Ca2+ signaling is a universal language used by cells to react and change. In skeletal muscle its patterns of interest cover multiple time scales: milliseconds -Ca2+ movements that determine contraction and relaxation-;seconds to minutes -when sustained activity may lead to myogenic fatigue- and hours to weeks -patterns that cause changes in gene expression and long-term adaptation-. This study is about inside its cellular store;its quantity, and its concentration, [Ca2+]SR, which conditions Ca2+ signals in every time scale. We ask (1) whether and how [Ca2+]SR controls Ca2+ release from the store, and (2) whether and how calsequestrin and triadin, two strategically located SR proteins, contribute to this control. A technical task, which we call "aim 0", is to image and measure [Ca2+]SR. This was accomplished in the current period and will continue in the next, using novel biosensors -molecules made by the cells themselves- and new hybrid monitors, consisting of high performance small synthetic sensors placed into cells manipulated to make special bio-anchors. To answer questions 1 and 2, we will respectively manipulate [Ca2+]SR while we measure it (aim 1) and force cells to change their endowment of calsequestrin and triadin (aim 2). These goals are now feasible in living animals thanks to a DNA transfection method that works with every protein and can be used also to prevent their synthesis. We propose that [Ca2+]SR -which decays when muscles fatigue- is sustained by SOCE, a universal Ca2+ entry pathway, crucial for mobilizing transcription factors that control gene expression. Using SOCE measures developed in the first period, we propose as aim 3 to define the role of newly discovered molecules of SOCE in the control of [Ca2+]SR. These molecules could be bulwarks against fatigue, and provide powerful tools for experimental alterations of [Ca2+]SR in iterative approaches to the main questions. Ca2+ signals deteriorate in disease, fatigue and aging. Fast Ca2+ signals fail in diseases like hypo-PP, MH susceptibility and central core and minicore, as well as in ageing muscle. Mid-range signaling is affected in fatigue and in an MH-like phenotype of mice lacking calsequestrin. Diseases of long term Ca2+ signals, which show striking parallels in muscle and the immune system, include SCID, a familial immune defect that combines loss of SOCE in lymphocytes and a myogenic myopathy. Our work will advance understanding of these deficits by evaluating roles of specific molecules and their interactions. While only fatigue will be specifically addressed in the present project, questions on the relationships among deficits of function, the intricate pathophysiology and the rational design of therapeutic corrections will be addressed better as we understand what controls stored calcium, and what the stored calcium controls.

Public Health Relevance

This project and our lab's work deal with movements of calcium inside muscle. So-called "calcium signaling" is a universal language used by cells to react and change. In skeletal muscle these signals make the difference between rest and motion, thus influencing multiple aspects of our life, including physical conditioning, metabolism, and even body weight. Because calcium is used for many tasks, including killing cells, the signals are dangerous and must be kept under control. In this project we ask what controls the movements of calcium, in particular focusing on roles of calcium storage areas inside the cell. We also ask how the intra-store calcium determines how much will move, or when the movement will cease. Therefore, our work should help understand why signals fail, or get out of hand. This occurs in diseases, including muscular dystrophies, malignant hyperthermia, and others. Signals also decay during fatigue -their decay actually causing fatigue-- and in old age. In muscle fatigue, the stores are depleted. We propose that two newly discovered molecules are important factors that delay this depletion. These molecules are known to play roles in organs and cells other than muscle, including cells that combat infections. As a result there will be multiple repercussions to their defects or failures. It can be anticipated, therefore, that our research will have consequences outside its field of muscle and exercise.

Agency
National Institute of Health (NIH)
Institute
National Institute of Arthritis and Musculoskeletal and Skin Diseases (NIAMS)
Type
Research Project (R01)
Project #
5R01AR049184-10
Application #
8464002
Study Section
Skeletal Muscle and Exercise Physiology Study Section (SMEP)
Program Officer
Boyce, Amanda T
Project Start
2003-01-01
Project End
2014-04-30
Budget Start
2013-05-01
Budget End
2014-04-30
Support Year
10
Fiscal Year
2013
Total Cost
$293,901
Indirect Cost
$90,753
Name
Rush University Medical Center
Department
Physiology
Type
Schools of Medicine
DUNS #
068610245
City
Chicago
State
IL
Country
United States
Zip Code
60612
Figueroa, Lourdes; Shkryl, Vyacheslav M; Blatter, Lothar A et al. (2013) Using two dyes with the same fluorophore to monitor cellular calcium concentration in an extended range. PLoS One 8:e55778
Rios, Eduardo (2013) On an early demonstration of the cell boundary theorem. J Physiol Sci 63:161
Manno, Carlo; Sztretye, Monika; Figueroa, Lourdes et al. (2013) Dynamic measurement of the calcium buffering properties of the sarcoplasmic reticulum in mouse skeletal muscle. J Physiol 591:423-42
Manno, Carlo; Figueroa, Lourdes; Fitts, Robert et al. (2013) Confocal imaging of transmembrane voltage by SEER of di-8-ANEPPS. J Gen Physiol 141:371-87
Manno, Carlo; Figueroa, Lourdes; Royer, Leandro et al. (2013) Altered Ca2+ concentration, permeability and buffering in the myofibre Ca2+ store of a mouse model of malignant hyperthermia. J Physiol 591:4439-57
Figueroa, Lourdes; Shkryl, Vyacheslav M; Zhou, Jingsong et al. (2012) Synthetic localized calcium transients directly probe signalling mechanisms in skeletal muscle. J Physiol 590:1389-411
Shkryl, Vyacheslav M; Blatter, Lothar A; Rios, Eduardo (2012) Properties of Ca2+ sparks revealed by four-dimensional confocal imaging of cardiac muscle. J Gen Physiol 139:189-207
Sztretye, Monika; Yi, Jianxun; Figueroa, Lourdes et al. (2011) D4cpv-calsequestrin: a sensitive ratiometric biosensor accurately targeted to the calcium store of skeletal muscle. J Gen Physiol 138:211-29
Yi, Jianxun; Ma, Changling; Li, Yan et al. (2011) Mitochondrial calcium uptake regulates rapid calcium transients in skeletal muscle during excitation-contraction (E-C) coupling. J Biol Chem 286:32436-43
Sztretye, Monika; Yi, Jianxun; Figueroa, Lourdes et al. (2011) Measurement of RyR permeability reveals a role of calsequestrin in termination of SR Ca(2+) release in skeletal muscle. J Gen Physiol 138:231-47

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