Importance of mitochondrial calcium uniporter in high glucose-induced endothelial cell dysfunction

Abstract

Objective: Mitochondrial Ca²⁺ overload is implicated in hyperglycaemia-induced endothelial cell dysfunction, but the key molecular events responsible remain unclear. We examined the involvement of mitochondrial calcium uniporter, which mediates mitochondrial Ca²⁺ uptake, in endothelial cell dysfunction resulting from high-glucose treatment.

Methods: Human umbilical vein endothelial cells were exposed to various glucose concentrations and to high glucose (30 mM) following mitochondrial calcium uniporter inhibition or activation with ruthenium red and spermine, respectively. Subsequently, mitochondrial calcium uniporter and mitochondrial calcium uniporter regulator 1 messenger RNA and protein expression was measured by real-time polymerase chain reaction and western blotting. Ca²⁺ concentrations were analysed by laser confocal microscopy, and cytoplasmic and mitochondrial oxidative stress was detected using 2',7'-dichlorofluorescein diacetate and MitoSOX Red, respectively. Apoptosis was assessed by annexin V-fluorescein isothiocyanate/propidium iodide staining, and a wound-healing assay was performed using an in vitro model.

Results: High glucose markedly upregulated mitochondrial calcium uniporter and mitochondrial calcium uniporter regulator 1 messenger RNA expression, as well as protein production, in a dose- and time-dependent manner with a maximum effect demonstrated at 72 h and 30 mM glucose concentration. Moreover, high-glucose treatment significantly raised both mitochondrial and cytoplasmic Ca²⁺ and reactive oxygen species levels, increased apoptosis and compromised wound healing (all p < 0.05). These effects were enhanced by spermine and completely negated by ruthenium red, which are known to activate and inhibit mitochondrial calcium uniporter, respectively.

Conclusion: Mitochondrial calcium uniporter plays an important role in hyperglycaemia-induced endothelial cell dysfunction and may constitute a therapeutic target to reduce vascular complications in diabetes.

Keywords: High glucose; endothelial cell; mitochondrial calcium uniporter; mitochondrial calcium uniporter regulator 1.

Figure 1.

MCU and MCUR1 mRNA and protein expression in HUVECs cultured in the presence of HG. (a) Cells were treated with different glucose concentrations (2, 5.5, 10, 20 and 30 mM) for 72 h, and HG (30 mM) for various periods (0, 12, 24, 48 and 72 h). (b) MCU, MCUR1 and β-actin (the latter as a loading control) mRNA and protein levels were evaluated by real-time PCR and western blotting, respectively. Data are expressed as the means ± SEM of three independent experiments. Results were analysed by unpaired t-test. *p < 0.05 versus the NG group.

Figure 2.

Cytoplasmic and mitochondrial calcium and oxidative stress levels in HUVECs cultured in the presence of HG. (a) Cells were treated with NG, HG or Mnt for 72 h, and calcium concentrations in the mitochondria ([Ca²⁺]mito) and cytoplasm ([Ca²⁺]cyt) were determined by laser confocal microscopy following exposure to the Ca²⁺ indicators Rhod-2 AM and Fluo-3 AM. (b) Total ROS and mitochondrial ${\mathrm{O}}_2^{-}$ levels were quantified by fluorescence microscopy following DCF-DA and MitoSOX Red staining, respectively. Data are expressed as the means ± SEM of three independent experiments. Results were analysed by one-way ANOVA followed by Tukey’s post hoc test. *p < 0.05 versus the NG group.

Figure 3.

Effects of HG on apoptosis of wound-healing ability of HUVECs. (a) Cells were treated with NG, HG or Mnt for 72 h, before being stained with both annexin V and PI and subjected to flow cytometric analysis. (b) Degree of wound closure after 0, 18 and 36 h for each treatment group was quantified using a microscope. Results are expressed as means ± SEM (n = 3). Results were analysed by one-way ANOVA followed by Tukey’s post hoc test. *p < 0.05 versus the NG group.

Figure 4.

MCU inhibition negates HG-induced oxidative stress and mitochondrial Ca²⁺ overload in HUVECs. HUVECs were pre-treated for 30 min with ruthenium red (RR) or spermine (sper), which, respectively, inhibits and activates MCU, and exposed to HG for 72 h. (a) [Ca²⁺]mito, [Ca²⁺]cyt, and (b) levels of total ROS and mitochondrial ${\mathrm{O}}_2^{-}$ were then quantified. Data are expressed as the means ± SEM from three independent experiments. Results were analysed by unpaired t-test. #p < 0.05 versus the HG group.

Figure 5.

MCU inhibition negates hyperglycaemia-induced HUVEC dysfunction. HUVECs were pre-treated for 30 min with ruthenium red (RR) or spermine (sper) before being subjected to HG treatment for 72 h. (a) The incidence of apoptosis and (b) degree of wound closure were quantified. Results are expressed as means ± SEM (n = 3). Results were analysed by unpaired t-test. #p < 0.05 versus the HG group.