MODULATION OF SARCOPLASMIC RETICULUM CALCIUM RELEASE
L Julio
Baylor College Of Medicinecity: Houston country: United States (us)
Grant 5R01AR041802-17 from National Institute Of Arthritis And Musculoskeletal And Skin Diseases
Keywords: Adult; Affinity; Amino Acids; Behavior; Binding (Molecular Function); Binding Sites; Calcineurin; calcineurin phosphatase; Calcium; Carboxylic Acids; Cardiovascular Diseases; Coupling; Depressed mood; design; Development; Diaphragm (Anatomy); Dose; Electroporation; Embryo; Failure (biologic function); Fatigue; Fiber; FK506; Growth; Growth Factor; Homeostasis; human FRAP1 protein; Immunofluorescence Immunologic; Immunophilins; Immunosuppressive Agents; improved; in vivo; Injury; Laboratories; Location; Lung diseases; Measurement; Mechanics; Medical; Membrane; Mus; Muscle; Muscle Fibers; Muscle function; mutant; Mutate; Mutation; Myopathy; myostatin; Natural immunosuppression; novel; novel therapeutic intervention; Pathway interactions; Patients; Pharmaceutical Preparations; Phosphotransferases; prevent; Probability; Production; Property; Proteins; Protocols documentation; receptor; Recovery; Rehabilitation therapy; Relative (related person); Research; Respiratory Failure; response; Role; Ryanodine Receptor Calcium Release Channel; Ryanodine Receptors; RyR1; RyR3; Sarcoplasmic Reticulum; sensor; Signal Transduction; Sirolimus; Skeletal muscle injury; Skeletal muscle structure; Staining method; Stains; Tacrolimus Binding Protein 1A; Tacrolimus Binding Proteins; Testing; Time; Transforming Growth Factors; Triad Acrylic Resin; Ventilator; Vesicle; voltage; Weaning; Work
Relevance: The research in this application will investigate the role of small immunophilins (FKBPs) in muscle Ca2+ homeostasis, fatigue, and recovery from injury. Diaphragm fatigue is a serious medical problem that contributes to respiratory failure in patients with skeletal muscle, cardiovascular and pulmonary diseases and to failure to wean patients from ventilators. Also, the rate of recovery of skeletal muscle from injury has profound effects on rehabilitation
Project start date: 1993-08-01
Project end date: 2014-11-30
Budget start date: 1-DEC-2011
Budget end date: 30-NOV-2012
5R01AR041802-17 (2012): $419698
Sponsored Links Excellgen http://Excellgen.com
Grants awarded to L Julio
EXCITATION-CONTRACTION COUPLING IN NORMAL AND DYSTROPHIC MAMMALIAN MUSCLE
L Julio, Professor
University Of California Los Angelescity: Los Angeles country: United States (us)
Grant 5R01AR047664-08 from National Institute Of Arthritis And Musculoskeletal And Skin Diseases
Abstract: The overall goal of this proposal is to obtain an in-depth understanding of the mechanistic links between alterations of the dystrophin glycoprotein complex (DGC) and impairment of the excitation-contraction coupling (ECC) process in mammalian skeletal muscle. This functional characterization will be achieved in muscle fibers from various animal models of human muscular dystrophies. We have found that Ca2+ release evoked by action potentials (APs) (or voltage-clamp pulses) in muscle fibers from two of such models, the adult mdx mouse and the phenotypic sarcospan (SSPN) overexpressing mouse (SSPN-Tg), is significantly smaller than in wild type fibers. We hypothesize that disruption of the DGC undermines the structural and functional support for the transverse tubular system (TTS) and the sarcoplasmic reticulum (SR), thus attenuating the ECC process. We will first investigate the mechanisms responsible for the impairment of Ca2+ release in mdx mice (Aim 1), the most prevalently used animal model for Duchenne Muscular Dystrophy (DMD), which lacks dystrophin in the DGC. However, since the phenotypic alterations in mdx mice are relatively benign, possibly due to utrophin substitution in the DGC, experiments will be also carried out in double knockout mdx/utrophin (mdx/utr-/-) mice that display a phenotype more comparable to that in DMD patients (Aim 2). To further characterize the link between the DGC integrity and a fully functional ECC, we will take advantage of our ability to express DGC proteins by in vivo electroporation and use transgenic animal models with other genetic conditions altering the DGC (e.g. SSPN-Tg, and Utr-TET). The last goal of the proposal is to investigate, using 2-photon laser scanning microscopy (TPLSM) the subcellular distribution of representative DGC protein components in order to assess if they are associated exclusively with the sarcolemma or if they have a more ubiquitous distribution in association with the Z-line and the TTS. This characterization will help us understand the function of the DGC in terms of ECC and sarcolemmal integrity (Aim 3). These investigations will be carried out by using electrophysiological and state-of-the-art optical methods, such as F"ster resonance energy transfer (FRET) and total internal reflection fluorescence microscopy (TIRFM), to also assess the nanoscale localization of DGC and ECC proteins with respect to the internal and external leaflets of the surface and TTS membranes. In Duchenne Muscular Dystrophy (DMD) the muscles lack the protein dystrophin, an integral component of a dystrophin-glycoprotein complex (DGC). We have discovered that the absence of dystrophin in the muscle fibers of the mdx mouse, a widely used animal model of DMD, impairs their ability to release Ca2+ from the sarcoplasmic reticulum in response to electrical stimulation, thus explaining the muscle weakness observed in the DGC pathology. We now propose to investigate the mechanisms that link DGC alterations with a deficient Ca2+ release. The results will significantly broaden our understanding of muscle disease mechanisms and will potentially provide therapeutic molecular tools which are deemed necessary for further advances in gene therapy
Keywords: Action Potentials; Adult; Affect; Animal Model; Animals; Attenuated; Benign; Buffers; Calsequestrin; Cells; Characteristics; Complex; Coupling; Data; Development; Dystroglycans; Dystrophin; Egtazic Acid; Electric Stimulation; Electroporation; Energy Transfer; Fiber; Fluorescence Microscopy; Fostering; Foundations; gene therapy; Glycoproteins; Goals; Health; Hereditary Disease; Human; human SSPN protein; Impairment; in vivo; Inbred mdx Mice; Investigation; Knock-out; Laser Scanning Microscopy; Life; Link; Measurement; Membrane; mini-dystrophin; Modeling; Molecular; Mus; Muscle; Muscle Fibers; Muscle Weakness; Muscular Dystrophies; Muscular Dystrophy, Duchenne; Mutation; Myopathy; nanoscale; novel; Optical Methods; Optics; Other Genetics; overexpression; Pathology; Patients; Phenotype; Photons; Physiologic pulse; Physiological; Positioning Attribute; Process; protein complex; Proteins; Relative (related person); research study; response; Role; RyR1; Sarcoglycans; Sarcolemma; Sarcoplasmic Reticulum; SERCA1; Signal Transduction; Skeletal muscle structure; Structure; Surface; System; Techniques; Testing; Therapeutic; tool; Transgenic Animals; Transgenic Organisms; Tubular formation; two-photon; Utrophin; voltage clamp
Project start date: 2001-04-01
Project end date: 2014-01-31
Budget start date: 1-FEB-2011
Budget end date: 31-JAN-2012
PFA/PA: PA-07-070
5R01AR047664-08 (2011): $301871
ROLE OF THE TRANSVERSE TUBULAR SYSTEM IN MAMMALIAN SKELETAL MUSCLE EXCITABILITY
L Julio, Professor
University Of California Los Angelescity: Los Angeles country: United States (us)
Grant 5R01AR054816-05 from National Institute Of Arthritis And Musculoskeletal And Skin Diseases
Abstract: The central hypothesis of this proposal is that the transverse tubular system (TTS) plays such a preponderant role in the overall properties of mammalian skeletal muscle that, in order to understand the pathophysiology of muscle channelopathies it will be necessary to carefully characterize the electrical properties of this membrane compartment. Changes in membrane potential of the TTS, which are mediated by the activation of ion channels, not only affect the electrical properties of the muscle fiber, but also are responsible for triggering the mechanisms of excitation-contraction coupling (ECC). We will use electrophysiological methods, state-of-the-art optical techniques (which permit to measure TTS voltage changes), and mathematical modeling of the radial spread of the depolarization in this compartment, in order to probe the detailed role that ionic conductances play in these processes. First, we will characterize the passive electrical properties and each of the major conductive pathways in normal mouse muscle fibers under voltage clamp conditions (Aim 1). We will then study the properties and limitations of the TTS propagation in fibers stimulated to elicit repetitive firing and test the effects that alterations in individual conductances (sodium and chloride in particular) have on these properties. The goal is to elucidate the potential role that K accumulation in the lumen of the TTS lumen may play in the phenomenology associated with channelopathies such as periodic paralysis and myotonia (Aim 2). Since a conundrum in the functional investigation of channelopathies is the tenuous demarcation between myotonia and paralysis, in Aim 3 we will investigate whether intricacies of the voltage regulation of the ECC can result in abolition or preservation of the Ca2+ release process depending on the pattern of electrical activity in the TTS. Finally, with the knowledge acquired in previous aims, we will investigate whether the pathogenesis observed in animal models of myotonia and hyperkaelemic periodic paralysis can be understood from alterations in the electrical propagation at the TTS (Aim 4). The knowledge gained with these investigations will not only be relevant towards the understanding of the pathophysiology of channelopathies, but since they will provide basic information about the physiological mechanisms of TTS electrical propagation, they will be of significance for understanding a number of muscle diseases
Keywords: 21+ years old; Accounting; Address; Adult; adult human (21+); Adynamia Episodica Hereditaria; Affect; Animal Model; Animal Models and Related Studies; Back; base; Biological Preservation; biological signal transduction; Cell Communication and Signaling; Cell Signaling; Characteristics; Chloride; Chloride Channels; Chloride Ion; Chlorides; Cl- element; Control Animal; Coupling; Dependence; Development; Disorder of muscle, unspecified; Dorsum; Dysfunction; Dystrophia Myotonica; electrical property; Electrodes, Miniaturized; Electrophysiology; Electrophysiology (science); experiment; experimental research; experimental study; Fiber; flexor digitorum brevis; Fluorescence Resonance Energy Transfer; Frequencies (time pattern); Frequency; FRET; Functional disorder; Genetic Alteration; Genetic Change; Genetic defect; genome mutation; Goals; Human; Human, Adult; Human, General; Hybrids; Hyperkalemic periodic paralysis; Individual; Intracellular Communication and Signaling; Investigation; Investigators; Ion Channel; Ion Channels, Chloride; Ionic Channels; Ions; Knock-in; Knock-in Mouse; Knock-out; Knockout; Knowledge; Length; Mammals, Mice; Man (Taxonomy); Man, Modern; Math Models; mathematical model; mathematical modeling; Measures; Mediating; Membrane; Membrane Channels; Membrane Potentials; membrane structure; Methods; Methods and Techniques; Methods, Other; Mice; Mice, Mutant Strains; Microelectrodes; model organism; Modeling; Molecular; mouse mutant; Murine; Mus; Muscle; Muscle Disease; Muscle disease or syndrome; Muscle Disorders; Muscle Fibers; Muscle Tissue; Muscle, Skeletal; Muscle, Voluntary; Muscular Diseases; muscular disorder; Mutant Strains Mice; Mutation; Myopathic Conditions; Myopathic disease or syndrome; Myopathic Diseases and Syndromes; Myopathy; Myopathy, unspecified; Myotonia; Myotonia Atrophica; Myotonia Dystrophica; Myotonic Dystrophy; Myotonic Periodic Paralysis; Myotubes; Na element; Nature; Neurophysiology / Electrophysiology; Optics; overexpression; Palsy; Paralysed; paralysis; Paralysis, Periodic, Hyperkalemic, Familial; paralytic; Pathogenesis; pathophysiology; pathway; Pathway interactions; Pattern; Physiologic; Physiological; Physiopathology; Play; Plegia; preservation; Preservation, Biologic; Preservation, Biological; Primary Hyperkalemic Periodic Paralysis; Process; programs; Programs (PT); Programs [Publication Type]; Property; Property, LOINC Axis 2; Radial; Records; Regulation; Relative; Relative (related person); Reporting; Research Personnel; research study; Researchers; response; Resting Potentials; Rhabdomyocyte; Role; salt; Sarcolemma; Signal Transduction; Signal Transduction Systems; Signaling; Simulate; Skeletal Fiber; Skeletal Muscle Cell; Skeletal Muscle Fiber; Skeletal muscle structure; Skeletal Muscle Tissue; Skeletal Myocytes; social role; Sodium; Sodium Chloride; Sodium chloride (NaCl); Staining method; Stainings; Stains; Steinert Disease; Surface; System; System, LOINC Axis 4; Techniques; Testing; Transgenic Animals; Transmembrane Potentials; Tubular; Tubular formation; V (voltage); Vacuum; voltage; voltage clamp
Project start date: 2007-04-11
Project end date: 2012-01-31
Budget start date: 1-FEB-2011
Budget end date: 31-JAN-2012
5R01AR054816-05 (2011): $289109