Skeletal Muscle. Ca Release Control Inside the Sarcoplasmic Reticulum.

Rush University Medical Center, Chicago IL

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Abstract

DESCRIPTION (provided by applicant): 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.