Separation of fluorescence signals from Ca2+ and NADH during cardioplegic arrest and cardiac ischemia

Determinations of intracellular [Ca(2+)](i) during ischemia using fluorescent indicators are hampered by overlapping cellular autofluorescence (AF), which largely depends on NADH. If Ca(2+) is to be determined under different kinds of ischemia, signal separation merits special attention. We used tri...

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Bibliographic Details
Published in:Cell calcium (Edinburgh) 2004-04, Vol.35 (4), p.381-391
Main Authors: Brachmanski, Monika, Gebhard, Martha Maria, Nobiling, Rainer
Format: Article
Language:English
Subjects:
Animals
Calcium - chemistry
Calcium - metabolism
Cardioplegic Solutions - administration & dosage
Female
Fluorescence
Fluorescent Dyes
Fura-2
Guinea Pigs
Heart Arrest, Induced
Male
Myocardial Ischemia - physiopathology
Myocardial Reperfusion
Myocardium - metabolism
NAD - chemistry
NAD - metabolism
Oxidation-Reduction
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Summary:Determinations of intracellular [Ca(2+)](i) during ischemia using fluorescent indicators are hampered by overlapping cellular autofluorescence (AF), which largely depends on NADH. If Ca(2+) is to be determined under different kinds of ischemia, signal separation merits special attention. We used triple wavelength excitation fluorescence to separate autofluorescence from [Ca(2+)]-dependent fura-2 fluorescence. Excitation at 360 nm served as third, Ca(2+)-insensitive wavelength. Using an appropriate evaluation procedure, we separated Ca(2+)-dependent signals from autofluorescence which is semiquantitatively associated with NADH, an indicator of the cellular redox state. We compared changes of [Ca(2+)](i) in isolated hearts during ischemia following cardioplegic arrest with those after transient stop of nutritive perfusion. We observed [Ca(2+)] transients in spontaneously beating hearts, persisting during ischemic episodes, and an increase of mean [Ca(2+)](i). In contrast, cardioplegic arrest stopped periodical [Ca(2+)](i) transients and heart beats simultaneously. [Ca(2+)](i) remained at diastolic values, tended to decrease during the first minutes of cardioplegic arrest and then increased slowly. Autofluorescence increased under both conditions. During ischemia, this increase was faster than in cardioplegia experiments. It started after the last heart beat despite persisting perfusion. Our measurements demonstrate that rhythmical heart beat is essential for sufficient perfusion. Reduced [Ca(2+)](i) under cardioplegic arrest may influence metabolism.
ISSN:0143-4160
DOI:10.1016/j.ceca.2003.10.001