Diet-induced obesity augments ischemic myopathy and functional decline in a murine model of peripheral artery disease.

Peripheral artery disease (PAD) causes an ischemic myopathy contributing to patient disability and mortality. Most preclinical models to date use young, healthy rodents with limited translatability to human disease. Although PAD incidence increases with age, and obesity is a common comorbidity, the pathophysiologic association between these risk factors and PAD myopathy is unknown. Using our murine model of PAD, we sought to elucidate the combined effect of age, diet-induced obesity and chronic hindlimb ischemia (HLI) on (1) mobility, (2) muscle contractility, and markers of muscle (3) mitochondrial content and function, (4) oxidative stress and inflammation, (5) proteolysis, and (6) cytoskeletal damage and fibrosis. Following 16-weeks of high-fat, high-sucrose, or low-fat, low-sucrose feeding, HLI was induced in 18-month-old C57BL/6J mice via the surgical ligation of the left femoral artery at 2 locations. Animals were euthanized 4-weeks post-ligation. Results indicate mice with and without obesity shared certain myopathic changes in response to chronic HLI, including impaired muscle contractility, altered mitochondrial electron transport chain complex content and function, and compromised antioxidant defense mechanisms. However, the extent of mitochondrial dysfunction and oxidative stress was significantly greater in obese ischemic muscle compared to non-obese ischemic muscle. Moreover, functional impediments, such as delayed post-surgical recovery of limb function and reduced 6-minute walking distance, as well as accelerated intramuscular protein breakdown, inflammation, cytoskeletal damage, and fibrosis were only evident in mice with obesity. As these features are consistent with human PAD myopathy, our model could be a valuable tool to test new therapeutics.

Expanding the Reactive Sulfur Metabolome: Intracellular and Efflux Measurements of Small Oxoacids of Sulfur (SOS) and H2S in Human Primary Vascular Cell Culture.

Hydrogen sulfide (H2S) is an endogenous signaling molecule which is important for cardiovascular health, but its mechanism of action remains poorly understood. Here, we report measurements of H2S as well as its oxidized metabolites, termed small oxoacids of sulfur (SOS = HSOH and HOSOH), in four human primary vascular cell lines: smooth muscle and endothelial cells derived from both human arterial and coronary tissues. We use a methodology that targets small molecular weight sulfur species; mass spectrometric analysis allows for species quantification to report cellular concentrations based on an H2S calibration curve. The production of H2S and SOS is orders of magnitude higher in smooth muscle (nanomolar) as compared to endothelial cell lines (picomolar). In all the primary lines measured, the distributions of these three species were HOSOH >H2S > HSOH, with much higher SOS than seen previously in non-vascular cell lines. H2S and SOS were effluxed from smooth muscle cells in higher concentrations than endothelial cells. Aortic smooth muscle cells were used to examine changes under hypoxic growth conditions. Hypoxia caused notable increases in HSOH and ROS, which we attribute to enhanced sulfide quinone oxidase activity that results in reverse electron transport.

Characterization of Endogenous and Extruded H2S and Small Oxoacids of Sulfur (SOS) in Cell Cultures.

This report characterizes and quantifies endogenous hydrogen sulfide (H2S) and small oxoacids of sulfur (SOS = HOSH, HOSOH) in a panel of cell lines including human cancer (A375 melanoma cells, HeLa cervical cells) and noncancer (HEK293 embryonic kidney cells), as well as E. coli DH5α and S. cerevisiae S288C. The methodology used is a translation of well-studied nucleophilic and electrophilic traps for cysteine and oxidized cysteines residues to target small molecular weight sulfur species; mass spectrometric analysis allows for species quantification. The observed intracellular concentrations of H2S and SOS vary in different cell types, from nanomolar to femtomolar, typically with H2S > HOSOH > HOSH. We propose the term sulfome, a subset of the metabolome, describing the nonproteinaceous metabolites of H2S; the sulfomic index is as a measure of the S-oxide redox status, which gives a profile of endogenous sulfur at different oxidation states. An important observation is that H2S and SOS were found to be continuously extruded into surrounding media against a concentration gradient, implying an active efflux process. Small molecule inhibition of several H2S generating enzymes suggest that SOS are not derived solely from H2S oxidation. Even after successful inhibition of H2S production, cells maintain constant efflux and repopulate H2S and SOS over time. This work proves that these small sulfur oxoacids are generated in cells of all types, and their efflux implies that they play a role in cell signaling and possibly other vascular physiology attributed to H2S.