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Published online 17 April 2006. doi:10.1083/jcb.200601160
The Rockefeller University Press, 0021-9525 $8.00
JCB, Volume 173, Number 2, 187-193
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Direct in vivo monitoring of sarcoplasmic reticulum Ca2+ and cytosolic cAMP dynamics in mouse skeletal muscle



Rüdiger Rudolf1,2, Paulo J. Magalhães1, and Tullio Pozzan1,2

1 Department of Biomedical Sciences, University of Padua, I-35121 Padua, Italy
2 Venetian Institute of Molecular Medicine, I-35129 Padua, Italy

Correspondence to Rüdiger Rudolf: ruediger.rudolf{at}itg.fzk.de

Skeletal muscle contraction depends on the release of Ca2+ from the sarcoplasmic reticulum (SR), but the dynamics of the SR free Ca2+ concentration ([Ca2+]SR), its modulation by physiological stimuli such as catecholamines, and the concomitant changes in cAMP handling have never been directly determined. We used two-photon microscopy imaging of GFP-based probes expressed in mouse skeletal muscles to monitor, for the first time in a live animal, the dynamics of [Ca2+]SR and cAMP. Our data, which were obtained in highly physiological conditions, suggest that free [Ca2+]SR decreases by ~50 µM during single twitches elicited through nerve stimulation. We also demonstrate that cAMP levels rise upon ß-adrenergic stimulation, leading to an increased efficacy of the Ca2+ release/reuptake cycle during motor nerve stimulation.

Abbreviations used in this paper: CPA, cyclopiazonic acid; DHPR, dihydropyridine receptor; Kd, apparent dissociation constant; PB, phosphate buffer; PKA, protein kinase A; RYR, ryanodine receptor; SERCA, sarcoendoplasmic reticulum Ca2+ ATPase; ROI, regions of interest; SR, sarcoplasmic reticulum; TA, tibialis anterior.


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