1993 — 1995 |
Dirksen, Robert T |
F32Activity Code Description: To provide postdoctoral research training to individuals to broaden their scientific background and extend their potential for research in specified health-related areas. |
Functional Analysis of Single Dihydrophyridine Receptors @ Colorado State University-Fort Collins |
0.937 |
1997 — 2011 |
Dirksen, Robert T |
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. R29Activity Code Description: Undocumented code - click on the grant title for more information. |
Control of Calcium Movements in Muscle @ University of Rochester
[unreadable] DESCRIPTION (provided by applicant): Central Core Disease (CCD), the most common congenital myopathy in humans, is characterized by fetal hypotonia, proximal muscle weakness, and inert core regions that lack functional mitochondria and oxidative enzyme activity in type 1 skeletal muscle fibers. Although CCD is known to be caused by mutations in the skeletal muscle Ca2+ release channel (RyR1), diagnosis still depends on painful and invasive muscle biopsies and there are currently no effective treatments. The long-term goal of this project is to characterize the pathophysiological mechanisms of muscle weakness in CCD and to develop novel therapeutic and diagnostic strategies. Motivated by this goal, we aim to drive discovery of novel and fundamental aspects of altered Ca2+ signaling in CCD, as well as test the validity of potential therapeutic (allele-specific gene silencing) and diagnostic (superoxide flashes as a CCD biomarker) strategies. Aim #1 will test the hypothesis that CCD mutations in different structural regions of RyR1 alter Ca2+ release during muscle contraction via fundamentally distinct molecular and biophysical mechanisms. Experiments will determine if CCD mutations in the M6 and pore/TM10 transmembrane regions of RyR1 alter Ca2+ permeation, channel gating, and/or regulation by specific RyR1 binding proteins. Aim #2 will assess the utility of allele-specific gene silencing approaches to correct defects in Ca2+ homeostasis, Ca2+ release, and mitochondrial function observed in knock-in mice heterozygous for either the Y522S or I4895T CCD mutations in RyR1. An important conceptual innovation of this project is to use shRNA-mediated mutant RyR1 allele-specific knockdown to reverse toxic gain-of-function effects produced by CCD mutations in RyR1 in heterozygous Y522S and I4895T knock-in mice. An important technological innovation is to take advantage of our recently developed mitochondrial-targeted superoxide sensor (mt-cpYFP) to assess changes in mitochondrial function (reflected in the incidence and properties of mitochondrial superoxide flashes) in normal and RyR1 knock-in mice. The fundamental pathophysiologic mechanisms identified and discoveries made as a result of this venture will provide extraordinary promise for the development of novel approaches, therapeutics, and diagnostics not only for CCD, but also other disorders of Ca2+ dysregulation and altered oxidative stress in related clinical arenas including cardiac arrhythmias, heart failure, hypertension, diabetes, neuro-degeneration and stroke. [unreadable] [unreadable] [unreadable]
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1 |
2010 — 2020 |
Dirksen, Robert T |
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. |
Molecular Mechanism and Functional Role of Soce in Skeletal Muscle @ University of Rochester
? DESCRIPTION (provided by applicant): Being first reported in 2001, store-operated Ca2+ entry (SOCE) is a relatively new phenomenon in skeletal muscle. SOCE is coordinated by coupling between two proteins: STIM1 calcium sensors in the sarcoplasmic reticulum (SR) and Ca2+-permeable Orai1 channels in the transverse tubule (TT) membrane. SOCE enhances muscle growth/development, limits fatigue, and promotes fatigue-resistant type I fiber specification. On the other hand, SOCE dysfunction contributes to muscle weakness/fatigue in aging, exacerbates muscular dystrophy, and mutations in STIM1 and Orai1 genes result in debilitating myopathies. The picture that emerges is that tight regulation of STIM1/Orai1-dependent SOCE activity is critical for optimal muscle performance such that increases or decreases in SOCE activity can lead to muscle fatigue, sarcopenia, and myopathy. While SOCE activity clearly impacts muscle performance, sites of STIM1-Orai1 coupling in muscle remain unclear. For this renewal, we developed inducible, muscle-specific Orai1 and STIM1 KO mice to determine the mechanism by which STIM1-Orai1 coupling limits fatigue. We provide exciting evidence that fatiguing exercise drives the formation of heretofore undescribed junctions between the TT and SR where STIM1-Orai1 coupling occurs, which we refer to as Ca2+ entry units (CEUs). CEUs are connected to, but distinct from, the triad or Ca2+ release unit. We provide preliminary data that Orai1 has a stronger impact on muscle fiber contractile function in female compared to male mice. We also provide preliminary collaborative immunoprecipitation and mass spectroscopy feasibility data for characterizing the STIM1 proteome before and after CEU formation. We will use these research tools, approaches, discoveries, and collaborations to advance understanding of the molecular determinants, subcellular location, and functional role of SOCE in skeletal muscle. Based on our published and preliminary data, we hypothesize that fatiguing exercise triggers formation of junctional extensions of the triad containing activated STIM1-Orai1 complexes that coordinate SOCE to enhance SR calcium refilling, limit muscle fatigue, and over the long-term, promote NFATc1 nuclear localization and type I fiber specification. We also hypothesize that fatigue-induced CEU formation in muscle involves a complex coordination of multiple protein components (including Bin1, STIM1, and Orai1). We propose to test these hypotheses according to the following two Specific Aims. Aim 1 will characterize the role of Orai1 in muscle fatigue and Type I fiber specification. Aim 2 will identif the subcellular location, molecular components, and stability of newly identified CEUs in adult skeletal muscle and determine the dependence of CEU formation and disassembly on the development of and recovery from fatigue. This project will: 1) provide novel mechanistic insights into the physiological role and subcellular location of SOCE in muscle, 2) use targeted and non-biased discovery approaches to identify and validate proteins involved CEU formation, and 3) determine the impact of gender on Orai1-dependent SOCE function, fatigue, fiber type specification, and CEU formation.
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1 |
2011 — 2015 |
Dirksen, Robert T Hamilton, Susan L [⬀] |
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. |
Basis of Muscle Dysfunction in Malignant Hyperthermia and Central Core Disease @ Baylor College of Medicine
DESCRIPTION (provided by applicant): Mutations in the RYR1 gene underlie several debilitating, life-threatening muscle diseases. In this application we are proposing to use a mouse model with a knockin mutation into RyR1 (Y524S) that has features of three of these disorders: malignant hyperthermia (MH) heat/exercise-induced exertional rhabdomyolysis (ER) and central core disease (CCD). Our long term goals are to define the mechanisms that underlie the decline in muscle function associated with these diseases and to develop new interventions. Our overall working hypothesis for the current proposal the Y524S mutation in RyR1 increases the temperature sensitivity of both excitation-contraction coupling (ECC) and RyR1 Ca2+ leak, producing the MH response and driving oxidative stress and mitochondrial destruction that leads to the myopathy. Our specific aims are to: 1) Elucidate the role of Ca2+ influx via CaV1.1 in the MH response and the development of the myopathy; 2) Delineate the mechanisms of core formation in YS mice; 3) Define the mechanism by which an activator (AICAR) of the energy sensing kinase AMPK prevents the MH response and evaluate AICAR's ability to slow the development of the myopathy in YS mice; and 4) Define the mechanism by which a non-immunosuppressive ligand for the protein FKBP12 prevents the MH response and evaluate SLF's effect on the myopathy in YS mice. The proposed work is highly significant because we directly couple the delineation of pathways underlying disease processes with the development of novel therapeutic interventions that have distinct mechanisms of action, allowing for some flexibility to tailor treatment strategies to different mutations associated with MH and CCD. The proposed research is innovative because of: 1) paradigm shifting hypotheses; 2) unique mouse models, 3) cutting edge technologies, and 4) therapeutic interventions never previously proposed as treatments for MH or other RyR1-linked myopathies.
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0.916 |
2011 |
Dirksen, Robert |
R13Activity Code Description: To support recipient sponsored and directed international, national or regional meetings, conferences and workshops. |
Mitochondrial Physiology and Medicine @ University of Rochester
DESCRIPTION (provided by applicant): This proposal requests funds to support an international symposium on "Mitochondrial Physiology and Medicine" to be held on September 7 - 11, 2011, at the Marine Biological Laboratory in Woods Hole, MA. This meeting will be the 65th Annual Symposium of the Society of General Physiologists (SGP). The annual symposium of the SGP has established its legacy as being the premier and most innovative meeting for physiologists, cell biologists, and biophysicists worldwide that span across all career stages and professional arenas. Recent groundbreaking discoveries demonstrating the pivotal role of mitochondria in controlling human physiology and disease have repositioned mitochondria back to the center stage of biomedical research. With typically d 200 participants, SGP symposia are large enough to provide detailed and in depth analyses of a focused area of research, while at the same time being small enough to maximize individual discussions and foster collaborative interactions between students, postdoctoral fellows, new investigators and established leaders within the field. An incredibly enriched and ambitious program has been developed for this meeting. Specifically, the meeting benefit greatly from two internationally-acclaimed leaders in mitochondrial research as keynote speakers: Dr. David Clapham (Harvard Medical School) and Dr. Douglas Wallace (Children's Hospital of Philadelphia). The program was developed by an impressive Organizing Committee consisting of recognized leaders in mitochondrial research including Drs. P. Bernardi (University of Padova), R. Dirksen (University of Rochester), R. Gottlieb (San Diego State University), G. HajnLczky (Thomas Jefferson University), and B O'Rourke (Johns Hopkins University). The symposium will focus on 5 overarching themes: (1) mitochondrial morphology, movement, and dynamics, (2) mitochondrial systems biology, (3) mitochondrial ion channels and transporters, (4) mitochondrial communication and signaling, and (5) mitochondria in cell death and disease. These topics represent an integration of mitochondrial physiology and medicine from molecular to human levels with a special emphasis on the role of mitochondria as agents and therapeutic targets in cardiovascular disease. We expect that this conference will attract approximately 150-200 basic and clinical scientists across all career stages working at the cutting edge of mitochondrial research. The Society of General Physiologists and the members of organization committee are poised to ensure the success of this symposium. It is our aim that through open dialogue and communication, new and important ideas and novel therapies will emerge as a direct result of this timely meeting.
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0.915 |
2013 — 2017 |
Dirksen, Robert T |
U54Activity Code Description: To support any part of the full range of research and development from very basic to clinical; may involve ancillary supportive activities such as protracted patient care necessary to the primary research or R&D effort. The spectrum of activities comprises a multidisciplinary attack on a specific disease entity or biomedical problem area. These differ from program project in that they are usually developed in response to an announcement of the programmatic needs of an Institute or Division and subsequently receive continuous attention from its staff. Centers may also serve as regional or national resources for special research purposes, with funding component staff helping to identify appropriate priority needs. |
Cardiac Pathogenesis and Theraphy of Dm1 @ University of Rochester
PROJECT SUMMARY (See Instructions): Myotonic dystrophy type 1 (DM1) is caused by expansion of a CTG repeat in the DM protein kinase (DMPK) gene. Transcripts from the mutant allele contain an expanded CUG repeat, and are retained in the nucleus. This results in reduction of the kinase protein and toxicity of the mutant RNA. One mechanism for RNA toxicity is that splicing factors in the Muscleblind-like (MBNL) family are sequestered by the CUG-expanded RNA. This leads to abnormal regulation of alternative splicing and defects of miRNA biogenesis. It appears that many aspects of DM1 result from RNA toxicity (myotonia, insulin resistance) but DMPK reduction may contribute to the cardiac phenotypes. Deletion of Dmpk in mice produces cardiac conduction defects that are similar to those observed human DM1. This suggests that cardiac phenotypes of DM1 may result from combined effects of DMPK deficiency and RNA toxicity, but this possibility has not been rigorously tested. Studies in transgenic mice indicate that features of DM1 in skeletal muscle are reversible by targeting the mutant RNA with antisense oligonucleotides (ASOs). However, it is unclear whether this approach will be effective in the heart. In skeletal muscle, the nuclear-retained mutant transcripts are sensitized to ASOs, providing a mechanism for preferential knockdown of RNA from the mutant allele. Whether ASOs provide a method for cardiac correction without exacerbating DMPK deficiency is unknown. Here we propose to study the impact of DMPK deficiency on cardiac function, in the presence and absence of CUG-expanded RNA, and the feasibility of using ASOs to target CUG-expanded transcripts in the heart. Aim 1 will characterize the effects of constitutive Dmpk deficiency on cardiac conduction and contractile function in mice. Acquired (ASO-mediated) Dmpk deficiency will also be examined. Aim 2 will use transgenic mice to examine the effects of CUG-expanded RNA on alternative splicing, miR-1 biogenesis, and cardiac function in vivo. Aim 3 will use mouse models to test for synergistic effects of RNA toxicity and Dmpk deficiency in the heart. Aim 4 will use mice with RNA toxicity and Dmpk deficiency - the condition that approximates the molecular lesion in human DM1 heart - to assess therapeutic effects of ASOs on cardiac function in vivo.
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1 |
2014 — 2015 |
Dirksen, Robert T |
R13Activity Code Description: To support recipient sponsored and directed international, national or regional meetings, conferences and workshops. |
2015 Muscle: Excitation/Contraction Coupling Gordon Research Conference & Gordon Research Seminar @ Gordon Research Conferences
? DESCRIPTION (provided by applicant): This is a proposal for partial support of the only major national or international meeting dedicated to excitation/contraction coupling and how defects in this critical process underlie muscle disease. The Muscle: Excitation/Contraction Coupling (ECC) Gordon Research Conference (GRC) will meet from May 30 - June 5, 2015 at the new Sunday River Resort GRC site in Newry, ME. The GRC program was developed by an Executive Organizing Committee consisting of leaders in ECC research including Drs. Robert T. Dirksen (University of Rochester), Susan Treves (Basel University Hospital), and Isaac N. Pessah (UC Davis). The two overarching objectives of the 2015 Muscle: Excitation/Contraction Coupling GRC are: Objective 1: To discuss and critically evaluate new breakthroughs regarding the molecular mechanisms that control ECC and how this process is altered in muscle fatigue, atrophy, sarcopenia, metabolic disease, and genetically-inherited forms of skeletal and cardiac muscle disease. Objective 2: To promote visibility and leadership of junior investigators in the field by providing a platform for students, postdoctoral fellows, and new independent investigators to present their work, as well as to interact and network with established senior investigators. The 2015 GRC conference will consist of 35 speakers, 18 Discussion Leaders, and 16 poster presentations. Scientific sessions will focus on structural studies of the ECC apparatus, new insights into the molecular mechanisms and regulation of ECC, identification of new therapeutic targets, and the development of innovative treatments for muscle fatigue, sarcopenia, and skeletal and cardiac muscle disorders that adversely impact millions of individuals in the US. For the first time for this GRC, the meeting will be preceded by a two-day Gordon Research Seminar (GRS) for graduate students and postdoctoral fellows. In addition, a different young investigator will be paired with a senior investigator to serve as Co-Discussion Leaders for sessions during the GRC. Innovative Rapid Poster Preview and This Just In speaker slots will be used to highlight late-breaking news. These innovative program elements will also maximize poster session visibility, as well as to enhance the integration and exposure of promising young investigators in the field during the GRC. We expect that this conference will attract 150-200 basic and clinical scientists across all career stages, genders, races, heritages, research disciplines, professional affiliations (academic, industrial, government), and geographical locations. Our overall aim is to promote: 1) open dialogue and communication, 2) exposure and career advancement of promising young investigators in the field, 3) fundamental understanding in the mechanisms of ECC and how defects in this process lead to compromised muscle performance, and 4) the development of novel and effective therapies to combat skeletal and cardiac muscle disorders.
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0.915 |
2016 |
Dirksen, Robert T Hamilton, Susan L [⬀] Jung, Sung Yun Rodney, George G |
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. |
Basis of Muscle Dysfunction in Malignant Hyperthermia & Central Core Disease @ Baylor College of Medicine
? DESCRIPTION (provided by applicant): Mutations in the type I ryanodine receptor (RyR1) are associated with a variety of human muscle diseases including malignant hyperthermia (MH), MH with cores, central core disease (CCD), multi-minicore disease, and others. RyR1-related myopathies are among the most common group of non-dystrophic muscle diseases and are associated with significant clinical disabilities, often including wheelchair dependence, severe scoliosis, respiratory failure, and can result in premature death in childhood. Currently there are no therapies for these devastating myopathies. MH mutations in RyR1 are frequently associated with heat stress, and/or exercise-induced heat stroke and/or rhabdomyolysis. CCD is a congenital myopathy associated with metabolically inactive central cores in skeletal muscle fibers, but the presence of cores is highly variable (despite the name) and the severity of the disease does not correlate with cores. We have shown that some CCD mutations increase Ca2+ leak from the sarcoplasmic reticulum, while others decrease Ca2+ permeation through RyR1. These finding raise the question of how opposing functional effects on RyR1 can result in related diseases. To answer this central, unresolved question and aid in the development of new interventions, we created mouse models of MH with cores (Y524S, YS) and CCD (I4895T, IT). Heterozygous YS mice are susceptible to anesthetic and heat-induced sudden death and also exhibit an age-dependent myopathy characterized by mitochondrial damage and the formation of amorphous cores. Heterozygous IT mice are not heat sensitive, but display decreased exercise capacity, deceased muscle fiber cross-sectional area, and a myopathy that increases with age. In this renewal application, we propose to define the cellular and molecular mechanisms by which functionally opposing RyR1 mutations produce these distinct phenotypes and then use these findings to develop new, mechanism-based therapeutic interventions for MH and CCD. Our working hypothesis is that the YS mutation drives the disease via increased RyR1 Ca2+ leak, activation of the mitochondrial permeability transition pore (mPTP), and oxidative stress, while the IT mutation enhances SR Ca2+ content, which leads to increased ER stress, p53 expression, mitochondrial damage and apoptosis. To test this hypothesis, we propose to: 1) Define the role of altered cytosolic, mitochondrial, and SR lumenal Ca2+ signaling and mPTP activation in the YS and IT myopathies. 2) Define the roles of RyR1 post- translational modifications. 3) Define the role of p53 in the IT myopathy and the enhanced heat sensitivity of YS mice. 4) Assess the potential of S107, NAC, 4PBA, and pifithrin-µ as interventions for MH and CCD. There are currently no therapies to treat RyR1-related myopathies. This multi-PI application is designed to address this need by carefully delineating specific cellular pathways that underlie disease processes and then to use this information to develop and test several novel therapeutic interventions with distinct mechanisms of action.
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0.916 |
2016 — 2017 |
Dirksen, Robert T |
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.) |
Orai1 as a Therapeutic Target For Muscular Dystrophy @ University of Rochester
Being first reported in 2001, store-operated Ca2+ entry (SOCE) is a relatively new phenomenon in skeletal muscle. SOCE is coordinated by coupling between two proteins: STIM1 calcium sensors in the sarcoplasmic reticulum (SR) and Ca2+-permeable Orai1 channels in the transverse tubule (TT) membrane. SOCE enhances muscle growth/development, limits fatigue, and promotes fatigue-resistant type I fiber specification. On the other hand, SOCE dysfunction contributes to muscle weakness/fatigue in aging, exacerbates muscular dystrophy, and mutations in STIM1 and Orai1 genes result in debilitating myopathies. The picture that emerges is that tight regulation of STIM1/Orai1-dependent SOCE activity is critical for optimal muscle performance such that increases or decreases in SOCE activity can lead to muscle fatigue, sarcopenia, and myopathy. Thus, Orai1-dependent SOCE represents a provocative potential therapeutic target for muscular dystrophy. We recently demonstrated that SOCE promotes skeletal muscle growth, limits muscle fatigue, and exacerbates the severity of muscular dystrophy in dystrophin- (mdx) and ?-sarcoglycan-deficient (sgcd-/-) mice. For this R21 application, we developed tamoxifen-inducible, muscle-specific Orai1 knockout mice in order to determine the specific role of Orai1-dependent Ca2+ entry in skeletal muscle in the dystrophic phenotypes observed in mdx and sgcd-/- mice, established mouse models of Duchene Muscular Dystrophy and Limb Girdle Muscular Dystrophy, respectively. We also established a collaboration with CalciMedica Inc. to evaluate the efficacy of systemic delivery of 4 potent new investigational SOCE channel inhibitors in mitigating the myopathic phenotypes of mdx and sgcd-/- mice. We will use these new research tools and collaborations, together with a comprehensive multi-disciplinary experimental approach, to evaluate the efficacy of inhibiting Orai1-dependent SOCE as a therapeutic intervention for muscular dystrophy. Based on our published and preliminary data, we hypothesize that partial inhibition of Orai1-dependent Ca2+ entry in skeletal muscle provides protection against myopathy in mouse models of muscular dystrophy without enhancing susceptibility to muscle fatigue. The validity of this central hypothesis will be rigorously tested in two Specific Aims. Aim 1 will use tamoxifen-inducible, muscle-specific Orai1 knockout mice to determine the impact of partial post-developmental, muscle-specific reduction of SOCE on muscular dystrophy. Aim 2 will determine the therapeutic efficacy of systemic administration of new generation Orai1 channel inhibitors obtained through a collaboration from CalciMedica Inc. to reduce the dystrophic phenotypes of mdx and sgcd-/- mice. The results of this project will provide needed preclinical evidence regarding the therapeutic potential of targeting Orai1-dependent Ca2+ entry as a treatment for muscular dystrophy.
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1 |
2017 — 2020 |
Dirksen, Robert T Hamilton, Susan L [⬀] Jung, Sung Yun Rodney, George G |
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. |
Basis of Muscle Dysfunction in Maligt Hyperthermia & Central Core Disease @ Baylor College of Medicine
? DESCRIPTION (provided by applicant): Mutations in the type I ryanodine receptor (RyR1) are associated with a variety of human muscle diseases including malignant hyperthermia (MH), MH with cores, central core disease (CCD), multi-minicore disease, and others. RyR1-related myopathies are among the most common group of non-dystrophic muscle diseases and are associated with significant clinical disabilities, often including wheelchair dependence, severe scoliosis, respiratory failure, and can result in premature death in childhood. Currently there are no therapies for these devastating myopathies. MH mutations in RyR1 are frequently associated with heat stress, and/or exercise-induced heat stroke and/or rhabdomyolysis. CCD is a congenital myopathy associated with metabolically inactive central cores in skeletal muscle fibers, but the presence of cores is highly variable (despite the name) and the severity of the disease does not correlate with cores. We have shown that some CCD mutations increase Ca2+ leak from the sarcoplasmic reticulum, while others decrease Ca2+ permeation through RyR1. These finding raise the question of how opposing functional effects on RyR1 can result in related diseases. To answer this central, unresolved question and aid in the development of new interventions, we created mouse models of MH with cores (Y524S, YS) and CCD (I4895T, IT). Heterozygous YS mice are susceptible to anesthetic and heat-induced sudden death and also exhibit an age-dependent myopathy characterized by mitochondrial damage and the formation of amorphous cores. Heterozygous IT mice are not heat sensitive, but display decreased exercise capacity, deceased muscle fiber cross-sectional area, and a myopathy that increases with age. In this renewal application, we propose to define the cellular and molecular mechanisms by which functionally opposing RyR1 mutations produce these distinct phenotypes and then use these findings to develop new, mechanism-based therapeutic interventions for MH and CCD. Our working hypothesis is that the YS mutation drives the disease via increased RyR1 Ca2+ leak, activation of the mitochondrial permeability transition pore (mPTP), and oxidative stress, while the IT mutation enhances SR Ca2+ content, which leads to increased ER stress, p53 expression, mitochondrial damage and apoptosis. To test this hypothesis, we propose to: 1) Define the role of altered cytosolic, mitochondrial, and SR lumenal Ca2+ signaling and mPTP activation in the YS and IT myopathies. 2) Define the roles of RyR1 post- translational modifications. 3) Define the role of p53 in the IT myopathy and the enhanced heat sensitivity of YS mice. 4) Assess the potential of S107, NAC, 4PBA, and pifithrin-µ as interventions for MH and CCD. There are currently no therapies to treat RyR1-related myopathies. This multi-PI application is designed to address this need by carefully delineating specific cellular pathways that underlie disease processes and then to use this information to develop and test several novel therapeutic interventions with distinct mechanisms of action.
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0.916 |
2018 — 2021 |
Dirksen, Robert T Hamilton, Susan L [⬀] Jung, Sung Yun |
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. |
Redefining the Role of Fkbp12 in Skeletal Muscle @ Baylor College of Medicine
Abstract Mice with a skeletal muscle (SkM)-specific decrease in the small 12 kDa FK506 binding protein, FKBP12, (FKD mice) display improved endurance, insulin-mediated glucose clearance, and bone mineral density, as well as decreased body fat and resistance to weight gain on a high fat diet. Low doses of rapamycin and SLF (synthetic ligand for FKBP12) that displace FKBP12 from its binding partners mimic the effects of FKBP12 deficiency in SkM, suggesting these drugs have potential as interventions to improve muscle function and metabolism. The primary target of FKBP12 in SkM is the sarcoplasmic reticulum (SR) Ca2+ release channel, RyR1, which controls the release of Ca2+ from intracellular stores during excitation-contraction coupling (ECC). Partial removal of FKBP12 from RyR1 (genetically or by treatment with low doses of rapamycin or SLF) increases both the amplitude of the myoplasmic Ca2+ transient and Ca2+ influx into the muscle fiber during repetitive stimulation. Both enhanced SR Ca2+ release and increased Ca2+ influx associated with partial removal of FKBP12 from RyR1 are blocked by inhibitors of calmodulin-dependent protein kinase II (CaMKII) and store-operated Ca2+ entry (SOCE). However, the mechanisms by which increases in Ca2+ release and influx result in improved muscle function and metabolism remain unknown. We hypothesize the existence of a tunable feedback loop that functionally couples ECC, SOCE, and mitochondrial Ca2+ uptake to modulate muscle function and metabolism. The specific aims of this application are to: A1. Define the roles of FKBP12 and RyR1 phosphorylation in regulating the amplitude of the Ca2+ transient during repetitive stimulation and improving SkM performance and metabolism. 2. Define the feedback loop that enhances Ca2+ store refilling and ATP production to sustain the improved muscle performance and metabolism in FKBP12 deficient mice. 3. Evaluate the therapeutic potential of SLF to improve muscle function and metabolism. Our long-term goal is to develop interventions to improve muscle function in people who cannot perform strenuous exercise due to age, muscle disease, obesity and/or have type II diabetes.
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0.916 |
2020 — 2021 |
Dirksen, Robert T Dowling, James J (co-PI) [⬀] |
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. |
Pathophysiology and Treatment of Recessive Ryr1 Related Myopathy @ University of Rochester
Mutations in the gene that encodes the skeletal muscle type I ryanodine receptor (RYR1) result in a wide range of muscle disorders that collectively comprise the most common cause of non-dystrophic myopathy. The most severe cases of RYR1-related myopathy (RYR1-RM) exhibit a recessive pattern of inheritance and present in infancy with muscle hypotrophy, weakness, respiratory insufficiency, short stature, and a marked reduction in RYR1 protein expression in muscle. Despite their severity, high prevalence and association with significant disability and early mortality, there are no treatments or disease-modifying therapies for RYR1-RM. A major barrier to therapy development has been the lack of an animal model that mirrors the early onset and clinical severity of recessive RYR1-RM. To overcome this barrier, we developed two mouse models of recessive RYR1-RM that pheno-copy key characteristics of the human disorder including myofiber hypotrophy, reduced muscle/body mass, muscle weakness, markedly reduced RYR1 expression, and premature death. The scientific premise of this proposal is that these new mouse models of RYR1-RM provide a unique opportunity to explore the underlying patho-mechanisms of RYR1-RM and test the therapeutic efficacy of mechanism-based interventions. The overall goal of the project is to elucidate the patho-mechanisms responsible for muscle dysfunction in recessive RYR1-RM and to develop and validate effective treatments. We hypothesize that reduced folding/stability of mutated RYR1 homotetramers results in increased RYR1 protein degradation that markedly reduces RYR1 expression, and that even a modest increase in either RYR1 expression or function will ameliorate the myopathy and prolong survival. Furthermore, we also hypothesize that reduced myofiber size in RYR1-RM is a key aspect of disease pathogenesis, that hypotrophy is due to epigenetic abnormalities, and that drugs that target the epigenome or promote muscle growth can ameliorate the disease phenotype. The validity of these hypotheses will rigorously evaluated in three specific aims. Aim 1 will characterize RYR1 expression, function and myopathy in two mouse models of severe, recessive RYR1-RM and assess the therapeutic potential of systemic treatment with ebselen, an FDA-approved drug and known RYR1 activator. Aim 2 will elucidate the mechanism(s) for reduced RYR1 expression in our mouse models of RYR1-RM mice and evaluate the therapeutic efficacy of systemic treatment with a chemical chaperone and ER stress inhibitor (4PBA). Aim 3 will determine the mechanisms leading to muscle hypotrophy in RYR1-RM mice and test the potential of treatment with either HDAC inhibitors or modulators of myofiber size. The results of these studies will provide novel insights into the patho-mechanisms responsible for reduced RYR1 expression and muscle fiber hypotrophy in recessive RYR1-RM and determine the therapeutic potential of several mechanism-based interventions designed to enhance RYR1 function, reduce RYR1 degradation, and limit muscle hypotrophy in pre-clinical models of recessive RYR1-RM.
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