Endogenous Gaseous Signaling Molecule Research Articles

Exogenous hydrogen sulfide prevents SOD2 degradation to safeguard renal function in diabetic kidney disease.

Diabetic kidney disease (DKD) is a major contributor to chronic kidney disease. Hydrogen sulfide (H2S) serves as an endogenous gaseous signaling molecule capable of safeguarding renal function within the context of DKD. However, the underlying mechanisms need to be elucidated. This study was undertaken to unveil the mechanisms by which H2S counteracts against DKD. Utilizing mice and human renal tubular epithelial (HK-2) cells, we demonstrated a reduction in cystathionine-γ-lyase/H2S levels within renal tissues of db/db mice and in HK-2 cells subjected to hyperglycemic and hyperlipidemic environments. Notably, we observed that sodium hydrosulfide (NaHS) supplementation could serve as an exogenous source of H2S. Exogenous H2S exhibited the capacity to mitigate the accumulation of reactive oxygen species and attenuate the degradation of superoxide dismutase 2 (SOD2) by Lon protease homolog 1 induced by hyperglycemia and hyperlipidemia, thus affording cellular protection against mitochondrial apoptosis. Consequently, NaHS treatment led to decreased serum levels of blood urea nitrogen and serum creatinine, reflecting alleviated renal damage and thereby preserving renal function in db/db mice. Based on these findings, we propose that exogenous H2S exerts a protective role against DKD by inhibiting SOD2 degradation.

CoOx‐Caged Metal‐Organic Frameworks for Sonocatalyzing CO2 to Co for Ultrasound‐Assisted Chemodynamic‐Gas Cancer Therapy

AbstractCarbon monoxide (CO) as an endogenous gaseous signaling molecule, is widely studied for its ability to inhibit cancer cell proliferation as gas therapy. However, the therapeutic efficiency of CO gas therapy is far from ideal due to the low availability of CO supply. Here, inspired by the band structure and Fenton‐related elements, MIL‐101(Cr)@CoOx nanoparticles are introduced for the first time to achieve ultrasound‐mediated synergistic therapy by combining CO gas therapy and chemodynamic therapy together effectively. MIL‐101(Cr)@CoOx can present substantial CO2 adsorption capacity for the BDC2− linkers and Co center, and enable to transformation of endogenous CO2 to CO efficiently under ultrasound irradiation due to the appropriate band structure and conduction band position. With the Co‐engaged Fenton‐like reaction, MIL‐101(Cr)@CoOx triggered the reactions to catalyze the over‐expressed intracellular H2O2 into cytotoxic hydroxyl radicals (·OH). The potential therapeutic outcome of chemodynamic therapy can be amplified by the ultrasonic cavitation‐induced conversion between Co3+ and Co2+ in vitro is demonstrated. The proliferation, ATP, and mitochondria functions of 4T1 mouse breast cancer cells are severely compromised by CO‐promoted ROS generation and the sono‐Fenton effect. Further, in vivo studies confirm that MIL‐101(Cr)@CoOx combined with ultrasound irradiation exhibits superior tumor suppression, and achieves the ultrasound‐mediated chemodynamic‐gas cancer therapy with higher precisions for personalized treatments. This new system offers a distinct concept for constructing smart ultrasound‐stimulus systems to realize synergetic cancer therapy.

Nitric Oxide‐Releasing Macromolecular Systems in Anticoagulant Therapeutics

AbstractNitric oxide (NO), carbon monoxide (CO), and hydrogen sulfide (H2S) are considered endogenous gaseous signaling molecules that play crucial roles in regulating physiological and pathological pathways. Among these molecules, NO has demonstrated unique advantages and clear efficacy in the treatment of blood coagulation in cardiovascular diseases. As a result, the development of NO‐releasing materials for anticoagulant therapeutics has garnered significant attention. However, unlike traditional anticoagulant treatments such as heparin and oral anticoagulants, NO, a gaseous molecule under ambient conditions, cannot be administered orally, through injection, or via the skin. Consequently, researchers have focused on developing NO‐releasing macromolecular systems by combining various donors with polymeric materials for use in anticoagulant therapeutics. This minireview aims to highlight the methods and advancements in constructing such NO‐releasing macromolecular systems and exploring their applications in anticoagulant therapeutics.

Responsive luminescent silver-based metal-organic frameworks for highly sensitive and selective detection of hydrogen sulfide in biological system via a self-assembled headspace separation device

As a highly toxic gas pollutant and also an endogenous gaseous signaling molecule existing in a variety of physiological processes, the rapid and accurate in-field detection of hydrogen sulfide is of great concern. Nevertheless, two drawbacks as for the optical probes for H2S detection, taking about a long time to reach the optical signal balance or the low selectivity, always exist. Herein, by using a highly photoluminescent and H2S-stimuli responsive silver-based metal-organic frameworks (MOFs): Ag-BDC (BDC = 1, 4-benzene dicarboxylate), we demonstrated that the luminescence intensity of Ag-BDC MOFs was inversely proportional to the concentration of H2S due to the Ag–S coordination and the obstruction of ligand-to-metal charge transfer (LMCT) transition process, and there was a quick response time of below 3.0 min. Combined with a simple customized device to separate H2S from the sample, the selectivity of the method for H2S detection could be greatly improved, and no interference would be caused even if the other sulfur-containing species coexisted. The luminescence probe presented a favorable sensitivity within a linear range of 0.1–1000 μM along with a detection limit of 23.7 nM. When employed to assay the endogenous sulfide level in the human serum and mouse brain tissue, the approach showed recoveries from 96.3% to 102% with relative standard derivation (RSD) less than 2.0%. By the integration of the responsive luminescent silver-based MOFs with a simple self-assembled headspace separation device, obviously the present strategy could be beneficial to the development and design of the in-field fast H2S measurement, possessing particular advantages in biological systems to eliminate the potential interferences.

Water-soluble fluorescent probe for specific detection of SO2 derivatives in food and cells

SO2, the fourth endogenous gaseous signaling molecule, plays an important role in cells. Besides, SO2 derivatives are used as preservatives in food and drinks. To understand the level of SO2 derivatives in body and food, we developed a FRET-based fluorescent probe CPO. CPO had high energy transfer efficiency and high selectivity toward SO2 derivatives. It could fast respond toward SO2 derivatives in an aqueous solution (3 min) and possessed a low detection limit (68 nM). Furthermore, the SO2 derivatives in tap water, sugar, vermicelli and cells were detected by probe CPO. In addition, probe CPO had a high colocalization coefficient (0.95) for lipid droplets.

Current Status and Future Prospects of Hydrogen Sulfide Donor‐Based Delivery Systems

AbstractNowadays, hydrogen sulfide (H2S) is regarded as an endogenous gaseous signaling molecule in mammals. As a gas transmitter, it induces important biological effects in many physiological and pathological processes, and possesses great therapeutic potentials. However, this bioactive gas has concentration‐related toxicity, rapid evaporation, and high reactivity, making it inconvenient to use as a therapeutic agent. As an exogenous source of H2S, various H2S donors are developed to release H2S after triggering the response under specific conditions, so as to supplement the level of H2S in the body. Meanwhile, to enhance the therapeutic efficacy and reduce the possible toxicities of the H2S donors, various new materials or methods for H2S delivery are studied, realizing sustained/controlled release profiles as well as achieving triggered, long‐term, local or targeted delivery approaches for the gas. This review summarizes the representative H2S donors and the latest progress of hydrogen sulfide donor‐based delivery systems, and the applications of such delivery systems in a series of target diseases that are suitable and expected to be treated by H2S, in order to provide reference for the design of secure and efficient H2S donor drug delivery system. In addition, the future of this field is also prospected.

CTH/H2S Regulates LPS-Induced Inflammation through IL-8 Signaling in MAC-T Cells.

Hydrogen sulfide (H2S), as an endogenous gaseous signaling molecule, plays an important role in the inflammatory process. Our previous study found that Cystathionine-γ-lyase (CTH) and H2S are correlated with the occurrence and development of Clinical Mastitis (CM) in Holstein cows. However, the functions and regulatory mechanisms of CTH/H2S are still unknown. In this study, the inflammatory mammary cell model based on the MAC-T cell line was established by Lipopolysaccharide (LPS)-induced manner to further explore the function and regulatory mechanism of CTH/H2S in cows with CM. In the inflammatory MAC-T cell, the CTH expression and H2S production were both repressed in an LPS-dose dependent manner, which demonstrated that CTH/H2S is related to the progression of inflammation. The inhibition of CTH/H2S using a selective CTH inhibitor, β-cyano-l-Alanine (BCA), promoted LPS-induced inflammation response and the expression of inflammatory cytokines. However, this was reversed by the H2S donor NaHS, demonstrating that H2S can protect cells from inflammatory damage. Intriguingly, interleukin-8 (IL-8) showed an inverse expression pattern correlated with the H2S-mediated cell protection effect during the inflammation process, and the inhibition test using a selective IL-8 receptor antagonist, SB225002, showed that IL-8 signaling plays a critical role in mediating endogenous H2S synthesis, and CTH/H2S exerts its anti-inflammation via IL-8-mediated signaling. This study provided support for the prevention and treatment of CM and the development of a novel anti-inflammatory strategy.

Ultrasound-Mediated Release of Gaseous Signaling Molecules for Biomedical Applications.

Although nitric oxide (NO), carbon monoxide (CO), and hydrogen sulfide (H2 S) have been considered as notorious gas pollutants for decades, they are considered as endogenous gaseous signaling molecules (GSMs), which have been widely recognized for their important signaling functions and prominent medical applications in human physiology. To achieve local delivery of GSMs to optimize therapeutic efficacy and reduce systemic side effects, stimuli-responsive nanocarriers have been successfully developed. Among them, ultrasound is considered as an attractive theranostic modality that can be used to track drug carriers, trigger drug release, and improve drug deposition, etc. In this minireview, recent achievements in designing ultrasound-responsive nanocarriers for the controlled delivery of GSMs and their biomedical applications are summarized. This emerging research direction enables the controlled delivery of GSMs to deep tissues, and the combination of ultrasound imaging techniques offers many possibilities for the fabrication of new theranostic platforms.

Sulphenylation of CypD at Cysteine 104: A Novel Mechanism by Which SO2 Inhibits Cardiomyocyte Apoptosis.

Objectives: The study was designed to explore the role of endogenous gaseous signaling molecule sulfur dioxide (SO2) in the control of cardiomyocyte apoptosis and its molecular mechanisms. Methods: Neonatal mouse cardiac myocytes (NMCMs) and H9c2 cells were used in the cell experiments. The endogenous SO2 pathway including SO2 level and the expression of SO2-generating enzyme aspartate aminotransferase 1/2 (AAT1/2) were detected in NMCMs. The apoptosis of cardiomyocytes was examined by a TUNEL assay. The cleavage and the activity of apoptotic proteins caspase9 and caspase3 were measured. The content of ATP, the opening of mitochondrial permeability transition pore (mPTP), and the cytochrome c (cytc) leakage were detected by immunofluorescence. The sulphenylation of cyclophilin-D (CypD) was detected by biotin switch analysis. The four CypD mutant plasmids in which cysteine sites were mutated to serine were constructed to identify the SO2-affected site in vitro. Results: ISO down-regulated the endogenous SO2/AAT pathway of cardiomyocytes in association with a significant increase in cardiomyocyte apoptosis, demonstrated by the increases in apoptosis, cleaved-caspase3/caspase3 ratio, and caspase3 activity. Furthermore, ISO significantly reduced ATP production in H9c2 cells, but the supplement of SO2 significantly restored the content of ATP. ISO stimulated mPTP opening, resulting in an increase in the release of cytc, which further increased the ratio of cleaved caspase9/caspase9 and enhanced the protein activity of caspase9. While, the supplementation of SO2 reversed the above effects. Mechanistically, SO2 did not affect CypD protein expression, but sulphenylated CypD and inhibited mPTP opening, resulting in an inhibition of cardiomyocyte apoptosis. The C104S mutation in CypD abolished SO2-induced sulphenylation of CypD, and thereby blocked the inhibitory effect of SO2 on the mPTP opening and cardiomyocyte apoptosis. Conclusion: Endogenous SO2 sulphenylated CypD at Cys104 to inhibit mPTP opening, and thus protected against cardiomyocyte apoptosis.

Endogenous hydrogen sulfide promotes human preimplantation embryonic development by regulating metabolism-related gene expression

Hydrogen sulfide (H2S) as an endogenous gaseous signaling molecule had been proved to play a vital role in gametes physiology, covering meiosis, maturation and aging. However, little is known about H2S involvement in embryonic development. The present study explored the positive effect of H2S on human early embryonic development. Results validated that the two H2S producing enzymes, CBS and CSE mRNA and proteins were identified in donated human cleavage and blastocyst-stage embryos. The l-cysteine incubation produced endogenous H2S in human blastocysts. NaHS positively affected in vitro blastulation. Single-cell RNA-seq analysis identified 228 differentially expressed genes (DEGs) after NaHS treatment versus the control. The Gene Ontology (GO) enrichment analysis of DEGs showed that genes for protein modification and metabolism were significantly enriched in the NaHS treatment group. For the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis, 2-oxocarboxylic acid metabolism, glycosaminoglycan biosynthesis-keratan sulfate, steroid biosynthesis, carbon metabolism, and biosynthesis of amino acids were significantly enriched. Six DEGs, including Neural EGFL like 1 (NELL1), aconitase 1 (ACO1), phosphoglycerate mutase 1 (PGAM1), TP53 induced glycolysis regulatory phosphatase (TIGAR), UDP-GlcNAc:betaGal beta-1,3-N-acetylglucosaminyltransferase 2 (B3GNT2), and carbohydrate Sulfotransferase 4 (CHST4) were validate by real-time RT-PCR. These findings suggest that H2S is a positive regulator of early embryonic development and may alter the transcription of embryonic genes for protein modification and metabolism in human embryos.

Engineering macromolecular nanocarriers for local delivery of gaseous signaling molecules.

In addition to being notorious air pollutants, nitric oxide (NO), carbon monoxide (CO), and hydrogen sulfide (H2S) have also been known as endogenous gaseous signaling molecules (GSMs). These GSMs play critical roles in maintaining the homeostasis of living organisms. Importantly, the occurrence and development of many diseases such as inflammation and cancer are highly associated with the concentration changes of GSMs. As such, GSMs could also be used as new therapeutic agents, showing great potential in the treatment of many formidable diseases. Although clinically it is possible to directly inhale GSMs, the precise control of the dose and concentration for local delivery of GSMs remains a substantial challenge. The development of gaseous signaling molecule-releasing molecules provides a great tool for the safe and convenient delivery of GSMs. In this review article, we primarily focus on the recent development of macromolecular nanocarriers for the local delivery of various GSMs. Learning from the chemistry of small molecule-based donors, the integration of these gaseous signaling molecule-releasing molecules into polymeric matrices through physical encapsulation, post-modification, or direct polymerization approach renders it possible to fabricate numerous macromolecular nanocarriers with optimized pharmacokinetics and pharmacodynamics, revealing improved therapeutic performance than the small molecule analogs. The development of GSMs represents a new means for many disease treatments with unique therapeutic outcomes.

Impact of Hydrogen Sulfide on Mitochondrial and Bacterial Bioenergetics.

This review focuses on the effects of hydrogen sulfide (H2S) on the unique bioenergetic molecular machines in mitochondria and bacteria—the protein complexes of electron transport chains and associated enzymes. H2S, along with nitric oxide and carbon monoxide, belongs to the class of endogenous gaseous signaling molecules. This compound plays critical roles in physiology and pathophysiology. Enzymes implicated in H2S metabolism and physiological actions are promising targets for novel pharmaceutical agents. The biological effects of H2S are biphasic, changing from cytoprotection to cytotoxicity through increasing the compound concentration. In mammals, H2S enhances the activity of FoF1-ATP (adenosine triphosphate) synthase and lactate dehydrogenase via their S-sulfhydration, thereby stimulating mitochondrial electron transport. H2S serves as an electron donor for the mitochondrial respiratory chain via sulfide quinone oxidoreductase and cytochrome c oxidase at low H2S levels. The latter enzyme is inhibited by high H2S concentrations, resulting in the reversible inhibition of electron transport and ATP production in mitochondria. In the branched respiratory chain of Escherichia coli, H2S inhibits the bo3 terminal oxidase but does not affect the alternative bd-type oxidases. Thus, in E. coli and presumably other bacteria, cytochrome bd permits respiration and cell growth in H2S-rich environments. A complete picture of the impact of H2S on bioenergetics is lacking, but this field is fast-moving, and active ongoing research on this topic will likely shed light on additional, yet unknown biological effects.

Gibberellic acid maintains postharvest quality of Agaricus bisporus mushroom by enhancing antioxidative system and hydrogen sulfide synthesis.

The application of gibberellic acid (GA3 ) treatment to the postharvest quality maintenance of white button mushroom (Agaricus bisporus) was investigated. The optimum concentration of exogenous GA3 was 100mg/L. At this concentration, the color change was inhibited, the firmness was maintained, and the weight loss and respiratory rates were reduced. The GA3 group had significantly lower malonaldehyde (MDA) content and membrane permeability. Reactive oxygen species accumulation was reduced due to the regulation of polyphenol oxidase (PPO), peroxidase (POD), and superoxide dismutase (SOD) enzyme activities. Moreover, the production of endogenous gaseous signaling molecule hydrogen sulfide (H2 S) was triggered by GA3 treatment, which enhanced cystathionine γ-lyase (AbCSE) and cystathionine β-synthase (AbCBS) activities alongside the corresponding gene expressions. The preservation of button mushroom postharvest storage quality by GA3 was most likely due to the regulation of reactive oxygen species metabolism and hydrogen sulfide biosynthesis. PRACTICAL APPLICATIONS: Mushroom is rich in nutrients and functional substances. However, due to the lack of cuticle, high respiration rate, and moisture content, mushroom's postharvest quality deteriorates rapidly. A safe and effective reagent that prevents the senescence and quality deterioration of harvested mushroom is urgently needed. The effects of plant hormone GA3 on the postharvest quality of edible fungi remain unclear. The present study provided convincing evidence that 100mgL-1 of GA3 effectively maintained postharvest button mushroom quality by regulating reactive oxygen species metabolism and hydrogen sulfide biosynthesis.

Nitric Oxide Releasing Delivery Platforms: Design, Detection, Biomedical Applications, and Future Possibilities.

Gasotransmitters belong to the subfamily of endogenous gaseous signaling molecules, which find a wide range of biomedical applications. Among the various gasotransmitters, nitric oxide (NO) has an enormous effect on the cardiovascular system. Apart from this, NO showed a pivotal role in neurological, respiratory, and immunological systems. Moreover, the paradoxical concentration-dependent activities make this gaseous signaling molecule more interesting. The gaseous NO has negligible stability in physiological conditions (37 °C, pH 7.4), which restricts their potential therapeutic applications. To overcome this issue, various NO delivering carriers were reported so far. Unfortunately, most of these NO donors have low stability, short half-life, or low NO payload. Herein, we review the synthesis of NO delivering motifs, development of macromolecular NO donors, their advantages/disadvantages, and biological applications. Various NO detection analytical techniques are discussed briefly, and finally, a viewpoint about the design of polymeric NO donors with improved physicochemical characteristics is predicted.

Design and application of a fluorescent probe for rapid detection of H2S

H2S is an endogenous gaseous signal molecule with cell protective function, which plays a critical part in many physiological and pathological processes. On the one hand, abnormal fluctuations of the concentration of hydrogen sulfide in the human body are closely related to diseases such as Down's syndrome, Alzheimer's, diabetes and cirrhosis of the liver. On the other hand, acts as an antioxidant in the body, hydrogen sulfide can protect cells from damage caused by reactive oxygen species. Therefore, it is indispensible to detect hydrogen sulfide in living organisms. A fluorescent probe—NX-H2S, was constructed using azide group as the recognition group and naphthalimide as the framework. The spectroscopic properties of the probe were explored comprehensively. The experiment results show that NX-H2S exhibits not only a fast response (5 min) but also excellent sensitivity and selectivity toward H2S. In addition to the rapid detection of H2S in vitro, NX-H2S can also be successfully applied to the fluorescence imaging analysis of endogenous and exogenous H2S in living cells.

Terminal Oxidase Cytochrome bd Protects Bacteria Against Hydrogen Sulfide Toxicity.

Hydrogen sulfide (H2S) is often called the third gasotransmitter (after nitric oxide and carbon monoxide), or endogenous gaseous signaling molecule. This compound plays important roles in organisms from different taxonomic groups, from bacteria to animals and humans. In mammalian cells, H2S has a cytoprotective effect at nanomolar concentrations, but becomes cytotoxic at higher concentrations. The primary target of H2S is mitochondria. At submicromolar concentrations, H2S inhibits mitochondrial heme-copper cytochrome c oxidase, thereby blocking aerobic respiration and oxidative phosphorylation and eventually leading to cell death. Since the concentration of H2S in the gut is extremely high, the question arises - how can gut bacteria maintain the functioning of their oxygen-dependent respiratory electron transport chains under such conditions? This review provides an answer to this question and discusses the key role of non-canonical bd-type terminal oxidases of the enterobacterium Escherichia coli, a component of the gut microbiota, in maintaining aerobic respiration and growth in the presence of toxic concentrations of H2S in the light of recent experimental data.

Rapid detection of SO2 in living cells and zebrafish by using an efficient near-infrared ratiometric fluorescent probe with large emission shift

Sulfur dioxide (SO2), a possible endogenous gaseous signaling molecule, has been found to be correlated with various diseases in living organisms. Thus, the development of effective methods for detection of SO2 are of great physiological and pathological significance. In this work, we report a ratiometric NIR fluorescent probe Cou-Rh for monitoring bisulfite on basis of Michael addition reaction. This probe is prepared from coumarin by acid-catalyzed cyclization reaction. The addition of bisulfite results in a significant decrease of fluorescence intensity at 695 nm and enhancement at 550 nm. The probe also displays high selectivity over various biologically related analytes with nanomolar limit of detection. What’s more, fluorescence imaging further confirms that the probe is capable of detecting bisulfite in living cells and zebrafish larvae. The results demonstrate that probe Cou-Rh can be employed for a useful scaffold to study the physiological function of sulfur dioxide.

Breathing Micelles for Combinatorial Treatment of Rheumatoid Arthritis

AbstractBreathing process involves inhalation and exhalation of different gases in animals. The gas exchange of the breathing process plays a critical role in maintaining the physiological functions of living organisms. Although artificial breathing materials exhibiting volume expansion and contraction upon alternate exposure to different gases have been well explored, those being able to realize the gas exchange remain elusive. Herein, we report breathing micelles (BM) capable of inhaling nitric oxide (NO) and exhaling carbon monoxide (CO), both of which are endogenous gaseous signaling molecules. We demonstrate that BM can simultaneously scavenge overproduced NO and attenuate proinflammatory cytokines in lipopolysaccharide (LPS)‐challenged macrophage cells. In vivo studies revealed that BM outperformed conventional nonsteroidal anti‐inflammatory drugs such as dexamethasone (Dexa) in treatment of rheumatoid arthritis (RA) in adjuvant‐induced arthritis (AIA) rats, likely due to the combinatorial effect of NO depletion, CO‐mediated deactivation of inducible NO synthase (iNOS) and activation of heme oxygenase‐1 (HO‐1). This work provides new insights into artificial BM for potential biomedical applications.

Breathing Micelles for Combinatorial Treatment of Rheumatoid Arthritis.

Breathing process involves inhalation and exhalation of different gases in animals. The gas exchange of the breathing process plays a critical role in maintaining the physiological functions of living organisms. Although artificial breathing materials exhibiting volume expansion and contraction upon alternate exposure to different gases have been well explored, those being able to realize the gas exchange remain elusive. Herein, we report breathing micelles (BM) capable of inhaling nitric oxide (NO) and exhaling carbon monoxide (CO), both of which are endogenous gaseous signaling molecules. We demonstrate that BM can simultaneously scavenge overproduced NO and attenuate proinflammatory cytokines in lipopolysaccharide (LPS)-challenged macrophage cells. In vivo studies revealed that BM outperformed conventional nonsteroidal anti-inflammatory drugs such as dexamethasone (Dexa) in treatment of rheumatoid arthritis (RA) in adjuvant-induced arthritis (AIA) rats, likely due to the combinatorial effect of NO depletion, CO-mediated deactivation of inducible NO synthase (iNOS) and activation of heme oxygenase-1 (HO-1). This work provides new insights into artificial BM for potential biomedical applications.

Abstract 501: Hydrogen Sulfide Protects the Heart Against Ferroptotic Cell Death in Diabetic Cardiomyopathy

Hyperglycemia-induced death of terminally differentiated cardiomyocytes in diabetic cardiomyopathy (DMCM) leads to heart failure. Ferroptosis is a newly discovered form of cell death triggered by intracellular iron and oxidative stress. While little is known about ferroptosis in DMCM, hyperinsulinemia stimulates intracellular iron uptake, which would be predicted to increase ferroptosis. H 2 S is an endogenous gaseous signaling molecule which may inhibit ferroptosis. H 2 S donors increase glutathione substrate for glutathione peroxidase 4 (GPX4), which removes lipid peroxidation and is the primary inhibitor of ferroptosis. However, no study has investigated the role of H 2 S in ferroptosis. We tested the hypothesis that increased ferroptosis contributes to DMCM, which is ameliorated by restoring H 2 S levels, by measuring ferroptosis in the hearts of db/db mice with DMCM and high glucose treated primary neonatal cardiomyocytes after H 2 S donor treatment. GPX4 expression was decreased (3.21±0.41 GPX4/total protein in db/+ control mice vs. 1.84±0.26 in db/db, P<0.05, n=6/group) and 4-HNE lipid peroxide was increased (7.37±0.98 μg 4-HNE/total protein in db/+ control mice vs. 8.73±0.84 in db/db, P<0.05, n=3/group) in the left ventricle of db/db mice indicating increased ferroptosis in DMCM. GPX4 activity was also decreased along with increased 4-HNE in high glucose cultured cardiomyocytes. Treatment with the ferroptosis inhibitor ferrostatin-1 prevented hyperglycemia induced ferroptosis. Treatment of cardiomyocytes with the H 2 S donor GYY4137 in hyperglycemia also decreased 4-HNE. We also validated the anti-ferroptotic potential of H 2 S by treating cardiomyocytes with the ferroptosis inducer RSL3 and GYY4137. H 2 S donor treatment reduced reactive oxygen species and 4-HNE lipid peroxide seen after ferroptosis induction with RSL3. This study establishes ferroptosis as a new, non-apoptotic, form of cell death in DMCM, and H 2 S as a novel regulator of cardiac ferroptosis.