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 per fused mouse heart with the red fluorescent dye rhod-2. In Langendorff perfu sed isolated mouse hearts, rhod-2 is bolused through the perfusate, resulti ng in a 6.2+/-1.9-fold increase in fluorescence over background, and calciu m transients with a transient amplitude to diastolic fluorescence ratio of 33+/-9%. Quantification of the relative amount of rhod-2 in the heart was d one by taking the ratio of absorbance at 524 nm (rhod-2 sensitive) to 589 n m (rhod-2 insensitive). Maximal calcium saturated fluorescence was measured during tetanization of the heart with calcium chloride (20 mM) and cyclopi azonic acid (10 muM). Electron microscopy was used to determine the subcell ular localization of rhod-2, by fixing rhod-2 in the heart with a carbodiim ide compound, and then using a double antibody technique to stain rhod-2. T hese images demonstrated prominent cytosolic rhod-2 localization. Fluoresce nce and confocal fluorescence microscopy were consistent with the electron microscopy data. Endothelial cell uptake of rhod-2 was shown with fluoresce nce microscopy, though functional studies with bradykinin infusion (3 muM), which increases endothelial cell calcium, had no effects on mean fluoresce nce (N = 4, p = NS), suggesting that endothelial uptake was small relative to total fluorescence. Calculated values of intracellular calcium were 686/-237 nM at peak systole, and 360+/-101 nM in diastole, and with high perfu sate calcium (3.5 mM) were 1199+/-215 and 544+/-53 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 contractil ity in genetically engineered mice. (C) 2001 Society of Photo-Optical instr umentation Engineers.