The novel mitochondria-targeted hydrogen sulfide (H2S) donors AP123 and AP39 protect against hyperglycemic injury in microvascular endothelial cells in vitro

Abstract

The development of diabetic vascular complications is initiated, at least in part, by mitochondrial reactive oxygen species (ROS) production in endothelial cells. Hyperglycemia induces superoxide production in the mitochondria and initiates changes in the mitochondrial membrane potential that leads to mitochondrial dysfunction. Hydrogen sulfide (H₂S) supplementation has been shown to reduce the mitochondrial oxidant production and shows efficacy against diabetic vascular damage in vivo. However, the half-life of H₂S is very short and it is not specific for the mitochondria. We have therefore evaluated two novel mitochondria-targeted anethole dithiolethione and hydroxythiobenzamide H₂S donors (AP39 and AP123 respectively) at preventing hyperglycemia-induced oxidative stress and metabolic changes in microvascular endothelial cells in vitro. Hyperglycemia (HG) induced significant increase in the activity of the citric acid cycle and led to elevated mitochondrial membrane potential. Mitochondrial oxidant production was increased and the mitochondrial electron transport decreased in hyperglycemic cells. AP39 and AP123 (30-300nM) decreased HG-induced hyperpolarisation of the mitochondrial membrane and inhibited the mitochondrial oxidant production. Both H₂S donors (30-300nM) increased the electron transport at respiratory complex III and improved the cellular metabolism. Targeting H₂S to mitochondria retained the cytoprotective effect of H₂S against glucose-induced damage in endothelial cells suggesting that the molecular target of H₂S action is within the mitochondria. Mitochondrial targeting of H₂S also induced >1000-fold increase in the potency of H₂S against hyperglycemia-induced injury. The high potency and long-lasting effect elicited by these H₂S donors strongly suggests that these compounds could be useful against diabetic vascular complications.

Keywords: Bioenergetics; Complex II; Electron transport; Endothelial cells; Hydrogen sulfide; Hyperglycemia; Oxidative stress; SQR; Superoxide.

Graphical abstract

Fig. 1

H₂S release by mitochondrial H₂S donors. A and B: The chemical structure of mitochondrial H₂S donors: the H₂S releasing groups anethole dithiolethione (ADT-OH) in AP39 (A) and 4-hydroxythiobenzamide (HTB) in AP123 (B) are bound by ester linkage to 10-carbon alkyl linker region and the triphenyl phosphonium mitochondrial targeting group. C-D: The total amount of H₂S released from non-mitochondrial (ADT-OH, HTB) and mitochondrial (AP39, AP123) H₂S donors (100–500 μM) was detected in cell culture medium (DMEM supplemented with 10% FBS) for 10 days. E-F: Daily H₂S release values are plotted with curve-fitting results to highlight the donor compound decomposition. G-H: The total amount of H₂S liberated from mitochondrial and respective non-mitochondrial H₂S donors over the first 3-day long period is shown.

Fig. 2

Localization of H₂S release. b.End3 microvascular endothelial cells were pre-treated with H₂S donor compounds (30 μM, ADT-OH, AP39, HTB and AP123), then loaded with fluorescent H₂S sensor AzMc and mitotracker stain. The mitochondria (mitotracker signal) are shown in red and the H₂S production (AzMc signal) in the cells is shown in green. The H₂S signal completely overlaps with the mitochondrial signal in mitochondrial H₂S donor treated cells (as displayed in the merged channels), while in the non-mitochondrial H₂S donor-treated cells higher non-mitochondrial H₂S signal is detectable.

Fig. 3

Tolerability of H₂S donors. b.End3 cells were treated with mitochondrial and non-mitochondrial H₂S donor compounds for 24 h. A: The cellular viability was measured by the MTT assay. B: LDH release was detected by measuring the LDH activity in the cell culture supernatant. The non-mitochondrial H₂S donors are better tolerated by the cells: the mitochondrial H₂S donors reduce the cell survival at lower concentrations.

Fig. 4

Mitochondrial H₂S donors protect against ROS production in hyperglycemic endothelial cells. A-B: b.End3 endothelial cells were exposed to high extracellular glucose for 7 days with a single AP39 (A) or AP123 (B) treatment on the 4th day of hyperglycemia. The mitochondrial membrane potential was measured by JC-1, the mitochondrial superoxide production by MitoSOX Red, and the cellular ROS production by CM-H₂DCFDA. AP39 and AP123 restored the mitochondrial membrane potential and reduced the ROS production. (#p < 0.05 high glucose induced significant increase in mitochondrial membrane potential or ROS production. *p <0.05 H₂S donor compounds significantly reduced the mitochondrial membrane potential or ROS production compared to hyperglycemic control cells.).

Fig. 5

Mitochondrial H₂S donors reduce the cellular hypermetabolism in hyperglycemic endothelial cells. A-B: b.End3 endothelial cells were exposed to high extracellular glucose for 7 days with a single AP39 (A) or AP123 (B) treatment on the 4^th day of hyperglycemia. The MTT reducing capacity, the total cellular LDH activity and the cellular ATP content were measured on the 7th day. (# p < 0.05 high glucose induced significant changes in the cellular MTT reducing capacity and ATP content. * p < 0.05 H₂S donor compounds significantly reduced the MTT reduction and increased the cellular ATP content.).

Fig. 6

Mitochondrial H₂S donors affect the cellular bioenergetics. b.End3 cells exposed to 7-day-long hyperglycemia were treated with AP39 (30 nM) or AP123 (100 nM) and the metabolic profile of the cells was studied by extracellular flux analysis. Sequential injections of Oligomycin (1 μg/ml), FCCP (0.3 μM) and antimycin A (2 μg/ml) were used to measure A: the cellular oxygen consumption rate (OCR) and B: the extracellular acidification rate (ECAR). C: Basal oxygen consumption, D: ATP production linked oxygen consumption (determined by oligomycin injection), E: total respiratory capacity (determined following the addition of FCCP) and F: the proton leak/basal respiration was determined. G: Acid production of basal metabolism and H: acid production during anaerobic compensation was determined. AP39 and AP123 increase the respiratory capacity of the cells. (n = 3, *p < 0.05 compared to hyperglycemic control).

Fig. 7

Mitochondrial H₂S donors increase the respiratory Complex II/III activity. A and B: Cytochrome c reduction was monitored in bovine heart mitochondria following Complex I and IV blockade by rotenone and KCN, respectively. A: AP39 was added at 10 nM–10 μM and complex II/III activity was measured kinetically, B: Mitochondria were treated with AP123 (10 nM–10 μM) and the respiratory complex activity was monitored. (*p < 0.05, H₂S donors significantly increased the respiratory complex activity).