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Quantitative measurement of calcium transients in perfused mouse heart using Rhod-2 fluorometry

We present a technique for optical detection of calcium transients in perfused mouse heart labeled with the calcium sensitive fluorescent dye Rhod-2. The isolated mouse-heart is placed in a water-jacketed chamber at 37 degrees Celsius, and is stimulated at 8 Hz, with 100 (mu) g of Rhod-2 bolused through the perfusate. After a 25-minute washout period, approximately 6 fold increase in fluorescence above background can be detected spectrofluorimetrically at 589 nm when excited at 524 nm. Both of these wavelengths are isobestic with regard to O2, thus minimizing interference due to changes in tissue oxygenation. Ca2+-dependent fluorescence transients are measured, as well as the corresponding left ventricular pressure signal. Our calcium transient signals represent 33 plus or minus 9% of diastolic fluorescence intensity. As the fluorescence emission is attenuated with the washout of Rhod-2 through the perfusate, the reflected absorbance between 524 nm (Rhod-2 sensitive) and 589 nm (Rhod- 2 insensitive) is used as a measure of dye concentration in tissue. The fluorescence-to-absorbance ratio measured from the perfused heart is verified to be insensitive to dye concentration, and thus can be used to determine the calcium concentration of the heart. Maximal calcium dependent fluorescence is calibrated in situ using high calcium and a SR Ca-ATPase inhibitor to tetanize the heart. The calculated cytosolic calcium concentrations for perfused mouse heart are 368 plus or minus 68 nM and 654 plus or minus 164 nM in diastole and systole, respectively. An effective method of minimizing changes in tissue scattering in the calcium quantification is also discussed.