FLUORESCENCE MEASUREMENT OF CALCIUM TRANSIENTS IN PERFUSED RABBIT HEART USING RHOD-2

Surface fluorescence spectroscopy of the beating heart. to measure cyt osolic calcium has been limited by the need to use ultraviolet excitat ion light for many of the commonly used calcium indicators. Ultraviole t light in the heart produces a high level of background fluorescence and is highly absorbed, limiting tissue penetration. Visible wavelengt h fluorescence dyes such as rhod 2 are available; however, the lack of spectral shift with calcium binding precludes the use of ratio techni ques to account for changes in cytosolic dye concentration, We have de veloped a method for in vivo quantitation of cytosolic rhod 2 concentr ation that in conjunction with calcium-dependent fluorescence measurem ents permits estimation of cytosolic calcium levels in perfused rabbit hearts. Reflective absorbance of excitation light by rhod 2 loaded in to myocardium was used as an index of dye concentration and the ratio of fluorescence intensity to absorbance as a measure of cytosolic calc ium concentration Endothelial cell loading of rhod 2 was found to be m inimal (< 5 %), and dye leak rate out of the cytosol was slow, with si milar to 5 % loss of dye fluorescence occurring between 10 and 30 min after dye loading. Rhod 2 loading into subcellular compartments, deter mined by manganese quenching, was also minimal (< 5 %). The dissociati on constant of rhod 2 for calcium was measured in vitro to be 500 nM, and this value increased to 710 nM in the presence of 0.5 mM myoglobin . On the basis of this value and in vivo fluorescence measurements, cy tosolic calcium concentration in the rabbit heart was found to be 229 +/- 90 nM at end diastole and 930 +/- 130 nM at peak systole, with pea k fluorescence preceding peak, ventricular pressure by similar to 40 m s. This technique should facilitate detailed analysis of calcium trans ients from the whole heart.