The use of CalciumOrange-5N as a specific marker of mitochondrial Ca2+ in mouse skeletal muscle fibers |
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Authors: | Pura Bolaños Alis Guillen Héctor Rojas Simona Boncompagni Carlo Caputo |
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Institution: | (1) Laboratorio de Fisiología Celular, Centro de Biofísica y Bioquímica, Instituto Venezolano de Investigaciones Científicas IVIC, Apartado 21827, Caracas, 1020A, Venezuela;(2) IIM Interuniversity Institute of Myology, Ce.S.I. Center of Research on Aging, University degli Studi G. d’Annunzio, 66013 Chieti, Italy |
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Abstract: | We report the use of the fluorescent dye CalciumOrange-5N (CaOr-5N) as a specific mitochondria Ca2+ marker in enzymatically dissociated mouse FBD muscle fibers. Using laser scanning confocal microscopy and the dyes Mitotracker
Green (MTG), di-8-ANEPPS and endoplasmic reticulum tracker green (ERTG), we determined the relative position of mitochondria,
transverse tubules and sarcoplasmic reticulum in the sarcomere. Comparison with electron micrographies showed that mitochondria
are mostly present at both sides of Z lines and near the triads located at the A-I band border. CaOr-5N fluorescence was mainly
distributed in mitochondria, highly co-localised with MTG and basically excluded from the A band space. ERTG localised mostly
between the two t-tubules present in each sarcomere. We studied the effect of the protonophore FCCP using CaOr-5N to measure
mitochondrial Ca2+ and JC-1 dye to measure mitochondria inner membrane potential (ΔΨ
m). After FCCP treatment, the CaOr-5N fluorescence diminished by about 33% in 80 s, while JC-1 fluorescence diminished by 36%
in 200 s. Our results show the loss of Ca2+ from mitochondria when ΔΨm is depolarised and demonstrate the usefulness of CaOr-5N to mark mitochondrial Ca2+]m.
Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users. |
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Keywords: | Calcium mitochondria Skeletal muscle fiber Mitochondria Confocal microscopy Calcium release |
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