Cytosolic calcium in the ischemic rabbit heart: Assessment by pH- and temperature-adjusted rhod-2 spectrofluorometry

Abstract

Objectives: Cytosolic calcium ([Ca2+]i) mediates ischemia-reperfusion (I/R) injury, but magnitude and time course of I/R-induced [Ca2+]i overload remain unclear. Fluorescent indicators are commonly used to measure [Ca2+]i, and have a dissociation constant (Kd) that depends on pH and temperature. We hypothesized that changes of Kd during I/R lead to misleading interpretations of [Ca2+]i recordings. Methods: (1) In isolated rabbit hearts (n=4 each), intracellular pH was measured during I/R at 37°C, 20°C, and 4°C with and without cardioplegic arrest by 31P-NMR-spectroscopy. (2) Kd for rhod-2 and calcium was determined at varying pH and temperature in in vitro experiments. (3) Isolated rabbit hearts were subjected to I/R, and [Ca2+]i was recorded by surface rhod-2 spectrofluorometry. Finally, [Ca2+] i was computed using either the conventional Kd, or the pH- and temperature-adjusted Kd. Results: Kd(Ca 2+Rhod-2) remained stable between pH 7.1 and 6.8, but increased exponentially with lower pH and/or temperature. Calculations using a static Kd indicated that [Ca2+]i rose only mildly during warm ischemia and did not rise during cardioplegic arrest, while a large Ca2+ influx appeared to occur during early reperfusion. When the pH and temperature-adjusted Kd was used for calculation, [Ca 2+]i rose significantly during ischemia (431±37% during 20 min 37°C ischemia, and 78±19% during 20 min cardioplegic arrest at 20°C). During early reperfusion, [Ca2+]i decreased rapidly, without significant further [Ca2+]i elevation. Conclusions: In contrast to previous reports, [Ca2+] i accumulation occurs during unprotected ischemia as well as hypothermic ischemia with cardioplegic arrest, without further net Ca 2+ influx on reperfusion. This finding has important implications for timing of protective strategies during myocardial ischemia. © 2003 European Society of Cardiology. Published by Elsevier B.V. All rights reserved.