Activation of skeletal muscle fibers underlies all bodily movement, and is initiated by electrical depolarization of their transverse tubules (TTs), which penetrate the fibers at each sarcomere. Membrane voltage sensors in the TT dihydropyridine receptor (DHPR) respond to TT depolarization, and trigger Ca2+ release via the abutting ryanodine receptor (RyR)/Ca2+ release channels in the adjacent sarcoplasmic reticulum membrane. In frog muscle, the "macroscopic" Ca2+ release caused by fiber depolarization has been shown by us and others to be composed of discrete local events, the Ca2+ sparks.
In Aim 1 we will use ultra high speed confocal imaging to characterize the gating properties of the few channels that underlie each spark, and to determine whether the group of channels always gates in unison, or may sometimes gate independently during a spark.
In Aim 2 we will examine Ca2+ sparks in mammalian muscle, where they do not seem to be the basic unit of physiological Ca2+ release, but instead appear under abnormal or pathological conditions and during muscle repair, possibly due to DHPR / RyR uncoupling. We will study the properties and possible functional roles of Ca2+ sparks in mammalian muscle using adult muscle fiber dedifferrentiation in culture as a model system for myoblast fusion and fiber remodeling, which is important for muscle repair in disease, in dystrophic (mdx) muscle and in muscle from aging mice.
In Aim 3 we will investigate the modulation of macroscopic Ca2+ release in mammalian muscle by endogenous protein ligands and by experimental RyR "domain" peptides. We will use high speed (<50 us/line) line- or band-scan confocal imaging of fibers containing Ca2+ indicators to monitor Ca2+ release during depolarization, together with acute or chronic application of proteins, protein fragments or chimeric constructs, including fluorescent tags for sarcomeric localization, or of small peptides that may disrupt the normal endogenous coupling process. Among the molecules to be investigated for participation in, or modulation of the physiologic (ie, voltage activated) release process will be the DHPR beta subunit, the Ca2+ binding protein S100A1 and "domain" peptides of the RyR. This project will elucidate basic molecular mechanisms regulating Ca2+ release in skeletal muscle and the roles of Ca2+ sparks in muscle disease, damage and repair, and will provide a better understanding of abnormal regulation of Ca2+ release channels in muscle disease.

Agency
National Institute of Health (NIH)
Institute
National Institute of Arthritis and Musculoskeletal and Skin Diseases (NIAMS)
Type
Research Project (R01)
Project #
5R01AR055099-27
Application #
8016088
Study Section
Skeletal Muscle and Exercise Physiology Study Section (SMEP)
Program Officer
Boyce, Amanda T
Project Start
1985-07-01
Project End
2012-01-31
Budget Start
2011-02-01
Budget End
2012-01-31
Support Year
27
Fiscal Year
2011
Total Cost
$267,634
Indirect Cost
Name
University of Maryland Baltimore
Department
Biochemistry
Type
Schools of Medicine
DUNS #
188435911
City
Baltimore
State
MD
Country
United States
Zip Code
21201
Hernández-Ochoa, Erick O; Schneider, Martin F (2012) Voltage clamp methods for the study of membrane currents and SR Ca(2+) release in adult skeletal muscle fibres. Prog Biophys Mol Biol 108:98-118
Wu, Fenfen; Mi, Wentao; Hernández-Ochoa, Erick O et al. (2012) A calcium channel mutant mouse model of hypokalemic periodic paralysis. J Clin Invest 122:4580-91
Hernández-Ochoa, Erick O; Robison, Patrick; Contreras, Minerva et al. (2012) Elevated extracellular glucose and uncontrolled type 1 diabetes enhance NFAT5 signaling and disrupt the transverse tubular network in mouse skeletal muscle. Exp Biol Med (Maywood) 237:1068-83
Liu, Yewei; Hernández-Ochoa, Erick O; Randall, William R et al. (2012) NOX2-dependent ROS is required for HDAC5 nuclear efflux and contributes to HDAC4 nuclear efflux during intense repetitive activity of fast skeletal muscle fibers. Am J Physiol Cell Physiol 303:C334-47
Yamaguchi, Naohiro; Prosser, Benjamin L; Ghassemi, Farshid et al. (2011) Modulation of sarcoplasmic reticulum Ca2+ release in skeletal muscle expressing ryanodine receptor impaired in regulation by calmodulin and S100A1. Am J Physiol Cell Physiol 300:C998-C1012
Prosser, Benjamin L; Hernández-Ochoa, Erick O; Schneider, Martin F (2011) S100A1 and calmodulin regulation of ryanodine receptor in striated muscle. Cell Calcium 50:323-31
Robison, Patrick; Hernández-Ochoa, Erick O; Schneider, Martin F (2011) Adherent primary cultures of mouse intercostal muscle fibers for isolated fiber studies. J Biomed Biotechnol 2011:393740
Olojo, Rotimi O; Hernández-Ochoa, Erick O; Ikemoto, Noriaki et al. (2011) Effects of conformational peptide probe DP4 on bidirectional signaling between DHPR and RyR1 calcium channels in voltage-clamped skeletal muscle fibers. Biophys J 100:2367-77
Olojo, Rotimi O; Ziman, Andrew P; Hernández-Ochoa, Erick O et al. (2011) Mice null for calsequestrin 1 exhibit deficits in functional performance and sarcoplasmic reticulum calcium handling. PLoS One 6:e27036
Prosser, Benjamin L; Hernandez-Ochoa, Erick O; Lovering, Richard M et al. (2010) S100A1 promotes action potential-initiated calcium release flux and force production in skeletal muscle. Am J Physiol Cell Physiol 299:C891-902

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