FHC Tn Mutations: Functional Consequences And Mechanisms
James Douglas Potter, Professor And Chairman
University Of Miami School Of Medicine 1507 Levante Avenue Coral Gables, Fl 33124
Grant 5R01HL067415-04 from National Heart, Lung, And Blood Institute IRG: CVA
Abstract: Familial Hypertrophic Cardiomyopathy (FHC) is an autosomal dominant disease, which has been associated with mutations in almost every major cardiac sarcomeric protein. Whereas individuals with myosin heavy chain (MHC) mutations, in general, have a higher level of cardiac hypertrophy, those with cardiac Troponin T (CTnT) and some of the reported Troponin I (CTnI) mutations have less hypertrophy and a higher incidence of sudden cardiac death (SCD). Most recently, a single mutation in Troponin C has been reported to be possibly associated with FHC. Although several mutations have been extensively characterized in vitro, it is still unclear how they cause cardiac hypertrophy or SCD. Our working hypothesis is that FHC troponin mutations alter the pCa-force and -ATPase relationships, the ability of the muscle to develop maximum force and ATPase activity, myosin cross-bridge kinetics, efficiency of contraction, and the ability of the muscle to do work. That mutations, which increase Ca2+- sensitivity, are more closely associated with sudden death, while mutations, which decrease the ability of the muscle to develop force, are more closely associated with hypertrophy. Recently reported transgenic mouse results along with our data from three transgenic mouse lines expressing the human CTnT (HCTnT) FHC mutations (I79N, F1101 and R278C) and HCTnI-R145G, support this hypothesis. The objective of this proposal is to comprehensively study the in vitro consequences (e.g. Ca2+-sensitivity of contraction, kinetics of force development/relaxation, impaired CTnI inhibitory function, etc.) of different FHC associated Troponin mutations in existing and new transgenic mice to identify the key mechanisms involved in the pathogenesis of FHC. This application brings together highly talented scientists at the University of Miami with varied backgrounds and represents a comprehensive approach to the study of these troponin mutations. Our goal is to correlate, the observed effects of mutations in CTnT, CTnI and CTnC on the Ca2+ regulation of cardiac muscle contraction in our animal models, with the pathogenesis of FHC in humans, especially in cases where sudden cardiac deaths have been reported. These studies will determine the functional consequences of different troponin T, troponin I and troponin C mutations under the same experimental conditions, and thus help identify key mechanism(s) involved in the pathogenesis of Troponin-linked FHC and lead to potential therapeutic strategies
Keywords: hypertrophic myocardiopathy, molecular pathology, muscle contraction, troponin, calcium flux, disease /disorder model, enzyme activity, enzyme mechanism, gene mutation, muscle protein, muscle relaxation, myocardium, myosin, protein structure function, sudden cardiac death, clinical research, genetically modified animal, histopathology, human tissue, laboratory mouse
Project start date: 2002-07-25
Project end date: 2007-06-30
5R01HL067415-04 (2005): $376438
Sponsored Links Excellgen http://Excellgen.com
FHC Tn Mutations: Functional Consequences And Mechanisms
James Douglas Potter, Professor And Chairman
University Of Miami School Of Medicine 1507 Levante Avenue Coral Gables, Fl 33124
Grant 5R01HL067415-03 from National Heart, Lung, And Blood Institute IRG: CVA
Abstract: Familial Hypertrophic Cardiomyopathy (FHC) is an autosomal dominant disease, which has been associated with mutations in almost every major cardiac sarcomeric protein. Whereas individuals with myosin heavy chain (MHC) mutations, in general, have a higher level of cardiac hypertrophy, those with cardiac Troponin T (CTnT) and some of the reported Troponin I (CTnI) mutations have less hypertrophy and a higher incidence of sudden cardiac death (SCD). Most recently, a single mutation in Troponin C has been reported to be possibly associated with FHC. Although several mutations have been extensively characterized in vitro, it is still unclear how they cause cardiac hypertrophy or SCD. Our working hypothesis is that FHC troponin mutations alter the pCa-force and -ATPase relationships, the ability of the muscle to develop maximum force and ATPase activity, myosin cross-bridge kinetics, efficiency of contraction, and the ability of the muscle to do work. That mutations, which increase Ca2+- sensitivity, are more closely associated with sudden death, while mutations, which decrease the ability of the muscle to develop force, are more closely associated with hypertrophy. Recently reported transgenic mouse results along with our data from three transgenic mouse lines expressing the human CTnT (HCTnT) FHC mutations (I79N, F1101 and R278C) and HCTnI-R145G, support this hypothesis. The objective of this proposal is to comprehensively study the in vitro consequences (e.g. Ca2+-sensitivity of contraction, kinetics of force development/relaxation, impaired CTnI inhibitory function, etc.) of different FHC associated Troponin mutations in existing and new transgenic mice to identify the key mechanisms involved in the pathogenesis of FHC. This application brings together highly talented scientists at the University of Miami with varied backgrounds and represents a comprehensive approach to the study of these troponin mutations. Our goal is to correlate, the observed effects of mutations in CTnT, CTnI and CTnC on the Ca2+ regulation of cardiac muscle contraction in our animal models, with the pathogenesis of FHC in humans, especially in cases where sudden cardiac deaths have been reported. These studies will determine the functional consequences of different troponin T, troponin I and troponin C mutations under the same experimental conditions, and thus help identify key mechanism(s) involved in the pathogenesis of Troponin-linked FHC and lead to potential therapeutic strategies
Keywords: hypertrophic myocardiopathy, molecular pathology, muscle contraction, troponin, calcium flux, disease /disorder model, enzyme activity, enzyme mechanism, gene mutation, muscle protein, muscle relaxation, myocardium, myosin, protein structure function, sudden cardiac death, clinical research, genetically modified animal, histopathology, human tissue, laboratory mouse
Project start date: 2002-07-25
Project end date: 2006-06-30
5R01HL067415-03 (2004): $376506
5R01HL067415-02 (2003): $376571
Grants awarded to James Douglas Potter
TRAINING PROGRAM IN CARDIOVASCULAR SIGNALING
James Douglas Potter, Professor / Chairman
University Of Miami School Of Medicine, Po Box 016960 (r-64), Miami, Fl 33101
Grant 5T32HL007188-34 from National Heart, Lung, And Blood Institute
Abstract: This postdoctoral training program proposal is designed to train fellows, for a period of 2 or 3 years each, in basic science research relevant to cardiopulmonary and renal responses to stress. We anticipate recruiting 20 postdoctoral trainees at experience level 7. The overall research focus of the trainees is on four inter-related levels of basic science investigation 1) cell and molecular immunobiology; 2) cellular basis of organ injury; 3) organ-system interaction; and 4) outcomes-based research. This approach underscores the essential basis of critical care medicine, requiring an integration of cell and molecular biology to organ-system monitoring, and following therapies to define their effects on socially relevant outcomes. These areas of research coincide with extramural research grants in which the faculty collaborate. The research plans are excellent vehicles for training because they ask broad questions on which precise, well-targeted individual research training efforts can be staged. The faculty include an experienced and dedicated group of senior academicians, and all have extramural research training support and training experience. Upon completion of the training program, the fellow will understand how to design, execute, and complete experiments to answer specific questions derived from critically ill patients. Thus, the fellow will be trained not only in advanced laboratory methodology, but also in the thought processes needed to apply future experimental problems as they relate to real life problems in critically ill patients. All trainees will take formal postgraduate courses offered by the basic science departments of the University of Pittsburgh. Some may complete coursework necessary to receive advanced degrees. Most training will take place in the laboratories of the principal trainers using a carefully thought-out version of the master-apprentice system. This training technique combines weekly meetings between the trainee and trainer as well as hour long research training seminars each week at which the trainer group, including of the principal trainer and the extended research trainer group, whose special skills and interests are chosen to supplement the principal trainer and concurrently guide and monitor the trainee´s research program. Furthermore, formal research presentations by the trainees will be given biannually to the local research community. This flexible but intense degree of supervision permits the simultaneous completion of efficient, cordial, and cooperative research and training. Affirmative action recruitment efforts are already in place. The training facilities can serve more fellows than will be funded by this training proposal
Keywords: Cardiovascular; Cardiovascular Body System; Cardiovascular system; Cardiovascular system (all sites); Cell Communication and Signaling; Cell Signaling; Intracellular Communication and Signaling; Organ System, Cardiovascular; Signal Transduction; Signal Transduction Systems; Signaling; Training Programs; Vascular, Heart; biological signal transduction; circulatory system
Project start date: 1976-07-01
Project end date: 2012-03-30
Budget start date: 1-APR-2010
Budget end date: 31-MAR-2011
5T32HL007188-34 (2010): $383696
5T32HL007188-33 (2009): $381273
2T32HL007188-31A1 (2007): $378875
MUTATION OF SKELETAL TROPONIN C FUNCTIONAL DOMAINS
James Douglas Potter, Professor And Chairman
Molecular And Cellular Pharmuniversity Of Miami School Of Medicine
1507 Levante Avenue
coral Gables, Fl 33124
Grant 5R01AR040727-04 from National Institute Of Arthritis And Musculoskeletal And Skin Diseases IRG: PHY
Abstract: The regulation of muscle contraction by Ca2+ is essential for normal muscle function. The penultimate event in the process leading to activation of striated muscle is the binding of Ca2+ to Troponin C (TnC), thereby altering the interaction of this protein with the other subunits of the troponin complex, troponin I and troponin T, and leading to contraction. Two isoforms of TnC have been identified, one present in fast skeletal muscle and one found in cardiac and slow skeletal muscle. The greatest sequence divergence between these two proteins is found in the amino terminal helix and in the first Ca2+ binding domain, which is inactive in the cardiac isoform. The goal of this project is to determine the relationship of the structure of fast skeletal muscle TnC to its function in regulating muscle contraction. Much is known about the Ca2+ binding properties, structure, and mechanism of action of TnC. However, there remain many questions regarding the role of the four CA2+ binding sites and the significance of the amino terminal differences between TnC isoforms in the physiological regulation of contraction which, until recently, could not be addressed directly. Two of the CA2+ binding sites have a high affinity for both CA2+ and Mg2+ (sites III and IV) and are thought to play a structural, rather than a regulatory, role. Sites III and IV are thought to be occupied by Mg2+ under normal physiological conditions. The remaining two sites also bind Ca2+, but with a lower affinity and a higher specificity (sites I and II, CA2+-specific sites). A great deal of experimental evidence indicates that the CA2+-specific sites play a critical role in the initiation of muscle contraction upon Ca2+ binding. The expression of a complementary DNA (cDNA) clone for chicken fast skeletal muscle TnC in bacteria will permit us to directly examine the role of each of these Ca2+ binding sites in mediating muscle contraction. Using site-specific mutagenesis techniques, the primary amino acid sequence of TnC will be altered in the Ca2+ binding domains. The physical and functional properties of the mutated proteins will be studied both in vitro and in skinned skeletal muscle fibers. Using this approach to eliminate CA2+ binding to each site both individually and in combination with other sites, while minimizing changes in protein conformation, we will address the following questions 1) Is Ca2+ binding to both sites I and II necessary for the initiation of muscle contraction?; 2) Are both Ca2+-Mg2+ sites required for the interaction of TnC with other components of the thin filament?; and 3) Do the Ca2+-Mg2+ sites (sites III and IV) play a regulatory function during periods of prolonged contraction when they may be occupied by Ca2+? Similarly, the functional significance of the amino terminal helix will be tested by examining the regulatory properties of a series of fast skeletal muscle TnC deletion mutants. A comparison of our results with complementary studies of chicken cardiac TnC from other laboratories should lead to an understanding of why cardiac TnC is functional with only one Ca2+-specific site
Project start date: 1986-07-01
Project end date: 1996-08-31
5R01AR040727-04 (1994): $207387
THE FUNCTION OF SLOW SKELETAL TNT IN MUSCLE CONTRACTION
James Douglas Potter, Professor / Chairman
University Of Miami School Of Medicine, Po Box 016960 (r-64), Miami, Fl 33101
Grant 5R01AR050199-05 from National Institute Of Arthritis And Musculoskeletal And Skin Diseases
Abstract: The overall goal of the proposed experiments is to determine the molecular mechanisms involved in the regulation of slow skeletal muscle contraction by troponin T (TnT) and to determine what regions of TnT are important for its physiological function. To accomplish this overall goal we will carry out two specific aims SPECIFIC AIM 1. THE ROLE OF SLOW SKELETAL TROPONIN T ISOFORMS IN THE REGULATION OF MUSCLE CONTRACTION. Essentially nothing is known about the regulation of slow skeletal muscle by troponin T (TnT) and this is the prime focus of our study which will thoroughly investigate the function of slow skeletal muscle TnT (SSTnT) in the regulation of striated muscle contraction. The main hypothesis to be tested here is that the various N-terminal SSTnT isoforms modulate Ca2+- sensitivity and the relaxation properties of slow skeletal muscle contraction. We will answer the following questions 1) Is the Ca2+-sensitivity of force development and/or ATPase activity affected by the N- or Cterminal regions of SSTnT? 2) Do SSTnT isoforms directly affect the affinity of TnC for Ca2+ or is there an indirect effect of SSTnT on the Ca2+ sensitivity of contraction (e.g., thin filament or crossbridge effect)? 3) Do the different SSTnT isoforms interact differently with tropomyosin? 4) Do SSTnT isoforms affect the activation and relaxation of force in skinned muscle fibers? 5) Are there other isoforms of SSTnT that have not been previously described? These studies will determine the role of the N-and C-terminal alternatively spliced regions of SSTnT on slow skeletal muscle contraction. SPECIFIC AIM 2. THE ROLE OF DIFFERENT REGIONS OF SLOW SKELETAL TROPONIN T IN THE PHYSIOLOGICAL FUNCTION OF TROPONIN T. To understand the role of different regions of TnT we will carry out the following 1) Characterization of the region of SSTnT that is important for Ca2+-independent ATPase activity. 2) Characterization of the region(s) of SSTnT that interacts with SSTnl, CTnC (same isoform in slow skeletal and cardiac muscle) and tropomyosin. 3) Investigation of the importance of SSTnl in the function of SSTnT. No functional studies on any region of SSTnT have so far been reported. All the Specific Aims listed above focus on gaining a more detailed understanding of the role of SSTnT in slow skeletal muscle contraction, including the molecular mechanisms of SSTnT-linked activation of muscle contraction
Keywords: ATP phosphohydrolase; ATPase; ATPase, Actin-Activated; ATPase, Actomyosin; Actins; Actomyosin Adenosinetriphosphatase; Address; Adenosine Triphosphatase; Adenosine Triphosphatase, Myosin; Adenosinetriphosphatase; Adenosinetriphosphatase, Actomyosin; Affect; Affinity; Alternate Splicing; Alternative Splicing; Amino Acids; Attention; C-terminal; Cardiac; Cardiac Myocytes; Cardiocyte; Complex; Development; Dysfunction; Exons; Functional disorder; Genes; Genetic Alteration; Genetic Change; Genetic defect; Goals; Heart; Heart myocyte; Human; Human, General; In Vitro; Investigation; Investigators; Isoforms; Kinetic; Kinetics; Laboratories; Libraries; Link; Man (Taxonomy); Man, Modern; Messenger RNA; Molecular; Muscle; Muscle Cell Contraction; Muscle Cells, Cardiac; Muscle Cells, Heart; Muscle Contraction; Muscle Fibers; Muscle Tissue; Muscle, Cardiac; Muscle, Heart; Muscle, Skeletal; Muscle, Voluntary; Muscular Contraction; Mutation; Myocardium; Myocytes, Cardiac; Myopathies, Nemaline; Myopathy, Rod; Myopathy, Rod-Body; Myosin ATPase; Myosin Adenosinetriphosphatase; Myosins; Myotubes; N-terminal; NH2-terminal; Nemaline Myopathies; Nucleotides; Physiologic; Physiological; Physiopathology; Process; Programs (PT); Programs [Publication Type]; Property; Property, LOINC Axis 2; Protein Isoforms; Proteins; Publications; RNA Splicing; RNA Splicing, Alternative; RNA, Messenger; Regulation; Relaxation; Reporting; Research Personnel; Researchers; Rhabdomyocyte; Role; Scientific Publication; Screening procedure; Site; Skeletal Fiber; Skeletal Muscle Cell; Skeletal Muscle Fiber; Skeletal Muscle Tissue; Skeletal Myocytes; Skeletal muscle structure; Skin; Splicing; Striated Muscle Tissue; Striated Muscles; TM-4 (tropomyosin); TM-4 gene product; TNT; Testing; Thin Filament; TnI; Transcript; Transgenic Organisms; Tropomyosin; Troponin; Troponin I; Troponin T; Work; aminoacid; base; cardiac muscle; cardiomyocyte; experiment; experimental research; experimental study; gene product; genome mutation; heart muscle; inhibitory troponin I; interest; mRNA; myosin ATP phosphohydrolase (actin translocating); pathophysiology; programs; research study; screening; screenings; skeletal; social role; transgenic; tropomyosin binding protein troponin T; tropomyosin-4
Project start date: 2005-03-01
Project end date: 2011-02-28
Budget start date: 1-MAR-2009
Budget end date: 28-FEB-2011
5R01AR050199-05 (2009): $309708
5R01AR050199-02 (2006): $323255
1R01AR050199-01A2 (2005): $295490
MECHANISM OF CARDIAC MUSCLE REGULATION BY TROPONIN
James Douglas Potter, Professor And Chairman
Molecular And Cellular Pharmuniversity Of Miami School Of Medicine
1507 Levante Avenue
coral Gables, Fl 33124
Grant 5R01HL042325-13 from National Heart, Lung, And Blood Institute IRG: CVA
Abstract: The overall goal of the proposed experiments is to determine the molecular mechanisms involved in the regulation of cardiac muscle contraction by troponin and to determine its role in the genesis of familial hypertrophic cardiomyopathy (FHC). There are two major projects In I., we will determine the fundamental role of cardiac troponin T (CTnT) in the regulation of cardiac muscle contraction. Traditionally, it has been thought that the primary role of TnT is to interact with and anchor the complex of Tnl and TnC to the actin- containing thin filaments through TnT´s interactions with tropomyosin (Tm) and Tnl. Recent results from our lab, with skeletal muscle, additionally suggest that 1) Ca/2+ binding to STnC causes an interaction between STnC and the C-terminus of STnT and causes an activation of the ATPase activity; and 2) the maximum level of ATPase activation is determined by the particular N-terminal TnT variant which is present. Due to the similarities in primary structure between CTnT and STnT, we hypothesize that CTnT may play a comparable or related role in cardiac muscle. Biochemical, molecular and physiological approaches will be used to test this hypothesis and to determine the fundamental role of HCTnT in the regulation of cardiac muscle contraction. Project II. FHC is an autosomal dominant disease which has been shown to be associated with mutations in HCTnT, and HCTnl. In this section we will attempt to learn how these mutations affect the biochemical and contractile properties of cardiac muscle and give rise to FHC. Our current working hypothesis is that mutations of CTnT and CTnI lead to changes in the interactions between the CTn subunits, or changes in their interactions with the other thin filament proteins, which in turn lead to changes in contractility and/or the Ca/2+ affinity of CTnC (which would change the intracellular [Ca/2+] transient), and that these changes could trigger the cellular mechanisms responsible for the hypertrophic process. To test this hypothesis, transgenic animal models, skinned fiber, myofibrillar, and actomyosin systems, combined with biochemical, molecular, biophysical and physiological techniques, will be utilized. The combined results from these two projects will yield important new information on the role of CTnT and CTnI in the regulation of cardiac contraction and in FHC
Keywords: calcium binding protein, heart contraction, hypertrophic myocardiopathy, myocardium, protein isoform, troponin actin, calcium flux, gene mutation, microfilament, myosin ATPase, protein binding, protein structure function laboratory mouse, transgenic animal
Project start date: 1989-04-01
Project end date: 2003-03-31
5R01HL042325-13 (2001): $304833
5R01HL042325-12 (2000): $298025
5R01HL042325-18 (2006): $367090
Sponsored Links Excellgen http://Excellgen.com
5R01HL042325-17 (2005): $375991
5R01HL042325-16 (2004): $373996
2R01HL042325-15 (2003): $366334
5R37HL042325-10 (1998): $251166
5R37HL042325-09 (1997): $242753
5R37HL042325-07 (1995): $209928
4R37HL042325-06 (1994): $203557
5R37HL042325-05 (1993): $186249
5R37HL042325-04 (1992): $197784
Sponsored Links Excellgen http://Excellgen.com
Physiological Role Of The Myosin Regulatory Light Chain
James Douglas Potter, Professor And Chairman
University Of Miami School Of Medicine 1507 Levante Avenue Coral Gables, Fl 33124
Grant 5R01AR045183-07 from National Institute Of Arthritis And Musculoskeletal And Skin Diseases IRG: ECS
Abstract: The long-term goal of the proposed studies is to determine the physiological role(s) of the regulatory light chains of myosin (RLC) in the regulation and/or modulation of skeletal muscle contraction-The central hypothesis is that the RLC play an important role in the regulation and/or modulation of striated muscle contraction. Specifically, the hypotheses to be tested are that 1) Ca2+ and/or Mg+ binding to the single Ca2+-Mg2+ binding site on the RLC and 2) the phosphorylation of Ser (by Ca2+- calmodulin activated myosin light chain kinase, MLCK) play important roles in the regulation and/or modulation of contraction. In the first three years of this grant considerable progress has been made in understanding the role of the RLC in contraction. We have shown that a) the RLC affect crossbridge cycling; b) phosphorylation of the RLC increases the Ca2+- sensitivity of both force development and thin filament activated myosin ATPase activity; c) phosphorylation of the RLC increases maximal force production and d) the Ca2+-Mg2+ binding site is required for the phosphorylation induced shift in the Ca2+- sensitivity of force development. The latter suggests that there is coupling between the Ca2+-Mg2+ binding site and the phosphorylation site. In addition, we have shown that the level of endogenous RLC phosphorylation, in addition to the role played by Troponin, is a crucial determinant of the Ca2+- sensitivity of force development in skeletal muscle, the magnitude of which was not previously appreciated. Although these in vitro results have told us much about the function of the RLC, it is still not totally clear what their in vivo function is. It is also true that the role of the RLC in skeletal muscle has been fraught with controversy and much of this has come from the fact that methods to study the function of the RLC have not been available. Unfortunately there have been no artifact-free methods for the selective extraction/replacement of the RLC in either myosin or in more complex systems, e.g., myofibrils and skinned muscle fibers and this has contributed to the controversy. A more unequivocal way of approaching the role of the RLC would be to develop systems whereby the RLC can be manipulated in a native setting. Several powerful approaches are available today that make this possible and these include transgenic and knock-in/out mouse models. To test the above hypotheses, transgenic and knock-in/out animal models, where various mutants of RLC will replace the endogenous mouse RLC, will be utilized. Sophisticated physiological studies on both intact muscle and skinned fibers from these animals will be performed to determine the role of the RLC in skeletal muscle contraction and regulation. The proposed studies will allow us to uniquely study the role of the RLC in striated muscle contraction and to determine their in vivo role.
Keywords: enzyme activity, muscle contraction, myosin, protein structure function, striated muscle, binding protein, binding site, calcium ion, magnesium ion, myosin light chain kinase, phosphorylation, protein binding, gene targeting, genetically modified animal, laboratory mouse, laboratory rabbit
Project start date: 1999-02-01
Project end date: 2007-06-30
5R01AR045183-07 (2005): $310575
5R01AR045183-06 (2004): $356025
5R01AR045183-05 (2003): $356025
PHYSIOLOGICAL ROLE OF THE MYOSIN REGULATORY LIGHT CHAINS
James Douglas Potter, Professor And Chairman
University Of Miami School Of Medicine 1507 Levante Avenue Coral Gables, Fl 33124
Grant 5R01AR045183-03 from National Institute Of Arthritis And Musculoskeletal And Skin Diseases IRG: PC
Abstract: The long-term goal of the proposed studies is to determine the physiological role(s) of the regulatory light chains of myosin (RLC) in the regulation and/or modulation of skeletal muscle contraction. The central hypothesis to be tested is that Ca2+ and/or Mg2+ binding to the single Ca2+ binding site on the RLC plays a role in the regulation and/or modulation of contraction. In order to test this hypothesis, the following Specific Aims will be pursued I. What are the Ca2+ and Mg2+ binding properties of the single Ca2+ binding site on the RLC in muscle? To fully understand how metal (Ca2+ and/or Mg2+) binding to RLC might affect contraction, the metal binding properties of RLC in muscle will be measured. II. Does Ca2+ binding to the RLC affect the rate of force development and/or relaxation and, if so, how? Several lines of evidence suggest that Ca2+ binding to the RLC Ca2+ binding site affects the rate of force development, by somehow altering cross-bridge kinetics. The proposed experiments will test this idea by determining whether Ca2+ binding influences the rate of force development and/or relaxation. If, as expected, metal binding to the RLC plays a role in contraction, then changing the metal binding properties of the RLC should change the metal dependency of any affected contractile process. A series of RLC Ca2+ binding site mutants (e.g., inactivated site, higher Ca2+ affinity/ specificity site, etc.) will be incorporated into skinned muscle fibers and tested for their effects on a) steady state force development, b) the Ca2+-dependence of force development, c) the rate of activation/ relaxation, and d) the Ca2+ dependence of the rate of force development. III Does phosphorylation of RLC by MLCK affect the Ca2+ dependence and/or kinetics of force development and relaxation, and does RLC phosphorylation interact functionally with RLC metal binding? Our Preliminary Studies have shown that the RLC are mostly phosphorylated in isolated fibers, and that changing the level of phosphorylation has a much larger effect on the Ca2+ dependence of force development than had been previously appreciated. Our results also suggest that Ca2+ binding to the RLC is required to observe these effects of RLC phosphorylation. These results will be confirmed and extended to ask the following questions 1) are the effects of phosphorylation on the Ca2+-dependence of force development accompanied by changes in the kinetics of force activation/relaxation; 2) are the metal binding properties of the RLC affected by phosphorylation and vice versa and 3) are the effects of phosphorylation direct, due to changes in RLC Ca2+ binding, or indirect, through cross-bridge effects on Tn Ca2+ affinity? The answers to these questions will determine the mechanism and the importance of MLCK phosphorylation of RLC in skeletal muscle contraction. In summary, our experiments will define in detail the potential role of the RLC in the regulation and/or modulation of skeletal muscle contraction.
Keywords: enzyme activity, muscle contraction, muscle relaxation, myosin ATPase, striated muscle, binding protein, binding site, calcium, magnesium, myosin light chain kinase, protein binding, chromatography, electrophoresis, laboratory rabbit
Project start date: 1999-02-01
Project end date: 2002-06-30
5R01AR045183-03 (2001): $351374
5R01AR045183-02 (2000): $343364
CALCIUM REGULATION OF MUSCLE CONTRACTION
James Douglas Potter, Professor And Chairman
Molecular And Cellular Pharmuniversity Of Miami School Of Medicine
1507 Levante Avenue
coral Gables, Fl 33124
Grant 5R01AR037701-08 from National Institute Of Arthritis And Musculoskeletal And Skin Diseases IRG: PHY
Abstract: During the past four years we have made major progress in the development of several model systems for studying the regulation of muscle contraction. These include reconstituted thin filaments, TnC depleted reconstituted skinned fibers and intact muscle fibers. These systems combined with the techniques of fluorescence spectroscopy, flash photolysis of caged compounds, time resolved X-ray diffraction and proteolytic fragments of the regulatory proteins have allowed us to discover many new features about muscle activation/relaxation. In the present application, we propose to pursue several major questions related to these processes and have divided the application into two major areas of study as follows I. The use of skinned and intact muscle fibers to study muscle activation and relaxation processes. The following specific aims will be pursued I-1. What are the temporal relationships between a) the binding of Ca2+ to the regulatory sites of TnC (measured by incorporated fluorescent TnC derivatives) and the activation of force development and stiffness of skinned fiber systems from rabbit, frog and barnacle muscle following rapid step changes in free Ca2+ (photolysis of caged Ca2+) which are not limited by diffusion processes and b) the dissociation of Ca2+ from the regulatory sites of TnC and force relaxation and stiffness following rapid step decreases in free Ca2+ (photolysis of the caged Ca2+ chelator, diazo-2)? I-2. What are the effects of different crossbridge states (e.g. increment[ATP], increment [ADP]/[ATP], increment[Pi], rigor (-ATP), weakly attached (N- phenylmaleimide treated) and numbers (e.g. different sarcomere lengths, length perturbations) on these processes? I-3. What is the time course for Ca2+ binding to TnC in intact single muscle fibers, particularly barnacle, when activated electrically? I-4. During relaxation, what is the relation between free Ca2+ and the attached crossbridge states? I-5. Can the rates of force development and force relaxation observed in electrically excited intact frog, barnacle and scallop (myosin regulated) fibers be increased by laser flash photolysis of caged calcium or caged calcium chelator injected directly into the cell? I-6. What is the Ca2+ affinity of rabbit light chain-2 of myosin in situ and can it play a role in muscle regulation? II. The use of proteolytic fragments of Tn and Tm to study muscle regulation. The following specific aims will be pursued II-1. What are the roles the different regions of TnC in the regulation of contraction? II-2. What are the roles of the head-to-tail interaction of Tm and the NH2-terminal region of TnT in the cooperative activation of muscle contraction? II-3. What are the interactions of the different regions of TnI with the other thin filament proteins and how are these related to the activation and relaxation of muscle contraction? Taken together these unique approaches should yield a much clearer view of the temporal and molecular events involved in muscle activation and relaxation
Keywords: calcium channel, hormone regulation /control mechanism, muscle contraction alternatives to animals in research, calcium binding protein, clathrate, heart contraction, muscle relaxation, striated muscle, troponin Anura, Cirripedia, X ray crystallography, flash photolysis, fluorescence spectrometry, laboratory rabbit
Project start date: 1991-09-20
Project end date: 1996-08-31
5R01AR037701-08 (1994): $307605
Training Program In Cardiovascular Signaling
James Douglas Potter, Professor And Chairman
University Of Miami School Of Medicine 1507 Levante Avenue Coral Gables, Fl 33124
Grant 5T32HL007188-30 from National Heart, Lung, And Blood Institute IRG: ZHL1
Abstract: Diseases of the heart and circulation are of enormous human and economic importance in the United States. A successful attack on this problem requires a new generation of scientists trained to understand the cardiovascular system at a variety of levels and from diverse technical perspectives. Using the approaches and techniques of modem biomedical science, this competing renewal of the University of Miami Training Program in Cardiovascular Signaling seeks to provide these scientists. The revolution in molecular biology has vastly increased our ability to identify specific ion channels, receptors, pumps, signaling pathways, and their effectors. Future cardiovascular scientists will need to have not only an understanding of these processes at the molecular level but will also need to be able to integrate this knowledge to understand their function at the intact cell, tissue, organ and whole animal level. The present Program Faculty has a demonstrated ability to train new investigators in all of these areas, utilizing the latest modem technologies, for positions in universities, medical schools, government laboratories and biopharmaceutical and biotechnology firms. The Program Director, Dr. James Potter, is a respected researcher in the cellular and molecular aspects of the regulation of cardiac and skeletal muscle contraction. Dr. Potter is an experienced departmental chairman who has trained many graduate students and postdoctoral fellows. The Program Faculty, drawn from several basic science and clinical departments, are an outstanding, well-funded group of scientists and teachers whose research interests comprise a cross-section of modem Cardiovascular Signaling. These faculty are proven leaders in graduate and postdoctoral education. The overall aim of the program is to guide predoctoral and postdoctoral trainees through the process of acquiring the research skills and the intellectual rigor needed to become independent cardiovascular scientists. The Program also seeks to provide undergraduate medical students with intensive, short-term research experiences. An excellent pool of trainee candidates exists for this Training Program and past trainees have shown a strong record of accomplishment. In summary, major strengths of the proposed renewal of the training program include 1) the research productivity, research funding, and training experience of the Program Faculty, as well as the proven administrative skill of the Program Director, 2) the strong credentials of the current students and postdoctoral fellows, and 3) the past accomplishments of the Program in producing trainees who have gone on to successful careers in biomedical research.
Project start date: 1976-07-01
Project end date: 2007-03-31
5T32HL007188-30 (2005): $312983
5T32HL007188-29 (2004): $343605
5T32HL007188-28 (2003): $396511
Sponsored Links Excellgen http://Excellgen.com
5T32HL007188-27 (2002): $374919
2T32HL007188-26 (2001): $351319
TROPONIN T AND THE REGULATION OF CONTRACTION
James Douglas Potter, Professor And Chairman
Molecular And Cellular Pharmuniversity Of Miami School Of Medicine
1507 Levante Avenue
coral Gables, Fl 33124
Grant 5R01AR045391-04 from National Institute Of Arthritis And Musculoskeletal And Skin Diseases IRG: PC
Abstract: application ) "Troponin T (TnT) is one of three proteins (including TnI and TnC) that form the troponin complex (Tn), which regulates skeletal muscle contraction. Sequence analysis of the TnT gene predicts as many as 64 different isoforms, due to the presence of 5 alternatively spliced exons at the N-terminus and 2 mutually exclusive alternatively spliced exons (alpha and beta) at the C- terminus. Previous studies have suggested that the role of TnT in the regulation of contraction is to interact with and anchor the complex of TnI and TnC to actin-containing filaments through TnT´s interaction with tropomyosin (Tm). Two recent results from our lab suggest that, in addition to this role, TnT is critically involved in the regulation of actomyosin ATPase activation. First, the C-terminus of TnT (containing alpha or beta) interacts with TnC, and this interaction seems to be required for the activation of actomyosin ATPase activity. This interaction in part determines the Ca2+ -sensitivity of contraction, which differs depending on whether alpha or beta is present. Second, the N-terminal region of TnT (containing various alternatively spliced exons) appears to determine the level of activation of actomyosin ATPase activity once this interaction with TnC occurs. Our recent observations implicate both alternatively spliced regions (N- and C-) of TnT in the regulation of actomyosin ATPase activation. The major question being addressed in this proposal is how do structural changes in TnT, conferred by alternative splicing in these two regions, affect this novel function of TnT? Our working hypothesis, which will be tested in the present proposal, is that Ca2+ binding to TnC causes an interaction between TnC and the C-terminus of TnT (containing alpha or beta) and causes an activation of the ATPase activity. The Ca2+ -sensitivity of this activation depends on the alpha/beta splice variants and the maximum level of activation is determined by the particular N-terminal TnT variant which is present."
Keywords: enzyme activity, muscle contraction, myosin ATPase, protein isoform, protein structure function, troponin RNA splicing, actin, calcium ion, chemical kinetics, myofibril, protein binding, protein sequence, striated muscle X ray crystallography, affinity chromatography, laboratory rabbit, mutant, surface plasmon resonance
Project start date: 1998-09-10
Project end date: 2003-08-31
5R01AR045391-04 (2001): $300062
5R01AR045391-03 (2000): $293437
1R01AR045391-01 (1998): $256552
HTS FOR REGULATED MUSCLE THIN FILAMENT FUNCTION.
James Douglas Potter, Professor / Chairman
University Of Miami School Of Medicine, Po Box 016960 (r-64), Miami, Fl 33101
Grant 3R21NS064821-01S1 from Office Of The Director, National Institutes Of Health
Abstract: A hallmark of sarcomeric gene mutations in cardiomyopathies is their ability to alter the calcium regulation of cardiac muscle contraction. In general, the Ca2+ sensitivity of contraction decreases in dilated (DCM) cardiomyopathy; whereas, in hypertrophic (HCM) and restrictive (RCM) cardiomyopathies, the sensitivity increases. Since multiple forms of cardiomyopathies exist, the identification of new drugs that sensitize (+) or desensitize (-) the Ca2+ sensitivity could potentially reverse (+ or -) these aberrant changes. Therefore, the goal of this proposal is to use high throughput screening (HTS) to identify small molecules that can modulate the Ca2+ sensitivity of cardiac muscle contraction. To achieve this, we will use a model system composed of cardiac muscle regulated thin filaments (RTF) which are comprised of F-actin, tropomyosin and troponin (Tn). The RTF in combination with myosin (thick filament) make up the major proteins found in the contractile apparatus. In the absence of myosin, the RTF retains all of the Ca2+ regulated functions critical for muscle activation and relaxation. The proposed assay will use cardiac Tn (CTn) complexes that contain fluorescently labeled troponin C (CTnC), the Ca2+ binding subunit of the CTn complex. This will allow us to monitor changes in RTF fluorescence that occurs when Ca2+ binds to the CTnC regulatory site. Therefore, detecting an increase or decrease (+ or -) in the labeled RTF fluorescence intensity at a fixed [Ca2+] and wavelength in response to a compound or "hit" from the HTS screen will indicate that a change (+ or -) in the apparent Ca2+ affinity of CTnC has occurred. Hits from the HTS will be further validated using two biological secondary screens. Based on the above, the RTF system can provide a robust, stable and physiological assay to identify compounds that specifically alter the RTF Ca2+ sensitivity and not the force via cross bridge-drug interactions. To achieve our goals, this proposal will pursue two Specific Aims. Knowledge gained from these studies can uncover potentially new pharmacological agents for the investigation and treatments of cardiomyopathies, hypertension and other forms of cardiovascular diseases. The genetic basis for three major types of inherited cardiomyopathies including dilated, hypertrophic and restrictive have been studied intently over the last decade. The studies proposed here will identify new low molecular weight compounds, using modern high throughput screening technology, which can modulate a key phenotype that has been observed in model systems of these diseases. Results from these studies will ultimately be beneficial in developing new therapeutic approaches for the treatment of these and potentially other forms of cardiovascular disease
Keywords: ATP phosphohydrolase; ATPase; ATPase, Actin-Activated; Adenosine Triphosphatase; Adenosine Triphosphatase, Myosin; Adenosinetriphosphatase; Affinity; Assay; Binding; Binding (Molecular Function); Bioassay; Biologic Assays; Biological; Biological Assay; Biological Models; Blood Coagulation Factor IV; Blood Pressure, High; Ca++ element; Calcium; Cardiac; Cardiac Muscle Contraction; Cardiomyopathies; Cardiovascular Diseases; Cell Communication and Signaling; Cell Signaling; Chemicals; Coagulation Factor IV; Complex; DNA Alteration; DNA mutation; Data; Dependence; Detection; Development; Disease; Disorder; Dose; Drug Interactions; Drugs; Evaluation; F-Actin; Factor IV; Fiber; Filamentous Actin; Fluorescence; Gene Alteration; Gene Mutation; Genetic; Genetic mutation; Goals; Guidelines; Hereditary; High Throughput Assay; Hypertension; Inherited; Intracellular Communication and Signaling; Investigation; Ionic Strengths; Knowledge; Label; Laboratories; Ligands; Measurement; Measures; Medication; Miniaturisations; Miniaturization; Miniaturizations; Model System; Models, Biologic; Molecular Interaction; Molecular Weight; Monitor; Muscle; Muscle Cell Contraction; Muscle Contraction; Muscle Fibers; Muscle Tissue; Muscle, Cardiac; Muscle, Heart; Muscular Contraction; Mycocardium Disease; Myocardial Diseases; Myocardial Disorder; Myocardiopathies; Myocardium; Myosin ATPase; Myosin Adenosinetriphosphatase; Myosins; Myotubes; Noise; Pharmaceutic Preparations; Pharmaceutical Preparations; Phenotype; Phosphates; Physiologic; Physiological; Probability; Process; Production; Proteins; Quality Control; Reaction; Regulation; Relaxation; Reproducibility; Rhabdomyocyte; Screening procedure; Sequence Alteration; Signal Transduction; Signal Transduction Systems; Signaling; Site; Skeletal Fiber; Skeletal Muscle Cell; Skeletal Muscle Fiber; Skeletal Myocytes; Staging; Strengths, Ionic; System; System, LOINC Axis 4; TNC; Technology; Temperature; Thick Filament; Thin Filament; Time; Tropomyosin; Troponin; Troponin C; Vascular Hypertensive Disease; Vascular Hypertensive Disorder; base; biological signal transduction; calcium binding troponin C; cardiac muscle; cardiovascular disorder; comparison group; cost; disease/disorder; drug/agent; experiment; experimental research; experimental study; gene product; heart muscle; high throughput screening; hyperpiesia; hyperpiesis; hypertensive disease; inorganic phosphate; instrument; miniaturize; myocardium disorder; myosin ATP phosphohydrolase (actin translocating); novel therapeutic intervention; public health relevance; research study; response; scale up; screening; screenings; small molecule
Relevance: The genetic basis for three major types of inherited cardiomyopathies including dilated, hypertrophic and restrictive have been studied intently over the last decade. The studies proposed here will identify new low molecular weight compounds, using modern high throughput screening technology, which can modulate a key phenotype that has been observed in model systems of these diseases. Results from these studies will ultimately be beneficial in developing new therapeutic approaches for the treatment of these and potentially other forms of cardiovascular disease
Project start date: 2008-09-30
Project end date: 2010-08-31
Budget start date: 30-SEP-2008
Budget end date: 31-AUG-2010
PFA/PA: PAR-08-024
3R21NS064821-01S1 (2010): $38250
1R21NS064821-01 (2008): $0
CARDIAC TROPONIN IN HEALTH AND DISEASE
James Douglas Potter, Professor / Chairman
University Of Miami School Of Medicine, Po Box 016960 (r-64), Miami, Fl 33101
Grant 5R01HL042325-21 from National Heart, Lung, And Blood Institute
Keywords: ATP phosphohydrolase; ATPase; ATPase, Actin-Activated; Actins; Address; Adenosine Triphosphatase; Adenosine Triphosphatase, Myosin; Adenosinetriphosphatase; Affinity; Age; Animal Model; Animal Models and Related Studies; Arrhythmia; Asymmetric Septal Hypertrophy, Familial; Autoregulation; Binding; Binding (Molecular Function); Biochemical; Blood Coagulation Factor IV; Body Tissues; Ca++ element; Calcium; Calcium Binding; Cardiac; Cardiac Arrhythmia; Cardiac Diseases; Cardiac Disorders; Cardiac Muscle Contraction; Cardiomyopathies; Cardiomyopathy, Dilated; Cardiomyopathy, Hypertrophic, Familial; Cardiomyopathy, Restrictive; Characteristics; Coagulation Factor IV; Congestive Cardiomyopathy; Data; Death, Sudden, Cardiac; Development; Dilated Cardiomyopathy; Disease; Disorder; Disorder of muscle, unspecified; Echocardiogram; Echocardiography; Engineering; Engineerings; Evaluation; Event; Exercise; Exercise, Physical; Explosion; Factor IV; Fiber; Fibrosis; Gender; Genetic; Genetic Alteration; Genetic Change; Genetic Condition; Genetic Diseases; Genetic defect; Genomics; Goals; Health; Heart; Heart Arrhythmias; Heart Diseases; Hereditary Disease; Histopathology; Homeostasis; Human; Human, General; In Vitro; Knock-in; Knock-in Mouse; Lead; Learning; Link; Mammals, Mice; Man (Taxonomy); Man, Modern; Measurement; Measures; Mediating; Methods and Techniques; Methods, Other; Mice; Modeling; Molecular; Molecular Disease; Molecular Interaction; Morphology; Murine; Mus; Muscle; Muscle Disease; Muscle Disorders; Muscle Fibers; Muscle Tissue; Muscle disease or syndrome; Muscle, Cardiac; Muscle, Heart; Muscular Diseases; Mutation; Mycocardium Disease; Myocardial Diseases; Myocardial Disorder; Myocardiopathies; Myocardium; Myopathic Conditions; Myopathic Diseases and Syndromes; Myopathic disease or syndrome; Myopathy; Myopathy, unspecified; Myosin ATPase; Myosin Adenosinetriphosphatase; Myosins; Myotubes; Papillary Muscles; Pb element; Phenotype; Physiologic; Physiological; Physiological Homeostasis; Preparation; Pressure; Pressure- physical agent; Probability; Process; Property; Property, LOINC Axis 2; Proteins; Regulation; Regulatory Protein; Relaxation; Research; Restrictive Cardiomyopathy; Rhabdomyocyte; Role; Secondary to; Severities; Skeletal Fiber; Skeletal Muscle Cell; Skeletal Muscle Fiber; Skeletal Myocytes; Skin; Sorting - Cell Movement; Starlings; Structure of papillary muscle; Sturnidae; Sturnus vulgaris; System; System, LOINC Axis 4; TNC; TNT; Techniques; Thin Filament; Time; Tissues; TnI; Transthoracic Echocardiography; Tropomyosin; Troponin; Troponin C; Troponin I; Troponin T; Ventricular Hypertrophy, Familial; Work; base; calcium binding troponin C; cardiac muscle; disease phenotype; disease/disorder; experiment; experimental research; experimental study; gene product; genetic disorder; genetic regulatory protein; genome mutation; heart disorder; heart muscle; heart sonography; heavy metal Pb; heavy metal lead; hereditary disorder; in vivo; inhibitory troponin I; innovate; innovation; innovative; insight; malignant phenotype; man; man`s; model organism; mouse model; muscular disorder; mutant; myocardium disorder; myosin ATP phosphohydrolase (actin translocating); new approaches; new therapeutics; next generation therapeutics; novel approaches; novel strategies; novel strategy; novel therapeutics; papillary muscle; pressure; protein complex; protein protein interaction; reconstitute; reconstitution; regulatory gene product; research study; social role; sorting; sound measurement; starling (bird); theories; tropomyosin binding protein troponin T
Project start date: 1989-04-01
Project end date: 2012-02-28
Budget start date: 1-MAR-2010
Budget end date: 28-FEB-2011
PFA/PA: PA-07-070
5R01HL042325-21 (2010): $382500
The Function Of Slow Skeletal TnT In Muscle Contraction
James Douglas Potter, Professor And Chairman
Molecular And Cellular Pharmuniversity Of Miami-medical School, 1507 Levante Avenue, Coral Gables, Fl 33124
Grant 5R01AR050199-03 from National Institute Of Arthritis And Musculoskeletal And Skin Diseases IRG: SMEP
Project start date: 2005-03-01
Project end date: 2010-02-28
5R01AR050199-03 (2007): $316029
Sponsored Links Excellgen http://Excellgen.com
MECHANISM OF CARDIAC MUSCLE REGULATION BY TROPONIN
James Douglas Potter, Professor And Chairman
University Of Miami School Of Medicine 1507 Levante Avenue Coral Gables, Fl 33124
Grant 5R37HL042325-08 from National Heart, Lung, And Blood Institute IRG: NSS
Project start date: 1989-04-01
Project end date: 1999-02-28
5R37HL042325-08 (1996): $237664