Oxidants increase intracellular free Zn2+ concentration in rabbit ventricular myocytes

Abstract

Oxidative stress may alter cardiac function by affecting intracellular free Zn2+ concentrations ([Zn2+]i). Rabbit ventricular myocytes loaded with fura 2 were used to fluorometrically measure resting [Zn2+]i (0.23 +/- 0.03 nM) and intracellular Ca2+ concentration ([Ca2+]i) (36 +/- 7 nM). Fluorescence quenching by the heavy metal chelator N,N,N',N'-tetrakis(2-pyridylmethyl)ethylenediamine was used to quantitate [Zn2+]i. The thiol-reactive oxidants hypochlorous acid (0.1 mM) and selenite (1 mM) increased [Zn2+]i to 7.7 +/- 1.7 and 6.1 +/- 1.7 nM, respectively, within 5 min. Dithiothreitol (0.5 mM), a disulfide-reducing agent, rapidly restored normal [Zn2+]i. The oxidants did not affect [Ca2+]i. However, depolarization-induced Ca2+ transients and Ca2+ currents were zinc dependent. [Zn2+]i-associated fluorescence was substantial and, if ignored, it led to overestimation of [Ca2+]i by approximately twofold before oxidant treatment and by approximately eightfold after oxidants. The results demonstrate that [Zn2+]i 1) can be greatly increased by thiol-reactive oxidants; 2) may contribute to oxidant-induced alterations of excitation-contraction coupling; and 3) has strong fura 2 fluorescence which, if overlooked, can lead to significant overestimation of [Ca2+]i.