Knee width, body weights, and muscle weights

All rats exposed to intraarticular knee injection of Freund’s incomplete adjuvant and Mycobacterium butyricum developed AIA, and the maximum diameter of the knee joint of AIA rats was significantly increased by 20 % compared to the control rats (11.8 ± 0.3 versus 9.7 ± 0.1 mm (n = 6); P < 0.01). The body weights of the rats with AIA were significantly lower than those of the control group (271 ± 9 versus 320 ± 6 g (n = 6); P < 0.01). In addition, absolute EDL muscle weight was ~10 % lower in the rats with AIA than that in controls (115 ± 5 versus 128 ± 3 mg (n = 6); P < 0.05), but this difference disappeared when muscle weight was normalized to body weight (0.4 ± 0.01 versus 0.4 ± 0.01 mg/g (n = 6); P > 0.05). For the soleus muscles, both the absolute and the normalized muscle weights were significantly lower in the rats with AIA than in the control rats (75 ± 6 versus 117 ± 2 mg (n = 6–7); P < 0.01; 0.28 ± 0.01 versus 0.36 ± 0.01 mg/g (n = 6–7); P < 0.01).

AIA causes a decreased actomyosin ATPase and SERCA activity

There was no difference in the total myofibrillar protein concentration in EDL muscles between the rats with AIA and control rats (130.0 ± 5.7 versus 120.0 ± 13.4 mg/g wet wt (n = 7–8); P > 0.05). A minor reduction in MyHC, but not actin, protein expression has been observed in CIA soleus muscle [5]. We therefore assessed the expression levels of MyHC and actin. Figure 1a shows a typical expression pattern of these myofibrillar proteins in control and AIA muscles. Neither the MyHC nor actin content was altered in AIA muscles compared with control muscles (Fig. 1b).

Fig. 1 Actomyosin ATPase and sarcoplasmic reticulum (SR) Ca2+-ATPase activities are decreased in AIA EDL muscles. Representative Coomassie brilliant blue staining of myofibrillar proteins (a), percentage distribution of myosin heavy chain (MyHC) and actin content in total myofibrillar proteins (b), and actomyosin ATPase activity (c). Electrophoretically separated MyHC isoforms (d) and percentage distribution of MyHC isoforms (e): I, slow isoform and IIa, IId/x, and IIb, fast isoforms. Representative Western blots of ryanodine receptor (RyR1), dihydropyridine receptor α2 subunit (DHPR), and SR Ca2+-ATPase (SERCA) 1 (f), the expression levels of these proteins normalized to actin content (g), and SERCA activity (h). Control (CNT), white bars; AIA, black bars. Data are presented as mean ± SEM for three to seven muscles in each group. *P < 0.05 versus CNT Full size image

In contrast, the actomyosin ATPase activity in muscles of AIA rats was reduced to ~70 % than that in controls (Fig. 1c). Rat EDL muscles are composed mainly of fast-twitch fibers (Fig. 1d). There were no significant differences in the distribution of the MyHC isoforms between the control rats and rats with AIA (Fig. 1e). Thus, the decreased actomyosin ATPase activity observed in our study was not due to changes in the expression of MyHC isoforms.

There were no changes in the expression levels of the Ca2+-handling proteins RyR1, DHPR, or SERCA1 (Fig. 1f, g), whereas SERCA2 was not detected in either group. In contrast, SERCA activity was ~30 % lower in AIA than that in control EDL muscles (Fig. 1h). Thus, AIA muscles display impaired intrinsic function of both SERCA and actomyosin, i.e., the two major ATPases in the skeletal muscle.

Antioxidant does not prevent arthritis but protects against AIA-induced muscle weakness

There was no significant difference in the absolute twitch force of the EDL muscle between the rats with AIA and control rats (0.38 ± 0.02 versus 0.37 ± 0.02 N (n = 6); P > 0.05) (Fig. 2a). In contrast, the rates of force development (dP/dt) and relaxation (–dP/dt) in twitches were decreased by 27 and 29 % in AIA EDL muscles, respectively (Fig. 2a, b). Absolute tetanic force was ~20 % lower in AIA EDL muscles than that in controls at stimulation frequencies from 70 to 120 Hz (Fig. 2c). Although the cross-sectional area of AIA EDL muscles was somewhat decreased, tetanic force per cross-sectional area (i.e., specific force) was ~15 % lower than in control muscles at 100 and 120 Hz (P < 0.05 and P < 0.01, respectively) (Fig. 2d). Moreover, there was a left-ward shift of the force-frequency relationship in AIA EDL muscles with the frequency giving 50 % of the maximum force being significantly lower than in control muscles (31.9 ± 1.7 versus 40.9 ± 1.6 Hz (n = 6); P < 0.05).

Fig. 2 Antioxidant treatment prevents contractile dysfunction in AIA EDL muscles. Representative original records of twitch (a) and 100 Hz tetanic (c) force from a control (CNT, dashed line) and an AIA (full line) EDL muscle. The peak rates of twitch force development (dP/dt) and relaxation (−dP/dt) (b) and specific force-frequency relationship (d) in CNT and AIA EDL muscles. Maximum knee diameter (e) and specific force-frequency relationship (f) in EDL muscles from CNT and AIA rats with or without treatment with EUK-134 (EUK). Data are presented as mean ± SEM for four to seven muscles in each group. *P < 0.05, **P < 0.01 versus CNT, ## P < 0.01 versus AIA Full size image

In addition, there was a marked reduction in the specific force in AIA soleus muscles at stimulation frequency from 10 to 120 Hz (ranging from −33 to −42 % (n = 6–7); P < 0.01). Moreover, twitches were slower in AIA soleus muscles with dP/dt and –dP/dt being decreased by 27 and 34 %, respectively (n = 5–7; P < 0.05). No further experiments were performed on the soleus muscles. Thus, decreased tetanic force production and slowed twitch contractions are observed in both fast-twitch and slow-twitch muscles of AIA rats, which is consistent with previous results from CIA mice [5, 6].

We and others have proposed redox protein modification as one of the mechanisms underlying the intrinsic contractile dysfunction of the skeletal muscle in inflammatory conditions [5, 6, 21]. Therefore, we studied whether antioxidant treatment could prevent the arthritis-induced muscle weakness. AIA rats were then treated with the SOD/catalase mimetic EUK-134, which was injected intraperitoneally once daily starting the day after induction of AIA. EUK-134 treatment had no obvious effect on the development of arthritis, and the increase in the maximum diameter of the knee joint was similar in EUK-134-treated and PBS-injected control AIA rats (Fig. 2e). Nevertheless, the decrease in specific tetanic force was prevented in the EUK-134-treated AIA rats (Fig. 2f).

Antioxidant inhibits redox modifications of actin in muscles from AIA rat

Due to previous results [5, 6, 21] and the present result that antioxidant treatment prevented muscle weakness in AIA rats, we next investigated whether the contractile defects were accompanied by signs of changed redox status in the AIA EDL muscle. MDA is a highly reactive by-product of lipid peroxidation. Western blotting showed several positive bands for MDA-protein adducts in both control and AIA muscles. Among these bands, EDL muscles from the rats with AIA showed a specific increase in MDA-protein adducts at ~150 kDa (Fig. 3a, d). 3-NT is considered to be a major product of peroxynitrite-derived radical interaction with tyrosine residues in proteins [9]. There was also a marked increase in 3-NT protein content at ~150 kDa in AIA muscles (Fig. 3b, d). It has previously been demonstrated that oxidative stress can induce the formation of actin aggregates [22]. In addition to the normal actin band seen at ~40 kDa, Western blots for actin showed a strong band at ~150 kDa in AIA muscles (Fig. 3c, d). It should be noted that the appearance of the ~150 kDa band in AIA muscles was not accompanied by any detectable decrease in the major ~40 kDa actin band, which shows that normally sized actin was still dominating. Intriguingly, the ~150 kDa bands for 3-NT and actin in AIA muscles disappeared when Western blotting was performed in the presence of the 2-mercaptoethanol (ME), which reduces disulfide bonds (Fig. 3b, c). Taken together, these results indicate the formation of ~150 kDa MDA- and 3-NT-containing and disulfide bond-dependent actin aggregates in AIA muscles.

Fig. 3 Malondialdehyde-protein adducts and 3-nitrotyrosine content are increased in actin aggregates from AIA EDL muscles. Representative Western blots for malondialdehyde (MDA)-protein adducts (a), 3-nitrotyrosine (3-NT) (b), and actin (c) in control (CNT) and AIA EDL muscles. In the 3-NT and actin blots, samples from AIA rats were run in the absence and presence of 2-mercaptoethanol (ME). d Intensities for the protein band at ~150 kDa (indicated by the arrows) in MDA-protein adducts, 3-NT, and actin were normalized to the troponin I (TnI) content. Data are presented as mean ± SEM for six muscles in each group. *P < 0.05, **P < 0.01 versus CNT Full size image

The contractile deficiency was prevented when AIA rats were treated with the antioxidant EUK-134 (see Fig. 2f) and AIA muscles displayed major redox-induced actin modifications. Thus, we studied the effect of EUK-134 treatment on actin aggregates in EDL muscles of AIA rats. Importantly, the results show that the increase in 3-NT formation and aggregation of actin in AIA EDL muscles was prevented by EUK-134 treatment (Fig. 4a–c).

Fig. 4 Antioxidant treatment of AIA rats prevents redox modifications of actin in EDL muscles. Representative Western blots for 3-nitrotyrosine (3-NT) (a) and actin (b) in EDL muscles from control (CNT) and AIA rats with or without treatment with EUK-134 (EUK). c Intensities for the protein band at ~150 kDa (indicated by arrows) in 3-NT and actin were normalized to the troponin I (TnI) content. Data are presented as mean ± SEM for four muscles in each group. **P < 0.01 versus CNT, ## P < 0.01 versus AIA Full size image

Increased amount of redox-related proteins and inflammatory mediators in muscles from AIA rats

ONOO− is formed by the rapid, diffusion-controlled reaction of NO and superoxide. To assess the molecular mechanism of the accelerated production of ONOO− in the AIA muscle, as indicated by the increased ~150 kDa 3-NT band, the expression of redox-related proteins were analyzed. The expressions of NOX2/gp91phox and nNOS, but not eNOS, were significantly increased in AIA EDL muscles (Fig. 5a, b). iNOS was not detected in either group, and the expression levels of SOD2 were similar in the two groups.

Fig. 5 Expressions of redox-related proteins and inflammatory mediators are altered in AIA EDL muscles. a Representative Western blots illustrating the levels of NADPH oxidase (NOX2/gp91phox), neuronal nitric oxide synthase (nNOS), endothelial NOS (eNOS), superoxide dismutase 2 (SOD2), tumor necrosis factor α (TNF-α), and high-mobility group box 1 (HMGB1) in control (CNT) and AIA EDL muscles. The inducible NOS was not detected in either group. b Quantification of the levels of redox-related proteins normalized to actin content. Data are presented as mean ± SEM for three to six muscles in each group. *P < 0.05, **P < 0.01 versus CNT Full size image

We also measured the protein expression of the inflammatory mediators TNF-α and HMGB1, both of which have been suggested to induce redox stress [3, 23]. The protein expressions of both TNF-α and HMGB1 were substantially higher in AIA than in control EDL muscles (Fig. 5a, b).