Rhod-2 based measurements of intracellular calcium in the perfused mouse heart: Cellular and subcellular localization and response to positive inotropy

We have demonstrated a method of measuring intracellular calcium in the perfused mouse heart with the red fluorescent dye rhod-2. In Langendorff perfused isolated mouse hearts, rhod-2 is bolused through the perfusate, resulting in a -fold increase in fluorescence over background, and calcium transients with a transient amplitude to diastolic fluorescence ratio of Quantification of the relative amount of rhod-2 in the heart was done by taking the ratio of absorbance at 524 nm (rhod-2 sensitive) to 589 nm (rhod-2 insensitive). Maximal calcium saturated fluorescence was measured during tetanization of the heart with calcium chloride (20 mM) and cyclopiazonic acid (10 M). Electron microscopy was used to determine the subcellular localization of rhod-2, by fixing rhod-2 in the heart with a carbodiimide compound, and then using a double antibody technique to stain rhod-2. These images demonstrated prominent cytosolic rhod-2 localization. Fluorescence and confocal fluorescence microscopy were consistent with the electron microscopy data. Endothelial cell uptake of rhod-2 was shown with fluorescence microscopy, though functional studies with bradykinin infusion (3 M), which increases endothelial cell calcium, had no effects on mean fluorescence suggesting that endothelial uptake was small relative to total fluorescence. Calculated values of intracellular calcium were nM at peak systole, and nM in diastole, and with high perfusate calcium (3.5 mM) were and nM, respectively. Thus, this appears a valid method of measuring cytosolic calcium in the perfused mouse heart, which will help determine the mechanisms of altered contractility in genetically engineered mice. ©