Abstract Ischemic heart disease is the main global cause of death in the world. Abnormal sulfide catabolism, especially hydrogen sulfide accumulation, impedes mitochondrial respiration and worsens the prognosis after ischemic insults, but the substantial therapeutic strategy has not been established. Non-thermal atmospheric pressure plasma irradiation therapy is attracted attention as it exerts beneficial effects by producing various reactive molecular species. Growing evidence has suggested that supersulfides, formed by catenation of sulfur atoms, contribute to various biological processes involving electron transfer in cells. Here, we report that non-thermal plasma-irradiated cysteine (Cys∗) protects mouse hearts against ischemia/reperfusion (I/R) injury by preventing supersulfide catabolism. Cys∗ has a weak but long-lasting supersulfide activity, and the treatment of rat cardiomyocytes with Cys∗ prevents mitochondrial dysfunction after hypoxic stress. Cys∗ increases sulfide-quinone oxidoreductase (SQOR), and silencing SQOR abolishes Cys∗-induced supersulfide formation and cytoprotection. Local administration of mouse hearts with Cys∗ significantly reduces infarct size with preserving supersulfide levels after I/R. These results suggest that maintaining supersulfide formation through SQOR underlies cardioprotection by Cys∗ against I/R injury. Keywords: Non-thermal plasma, Supersulfides, Mitochondrial energy metabolism, SQOR, Ischemia/reperfusion Graphical abstract Image 1 [45]Open in a new tab 1. Introduction Ischemic heart disease remains the leading cause of cardiovascular-related mortality and chronic heart failure worldwide, despite significant advances in the physician's ability to initiate myocardial reperfusion and salvage heart tissue. Many therapeutic strategies, especially medical interventions to potentiate the heart's resistance to ischemic injury have been intensively investigated, but an efficacious therapy has yet to be successfully implemented in the clinical arena [[46]1]. It remains an urgent issue how to preserve the energy content of cardiomyocytes during ischemia. In an anaerobic environment, NO[3]^−, NO[2]^−, Fe^3+, SO[4]^2−, and CO[2] are used as terminal electron acceptors for microbial respirations [[47][2], [48][3], [49][4], [50][5], [51][6]]. In mammalian cells, fumarate reportedly acts as a terminal electron acceptor to sustain a circuit of electron flow in the electron transport chain (ETC) that maintains mitochondrial functions under hypoxia [[52]7]. However, the heart and skeletal muscle have less fumarate reduction activity and will rapidly suppress mitochondrial functions under oxygen limitation. Therefore, to find an oxygen-independent electron transporting mechanism in myocardial mitochondria is expected to establish a new therapeutic strategy for ischemic heart disease. Supersulfides, in which sulfur atom is catenated, have recently attracted attention as true biomolecules with high electron transfer (i.e., redox) reactivity to support energy metabolism and signal transduction [[53]8,[54]9]. Supersulfides emerge both electrophilic and nucleophilic properties, and form hydropolysulfides (R–S(S)[n]H; n ≥ 1 and R ≠ H) and polysulfides (R–S(S)[n]S-R) in cells. In particular, cysteine persulfide (CysSSH) and glutathione persulfide (GSSH) have been found to exist in the micromolar range within the organism and act as antioxidants through their potent nucleophilicity [[55]10]. Increased level of persulfides is implicated in lifespan extension across species [[56]11,[57]12]. We have previously reported that protein-SSH is mainly formed by mitochondria-localized cysteinyl-tRNA synthetase (CARS) 2 by dual functions: CysSSH synthase and cysteinyl-tRNA synthetase activities [[58]8]. CysSSH is also produced by cystathionine-β-synthase (CBS) and Cystathionine-γ-lyase (CSE) using cystine as a substrate [[59]10]. In contrast, supersulfides are catabolized to form H[2]S at mitochondrial ETC [[60]8,[61]13,[62]14] and H[2]S overproduction leads to mitochondrial dysfunction by inhibiting complex IV [[63]15]. We have also reported that supersulfide catabolism is enhanced in failing cardiomyocytes and reduced supersulfides disrupt mitochondrial quality and function by decreasing protein polysulfidation, suggesting the critical role of supersulfides in maintaining cardiac homeostasis and robustness [[64][16], [65][17], [66][18]]. Non-thermal plasma at atmospheric pressure has been successfully employed for potential therapeutic purposes, such as selective elimination of cancer cells [[67]19], promotion of wound healing [[68]20], and gene transfection [[69]21]. Plasma irradiation is accompanied by dissociation/reconstruction of molecular bonds, in a way that is distinct from ion interactions in liquids or the behavior of shared ions in metals. Thus, non-thermal plasma irradiation is expected to produce unique reactive species with unprecedented biochemical properties [[70]20]. Until recently, since hydrogen peroxide is abundantly produced in non-thermal plasma-irradiated culture media, the biological effect of non-thermal plasma irradiation has been widely regarded as the inducer of oxidative stress [[71]22]. However, it has been demonstrated that non-thermal plasma-irradiated solution can also stimulate reactive oxygen species (ROS)-independent signaling pathways to eliminate cancer cells, when starting materials are optimized (i.e. lactate) [[72]23]. Additionally, when HEPES-containing buffer is treated with non-thermal plasma irradiation, short-lived reactive species are estimated to be produced that elicit cytoplasmic Ca^2+ influx through a ROS-independent mechanism [[73]24,[74]25]. In this study, we report that non-thermal plasma-irradiated cysteine (Cys) solution protects mouse hearts against ischemia/reperfusion (I/R) injury and rat cardiomyocytes against oxygen glucose deprivation/reoxygenation (OGD/R) injury by preserving supersulfide metabolism. Although the chemical supersulfide activity in plasma-irradiated Cys is estimated only ∼80 nM of inorganic sulfane sulfur Na[2]S[4], the cardioprotective effect of plasma-irradiated Cys can be maintained much longer than that of Na[2]S[4]. We also show that sulfide-quinone oxidoreductase (SQOR) participates in plasma-irradiated Cys-induced cardioprotection against I/R injury by preserving supersulfide metabolism, suggesting a potential therapeutic strategy for ischemic heart disease. 2. Results 2.1. Plasma-irradiated culture medium containing Cys has a cardioprotective effect Using the setup for non-thermal plasma irradiation using helium as the carrier gas as previously described ([75]Fig. 1A) [[76]26] we first examined whether plasma-irradiated culture medium has a cardioprotective effect. To this end, we treated cardiomyocytes with plasma-irradiated DMEM (DMEM∗), and evaluated its effect on the survival rate following OGD/R stress [[77]27]. After plasma irradiation, a huge variety of short-lived reactive species have been generated, leading to cytotoxicity [[78]28,[79]29]. To eliminate these short-lived reactive species, DMEM∗ was incubated for 6 h at 37 °C. Because the incubated plasma solution still has a high amount of hydrogen peroxide (H[2]O[2]), DMEM∗ was then preincubated with catalase to remove H[2]O[2] ([80]Fig. 1B). This DMEM∗ containing stable plasma products was used for cardioprotection assay. OGD/R stress increased propidium iodide (PI)-positive cardiomyocyte death, which was inhibited by DMEM∗ treatment ([81]Fig. 1C). When irradiation time is shorter (1 s, 3 s and 10 s), the increase in cell death rate by OGD/R was significantly attenuated by the application of DMEM∗ ([82]Fig. 1D). On the other hand, there was no significant difference between the control group when cells were treated with 30 sec-irradiated DMEM ([83]Fig. 1D). These results suggest that the cardioprotective effect of DMEM∗ is dependent on irradiation time. Fig. 1. [84]Fig. 1 [85]Open in a new tab Cardioprotective effect of plasma-irradiated Cys medium (A) Experimental setup for the helium atmospheric-pressure plasma (He-APP) irradiation. Inlet: plasma plume reaching the target solution (250 μL) placed on a glass bottom dish. (B) Protocol of applying plasma-irradiated solutions to cultured cardiomyocytes. Several types of sample solutions were plasma-irradiated and incubated for 6 h at 37 °C before dilution with methionine and cysteine-free DMEM (ΔCM). Catalase and HEPES were supplemented in all media right before cellular application. ΔCM(Cys), ΔCM supplemented with cysteine; ΔCM(Met), ΔCM supplemented with methionine; Buf, carbonate buffer; Buf (Cys), Buf supplemented with cysteine. (C) Effect of plasma-irradiated DMEM (DMEM∗) on cardiomyocyte injury under normoxia or oxygen glucose deprivation/reoxygenation (OGD/R) condition. Cardiomyocytes were stained by PI (red) and Hoechst (blue). Plasma irradiation time, 3 s. Scale bar, 400 μm. (D) Cell death rate of cardiomyocytes treated with DMEM∗ with different irradiation times (1–30 s) (n = 4–5 independent experiments). (E) Cell death rate of cardiomyocytes treated with plasma-irradiated methionine and cysteine-free DMEM (ΔCM∗) under normoxia (Nor) or OGD/R (n = 5 independent experiments). (F, G) Cell death rate of cardiomyocytes treated with plasma-irradiated ΔCM supplemented with cysteine or methionine (ΔCM(Cys)∗or ΔCM(Met)∗, respectively) (n = 5 independent experiments). (H, I) Cell death rate of cardiomyocytes treated with plasma-irradiated carbonate buffer supplemented without or with cysteine (Buf∗ or Buf (Cys)∗, respectively) (n = 3 independent experiments). Data are shown as the means ± SEM. ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001 by one-way ANOVA. “ns” indicates not significant. (For interpretation of the references to