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The Zn2+‐Transporter ZnT6 Plays Important Role on Regulation of Cytosolic Free Zn2+ Through Modulation of Mitochondrial Zn2+ in Hyperglycemic Cardiomyocytes

Free zinc ion (Zn2+), have physiological importance for mammals and is found to be less than 1‐nM in cytosol. However, intracellular free Zn2+ levels ([Zn2+]i) can increase dramatically under pathological conditions including hyperglycemia and cancer. Any increase in [Zn2+]i can also cause significant changes in several intracellular signaling pathways. However, that [Zn2+]i is well regulated by various ways in physiological conditions and that value can fluctuate dynamically in cardiomyocytes. It is accepted that there are two‐types of Zn2+‐transporters, one group of them is responsible Zn2+‐influx from extracellular space and/or organelles to cytosol (ZIPs) and the other group is responsible Zn2+‐efflux from cytosol (ZnTs). Since it is not known whether the Zn2+‐transporter ZnT6 has a role in the regulation of [Zn2+]i in cardiomyocytes under hyperglycemia, we used control and ZnT6 overexpressed H9c2 cells (rat embryonic ventricular cell lines) to investigate its role. In addition, H9c2 cells are incubated with normal (5.5‐mM; control) and high glucose (25‐mM; hyperglycemic) for 24‐h. Following the treatment, control and ZnT6‐overexpressed cells are transfected with Zn+2‐sensitive as well as cytosole, sarco/endoplasmic reticulum S(ER) or mitochondria targeted Förster resonance energy transfer (FRET) sensors to measure cytosolic, S(ER) and mitochondrial [Zn2+], respectively. Furthermore, we examined the mRNA, protein expression levels and intracellular localizations by using molecular, biochemical and immunofluorescence methods. Our results demonstrated that ZnT6 but not ZnT5 mRNA and protein level is increased under hyperglycemia, while ZnT6 overexpression caused increase of ZnT5 protein level. Our immunofluorescence/precipitation experiments showed that ZnT6 localized on mitochondria together with ZnT5 in H9c2 cells. Furthermore, our [Zn2+] measurements by fluorescence imaging showed that cytosolic and mitochondrial [Zn2+] are increased and S(E)R [Zn2+] is decreased in hyperglycemic H9c2 cells. We also found that, ZnT6‐overexpression caused mitochondrial [Zn2+] increase and decrease in cytosolic [Zn2+], without changing the S(E)R [Zn2+]. To understand the role of excess [Zn2+] on mitochondrial function, we used ZnT6 overexpressed or high Zn2+ incubated H9c2 cell lines to measure mitochondrial‐membrane‐potential (MMP), ROS and ATP production. We found that excess intracellular or mitochondrial [Zn2+] caused depolarization of MMP, increase in ROS and decrease in ATP production. Overall, our results showed that ZnT6 has a significant role on [Zn2+]i by modulating mitochondrial [Zn2+] and function in hyperglycemia.