title: New method for determining total calcium content in tissue applied to skeletal muscle with and without calsequestrin.
abstract: We describe a new method for determining the concentration of total Ca in whole skeletal muscle samples ([CaT]WM in units of mmoles/kg wet weight) using the Ca-dependent UV absorbance spectra of the Ca chelator BAPTA (1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid). Muscle tissue was homogenized in a solution containing 0.15 mM BAPTA and 0.5% sodium dodecyl sulfate (to permeabilize membranes and denature proteins) and then centrifuged. The solution volume was adjusted so that BAPTA captured essentially all of the Ca. [CaT]WM was obtained with Beer's law from the absorbance change produced by adding 1 mM EGTA to capture Ca from BAPTA. Results from mouse, rat, and frog muscles were reasonably consistent with results obtained using other methods for estimating total [Ca] in whole muscles and in single muscle fibers. Results with external Ca removed before determining [CaT]WM indicate that most of the Ca was intracellular, indicative of a lack of bound Ca in the extracellular space. In both fast-twitch (extensor digitorum longus, EDL) and slow-twitch (soleus) muscles from mice, [CaT]WM increased approximately linearly with decreasing muscle weight, increasing approximately twofold with a twofold decrease in muscle weight. This suggests that the Ca concentration of smaller muscles might be increased relative to that in larger muscles, thereby increasing the specific force to compensate for the smaller mass. Knocking out the high capacity Ca-binding protein calsequestrin (CSQ) did not significantly reduce [CaT]WM in mouse EDL or soleus muscle. However, in EDL muscles lacking CSQ, muscle weights were significantly lower than in wild-type (WT) muscles and the values of [CaT]WM were, on average, about half the expected WT values, taking into account the above [CaT]WM versus muscle weight relationship. Because greater reductions in [CaT]WM would be predicted in both muscle types, we hypothesize that there is a substantial increase in Ca bound to other sites in the CSQ knockout muscles.
authors.affiliation: Institute of Sport, Exercise and Active Living, Victoria University, Melbourne, Victoria 8001, Australia.
authors.forename: Cédric R H
authors.initials: CR
authors.lastname: Lamboley
authors.affiliation: Département de physiologie et biophysique, Université de Sherbrooke Faculté de Médicine et des Sciences de la Santé, Sherbrooke, Québec J1H5N4, Canada.
authors.forename: Sandrine A
authors.initials: SA
authors.lastname: Kake Guena
authors.affiliation: Département de physiologie et biophysique, Université de Sherbrooke Faculté de Médicine et des Sciences de la Santé, Sherbrooke, Québec J1H5N4, Canada.
authors.forename: Fatou
authors.initials: F
authors.lastname: Touré
authors.affiliation: Département de physiologie et biophysique, Université de Sherbrooke Faculté de Médicine et des Sciences de la Santé, Sherbrooke, Québec J1H5N4, Canada.
authors.forename: Camille
authors.initials: C
authors.lastname: Hébert
authors.affiliation: Département de physiologie et biophysique, Université de Sherbrooke Faculté de Médicine et des Sciences de la Santé, Sherbrooke, Québec J1H5N4, Canada.
authors.forename: Louiza
authors.initials: L
authors.lastname: Yaddaden
authors.affiliation: Département de physiologie et biophysique, Université de Sherbrooke Faculté de Médicine et des Sciences de la Santé, Sherbrooke, Québec J1H5N4, Canada.
authors.forename: Stephanie
authors.initials: S
authors.lastname: Nadeau
authors.affiliation: Département de Réadaptation, Université Laval, Québec G1K 7P4, Canada.
authors.forename: Patrice
authors.initials: P
authors.lastname: Bouchard
authors.affiliation: Department of Pharmacology and Physiology, University of Rochester Medical Center, Rochester, NY 14642.
authors.forename: Lan
authors.initials: L
authors.lastname: Wei-LaPierre
authors.affiliation: Département de physiologie et biophysique, Université de Sherbrooke Faculté de Médicine et des Sciences de la Santé, Sherbrooke, Québec J1H5N4, Canada.
authors.forename: Jean
authors.initials: J
authors.lastname: Lainé
authors.affiliation: Département de physiologie et biophysique, Université de Sherbrooke Faculté de Médicine et des Sciences de la Santé, Sherbrooke, Québec J1H5N4, Canada.
authors.forename: Eric C
authors.initials: EC
authors.lastname: Rousseau
authors.affiliation: Département de Réadaptation, Université Laval, Québec G1K 7P4, Canada.
authors.forename: Jérôme
authors.initials: J
authors.lastname: Frenette
authors.affiliation: Center for Research on Aging and Department of Neuroscience, Imaging and Clinical Sciences, "G. d'Annunzio" University of Chieti-Pescara, I-66100 Chieti, Italy.
authors.forename: Feliciano
authors.initials: F
authors.lastname: Protasi
authors.affiliation: Department of Pharmacology and Physiology, University of Rochester Medical Center, Rochester, NY 14642.
authors.forename: Robert T
authors.initials: RT
authors.lastname: Dirksen
authors.affiliation: Département de physiologie et biophysique, Université de Sherbrooke Faculté de Médicine et des Sciences de la Santé, Sherbrooke, Québec J1H5N4, Canada Paul.Pape@USherbrooke.ca.
authors.forename: Paul C
authors.initials: PC
authors.lastname: Pape
mesh_terms: D000818:Animals; D001001:Anura; D002118:Calcium / Q000378:metabolism*; D065096:Calcium Chelating Agents / Q000737:chemistry; D002155:Calsequestrin / Q000378:metabolism*; D004533:Egtazic Acid / Q000031:analogs & derivatives / Q000737:chemistry; D051379:Mice; D018482:Muscle, Skeletal / Q000378:metabolism*; D051381:Rats; D013050:Spectrometry, Fluorescence / Q000379:methods
chemical_list: D065096:Calcium Chelating Agents; D002155:Calsequestrin; D004533:Egtazic Acid; C025603:1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid; D002118:Calcium