Intracellular free zinc during cardiac excitation- contraction cycle: calcium and redox dependencies

Aims Zinc exists in biological systems as bound and histochemically reactive free Zn2+. It is an essential structural constituent of many proteins, including enzymes from cellular signalling pathways, in which it functions as a signalling molecule. In cardiomyocytes at rest, Zn2+ concentration is in the nanomolar range. Very little is known about precise mechanisms controlling the intracellular distribution of Zn2+ and its variations during cardiac function. Methods and results Live-cell detection of intracellular Zn2+ has become feasible through the recent development of Zn2+-sensitive and –selective fluorophores able to distinguish Zn2+ from Ca2+. Here, in freshly isolated rat cardiomyocytes, we investigated the rapid changes in Zn2+ homeostasis using the Zn2+-specific fluorescent dye, FluoZin-3, in comparison to Ca2+-dependent fluo-3 fluorescence. Zn2+ sparks and Zn2+ transients, in quiescent and electrically stimulated cardiomyocytes, respectively, were visualized in a similar manner to known rapid Ca2+ changes. Both Zn2+ sparks and Zn2+ transients required Ca2+ entry. Inhibiting the sarcoplasmic reticulum Ca2+ release or increasing the Ca2+ load in a low-Na+ solution suppressed or increased Zn2+ movements, respectively. Mitochondrial inhibitors slightly reduced both Zn2+ sparks and Zn2+ transients. Oxidation by H2O2 facilitated and acidic pH inhibited the Ca2+-dependent Zn2+ release. Conclusion It is proposed that Zn2+ release during the cardiac cycle results mostly from intracellular free Ca2+ increase, triggering production of reactive oxygen species that induce changes in metal-binding properties of metallothioneins and other redox-active proteins, aside from ionic exchange on these proteins.