229 - RT01, A Novel Derivative of the Mitochondria-targeted Hydrogen Sulfide Donor AP39, Reversed Hyperglycaemia-induced Mitochondrial Dysfunction in Murine Brain Microvascular Endothelial Cells

Abstract

Diabetes is an increasingly prevalent condition. One hallmark of this disease is vascular endothelial dysfunction (VED) and angiopathy due, at least in part, to hyperglycaemia (HG)-induced overproduction of mitochondrial oxidants and accompanying mitochondrial damage. Hydrogen sulfide (H2S) is a physiological mediator, shown to decrease mitochondrial reactive oxygen species during HG. Lower H2S bioavailability has been reported in patients with diabetes, as well as several established animal models of diabetes and supplementation of cells and diabetic animals with very high concentrations of sulfide (as NaSH) has been shown to prevent, but not rescue, HG-induced metabolic changes to mitochondria in vascular endothelial cells. We have therefore, hypothesised that diabetes is a disease of “H2S deficiency” and strategies to target mitochondria to overcome this “deficiency” may represent a novel approach to treat/prevent diabetic VED and angiopathy. To investigate this hypothesis, we have synthesised novel mitochondria-targeted H2S donors containing triphenyl-phoshonium (TPP+) such as AP39 and AP123, which are highly efficacious in animal models of mitochondrial injury (e.g. stroke, myocardial infarction, and ischaemia-reperfusion injury etc). In this study, we have synthesised and evaluated a novel H2S releasing moiety (RT02) and coupled this to TPP+ decanoate. As a model of the ‘diabetic vascular endothelium’, we exposed murine B.End3 microvascular endothelial cells to either constant or intermittent HG for 7 days and then added RT01 (3-300 nM) with HG/intermittent HG for 3 further days. After this time, we determined the ability of RT01 to reverse HG-induced metabolic changes; specifically, mitochondrial ΔΨm (JC-1), mitochondrial superoxide (mitosox), ATP synthesis (by luminescence) and cell viability (LDH). Mitochondrial H2S production was also determined by fluorescence microscopy using AzMC and MitoTracker Red. RT01 caused a concentration-dependent (3-300 nM) increase in mitochondrial H2S levels and released H2S at a slower rate and for a shorter period of time than AP39. At all concentrations tested RT01 (and AP39) reversed HG-induced mitochondrial hyperpolarisation and oxidant production, and restored ATP synthesis. These effects were not observed with equimolar concentrations of the control compounds; RT02 (non-targeted H2S donor) or TPP-decanoate (lacking the H2S donor) indicating that mitochondrial H2S delivery was responsible for ‘mitochondrial protection’. This study further suggests that strategies which target H2S to mitochondria may be a useful therapeutic approach for preventing/inhibiting HG-induced VED and angiopathy, as well as in other indications in which there is mitochondrial dysfunction.