Background/Purpose: Systemic lupus erythematosus (SLE) peripheral blood lymphocytes (PBL) show mitochondrial dysfunction, characterized by elevated mitochondrial transmembrane potential (Δψ_m) and mass and low ATP, attributed to elevated production of nitric oxide (NO) and reduced glutathione (GSH).  To understand the molecular bases of mitochondrial dysfunction we tested activity of the electron transport chain (ETC) complexes I and IV.  As N-acetylcysteine (NAC) reversed GSH depletion and improved disease activity in SLE, its target of impact in mitochondria was also investigated.

Methods: PBL from 65 SLE subjects and 30 healthy controls, matched for patients’ age within ten years, gender, and ethnicity were studied. Mitochondrial respiration was measured using a Clark-type O₂ electrode (Oxygraph, Hansatech, Norfok, UK) in 1) freshly isolated PBL, 2) PBL rested overnight, 3) PBL exposed to NO donors NOC-9 or NOC-18, 4) PBL exposed to NAC, 5) PBL exposed to β-mercaptoethanol (BME). S-nitrosylation was assessed with a biotin switch kit (Cayman, Ann Arbor, MI). Δψ_m, mass, NO, ONOO^–, Ca²⁺, and H₂O₂ stress were assessed by flow cytometry (Meth. Mol. Biol. 900:61-89, 2012).

Results: SLE PBL have increased respiration upon T cell activation (SLE: 4.157nmol/ml/min ±0.186, control: 3.655nmol/ml/min ±0.167; p=0.012). Digitonin-permeabilized SLE PBL also  show increased respiration without exogenous substrates (SLE: 2.492 nmol/ml/min ± 0.196, control: 2.137 nmol/ml/min ± 0.153; p = 0.027) and with substrates of ETC complex IV (SLE: 7.722 nmol/ml/min ± 0.419, control: 7.006 nmol/ml/min ± 0.505; p = 0.028). SLE PBL had elevated mitochondrial mass (+10% ± 3% p = 0.002). When normalized to mitochondrial mass, SLE PBL exhibit 33% reduced respiration through complex I (p=0.036) and 81% enhanced respiration through complex IV (p = 0.036). As expected, exposure to NOC-18 increased NO (+158% ± 51% p = 0.005) and mitochondrial mass in normal PBL (+162% ± 68% p = 0.016). NO exposure selectively increased O₂ consumption by SLE PBL through complex I relative to healthy controls (SLE: 1.405 nmol/ml/min ± 0.206; Controls: 1.277 nmol/ml/min ± 0.150; p = 0.026). In contrast, NAC decreased both ONOO^– (-37% ±0.04 p=0.0001) and H₂O₂ levels without (-45% ± 5% p=0.0005) or with co-incubation with NOC-18 (ONOO^-: -38% ±7% p=0.002; H₂O₂: -71% ±2% p=0.000004). Compared to NOC-18 alone, co-incubation with NAC also decreased NO (-92% p=0.014), ONOO^– (-38% p=0.006), mitochondrial mass (-87% p=0.017), and Ca²⁺ (-52% p=0.042). Direct addition of NAC into the Oxygraph chamber inhibited respiration by 53% through complex I (p=0.004) but not complex IV. In contrast, BME failed to affect respiration at complex I, but it reduced respiration by 80% at complex IV (p=0.031). NAC also blocked respiration through complex I by 56% in SLE PBL (p=0.0001). Upon NO exposure, S-nitrosylation is increased in freshly isolated lupus PBL relative to matched healthy control PBL (p=0.002).

Conclusion: The results of this data suggest that SLE PBL have defective ETC activity at complex I. NAC helps maintain a reducing environment, possibly by directly blocking respiration at complex I.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/abnormal-mitochondrial-electron-transport-chain-activity-at-complex-i-is-regulated-by-nitric-oxide-and-n-acetylcysteine-in-lupus-lymphocytes/