Abstract
Contrasting information suggests either almost complete depletion of sarcoplasmic reticulum (SR) Ca2+ or significant residual Ca2+ concentration after prolonged depolarization of the skeletal muscle fiber. The primary obstacle to resolving this controversy is the lack of genetically encoded Ca2+ indicators targeted to the SR that exhibit low-Ca2+ affinity, a fast biosensor: Ca2+ off-rate reaction, and can be expressed in myofibers from adult and older adult mammalian species. This work used the recently designed low-affinity Ca2+ sensor (Kd = 1.66 mM in the myofiber) CatchER (calcium sensor for detecting high concentrations in the ER) targeted to the SR, to investigate whether prolonged skeletal muscle fiber depolarization significantly alters residual SR Ca2+ with aging. We found CatchER a proper tool to investigate SR Ca2+ depletion in young adult and older adult mice, consistently tracking SR luminal Ca2+ release in response to brief and repetitive stimulation. We evoked SR Ca2+ release in whole-cell voltage-clamped flexor digitorum brevis muscle fibers from young and old FVB mice and tested the maximal SR Ca2+ release by directly activating the ryanodine receptor (RyR1) with 4-chloro-m-cresol in the same myofibers. Here, we report for the first time that the Ca2+ remaining in the SR after prolonged depolarization (2 s) in myofibers from aging (~220 μM) was larger than young (~132 μM) mice. These experiments indicate that SR Ca2+ is far from fully depleted under physiological conditions throughout life, and support the concept of excitation–contraction uncoupling in functional senescent myofibers.
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Abbreviations
- ER/SR:
-
Endoplasmic/sarcoplasmic reticulum
- EGFP:
-
Enhanced green fluorescence protein
- FDB:
-
Flexor digitorum brevis
- RyR1:
-
Ryanodine receptor-isoform-1
- 4-CmC:
-
4-Chloro-m-cresol
- TEA:
-
Tetraethylammonium
- di-8-ANEPPS:
-
di-8-amino naphthyl ethenyl pyridinium
- CPA:
-
Cyclopiazonic acid
- CatchER:
-
Calcium sensor for detecting high concentrations in the ER
- GECI:
-
Genetically encoded Ca2+ biosensor
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Acknowledgments
The present study was supported by grants from the National Institutes of Health/National Institute on Aging AG07157, AG33385, and AG15820 to Osvaldo Delbono; and EB007268 and GM081749 to Jenny Yang, and the Wake Forest University Claude D. Pepper Older Americans Independence Center (P30-AG21332).
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Wang, ZM., Tang, S., Messi, M.L. et al. Residual sarcoplasmic reticulum Ca2+ concentration after Ca2+ release in skeletal myofibers from young adult and old mice. Pflugers Arch - Eur J Physiol 463, 615–624 (2012). https://doi.org/10.1007/s00424-012-1073-3
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DOI: https://doi.org/10.1007/s00424-012-1073-3