Pacing rate, halothane, and BDM affect fura 2 reporting of [Ca2+]i in intact rat trabeculae

Experiments were done on intact trabeculae from rats. Fura 2 in the salt form was microinjected directly into the myoplasm. The experiments were conducted at 30 degrees C, with 2 mM extracellular Ca2+ concentration and pacing at either 0.5 or 5 Hz. The aims were to establish a new method for in vivo calibration of fura 2 and to determine the effect of autofluorescence changes on intracellular Ca2+ concentration ([Ca2+]i) reported by fura 2. Autofluorescence was recorded under optimal conditions for fura 2 fluorescence (emission at 510 nm). By alteration of the oxidation-reduction state, it was shown that NADH is the main component of autofluorescence in heart. An increase in pacing frequency caused a decrease in autofluorescence. Both halothane and 2,3-butanedione monoxime (BDM) at 5-Hz pacing produced a substantial rise in autofluorescence, approaching the levels observed at 0.5-Hz pacing. The values for the dissociation constant (678 nM) and maximum fluorescence ratio of fura 2 for Ca2+ for the in vivo calibration are 3.4 times larger and 2.6 times smaller, respectively, than those found in vitro. Using the parameters obtained in vivo, we found that the diastolic and systolic [Ca2+]i of a twitch at 30 degrees C were 0.2 and 2.4 microM, respectively. Proper correction of the autofluorescence change unmasks the [Ca2+]i elevation caused by 5-Hz pacing. It was concluded that autofluorescence is not constant and that interventions affecting autofluorescence need correction if fura 2 is used to report [Ca2+]i.