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Excitation-Contraction Coupling in Normal and Dystrophic Mammalian Muscle

Supported by grant NIH/NIAMS R01-AR07664: Julio L. Vergara, PI & Marino DiFranco, Co-PI.

    The overall goal of this project 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 (EC) coupling process in mammalian skeletal muscle. We have found that Ca2+ release evoked by action potentials (APs) (or voltage-clamp pulses) in muscle fibers from two animal models, the adult mdx mouse and a phenotypic sarcospan overexpressing mouse (collaboration with Dr. Rachelle Crosbie, Dept. of Physiological Science), is significantly smaller than that in wild type (wt) fibers. We hypothesize that disruption of the DGC undermines the structural and/or the functional properties of the transverse tubular system (T-system) and the sarcoplasmic reticulum (SR), thus attenuating the EC coupling process. We are currently focusing on the mechanisms responsible for the impairment of Ca2+ release in mdx mice, 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 are also carried out in double knockout mdx/utrophin (mdx/utr-/-) mice that display a phenotype more comparable to that in DMD patients. To further characterize the link between the DGC integrity and a fully functional EC coupling, we 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). Another goal 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, which helps us understand the function of the DGC in terms of EC coupling and sarcolemmal integrity, is also carried out by using electrophysiological and state-of-the-art optical methods, such as Föster 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.

Role of the Transverse Tubular System in Mammalian Skeletal Muscle Excitability

Supported by grant NIH/NIAMS R01-AR054816: Julio L. Vergara, PI & Marino DiFranco, Co-PI.
   The underlying hypothesis on which this project is based is that the transverse tubular system (TTS) plays such a preponderant role in the overall electrical properties of mammalian skeletal muscle fibers that, before understanding the pathophysiology of muscle channelopathies, it is necessary to characterize the role that various conductances present in 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 use electrophysiological methods, state-of-the-art optical techniques (which permit to measure TTS voltage changes) and mathematical modeling in order to probe the role that individual ionic conductances play in the radial spread of the depolarization in the TTS membrane compartment. We are currently characterizing the passive electrical properties of the TTS and the role of each conductive pathway in normal mouse muscle fibers under voltage clamp conditions. Furthermore, we are studying the properties and limitations of the TTS propagation in fibers stimulated to elicit repetitive firing and testing 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. Since a conundrum in the functional investigation of channelopathies is the tenuous demarcation between myotonia and paralysis, we will soon 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 previously, we will investigate whether the pathogenesis observed in animal models of myotonia, hypokalemic (hypo-PP), and hyperkalemic periodic paralysis (hyper-PP), can be understood from alterations in the electrical propagation at the TTS.

Modulation of Sarcoplasmic Reticulum Calcium Release

Collaboration with Dr. Susan Hamilton, Department of Molecular Physiology and Biophysics, Baylor College of Medicine.

    We are studying the effects of FKBP12 on the gain of EC coupling process. FKBP12 is a small immunophilin that binds with high affinity to sarcoplasmic reticulum Ca2+ release channels (ryanodine receptors, RyRs) and transforming growth factor β1 receptors (TβR). Our goal is to demonstrate that FKBP12 binding to RyR1 controls the gain of EC coupling in a biphasic manner, thereby, modulating Ca2+ stores, force production, fatigue, and recovery from injury. To this end, we transfect muscle fibers in vivo with plasmids that encode for FKBP12, and correlate the observed alterations in the Ca2+ release (single pulse and tetanic stimulation) with the level of expression of this protein. We are further evaluating the ability TβR activation to increase the gain of EC coupling via FKBP12 and the ability of drugs that decrease FKBP12 binding to RyR1 to slow the development of fatigue and enhance recovery from injury.

Development of optical methods for monitoring voltage in groups of neuroanatomically-defined neurons

Supported by McKnight Foundation Technological Innovations in Neuroscience Awards: Thomas Otis, Department of Neurobiology (PI) & Julio L. Vergara, Department of Physiology (co-PI).
    Our ultimate goal is to establish a two photon laser-based optical methodology for measuring membrane potential with high time resolution from many neurons in a circuit. Our optical strategy is to measure membrane potential based on FRET; as a variation of one first suggested by Gonzalez and Tsien (Gonzalez and Tsien, 1995). On a recent sabbatical, one of us (Otis) tested his idea that the carbocyanine-based neuronal tracer dye DiO could be co-opted to report large changes in membrane potential when combined with dipicrylamine (DPA), a non-fluorescent molecule which absorbs blue-green light and whose membrane partitioning is voltage-sensitive (Chanda et al., 2005; DiFranco et al., 2007). Utilization of DiO as a donor has unique advantages since carbocyanine dyes have been used extensively to label specific neuronal pathways by targeted microinjection or by “diolistic” methods. Prior work by the Vergara lab has demonstrated that shift of the donor spectrum from green to blue can double the signal size observable with DPA/membrane tethered fluorescent protein pairs (DiFranco et al., 2007). We will compare the performance of DiB/DPA with that of DiO/DPA and will evaluate whether DiB/DPA signals can be generated with two-photon illumination. The specific objectives of this project are: 1) To determine whether a FRET-based sensor system can be further improved by substituting blue carbocyanine dyes for the green DiO. To this end, we compare the performance of DiB/DPA with that of DiO/DPA and will evaluate whether DiB/DPA signals can be generated with two-photon illumination. 2) To optimize in vivo tract labeling approaches. We have preliminary found that groups of neurons can be labeled via either diolistic approaches or by in vivo injection. We are currently attempting to optimize these methods by injecting DiO in the inferior olive. 3) Use SLM-based illumination methods combined with fast CCD camera detection to record DiO/DPA and/or DiB/DPA signals from multiple neurons in parallel. We are currently implementing a Spatial Light Modulator (SLM)-based method for delivering user-defined patterned illumination in order to confine laser illumination to small regions of interest and/or to illuminate multiple targets simultaneously. This will be combined with a high speed camera as a detector so as to optimize optical monitoring of parallel activity in small neural circuits.
Calcium transients induced by AMPA receptor activation in hypoglossal motoneurons

Consuelo Morgado-Valle, Departamento de Neurociencias, Universidad Veracruzana, Mexico, and Julio L. Vergara, UCLA.
    Our recent work on Ca2+ dynamics in the respiratory rhythm generating preBötC neurons suggest that somatic Ca2+ transients result from activation of voltage-gate Ca2+ channels (VGCC) due to action potential (AP) backpropagation (Morgado-Valle et al., 2008). We are using electrophysiology, microfluorometry, and pharmacology to investigate whether AMPA (α-amino-3-hydroxyl-5-methyl-4-isoxazole-propionate) receptors (AMPAR), known to mediate inspiratory-modulated synaptic activity, generate Ca2+ transients as a result from influx via their intrinsic Ca2+ permeability, or due to secondary voltage-gated Ca2+ channel (VGCC) activation, or both. We are also investigating whether AMPAR activation induces Ca2+-induced-Ca2+ release (CIRC). In order to advance these studies, we use transverse brainstem slices (containing the preBötC) that generate respiratory-related motor output are prepared from neonatal rodents (0-5 days old), monitor electrically the AMPAR activation, and discern between Ca2+ influx of synaptic origin from that due to action potential (AP) back propagation or CICR.
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