AP39, a novel mitochondria-targeted hydrogen sulfide donor, stimulates cellular bioenergetics, exerts cytoprotective effects and protects against the loss of mitochondrial DNA integrity in oxidatively stressed endothelial cells in vitro

Abstract

The purpose of the current study was to investigate the effect of the recently synthesized mitochondrially-targeted H2S donor, AP39 [10-oxo-10-(4-(3-thioxo-3H-1,2-dithiol-5yl)phenoxy)decyl) triphenylphosphonium bromide], on bioenergetics, viability, and mitochondrial DNA integrity in bEnd.3 murine microvascular endothelial cells in vitro, under normal conditions, and during oxidative stress. Intracellular H2S was assessed by the fluorescent dye 7-azido-4-methylcoumarin. For the measurement of bioenergetic function, the XF24 Extracellular Flux Analyzer was used. Cell viability was estimated by the combination of the MTT and LDH methods. Oxidative protein modifications were measured by the Oxyblot method. Reactive oxygen species production was monitored by the MitoSOX method. Mitochondrial and nuclear DNA integrity were assayed by the Long Amplicon PCR method. Oxidative stress was induced by addition of glucose oxidase. AP39 (30 – 300 nM) to bEnd.3 cells increased intracellular H2S levels, with a preferential response in the mitochondrial regions. AP39 exerted a concentration-dependent effect on mitochondrial activity, which consisted of a stimulation of mitochondrial electron transport and cellular bioenergetic function at lower concentrations (30–100 nM) and an inhibitory effect at the higher concentration of 300 nM. Under oxidative stress conditions induced by glucose oxidase, an increase in oxidative protein modification and an enhancement in MitoSOX oxidation was noted, coupled with an inhibition of cellular bioenergetic function and a reduction in cell viability. AP39 pretreatment attenuated these responses. Glucose oxidase induced a preferential damage to the mitochondrial DNA; AP39 (100 nM) pretreatment protected against it. In conclusion, the current paper documents antioxidant and cytoprotective effects of AP39 under oxidative stress conditions, including a protection against oxidative mitochondrial DNA damage.

Regulation of Vascular Tone, Angiogenesis and Cellular Bioenergetics by the 3-Mercaptopyruvate Sulfurtransferase/H2S Pathway: Functional Impairment by Hyperglycemia and Restoration by dl-α-Lipoic Acid

“…Cells were loaded with 10 μmol/L of the fluorescent H 2 S probe 7-azido-4-methylcoumarin (AzMC) for 30 min as described (40). 3-MP was added at various concentrations and cells were further incubated for 1 h. Cells were washed three times with phosphatebuffered saline (PBS), and the specific fluorescence of the dye was visualized using Nikon Eclipse 80i inverted microscope and NIS-Elements software.…”

“…Whole blood was collected 30 min later in K 2 EDTA blood collection tubes and centrifuged at 4°C for 15 min at 2000g. Plasma was isolated and H 2 S levels were measured with the fluorescent H 2 S probe 7-azido-4-methylcoumarin (AzMC) (40). After incubation of 200 μL of plasma with 10 μmol/L AzMC, the fluorescence was measured using SpectraMax M2 microplate reader (Molecular Devices, Sunnyvale, CA, USA.)…”

“…We have recently demonstrated that 3-MST activity is inhibited by oxidative stress in vitro and speculated that this may exert adverse effects on cellular homeostasis (20,21). Given the importance of H 2 S as a vasorelaxant (22)(23)(24)(25)(26)(27)(28)(29), proangiogenic agent (27,(30)(31)(32)(33)(34)(35)(36) and mitochondrial/bioenergetic modulator (20,21,35,(37)(38)(39)(40)(41)(42), here we examined whether the 3-MST/H 2 S system plays physiological regulatory roles in endothelial cells. Next, we examined whether a dysfunction of the 3-MST/H 2 S system develops in hyperglycemia/diabetes and tested whether this dysfunction can be corrected by DL-α-lipoic acid (LA), a known antioxidant and 3-MST cofactor (43)(44)(45).…”

Working with nitric oxide and hydrogen sulfide in biological systems

“…AP39, a sulfide donor with a mitochondria-targeting moiety, has been shown to increase intracellular H 2 S in these organelles as detected by the H 2 S probe 7-azido-4-methylcoumarin. It was also shown that AP39, at 100 nM, was able to prevent glucose oxidase-induced mitochondrial oxidative stress (113). Table 1 …”

Methane bioactivity and interactions with other biological gases

“…In the case of NO or H 2 S, precursors stimulating endogenous release of the gas can be administered or enzymatic synthesis of the compounds can be induced (Szczesny et al, 2014;Gero et al, 2016). However, with CH 4 , direct delivery is the only feasible option.…”