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Endogenous peroxynitrite mediates mitochondrial dysfunction in rat diaphragm during endotoxemia
J Boczkowski1, C L Lisdero, S Lanone
1Institut National de la Santé et de la Recherche Médicale (INSERM) U408 and IFR 02, Faculté X. Bichat, Paris, France. jbb2@bichat.inserm.fr
Abstract:
It has been shown that nitric oxide (NO), synthesized by the inducible NO synthase (iNOS) expressed in the diaphragm during endotoxemia, participates in the development of muscular contractile failure. The aim of the present study was to investigate whether this deleterious action of NO was related to its effects on cellular oxidative pathways. Rats were inoculated with E. coli lipopolysaccharide (LPS) or sterile saline solution (controls) and studied at 3 and 6 h after inoculation. iNOS protein and activity could be detected in the rat diaphragm as early as 3 h after LPS, with a sustained steady-state concentration of 0.5 microM NO in the muscle associated with increased detection of hydrogen peroxide (H(2)O(2)). In vitro, the same NO concentration produced a marked increase in H(2)O(2) production by isolated control diaphragm mitochondria, thus reflecting a higher intramitochondrial concentration of nondiffusible superoxide anion (O(2)(-.)). In a similar way, whole diaphragmatic muscle and diaphragm mitochondria from endotoxemic rats showed a progressive increase in H(2)O(2) production associated with uncoupling and decreased phosphorylating capacity. Simultaneous with the maximal impairment in respiration (6 h after LPS), nitration of mitochondrial proteins (a peroxynitrite footprint) was detected and diaphragmatic force was reduced. Functional mitochondrial abnormalities, nitration of mitochondrial proteins, and the decrease in force were significantly attenuated by administration of the NOS inhibitor L-NMMA. These results show that increased and sustained NO levels lead to a consecutive formation of O(2)(-.) that reacts with NO to form peroxynitrite, which in turn impairs mitochondrial function, which probably contributes to the impairment of muscle contractility. during endotoxemia.
Insights
Endotoxemia increases nitric oxide (NO) in diaphragm muscles, leading to mitochondrial dysfunction and muscle weakness. Inhibiting NO synthesis improves mitochondrial function and muscle strength during sepsis.
Area of Science:
- Physiology
- Biochemistry
- Pathology
Background:
- Inducible nitric oxide synthase (iNOS) in the diaphragm contributes to muscle contractile failure during endotoxemia.
- The role of nitric oxide (NO) in endotoxemia-induced muscle dysfunction and its relation to cellular oxidative pathways requires further investigation.
Purpose of the Study:
- To investigate the link between NO, cellular oxidative pathways, and diaphragmatic contractile failure during endotoxemia.
- To determine the impact of NO on mitochondrial function and oxidative stress in the diaphragm.
Main Methods:
- Rats were induced with endotoxemia using E. coli lipopolysaccharide (LPS).
- Diaphragm iNOS expression, NO levels, hydrogen peroxide (H(2)O(2)) production, and mitochondrial function were assessed.
- Mitochondrial protein nitration and diaphragmatic force were measured.
- The effect of the NOS inhibitor L-NMMA was evaluated.
Main Results:
- LPS-induced endotoxemia increased diaphragm iNOS, NO, and H(2)O(2) levels.
- Elevated NO impaired mitochondrial function, evidenced by increased H(2)O(2) and decreased phosphorylating capacity.
- Mitochondrial protein nitration and reduced diaphragmatic force were observed.
- L-NMMA administration attenuated mitochondrial dysfunction and improved diaphragmatic force.
Conclusions:
- Increased NO during endotoxemia leads to mitochondrial dysfunction via peroxynitrite formation.
- Impaired mitochondrial function contributes to diaphragmatic contractile failure in endotoxemia.
- Targeting NO synthesis may be a therapeutic strategy for sepsis-induced muscle dysfunction.