1999 — 2009 |
Regnier, Michael |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Cardiac Contractile Kinetics @ University of Washington
DESCRIPTION (provided by applicant): Heart Disease is the leading cause of death in the United States and pathologies such as diabetes, hypertrophic cardiomyopathy, hypothyroidism and heart failure, as well as ischemia/reperfusion injury, involve alterations in myocardial contractile and regulatory proteins. The long range goal of our research is to understand the molecular mechanisms of contractile activation in cardiac and skeletal muscle. Contractile activation in both muscle types depends on 1) calcium binding to the thin filament and 2) myosin crossbridges that promote continued thin filament activation. However, evidence from this grant suggests the relative contribution of the two activation processes differs between skeletal and cardiac muscle, with skeletal muscle more dependent on calcium activation while cardiac muscle is more dependent on activation by strongly-bound crossbridges. These differences likely provide a mechanism for greater cellular level contractile control that is required for beat-to-beat regulation of cardiac output. Direct evidence to support this idea is difficult to obtain, in part due to an inability to alter crossbridge binding and calcium binding properties independently of one another. Our proposed experiments are specifically designed to separate calcium- and crossbridge-dependent components of activation. An additional strength of our research is that, under these conditions, we us a multiplicity of approaches to study the kinetic interactions between contractile and regulatory proteins in muscle cells. These include genetic engineering techniques, pharmaceutical interventions that alter the kinetics of Ca2+ binding to troponin C (TnC) or trap myosin in different crossbridge states, and use of mechanical transients and caged compounds to measure rates of chemo-mechanical steps in the CB cycle and isolated protein mechanical measurements. Additionally, x-ray diffraction will monitor myofilament structural changes and be coupled with mechanical measurements to determine how altered crossbridge kinetics influence activation processes. Specific aims of the proposal will investigate cardiac vs. skeletal differences in 1) the relative ability of strongly-bound cross-bridges to promote thin filament activation and force development kinetics;2) the influence of calcium binding kinetics on thin filament activation and the rate force develops;3) the dependence of activation processes on troponin isoforms;and 4) the dependence of activation on-tropomyosin isoforms. Results from this work will provide novel information regarding activation processes in cardiac muscle that allow fine-tuning of contractility at the cellular level in response to beat-to-beat alterations in venous return that can fail in cardiomyopathies.
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1 |
2001 — 2012 |
Regnier, Michael |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Cooperative Thin Filament Activation in Striated Muscle @ University of Washington
DESCRIPTION (provided by applicant): Heart disease is the leading cause of death in the United States. To develop targeted therapeutic approaches for heart and skeletal muscle diseases it is critical to first understand the molecular mechanisms regulating contractile activation and relaxation. We study how different isoforms of myofilament proteins manifest in the different functional properties of the two striated muscle types and how alterations in these proteins lead to changes in function. Progress from this award has clearly demonstrated mechanistic differences in the cooperative activation of cardiac vs. skeletal muscle that suggest different strategies may be needed to improving contraction in impaired tissue. These differences may allow for greater cellular level of controlling force in cardiac muscle, which is required to match stroke volume with venous return on a beat-to-beat basis, i.e. the Frank-Starling Law of the Heart. Additionally, two approaches developed in this award have led to gene delivery-based methods of improving cardiomyocyte function, which is the subject of a new R21 award. In the current proposal we continue to use parallel experiments with cardiac and skeletal systems to study troponin subunit interactions and their role in setting the Ca2+sensitivity of myofilament contraction, and will look for additional mutants that improve cardiac and skeletal muscle function. Our experiments use a variety of recombinant protein mutants, sophisticated solution biochemical techniques and functional analysis at the level of individual myofilaments, myofibrils cells and tissue. In addition to activation and SL- dependence of contraction, we will now study how troponin subunit interaction properties affect relaxation. This is an understudied but quite important question, in as much as 50% of heart failure may be attributable to diastolic dysfunction. Another new area for the proposal is to study how tropomyosin and myosin isoform composition may influence to coordinated activity of myofilament proteins in determining activation and relaxation kinetics. Finally we will develop a novel 3D-myofilament half- sarcomere model that is 1) spatially explicit and contains the stochastic kinetics of cycling crossbridges and thin filament dynamics and 2) incorporates radial forces associated with actomyosin interaction for more realistic simulation of the SL-dependence of force and understanding of the mechanic-kinetic aspects of relaxation. PUBLIC HEALTH RELEVANCE: A myriad of cardiac and skeletal muscle diseases involve compromised contractility. Our work focuses on understanding the detailed mechanisms of contractile activation and regulation in these two muscle types, with the long-term goal of finding targeted approaches to improve contractile function. The work uses a combination of recombinant DNA technology, sophisticated protein solution characterization techniques, and mechanical measurements of isolated myofilament, myofibrils and muscle cells to study these mechanisms and develop approaches that improve contractile performance.
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1 |
2004 — 2019 |
Regnier, Michael |
T32Activity Code Description: To enable institutions to make National Research Service Awards to individuals selected by them for predoctoral and postdoctoral research training in specified shortage areas. |
Bioengineering Cardiovascular Training Grant @ University of Washington
[unreadable] DESCRIPTION (provided by applicant): The purpose of this program is to provide a broad spectrum of training for individuals committed to research careers in Bioengineering. The Bioengineering program at the University of Washington has a long-standing, strong tradition of training graduate students. This proposal builds on the 25 year long Bioengineering training program history in cardiovascular studies, involving a high degree of interaction with Bioengineering's clinical and research colleagues at the University of Washington. Cardiovascular diseases are the leading cause of mortality in the United States. To address this health problem will require training outstanding scientists with a variety of backgrounds and strengths that can provide new and innovative approaches to the study, diagnosis and treatment of cardiovascular disease. This application focuses specifically on four areas of training opportunities that are established strengths at the University of Washington: 1) Computational and Integrative Bioengineering; 2.) Diagnostic Imaging; 3.) Integrated Physiological Function, and; 4.) Therapeutic Technology Development. We are requesting support for six graduate students and five post-doctoral fellows or physician scientists interested in applying engineering and quantitative approaches to cardiovascular research. Predoctoral applicants will be selected from amongst highly qualified students, mainly in Bioengineering and basic health sciences, after they have passed their Qualifying Examinations. However, the program is also open to all individuals with background training in physics, mathematics, chemistry, biology, medicine and the life sciences. The goal is to provide the research, communication and professional skills required to develop careers as independent, laboratory-based principal investigators. [unreadable] [unreadable] A multi-disciplinary group of twenty-four faculty comprises the training team. These faculty have an exceptional record of interdisciplinary research and a long-term commitment to student training. Several outstanding junior faculty compliment the team. In addition to training students and post-doctoral fellows, mentoring these junior faculty is critical to the successful establishment, development and expansion of our program. The administration of the program will be mainly through the Department of Bioengineering, which is jointly in the School of Medicine and the College of Engineering. [unreadable] [unreadable]
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1 |
2008 — 2009 |
Murry, Charles E (co-PI) [⬀] Regnier, Michael |
R21Activity Code Description: To encourage the development of new research activities in categorical program areas. (Support generally is restricted in level of support and in time.) |
Calcium-Independent Enhancement of Contractility in Normal and Failing Cardiomyoc @ University of Washington
DESCRIPTION (provided by applicant): Heart disease is the leading cause of death in the United States and has been rising dramatically around the world. This is an exploratory application to develop strategies for improving cardiac performance by targeting the contractile apparatus of cardiomyocyte sarcomeres. While the current application focuses on in vitro studies, the long term goal is to enhance systolic function without compromising diastolic function of the heart, and still allow responsiveness to adrenergic stimulation. We will target the cardiac thin filament activation and the actin-myosin `crossbridge'cycle directly, such that cardiomyocyte contraction (but not relaxation) is enhanced without the need for increased intracellular [Ca2+]. To increase thin filament activation at a given [Ca2+] we will replace native troponin C in myofilaments with a mutant TnC (L48Q) that has enhanced Ca2+ binding properties (aim 1). To enhance crossbridge cycling we will increase the cellular production of 2 deoxy-ATP (dATP) via increased expression of the enzyme that converts ATP to dATP in cardiomyocytes (ribonucleotide reductase;RR) (aim 2). We have provided significant data demonstrating these approaches improve contractility in demembranated (skinned) cardiac tissue, without affecting relaxation kinetics or resting stiffness. For the proposed studies we will use a viral transfection strategy to determine if similar increases in cardiac contractility occur in intact adult cardiomyocytes in culture. We will also determine whether these approaches affect sarcomere contractile protein isoform and phosphorylation profiles or the level of intracellular [Ca2+] during stimulated activation and in relaxation. In the second year of the proposal we will begin development of animal models for an expanded proposal to study how these manipulations influence whole heart function in situ and in vitro in normal hearts and under pathological conditions. PUBLIC HEALTH RELEVANCE: Heart failure at its base is a reduction in cardiac myofilament contractility. Most current therapies focus on mechanisms that enhance intracellular Ca2+ during systole which, among other things, can affect diastolic function. To avoid this, our proposal targets myofilaments directly to enhance contraction without the need for increased intracellular Ca2+.
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2011 |
Regnier, Michael |
P41Activity Code Description: Undocumented code - click on the grant title for more information. |
Role of Phosphorylation of Ctnl in the Regulation of Myocardium @ Illinois Institute of Technology
This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. [unreadable]-adrenergic modulation of cardiac contractility occurs, in part, through protein kinase A (PKA) myofibrillar protein phosphorylation. PKA targets the N-terminus of cardiac troponin I (cTnI), cardiac myosin-binding protein C (cMyBP-C) and titin. To isolate the effects of cTnI phosphorylation from cMyBP-C/titin phosphorylation on force-[Ca2+] relations, endogenous cardiac troponin (cTn) in rat skinned trabeculae was passively exchanged with either WT cTn, cTn containing a non-phosphorylatable cTnI(S23/24A) mutant, or a phosphomimetic cTnI(S23/24D) mutant. PKA cannot phosphorylate either cTnI mutant, thus leaving MyBP-C and titin as the sole PKA targets in those preparations. Force-[Ca2+] relationships and Ca2+-sensitivity (pCa50) were measured at 2.3 and 2.0 [unreadable]m SL. pCa50 was similar between WT Tn and Tn containing cTnI(S23/24A) trabeculae at both long and short SL, with decreased pCa50 at short SL. PKA treatment of WT and cTnI(S23,24A) exchanged trabeculae reduced pCa50 at both SL, but to a greater extent 2.3 um SL, eliminating the SL-dependence of pCa50 for both conditions. Exchange with cTn containing cTnI(S23/24D) reduced pCa50 at both SL (compared to WT and cTnI(S23,24A)) and also eliminated the influence of SL on pCa50. In summary, phosphorylation of cTnI and cardiac C-protein/titin both independently reduced pCa50, but to greater extents at longer SLs, thus reducing length dependence of Ca2+ sensitivity of force. In order to get insight into why this is so, small-angle x-ray diffraction was performed to determine whether these shifts in pCa50 were associated with changes in myofilament spacing or interaction.
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0.912 |
2012 — 2014 |
Regnier, Michael |
R25Activity Code Description: For support to develop and/or implement a program as it relates to a category in one or more of the areas of education, information, training, technical assistance, coordination, or evaluation. |
Building Bridges to Bioengineering @ University of Washington
DESCRIPTION (provided by applicant): Building Bioengineering Bridges (B3) will work with underrepresented minority students from Seattle Community College as they transition to baccalaureate degree programs at the University of Washington (UW) and other four-year universities. The B3 program will focus on how bioengineering and biotechnology can be used to solve global health problems. The B3 program will provide both academic opportunities and mentored research experiences for students and will include workshops and seminars to provide students will experience giving scientific presentations. The program will provide students with the background and experiences necessary to successfully transition from their community college to four-year universities. Through this effort, the partnering institutions will provide a source of outstanding URM students' interested biomedical research to a variety of departments within the UW and other universities. The B3 program will involve 10-20 African American, Hispanic American, Native American, and/or Natives of the US Pacific Islands students who are currently enrolled at Seattle Central Community College (SCCC). These students will enroll in a new course Biotechnology & World Health. Students will also attend workshops and seminars presentations to gain experience presenting scientific research to various audiences. These experiences will provide students with the skills necessary to give scientific poster and oral presentations and to write for technical journals. A cohort of students who complete this course will be offered mentored-laboratory experiences within laboratories in the Department of Bioengineering, Material Sciences or other departments at the UW. Students who participate in the mentored laboratory program will present their work with other undergraduate students enrolled in other UW programs during a summer research symposium. We expect that in five years: a) the overall institutional transfer rate of students from targeted groups/populations from the participating associate degree-granting institution(s) to baccalaureate degree programs in biomedical/behavioral sciences will increase by 50%; b) at least 70% of the Bridges students, upon or before graduation from the associate degree program, will transfer to baccalaureate degree programs in biomedical/behavioral sciences; and c) at least 75% of the transferring Bridges students will successfully complete their bachelor's degrees in biomedical/behavioral sciences.
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2012 — 2015 |
Regnier, Michael |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Effect of R1r2 Over-Expression On Cardiac Function @ University of Washington
DESCRIPTION (provided by applicant): The goal of this project is to determine whether increased cardiomyocyte 2-deoxy ATP content [dATP], via over-expression of Ribonucleotide Reductase (R1R2), can be beneficial in potentiating cardiac function and treating heart failure. A multi-disciplinary team of investigators with considerable experience in the areas of cardiac contractile function, metabolism, electrophysiology and viral-mediated gene delivery has been assembled. Dr. Regnier (PI) previously demonstrated that dATP enhances contraction in demembranated cardiac muscle by increasing myosin binding to actin (crossbridge formation) and crossbridge cycling. In a recent paper (2011, JMCC. 51:894-901; Appendix 1), we demonstrated that increasing [dATP] in cultured adult rat cardiomyocytes by over-expression of R1R2 enhances contraction, speeds relaxation and has no effect on intracellular Ca2+ transient amplitude but speeds it's decay. In the same JMCC issue an editorial (2011, JMCC 51;883-4) urges that this novel approach be tested in animals to determine its potential for treating heart failure. This is the purpose of our proposal. Importantly, we present preliminary data demonstrating increasing [dATP] rescues depressed contractility and Ca2+ transients of cardiomyocytes from infarcted hearts. Additionally, we demonstrate that transgenic R1R2 over-expression mice (TG-R1R2) have elevated left ventricular (LV) function, measured by echocardiography and Langendorff perfusion. Here we propose a translational approach, i.e. delivery of an adeno-associated viral vector with a cardiac specific promoter (AAV6- R1R2cTnT455). We demonstrate that it results in R1R2 over-expression in the heart, but not skeletal muscle or lung, and that the AAV6 vector has sustained activity for at least 20 months in mice. We also report that a ~10x dose range of AAV6-R1R2cTnT455 injection is effective in increasing LV function (out to 6 weeks thus far). We will study AAV6-R1R2cTnT455 injected mice and TG-R1R2 mice under normal conditions (Aim 1) and in an acute (Aim 2) and chronic (Aim 3) infarct model. In vivo and in vitro whole heart studies will be complimented by trabeculae, intact cardiomyocyte and myofibril mechanical assessments during Ca2+ activated contraction. Because dATP increases crossbridge cycling and may affect other cellular ATPases, we will measure cardiac ATP synthesis and mitochondrial respiration, as well as high energy phosphate utilization, energetic reserve and oxygen consumption under basal conditions and with ¿-adrenergic stress using NMR spectroscopy. We will also study action potential and Ca2+ transient behavior and assess hearts and mice for potential pathological condition. Mechanistic interpretations will be aided by proteomic analysis of myofilament and membrane proteins to assess isoform and phosphorylation profiles. We will also use computational models (in collaboration with Dr. Andrew McCulloch, UCSD) to integrate the multi-scale data and provide mechanistic insight. Results from these studies will provide valuable insight on whether sustained enhancement of myofilament contractility (with dATP) has potential for treatment of heart failure in animal models and humans. PUBLIC HEALTH RELEVANCE: Experiments proposed in this application will study a novel gene therapy based approach to improving cardiac function following myocardial infarct (heart attack). Our approach will be to use an adeno-associated (AAV6) viral vector with a cardiac specific promoter to increase cellular 2-deoxy ATP (dATP), which we have shown increase heart cell contraction. We will study the effectiveness of this approach in normal heart function and following coronary occlusion heart attack models to improve cardiac pump function via enhanced myofilament contractility.
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2017 |
Regnier, Michael |
R56Activity Code Description: To provide limited interim research support based on the merit of a pending R01 application while applicant gathers additional data to revise a new or competing renewal application. This grant will underwrite highly meritorious applications that if given the opportunity to revise their application could meet IC recommended standards and would be missed opportunities if not funded. Interim funded ends when the applicant succeeds in obtaining an R01 or other competing award built on the R56 grant. These awards are not renewable. |
Myocardial Infarct in Aging Animals and Datp Therapy @ University of Washington
ABSTRACT The goal of this project is two-fold: 1) to determine how age compounds the effect of myocardial infarct (MI) on heart function and how this affects skeletal muscle function and exercise tolerance; and 2) to determine the ability of 2 deoxy-ATP (dATP) to affect heart and skeletal muscle performance, metabolism and exercise tolerance in age and MI induced heart failure. Most studies of performance decline with MI are done with young animal models, to look at infarct specific effects. However, MI occurs most often in the elderly, and it is not clear the extent to which this compounds pathologic effects at the system, organ and cell levels. Thus, we will study how age impacts the effects of MI on heart and skeletal muscle contractile and metabolic function. We will then use this model to study cardiac-specific vs. cardiac + skeletal muscle elevation of dATP. We have previously reported that dATP enhances contraction in demembranated cardiac and skeletal muscle by increasing myosin binding to actin (crossbridge formation) and crossbridge cycling. We have also reported that cellular levels of dATP can be elevated in the heart and skeletal muscle via transgenic or viral vector-mediated or over-expression of the enzyme Ribonucleotide Reductase (RNR). Both of these approaches enhance left ventricular (LV) heart function and increases the magnitude and speed of cardiomyocyte contraction & relaxation in normal hearts and rescues LV function of infarcted hearts of rodents and pigs. These previous MI studies were done in young adult animal and we did not determine how cardiac-specific elevation of dATP affected skeletal muscle or exercise tolerance. In preliminary data we demonstrate that 1) both demembranated cardiac and skeletal muscle from old mice have enhanced contraction when dATP (vs. ATP) is the substrate for contraction, 2) transgenic young mice with elevated heart and skeletal muscle [dATP] have greater exercise capacity, faster treadmill running and fatigue resistance, and 3) elevated cardiac RNR and dATP may protect against transition from an oxidative to glycolytic cardiac metabolic profile following MI (in young mice) and rescue this `more youthful' oxidative profile in old mice. In the proposed experiments we will determine if AAV-RNR vectors can improve cardiac and skeletal muscle performance, exercise capacity and metabolic performance of old mice with and without MI. We will compare vectors with cardiac-specific vs. striated muscle-specific expression of RNR. Thus our proposal offers a unique model to study how specifically targeting the heart to improve its performance can have secondary beneficial effects in skeletal muscle. Function will be measured at whole organ, cell and myofibril levels for both cardiac and skeletal muscle and coupled with measures of metabolic efficiency and activity, mitochondrial function, and metabolomics and proteomic analysis. This multi- scale analysis, using interdisciplinary approaches, will provide information for mechanistic interpretations. The results from these studies will determine feasibility of our approach (AAV-RNR mediated elevation of dATP in muscle) for treatment of heart failure and other age-related declines in cardiac function and exercise tolerance.
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2018 — 2021 |
Murry, Charles E (co-PI) [⬀] Regnier, Michael |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Engineered Stem Cells For Cardiac Repair @ University of Washington
ABSTRACT. This project is built around years of collaborative work between Drs. Murry and Regnier studying human embryonic stem cell derived cardiomyocytes (hESC-CMs) as a potential cell replacement strategy for cardiac repair following myocardial infarction (MI). Our group has shown that hESC-CMs and human inducible pluripotent SC-CMs (hiPS-CMs) can be produced at a scale and purity that permit testing in rodent models and the animal most likely to predict the human response: the non-human primate (NHP; Macaca nemestrina). We have demonstrated the ability of these cells to engraft in rodent models, covering the entire scar, and electrically integrate with host tissue to improve left ventricular performance. This project is based on two fundamental discoveries: 1) 2-deoxy ATP (dATP) is a potent natural nucleotide stimulant of cardiac contractility (via improved myosin binding to actin & faster detachment after the power stroke), and 2) hiPSC-CMs that overexpress the rate-limiting enzyme for dATP synthesis, ribonucleotide reductase (RNR), have both increased contractility and deliver dATP to the rest of the heart via gap junctions. Thus we will test the hypothesis that engineering hiPSC-CMs to elevate RNR (RNR-hiPSC-CMs) will improve outcomes in cell replacement therapy for MI (compared with control hiPSC-CMs), improving contractility of both graft and native myocardium. There are several highly novel aspects to our approach. 1) It is the first proposed use of cellular nucleotide manipulation to improve in vivo cardiac function. 2) The approach is not limited to replacement of lost tissue (with hiPSC-CMs) with a better functioning graft, but may also substantially benefit the post-MI depressed function of native myocardium. 3) The first use of engineered hiPSC-CMs to deliver what is effectively a small molecule therapy (dATP), a natural compound that improves heart muscle contraction. This effectively makes hiPSC-CMs a drug delivery device with cardiac specific delivery and effects. Aim 1 will develop and test engineered mutations in RNR that increase it's stability and activity in cardiomyocytes and their ability to titrate increasing levels of dATP produced in hiPSC-CMs. Aim 2 will use AAV vectors for RNR variants, selected from Aim 1, to investigate their capacity to improve cardiac function in a mouse model of myocardial infarct and heart failure. Aim 3 will produce engineered hiPS cell lines that will act as dATP `donor cells' following differentiation, for transplantation into acute MI and more challenging chronic MI athymic rat models to determine their capacity to improve function beyond transplantation of non- engineered hiPSC-CMs. We will evaluate the persistence of these effects and determine the long-term stability and viability of these cell lines. We expect significant contractile improvement of both the graft and native myocardium with RNR-hiPSC-CMs vs. hiPSC-CMs and this effect will be modulated by the dATP producing capacity of the transplanted cells. Results from these studies will elucidate the potential of this combination cell- and small molecule therapy to ameliorate or even improve pump function in failing hearts.
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2019 — 2021 |
Regnier, Michael |
P30Activity Code Description: To support shared resources and facilities for categorical research by a number of investigators from different disciplines who provide a multidisciplinary approach to a joint research effort or from the same discipline who focus on a common research problem. The core grant is integrated with the center's component projects or program projects, though funded independently from them. This support, by providing more accessible resources, is expected to assure a greater productivity than from the separate projects and program projects. |
Administration and Enrichment @ University of Washington
ABSTRACT ? Core A The goal of Core A (Administration and Enrichment and) will be to ensure that the Resource Center for Translational Muscle Research (CTMR) successfully accomplishes its objectives of enhancing skeletal muscle research efficiency, develop new tools and therapeutic approaches and recruits new investigators into skeletal muscle research at the University of Washington. The approach of Core A will be to provide administrative support, evaluation of core functionality, and coordinate enrichment activities. These responsibilities are divided into six aims to provide 1) leadership, 2) management and evaluation, 3) recruit new members and promote collaboration, 4) provide pilot grants to promote project development and have a funding mechanism for high risk - high reward research, 5) facilitate communication and translation, and 6) provide enrichment activities, training opportunities and other mechanisms to promote translational muscle research at the University of Washington.
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2019 — 2021 |
Bernstein, Daniel Mack, David Lee Pruitt, Beth L (co-PI) [⬀] Regnier, Michael Wu, Sean M |
RM1Activity Code Description: To support a large-scale research project with a complex structure that cannot be appropriately categorized into an available single component activity code. The performance period may extend up to seven years but only through the established deviation request process. ICs desiring to use this activity code for programs greater than 5 years must receive OPERA prior approval through the deviation request process. |
From Proteins to Cells to Tissues: a Multi-Scale Assessment of Biomechanical Regulation by the Myosin Molecular Motor
PROJECT SUMMARY/ABSTRACT The overarching goal of this project is to use myosin as a model system in which to address the fundamental biological question of how alterations in tissue organization and function can arise from often subtle changes in function at the molecular level. Force generation by myosin is required not only for the physiological functions of skeletal muscle and the heart, but also for the proper development and maintenance of these tissues during embryogenesis and beyond. Our team aims to develop a detailed mechanistic understanding of how force generation by myosin acts to regulate muscle tissue development and homeostasis. We examine this general question through the lens of asking how seemingly small changes in the activity of individual myosin molecules can drive dramatic changes in tissue-level organization and function, for example in the context of inherited disease. In Aim 1, we will determine how structural changes in myosin affect the chemo-mechanical properties of the myosin-actin interaction for individual and small assemblies of motor proteins. This aim will leverage innovative techniques developed by our team to quantify biomechanical changes induced by myosin mutations at the single molecule level and the corresponding consequences for sarcomere-level structure and function. In Aims 2 and 3, we will determine how changes in myosin kinetics and force production influence the growth, maturation, and function of cells and tissues, using cardiomyocytes and skeletal myocytes as model systems. These aims will leverage CRISPR-editing to introduce myosin mutations in isogenic hiPSC-derived cardiac and skeletal myocytes. We will then be able to compare biomechanical alterations at the individual molecule level with those in sub-cellular organelles (myofibrils), cells and micro-tissues. We expect to answer basic mechanistic questions as to how alterations in protein structure and function affect cell and tissue function, changing force and plasticity, and provide a window into understanding how cells adapt to alterations in changing mechanical forces. We will then be positioned to utilize our hiPSC platforms for high-throughput screens to develop novel therapies targeted to phenotypic subgroups of myosin mutations. Another major goal of our Research Program is to support Early Stage Investigators (ESI). We will support pilot studies from ESI investigators that explore innovative research questions relevant to our Research Program. Critical to the NIGMS mission, our team?s multi-disciplinary integrated approach, spanning the scale from individual molecules to sub-cellular structures to whole cells to engineered micro-tissues, will serve as a prototype for teams undertaking future studies using hiPSCs to explore other biological protein assemblies, using human disease-producing mutations as perturbations to define their molecular and functional mechanisms across organ systems.
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0.954 |
2019 — 2021 |
Regnier, Michael |
P30Activity Code Description: To support shared resources and facilities for categorical research by a number of investigators from different disciplines who provide a multidisciplinary approach to a joint research effort or from the same discipline who focus on a common research problem. The core grant is integrated with the center's component projects or program projects, though funded independently from them. This support, by providing more accessible resources, is expected to assure a greater productivity than from the separate projects and program projects. |
Mechanics and Devices @ University of Washington
ABSTRACT ? Core B The goals of Core B is to provide 1) state of the art instrumentation and other resources to investigators studying skeletal muscle development and diseases, 2) assays for maturation of early stage muscle cells, and 3) testing and developing novel therapeutic interventions. The Core has instrumentation to assess contractile function at the protein, filament, cell, tissue and organ levels, and assays for measuring the developing properties of early stage muscle from animal and human sources such as inducible pluripotent stem cell derived muscle cells. The Core will train investigators individually and with workshop, and work with them to develop novel research assays for drug and small molecule testing, and investigating engineered cells and tissues and gene therapies that are being developed for clinical application and commercialization.
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1 |
2020 — 2021 |
Daniel, Thomas L. (co-PI) [⬀] Raftery, Daniel Regnier, Michael |
P30Activity Code Description: To support shared resources and facilities for categorical research by a number of investigators from different disciplines who provide a multidisciplinary approach to a joint research effort or from the same discipline who focus on a common research problem. The core grant is integrated with the center's component projects or program projects, though funded independently from them. This support, by providing more accessible resources, is expected to assure a greater productivity than from the separate projects and program projects. |
Uw Center For Translational Muscle Research (Overall Application) @ University of Washington
ABSTRACT - Overall The University of Washington (UW) is internationally known for its excellence in all aspects of skeletal muscle biology, especially in mechanistic and translational research of diseases. However, there is currently no centralizing resource that brings together all the muscle investigators from multiple departments for the benefit of facilitating and accelerating their efforts. Therefore we propose the UW Resource Center for Translational Muscle Research (CTMR) to provide a unifying resource and state of the art approaches to enhance skeletal muscle research at the UW. The proposed Center will offer tools, facilities and expertise in a combination available only at the UW to facilitate novel insights to muscle pathologies and move new therapeutics towards the clinic and the marketplace. This new Center will offer 4 cores (one administrative and three research resource) to provide tools and expertise in several areas of interest to current principal investigators. The Administrative Core (A) will provide program management and enrichment by providing a pilot project program, workshops, a seminar series, training and educational opportunities for new investigators and more experienced investigators moving into muscle research. The Mechanics and Devices Core (B) will provide state of the art measurements of muscle biomechanics at multiple levels of integration, and develop new assays for maturation and assessment of early stage muscle. The Metabolism and Energetics Core (C) will provide tools for in depth measures and analysis of metabolomics, energetics, cell respiration and mitochondrial function. The Computational and Quantitative Analysis Core (D) will provide computational and statistical tools for understanding disease, suggesting new therapeutic targets and understanding mechanisms. The CTMR will offer an environment to facilitate using integrative analysis, from single molecule dynamics through muscle structure-function relationships, with interdisciplinary approaches to advance skeletal muscle disease research and therapeutics development.
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