Mitochondrial calcium transients in adult rabbit cardiac myocytes: inhibition by ruthenium red and artifacts caused by lysosomal loading of Ca(2+)-indicating fluorophores.
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 Ca(2+) uniporter, inhibited mitochondrial Rhod 2 fluorescence transients but not cytosolic Fluo 3 transients. Thus the ruthenium red-sensitive mitochondrial Ca(2+) uniporter catalyzes Ca(2+) uptake during beat-to-beat transients of mitochondrial free Ca(2+), which in turn may help match mitochondrial ATP production to myocardial ATP demand. After ester loading, substantial amounts of Ca(2+)-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.
Trollinger
D R
DR
Department of Cell Biology and Anatomy, School of Medicine, University of North Carolina, Chapel Hill, North Carolina 27599-7090 USA.
Cascio
W E
WE
Lemasters
J J
JJ
D000255:Adenosine Triphosphate / Q000378:metabolism; D000814:Aniline Compounds; D000818:Animals; D016477:Artifacts; D002118:Calcium / Q000378:metabolism*; D015220:Calcium Channels; D002135:Calcium-Binding Proteins / Q000187:drug effects / Q000378:metabolism; D002478:Cells, Cultured; D003600:Cytosol / Q000378:metabolism; D004952:Esters / Q000378:metabolism; D005456:Fluorescent Dyes / Q000378:metabolism / Q000494:pharmacology; D006321:Heart / Q000187:drug effects; D006575:Heterocyclic Compounds, 3-Ring; D007202:Indicators and Reagents / Q000494:pharmacology; D017136:Ion Transport / Q000187:drug effects; D008247:Lysosomes / Q000378:metabolism*; D008928:Mitochondria / Q000187:drug effects / Q000378:metabolism*; D009119:Muscle Contraction / Q000187:drug effects; D009206:Myocardium / Q000166:cytology / Q000378:metabolism*; D011817:Rabbits; D012430:Ruthenium Red / Q000494:pharmacology*; D013696:Temperature; D014966:Xanthenes
D000814:Aniline Compounds; D015220:Calcium Channels; D002135:Calcium-Binding Proteins; D004952:Esters; D005456:Fluorescent Dyes; D006575:Heterocyclic Compounds, 3-Ring; D007202:Indicators and Reagents; D014966:Xanthenes; C034703:mitochondrial calcium uniporter; C068483:rhod-2; D012430:Ruthenium Red; C059715:Fluo-3; D000255:Adenosine Triphosphate; D002118:Calcium