Overexpression of Slc30a7/ZnT7 increases the mitochondrial matrix levels of labile Zn2+ and modifies histone modification in hyperinsulinemic cardiomyoblasts

Abstract

Background: Cellular free Zn²⁺ concentrations ([Zn²⁺]) are primarily coordinated by Zn²⁺-transporters, although their roles are not well established in cardiomyocytes. Since we previously showed the important contribution of a Zn²⁺-transporter ZnT7 to [Zn²⁺]_i regulation in hyperglycemic cardiomyocytes, here, we aimed to examine a possible regulatory role of ZnT7 not only on [Zn²⁺]_i but also both the mitochondrial-free Zn²⁺ and/or Ca²⁺ in cardiomyocytes, focusing on the contribution of its overexpression to the mitochondrial function.

Methods: We mimicked either hyperinsulinemia (by 50-μM palmitic acid, PA-cells, for 24-h) or overexpressed ZnT7 (ZnT7OE-cells) in H9c2 cardiomyoblasts.

Results: Opposite to PA-cells, the [Zn²⁺]_i in ZnT7OE-cells was not different from untreated H9c2-cells. An investigation of immunofluorescence imaging by confocal microscopy demonstrated a ZnT7 localization on the mitochondrial matrix. We demonstrated the ZnT7 localization on the mitochondrial matrix by using immunofluorescence imaging. Later, we determined the mitochondrial levels of [Zn²⁺]_Mit and [Ca²⁺]_Mit by using the Zn²⁺ and Ca²⁺ sensitive FRET probe and a Ca²⁺-sensitive dye Fluo4, respectively. The [Zn²⁺]_Mit was found to increase significantly in ZnT7OE-cells, similar to the PA-cells while no significant changes in the [Ca²⁺]_Mit in these cells. To examine the contribution of ZnT7 overexpression on the mitochondria function, we determined the level of reactive oxygen species (ROS) and the mitochondrial membrane potential (MMP) in these cells in comparison to the PA-cells. There were significantly increased production of ROS and depolarization in MMP and increases in marker proteins of mitochondria-associated apoptosis and autophagy in ZnT7-OE cells, similar to the PA-cells, parallel to increases in K-acetylation. Moreover, we determined significant increases in trimethylation of histone H3 lysine27, H3K27me3, and the mono-methylation of histone H3 lysine36, H3K36 in the ZnT7OE-cells, demonstrating the role of [Zn²⁺]_Mit in epigenetic regulation of cardiomyocytes under hyperinsulinemia through histone modification.

Conclusions: Overall, our data have shown an important contribution of high expression of ZnT7-OE, through its buffering and muffling capacity in cardiomyocytes, on the regulation of not only [Zn²⁺_]i but also both [Zn²⁺]_Mit and [Ca²⁺]_Mit affecting mitochondria function, in part, via histone modification.

Keywords: Cardiomyocytes; Insulin resistance; Mitochondrial zinc; Reactive oxygen species; Zinc transporters.