Estimation of Systolic and Diastolic Free Intracellular Ca²⁺ by Titration of Ca²⁺ Buffering in the Ferret Heart

Spectroscopic Ca²⁺-indicators are thought to report values of free intracellular Ca²⁺ concentration ([Ca²⁺]_i) that may differ from unperturbed values because they add to the buffering capacity of the tissue. To check this for the heart we have synthesized a new 19F-labelled NMR calcium indicator, 1,2- bis-[2-bis(carboxymethyl)amino-4,5-difluorophenoxy] ethane (4,5FBAPTA) with a low affinity (K_d: 2950nM). The new indicator and four previously described 19F-NMR Ca²⁺ indicators, 1,2-bis-[2- bis(carboxymethyl)amino-5-fluorophenoxy]ethane (5-FBAPTA), 1,2-bis-[2-(1-carboxyethyl)(carboxymethyl) amino-5-fluorophenoxy]ethane (DiMe-5FBAPTA), 1,2-bis-[2-(1-carboxyethyl)(carboxymethyl) amino-4-fluorophenoxy]ethane (DiMe-4FBAPTA) and 1,2-bis-[2-bis(carboxymethyl)amino-5-fluoro-4-methylphenoxy]ethane (MFBAPTA), with dissociation constants for Ca²⁺ ranging from 46 to 537nM have been used to measure [Ca²⁺]_i, over the range <100nM to >3µM, in Langendorff-perfused ferret hearts (30°C, pH 7.4, paced at 1.0Hz) by 19F-NMR spectroscopy. Loading hearts within dicators resulted in buffering of the Ca²⁺ transient. The measured end-diastolic and peak-systolic [Ca²⁺]_i were both positively correlated with indicator K_d. The positive correlations between indicator K_d and the measured end-diastolic and peak-systolic [Ca²⁺]_i were used to estimate the unperturbed enddiastolic and peak-systolic [Ca²⁺]_i by extrapolation to K_d = 0 (diastolic) and to K_d = ∞ (systolic) respectively. The extrapolated values in the intact beating heart were 161nM for end-diastolic [Ca²⁺]_i and 2650nM for peak systolic [Ca²⁺]_i which agree well with values determined from single cells and muscle strips.