Julio L. Vergara - US grants

The research project proposed is aimed at the description, characterization and correlation of events involved in excitation-contraction coupling in twitch muscle fibers. Experiments are proposed for studying events occurring in tubular system membranes, triad and sacroplamsic reticulum membranes in addition to calcium concentration changes in thh sarcoplasm. The membrane potential changes of the tubular system and the sarcoplasmic reticulum will be monitored by voltage-sensing-dyes (fluorescence and absorption). Triadic function will be studied in correlation to charge movements. Calcium concentration changes will be monitored by calcium-sensing-dyes. Modifiers of excitation-contraction coupling will be tested to assess the role of each of the above processes in the physiology of muscle contractions. We will use a cut fiber preparation and double vaseline gap voltage clamp technique allowing modification of the internal medium of the muscle fiber, control of the surface membrane potential and measurement of optical signals. Our experiments are expected to provide new information on 1) the steps involved in excitation-contraction coupling, and 2) the links between consecutive steps. These studies are expected to be of importance in the physiology of striated muscle, including heart muscle.

The possibility that a chemical transmission mechanism, involving inositol (1,4,5) trisphosphate (InsP3) as a second messenger, is responsible for excitation-contraction coupling in skeletal muscle will be studied in physiological/biochemical experiments. The release of phosphoinositide-derived inositol phosphates induced by the electrical activation of skeletal muscle will be studied by the biochemical analysis of metabolic constituents obtained from rapidly frozen muscles. The ability of InsP3 and other inositol phosphates (cyclic InsP3) to rapidly induce release from the sarcoplasmic reticulum (SR) will be assessed using optical techniques in skinned skeletal muscle fibers. The modulation of this ability by several agents will also be investigated. Novel techniques to produce rapid release of Ca2+ and inositol phosphates (using photoreleasable derivatives) will be developed in order to investigate the kinetic response of the SR to putative agonists. These studies will give important kinetic information on the chemical modulation of the physiological process of Ca2+ release and they will complement our new findings about the nature of the transmission delay in E-C coupling. The role of Na and K conductances in the T-tubule depolarization and in possible ion depletion/accumulation phenomena will be studied with the aid of potentiometric dyes. The possibility that voltage dependent phospholipase C activity is endogenously present in the T tubule membranes will be directly tested in vitro. Studies in barnacle muscle fibers will allow us to evaluate the role of inositol phosphates on the E-C coupling of invertebrate muscle and will provide insights into the role of extracellular Ca in the regulation of this process.

This award supports a workshop on Transduction in Biological Systems to be held in Chile hosted by ten Chilean participants from the University of Chile, Centro de Estudios Cientificos de Santiago (C.E.C.S.), where the workshop will be held, and the Catholic University. The Chilean co-organizers are J. Bacigalupo, C. Hidalgo, E. Jaimovich and C. Vergara. The workshop will bring together people from several countries working on intracellular messengers, coupling in skeletal muscle and gland cells, visual, olfactory and auditory systems. The Chilean biophysicists represent an area of research strength and have produced excellent students, some now in the U.S. so the interactions should be considerable and of benefit to both sides. Three other Latin American countries: Argentina, Mexico and Venezuela will be represented as well as the U.K. and France. Experts from all the fields to be covered in the workshop will attend from the U.S. so personal connections will be made or reinforced, information will be freely exchanged and opportunities for future collaborations will be explored.

DESCRIPTION (Adapted from the Applicant's Abstract): It is currently believed that the release of Ca2+ from the sarcoplasmic reticulum (SR) in skeletal muscle fibers is triggered in responses to depolarization sensed by specialized voltage sensors located in the transverse tubular system (t-system) membranes. The main goal of this application is to understand the dynamic properties of the voltage dependent signal transduction process involving the junction between T-system and the SR. They propose to investigate this process on the same millisecond time scale as the physiological process of T-tubule action potential-elicited Ca2+ release. In order to do so it will be necessary to advance the standard voltage-clamp methodology using a newly developed methodology, supercharging command pulses, that boosts the rate of depolarization in the T-system. T-tubule depolarization will be measured with potentiometric dyes to determine the optimal parameters for supercharging waveforms such that they yield quasi-step depolarizations of the T-system. Ca2+ release from the SR will be measured with a low affinity indicator able to track the rapid kinetics of the release process. The voltage dependence of the transduction process in response to fast T-tubule depolarizations will be investigated in edge segments of muscle fibers with epifluorescence illumination and within localized regions of individual sarcomeres using the confocal spot detection methodology. They will also investigate the mechanisms of stochastic recruitment of Ca2+ release channels by analyzing elementary fluorescence fluctuations related to the opening of RyR channels in both mature muscle fibers and in lipid bilayers. The experimental results are expected to provide new data about the true dynamics and voltage-sensitivity of the Ca2+ release process, leading to a clarified picture of physiological excitation-contraction coupling in skeletal muscle. Since this application deals with general questions regarding voltage propagation in T-tubules, intracellular Ca2+ regulation, voltage dependence of SR Ca2+ release, and fluorescence fluctuations associated with elementary channel openings, its conclusions apply not only to skeletal muscle, but to cardiac muscle, smooth muscle, and to almost every other cell in which Ca2+ regulation processes operate as well.

[unreadable] DESCRIPTION (provided by applicant): Abnormalities in the mechanisms of calcium regulation and excitation-contraction (EC) coupling that may be linked to the degeneration of skeletal muscle fibers in Becker Muscular Dystrophy (BMD) and in Duchenne Muscular Dystrophy (DMD) will be investigated using isolated muscle fibers from mdx mice. Cells from this animal model, like those of dystrophic patients, have deficiencies in the expression of the protein dystrophin. Although there is substantial biochemical evidence demonstrating the association of the dystrophin-glycoprotein complex with transmembrane- and membrane-bound muscle proteins, little is known about its specific role in the physiological aspects of a muscle fiber. The main goal of this proposal is to obtain critical experimental evidence linking the absence of dystrophin with specific alterations in the electrical propagation in the transverse tubular system and calcium signaling machinery. Several possibilities that may explain these observations will be explored experimentally. Changes in intracellular calcium concentration triggered by electrical activity of the muscle fibers will be recorded with the aid of low affinity calcium sensitive fluorescent indicators and membrane potential changes in the transverse tubules will be monitored with potentiometric indicators. The investigations will be carried out using high-resolution optical methods that permit to assess the functional state of these critical steps of the EC coupling process, not only at the cellular level, but also within sub-regions of the muscle fiber and even within a single sarcomere. We will perform these measurements across three different age groups of the mdx mouse in order to understand the progression of the disease with time. We will also test if muscle fibers from a utrophin/dystrophin-lacking double mutant mouse, which exhibits a harsher pathology (similar to DMD), show signs of more pronounced defects in EC coupling. These types of experiments are necessary to unravel the mysterious role that dystrophin may play in the normal regulation of calcium metabolism in skeletal muscle. The knowledge gained in the proposed studies will help to elucidate the functional role of dystrophin in mammalian skeletal muscle, to this date the most fundamental and elusive problem in muscular dystrophy research. The enhanced methods proposed to detect defective steps in the EC coupling mechanisms within localized submicroscopic regions of mammalian muscle fibers may become the optimal choice for the future evaluation of genetic therapeutic procedures in sub-regions of a single muscle cell. [unreadable] [unreadable]

[unreadable] DESCRIPTION (provided by applicant): This R21 exploratory research grant responds to the NIGMS call for the development of novel eukaryotic expression systems capable of generating pure membrane proteins in sufficient quantities to afford crystallization trials. The lack of such a system has critically limited the number of structural studies of integral membrane proteins, such as ion channels and transporters, with atomic level resolution. The current application aims to cope with this problem by investigating the potential use of mammalian skeletal muscle to produce large quantities of recombinant membrane proteins when transfected in vivo with genetically engineered DNA plasmids. We propose that skeletal muscle may be an ideal preparation not only for the massive synthesis of cytosolic proteins, but most importantly, for the production and exportation of large quantities of transmembrane proteins. We plan to pursue these ideas experimentally in two separate, but intrinsically connected specific aims. Specific Aim 1 will test whether in vivo electroporation of muscle tissue with piasmids engineered for mammalian expression of six-histidine (6His) tagged EGFP and ECFP allows us to purify them to homogeneity (using standard chromatographic methods) in quantities compatible with crystallization protocols. With this knowledge, we will attempt to purify a very relevant cytosolic muscle protein, the beta-1a subunit of the skeletal muscle dihydropyridine receptor (DHPR), whose structure has not yet being determined with atomic resolution because of limitations in its synthesis. In Specific Aim 2, we will first electroporate muscle with plasmids encoding fluorescently-tagged transmembrane proteins and invest gate which of the observed expression patterns for these proteins leads to an efficient membrane protein extraction protocol. We will focus on the expression of the following integral membrane proteins (ionic channels): the alpha-15 subunit of the skeletal muscle DHPR, the Shaker K channel, and the mammalian skeletal ryanodine receptor channel. Later on, plasmids will be engineered for the expression of 6His-tagged membrane proteins in order to ensure that each of them can be isolated and purified to homogeneity in sufficient amount to permit their crystallization. [unreadable] [unreadable]

DESCRIPTION (provided by applicant): 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.

DESCRIPTION (provided by applicant): 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 Fster 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. PUBLIC HEALTH RELEVANCE: 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.

FKBP12 is a small immunophilin that binds with high affinity to sarcoplasmic reticulum Ca2+ release channels (ryanodine receptors, RyRs) and transforming growth factor ¿1 receptors (T¿I RI). In this application we are going to test the hypothesis that the gain of E-C coupling is regulated in a biphasic manner by FKBP concentration. Specifically we will: 1) Demonstrate that FKBP12 binding to RyR1 controls the gain of E-C coupling in a biphasic manner, thereby, modulating Ca2+ stores, force production, fatigue, and recovery from injury; 2) Evaluate the ability T¿RI activation to increase the gain of E-C coupling via FKBP12; 3) Evaluate the ability of drugs that decrease FKBP12 binding to RyR1 to slow the development of fatigue and enhance recovery from injury and 4) Identify the amino acids on RyR1 that contribute to FKBP12 binding. The research described in this application will clarify the role of FKBP12 in skeletal muscle function and lay the groundwork for the development of new therapeutic interventions to slow diaphragm fatigue and enhance recovery from injury.

DESCRIPTION (provided by applicant): Several conditions such as diseases or accidental and chirurgical trauma can lead to the loss of nerve control of skeletal muscles. Optogenetic control of excitability, which has revolutionized neurobiological research but has never been attempted in adult skeletal muscles, is a promising alternative for the recovery of muscle function. However, several basic questions need to be answered prior to the practical implementation of optogenetic approaches in vivo. In Aim 1 of this proposal we will investigate whether activating (e.g. channelrhodopsin 2) and silencing actuators (e.g. archeorhodopsin 3) are expressed in sufficient quantities in adult skeletal muscle fibers so that pulses of illuminatin can either elicit action potentials (APs), or inhibit electrically elicited APs, respectively. This information will allow us to comparatively test (in Aim 2) the ability of light and electrical stimulation to attain fine control of the mechanical activity of intact muscles expressing optical actuators. We will use two approaches for the specific transfection of muscle with plasmids encoding for opsins: electroporation and adeno-associated virus. This will enable us to investigate the feasibility of using novel approaches using wireless optoelectronic implantable devices for the fine control of muscles in the intact animal. The outcome of the innovative research approaches proposed in this grant are expected to pave the way for the practical use of optogenetics tools to restore the control of muscle output when they are deemed inactive due to the absence of nerve inputs.