of muscle contraction. s are requesting three positions for pre- graduate trainees, who will join the existing Ph.D. program within the Department of Molecular Physiology and Biophysics for a 4-5 year program. In addition, they are seeking support for 4 postdoctoral fellows to join individual laboratories, and to facilitate the interactivity of the group by developing projects that span different labs.
Project start date: 2000-09-01
Project end date: 2006-03-31
5T32HL007944-05 (2004): $89961
Sponsored Links Excellgen http://Excellgen.com
David M Warshaw
University Of Vermont & St Agric College
Project start date: 2000-02-01
Project end date: 2015-01-31
SMOOTH MUSCLE CROSSBRIDGES--MOLECULAR MECHANICS
David M Warshaw, Professor And Chariman
Physiology And Biophysicsuniversity Of Vermont & St Agric College
85 South Prospect Street
burlington, Vt 05405
Grant 5R01AR042231-04 from National Institute Of Arthritis And Musculoskeletal And Skin Diseases IRG: PHY
Project start date: 1993-08-01
Project end date: 1998-07-31
5R01AR042231-04 (1996): $247153