Null mutation of gp91phox reduces muscle membrane lysis during muscle inflammation in mice

Abstract

Muscle inflammation is a common feature in muscle injury and disease. Recently, investigators have speculated that inflammatory cells may increase or decrease muscle damage following modified muscle use, although there are few experimental observations to confirm either possibility. In the present study, a null mutation of gp91phox in neutrophils prevented superoxide production in cytotoxicity assays in which muscle cells were targets, and prevented most neutrophil-mediated cytolysis of muscle cells in comparison to wild-type neutrophils in vitro. We further tested whether deficiency in superoxide production caused a decrease in muscle membrane damage in vivo during modified muscle use. Gp91phox null mutant mice and wild-type mice were subjected to 10 days of muscle hindlimb unloading followed by reloading through return to normal locomotion, which induced muscle membrane lesions and muscle inflammation. Membrane lesions were quantified by measuring the presence of extracellular marker dye in reloaded soleus muscle fibres. There was a 90 % reduction in the number of fibres showing extensive membrane injury in gp91phox null mice compared to controls. Mutation of gp91phox did not change the concentration of neutrophils or macrophages in the reloaded muscle. Furthermore, muscle fibre growth during the reloading period was unaffected by the reduction in membrane injury. Together, these findings show that neutrophils can induce muscle membrane lysis through superoxide-mediated events, and indicate that superoxide-mediated membrane damage in vivo is not required for myeloid cell chemotaxis or muscle growth during muscle reloading.

Figure 2. The null mutation of gp91^phox greatly reduces muscle cell lysis by neutrophils in vitro

Neutrophils at 25 000 neutrophils mm⁻² from C57 control mice or gp91^phox null mutant mice that are activated by receptor-mediated (FMLP) or receptor-independent (PMA) mechanisms were used as effectors in cytotoxicity assays with C2C12 myotubes as targets. *Significantly different from the value for C57 neutrophils under same culture conditions (P < 0.05). Bars = s.e.m.

Figure 1. Neutrophils that are null mutants for the 58 kDa subunit of NADPH oxidase lack inducible superoxide production

Western analysis was used to confirm that peritoneal cells collected from C57 mice expressed the 58 kDa subunit of NADPH oxidase (A; arrowhead indicates 58 kDa) and that gp91^phox null mutant mice did not express the 58 kDa subunit (B). In addition, assays of superoxide production were performed on C57 neutrophils and gp91^phox null neutrophils (C) to confirm that the mutation prevented the production of superoxide that was induced by FMLP or by PMA. Superoxide release assays were performed using 10 000 neutrophils mm⁻². *Significantly different from the value for C57 neutrophils under the same culture conditions (P < 0.05). Bars = s.e.m.

Figure 4. A null mutation of gp91^phox decreases the concentration of injured fibres in reloaded muscle

The concentration of brightly fluorescent fibres (see Fig. 3B) in cross-sections of soleus muscles that were incubated in procion orange was determined. AMB, control ambulatory group (i.e. did not undergo hindlimb unloading); UNLOAD, animals were subjected to hindlimb unloading and then immediately sacrificed; RELOAD, animals were subjected to hindlimb unloading followed by 6 or 24 h of reloading before being sacrificed. #Concentrations that differed significantly from unloaded muscles of same genotype (P < 0.05); *concentrations that differed significantly from C57 muscles in the same treatment group (P < 0.05). Bars = s.e.m.

Figure 3. Identification of muscle fibre membrane lesions by the presence of intracellular procion orange

A, cross-section of a control soleus muscle that was incubated with the extracellular marker dye procion orange. In healthy control muscle the procion orange is located in the extracellular space that outlines each muscle fibre. B, cross-section of a soleus muscle from a C57 mouse that experienced 24 h of muscle reloading after 10 days of unloading. Two fibres are brightly fluorescent because of the entry of procion orange through membrane lesions. Note also that the general level of intracellular fluorescence in muscle fibres in reloaded soleus (B) is higher than in control fibres (A). Bar = 100 μm.

Figure 5. Frequency distributions of muscle fibres over the range of intensities of intracellular fluorescence

Intracellular fluorescence was measured for individual fibres in entire cross-sections of soleus muscles from each treatment group (six animals per treatment group). Treatments consisted of ambulatory controls, animals experiencing 10 days of hindlimb unloading only (0 h), or 10 days unloading followed by 6 h (6 h) or 24 h reloading (24 h). A rightward shift of peaks on the abscissa indicates an increase in the frequency of fibres with muscle membrane lesions.

Figure 6. Myeloid cell invasion is not affected by the gp91^phox null mutation

Muscles from ambulatory control mice experiencing 10 days unloading only, or unloading followed by 6 h or 24 h reloading were assayed for neutrophil concentration (A) and macrophage concentration (B). Each treatment group included six animals. *Concentrations that differed significantly from unloaded muscles of the same genotype (P < 0.05). No significant differences were found between gp91^phox and C57 mice in any experimental group. Bars = s.e.m.

Figure 7. Rates of muscle atrophy and growth are not affected by the gp91^phox null mutation

Values are means ±s.e.m. of soleus muscle fibre cross-sectional areas for all fibres in the entire cross-section for each treatment group (six animals per treatment group). *Significant difference from unloaded muscles of same genotype (P < 0.05). No significant differences were found between gp91^phox and C57 mice in any experimental group.