The use of CalciumOrange-5N as a specific marker of mitochondrial Ca2+ in mouse skeletal muscle fibers.
We report the use of the fluorescent dye CalciumOrange-5N (CaOr-5N) as a specific mitochondria Ca(2+) 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 Ca(2+) and JC-1 dye to measure mitochondria inner membrane potential (DeltaPsi(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 Ca(2+) from mitochondria when DeltaPsi(m) is depolarised and demonstrate the usefulness of CaOr-5N to mark mitochondrial [Ca(2+)](m).
Bolaños
Pura
P
Laboratorio de Fisiología Celular, Centro de Biofísica y Bioquímica, Instituto Venezolano de Investigaciones Científicas IVIC, Apartado 21827, Caracas 1020A, Venezuela.
Guillen
Alis
A
Rojas
Héctor
H
Boncompagni
Simona
S
Caputo
Carlo
C
D000447:Aldehydes; D000818:Animals; D001562:Benzimidazoles; D002118:Calcium / Q000378:metabolism*; D002232:Carbocyanines; D002259:Carbonyl Cyanide p-Trifluoromethoxyphenylhydrazone / Q000494:pharmacology; D005456:Fluorescent Dyes; D053078:Membrane Potential, Mitochondrial / Q000187:drug effects; D051379:Mice; D018613:Microscopy, Confocal; D008931:Mitochondria, Muscle / Q000378:metabolism* / Q000648:ultrastructure; D018485:Muscle Fibers, Skeletal / Q000378:metabolism* / Q000648:ultrastructure; D018482:Muscle, Skeletal / Q000378:metabolism* / Q000648:ultrastructure; D009930:Organic Chemicals; D011726:Pyridinium Compounds; D012518:Sarcomeres / Q000378:metabolism; D012519:Sarcoplasmic Reticulum / Q000378:metabolism; D013194:Staining and Labeling / Q000379:methods*; D013997:Time Factors; D014475:Uncoupling Agents / Q000494:pharmacology
C089669:1-(3-sulfonatopropyl)-4-(beta-(2-(di-n-octylamino)-6-naphthyl)vinyl)pyridinium betaine; D000447:Aldehydes; D001562:Benzimidazoles; C093686:CaOrange-5N; D002232:Carbocyanines; D005456:Fluorescent Dyes; D009930:Organic Chemicals; D011726:Pyridinium Compounds; D014475:Uncoupling Agents; C111472:mitotracker green FM; C068624:5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolocarbocyanine; D002259:Carbonyl Cyanide p-Trifluoromethoxyphenylhydrazone; D002118:Calcium
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