Fluorescence measurement of calcium transients in perfused rabbit heart using rhod 2

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Abstract
Surface fluorescence spectroscopy of the beating heart to measure cytosolic calcium has been limited by the need to use ultraviolet excitation light for many of the commonly used calcium indicators. Ultraviolet light in the heart produces a high level of background fluorescence and is highly absorbed, limiting tissue penetration. Visible wavelength fluorescence dyes such as rhod 2 are available; however, the lack of spectral shift with calcium binding precludes the use of ratio techniques to account for changes in cytosolic dye concentration. We have developed a method for in vivo quantitation of cytosolic rhod 2 concentration that in conjunction with calcium-dependent fluorescence measurements permits estimation of cytosolic calcium levels in perfused rabbit hearts. Reflective absorbance of excitation light by rhod 2 loaded into myocardium was used as an index of dye concentration and the ratio of fluorescence intensity to absorbance as a measure of cytosolic calcium concentration. Endothelial cell loading of rhod 2 was found to be minimal (<5%), and dye leak rate out of the cytosol was slow, with ∼5% loss of dye fluorescence occurring between 10 and 30 min after dye loading. Rhod 2 loading into subcellular compartments, determined by manganese quenching, was also minimal (<5%). The dissociation 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, cytosolic calcium concentration in the rabbit heart was found to be 229 ± 90 nM at end diastole and 930 ± 130 nM at peak systole, with peak fluorescence preceding peak ventricular pressure by ∼40 ms. This technique should facilitate detailed analysis of calcium transients from the whole heart.

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