362 - Both Reactive ROS and RNS Contribute to Intracellular Free Zn2+ Regulation in Cardiomyocytes Via Zinc Transporter ZIP7 Under Hyperglycemia

Abstract

Background and Aim: It has been shown that Zn 2+ release during cardiac cycle results mostly increases in intracellular free Zn 2+ levels ([Zn 2+ ] i ),which is further triggering reactive oxygen species (ROS) and reactive nitrogen species (RNS) production. Very small cytosolic free Zn 2+ level in cardiomyocytes can increase with pathological stimuli, inducing further oxidations/phosphorylation in proteins as well as changes in protein expression levels of Zn 2+ –transporters. In here, we aimed to examine the possible correlation between the changes in the level of either [Zn 2+ ] cyt , [Zn 2+ ] ER or [Zn 2+ ] mit and production of ROS and RNS in cardiomyocytes under hyperglycemic condition. Methods and Results: By using confocal imaging, we monitored [Zn 2+ ] cyt , [Zn 2+ ] ER and [Zn 2+ ] mit changes, as well as production of ROS and RNS and changes in mitochondrial membrane potential (MMP) by using Zn 2+ sensitive eCALWY FRET probes and specific fluorescence dyes, DAF, DCDFA and JC-1, respectively, in cardiac ventricular cell line H9c2 cells. We also measured cytosolic, ER and mitochondrial free Zn 2+ levels in H9c2 cells with adenovirally infected eCALWY FRET probes. Results We measured high [Zn 2+ ] cyt and [Zn 2+ ] mit and low [Zn 2+ ] ER in high glucose incubated cells comparison to those of controls. The basal levels of ROS and RNS are significantly high in hyperglycemic cardiomyocytes compared to the controls. When either ROS or RNS is acutely increased, it turns to induce marked increase in total intracellular free [Zn 2+ ] i , while those can be reversed by a thiol reducing agent DTT or a Zn 2+ -chelator TPEN. Moreover, an important depolarization of MMP is observed with increases in [Zn 2+ ] i via an increase in either ROS or RNS production as well as decrease in ATP production. Furthermore, Western blot analysis showed that Zn 2+ –transporter ZIP7 is increased and hyperphosphorylated with a marked decrease in ZnT7, both are localized on ER, in hyperglycemic cardiomyocytes. Additionally, we measured markedly high phosphorylation in some members of macromolecular protein complex of cardiac ryanodine receptors such as PKA, CaMKII and PP2A in these cells, which are similar to those of obtained with high [Zn 2+ ]. Discussion Overall, our present data demonstrate that hyperglycemia causes increase of [Zn 2+ ] cyt and [Zn 2+ ] mit , with a marked decrease in [Zn 2+ ] ER via changes in ZnT7 and ZIP7 expression levels, which further causes depolarization of MMP and increases of ROS and RNS production in H9c2 cells under hyperglycemia. These changes altogether can contribute, in part, to hyperglycemia-induced cardiac dysfunction. Supported by TUBITAK-SBAG-113S466, COSTAction-TD1304 and EFSD-Fellowships