Mitochondrial damage is the main source of cellular injury upon
ischemia-reperfusion, and
calcium loading has been implicated in this phenomenon. The use of optical probes for
calcium monitoring of the intact heart is hampered by internal filter effects of intracellular hemoproteins, endogenous fluorescence, and their sensitivity to pH. We describe here a method for measurement of intracellular free
calcium in isolated
myoglobin-deficient perfused mouse hearts under conditions of large intracellular pH fluctuations by simultaneous fluorescence monitoring of the
calcium-probe
Fura-2 and the pH probe
BCECF through dual wavelength excitation of both probes. In
myoglobin-containing mouse heart endogenous chromophores interfere with
Fura-2 fluorometry. It is shown that a paradoxical decrease in
Fura-2 fluorescence occurs during
ischemia in isolated mouse hearts. Simultaneous recording of
BCECF fluorescence (calibrated against pH measurement with
phosphorus NMR) and data reduction based on continual recalculation of the apparent dissociation constant of the
calcium-probe complex revealed that a marked increase in intracellular free
calcium occurs, and that the
Fura-2 fluorescence decrease was caused by an increase in dissociation constant due to intracellular acidification. Intracellular free
calcium rose almost linearly during a 20-min period of
ischemia and returned to basal values rapidly upon the commencement of perfusion.