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Biophys J, July 2000, p. 39-50, Vol. 79, No. 1
and
Departments of *Cell Biology and Anatomy and
Medicine, School of Medicine, University of North
Carolina, Chapel Hill, North Carolina 27599-7090 USA
A cold/warm loading protocol was used to ester-load Rhod
2 into mitochondria and other organelles and Fluo 3 into the
cytosol of adult rabbit cardiac myocytes for confocal fluorescence
imaging. Transient increases in both cytosolic Fluo 3 and mitochondrial Rhod 2 fluorescence occurred after electrical stimulation. Ruthenium red, a blocker of the mitochondrial Ca2+ uniporter,
inhibited mitochondrial Rhod 2 fluorescence transients but not
cytosolic Fluo 3 transients. Thus the ruthenium red-sensitive mitochondrial Ca2+ uniporter catalyzes Ca2+
uptake during beat-to-beat transients of mitochondrial free
Ca2+, which in turn may help match mitochondrial ATP
production to myocardial ATP demand. After ester loading, substantial
amounts of Ca2+-indicating fluorophores localized into an
acidic lysosomal/endosomal compartment. This lysosomal fluorescence did
not respond to electrical stimulation. Because fluorescence arose
predominantly from lysosomes after the cold loading/warm incubation
procedure, total cellular fluorescence failed to track beat-to-beat
changes of mitochondrial fluorescence. Only three-dimensionally
resolved confocal imaging distinguished the relatively weak
mitochondrial signal from the bright lysosomal fluorescence.
Biophys J, July 2000, p. 39-50, Vol. 79, No. 1
© 2000 by the Biophysical Society 0006-3495/00/07/39/12 $2.00
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