title: Assessment of calcium sparks in intact skeletal muscle fibers.
abstract: Maintaining homeostatic Ca(2+) signaling is a fundamental physiological process in living cells. Ca(2+) sparks are the elementary units of Ca(2+) signaling in the striated muscle fibers that appear as highly localized Ca(2+) release events mediated by ryanodine receptor (RyR) Ca(2+) release channels on the sarcoplasmic reticulum (SR) membrane. Proper assessment of muscle Ca(2+) sparks could provide information on the intracellular Ca(2+) handling properties of healthy and diseased striated muscles. Although Ca(2+) sparks events are commonly seen in resting cardiomyocytes, they are rarely observed in resting skeletal muscle fibers; thus there is a need for methods to generate and analyze sparks in skeletal muscle fibers. Detailed here is an experimental protocol for measuring Ca(2+) sparks in isolated flexor digitorm brevis (FDB) muscle fibers using fluorescent Ca(2+) indictors and laser scanning confocal microscopy. In this approach, isolated FDB fibers are exposed to transient hypoosmotic stress followed by a return to isotonic physiological solution. Under these conditions, a robust Ca(2+) sparks response is detected adjacent to the sarcolemmal membrane in young healthy FDB muscle fibers. Altered Ca(2+) sparks response is detected in dystrophic or aged skeletal muscle fibers. This approach has recently demonstrated that membrane-delimited signaling involving cross-talk between inositol (1,4,5)-triphosphate receptor (IP3R) and RyR contributes to Ca(2+) spark activation in skeletal muscle. In summary, our studies using osmotic stress induced Ca(2+) sparks showed that this intracellular response reflects a muscle signaling mechanism in physiology and aging/disease states, including mouse models of muscle dystrophy (mdx mice) or amyotrophic lateral sclerosis (ALS model).
authors.affiliation: Department of Surgery, Davis Heart and Lung Research Institute, The Ohio State University Wexner Medical Center.
authors.forename: Ki Ho
authors.initials: KH
authors.lastname: Park
authors.affiliation: Department of Physiology and Cell Biology, Davis Heart and Lung Research Institute, The Ohio State University Wexner Medical Center.
authors.forename: Noah
authors.initials: N
authors.lastname: Weisleder
authors.affiliation: Department of Molecular Biophysics and Physiology, Rush University Medical Center.
authors.forename: Jingsong
authors.initials: J
authors.lastname: Zhou
authors.affiliation: Department of Surgery, Davis Heart and Lung Research Institute, The Ohio State University Wexner Medical Center.
authors.forename: Kristyn
authors.initials: K
authors.lastname: Gumpper
authors.affiliation: Department of Surgery, Davis Heart and Lung Research Institute, The Ohio State University Wexner Medical Center.
authors.forename: Xinyu
authors.initials: X
authors.lastname: Zhou
authors.affiliation: Department of Internal Medicine, Davis Heart and Lung Research Institute, The Ohio State University Wexner Medical Center.
authors.forename: Pu
authors.initials: P
authors.lastname: Duann
authors.affiliation: Department of Surgery, Davis Heart and Lung Research Institute, The Ohio State University Wexner Medical Center.
authors.forename: Jianjie
authors.initials: J
authors.lastname: Ma
authors.affiliation: Department of Surgery, Davis Heart and Lung Research Institute, The Ohio State University Wexner Medical Center; pei-hui.lin@osumc.edu.
authors.forename: Pei-Hui
authors.initials: PH
authors.lastname: Lin
mesh_terms: D000690:Amyotrophic Lateral Sclerosis / Q000378:metabolism; D000818:Animals; D002118:Calcium / Q000032:analysis / Q000378:metabolism*; D020013:Calcium Signaling; D004195:Disease Models, Animal; D051379:Mice; D018485:Muscle Fibers, Skeletal / Q000737:chemistry / Q000378:metabolism*; D009136:Muscular Dystrophies / Q000378:metabolism; D009997:Osmotic Pressure; D012519:Sarcoplasmic Reticulum / Q000737:chemistry / Q000378:metabolism
chemical_list: D002118:Calcium