Lack of transcriptionally active Nrf2 enhances skeletal muscle degeneration after CTX-induced injury
To analyse the effect of Nrf2 transcriptional deficiency during acute muscle damage, we examined inflammatory reaction and muscle degeneration as well as regeneration in the model of CTX-induced myoinjury. In Nrf2tKO mice, the level of muscle degeneration and inflammatory infiltration evaluated based on H&E staining (Fig. 1 A, B) was significantly higher in Nrf2tKO mice on the 3rd day after muscle damage. Although the activity of CK (Fig. 1 C) was increased in Nrf2tKO animals on day 1 after injection, a statistically significant difference between WT and Nrf2tKO mice was not evident. On the other hand, the level of LDH (Fig. 1 D) was significantly elevated in mice of both genotypes, and additionally, it was much higher in Nrf2tKO animals in comparison to WT on the 1st day after CTX injection. Moreover, we have shown increased protein level of pro-inflammatory cytokine MCP-1 (Fig. 1 E) and mRNA level of Hmox1 (Fig. 1 F), Il1b (Fig. 1 G) and Il6 (Fig. 1 H) on the 1st day after CTX injection in both genotypes. Furthermore, IHF analysis of necrotic fibers on the 3rd day after myoinjury did not reveal differences between genotypes (Fig. 1 I, J).
Muscle regeneration is not affected in the absence of transcriptionally active Nrf2 following CTX-induced injury
To assess the role of Nrf2 during muscle regeneration following the acute muscle injury caused by CTX injection, we examined the mRNA level of Myh3 and the number of eMyHC+ myofibers. Following muscle damage we observed a higher level of Myh3 in both WT and Nrf2tKO animals on the 7th day after injury, however, there were no differences among genotypes in all analysed time-points (Fig. 2 A). Accordingly, the number of eMyHC+ fibers (Fig. 2 B, C), CSA (Fig. 2 D), and mean fiber area (Fig. 2 E) were similar on day 7 after injury.
Transcriptional deficiency of Nrf2 does not aggravate dystrophic phenotype in mdx mice
To investigate the role of Nrf2 in chronic muscle injury, we generated dystrophic mice lacking the transcriptional activity of Nrf2 (Nrf2tKOmdx). In order to determine whether the lack of Nrf2 can affect exercise performance, mice were subjected to a downhill treadmill run to exhaustion. As shown by us (9) and others (24) previously, and confirmed in the present study, mdx mice were able to run a shorter distance than WT (Fig. 3 A). However, we did not observe an effect of the transcriptional deficiency of Nrf2 on the running pattern. The exercise capacity of Nrf2tKO animals was comparable to WT mice and Nrf2tKOmdx mice run similar distance as mdx counterparts (Fig. 3 A). Body weight (Fig. 3 B) and GM mass (Fig. 3 C) were significantly increased in mdx mice in comparison to healthy animals, while in Nrf2tKOmdx no striking differences compared with age-matched mdx mice were found.
Transcriptional knockout of Nrf2 does not exacerbate muscle degeneration in mdx mice
Muscle degeneration was evaluated based on the percentage of necrotic fibers in GM as well as the plasma level of CK and LDH, typical markers of muscle damage. Neither the number of necrotic fibers (Fig. 4 A, B) nor LDH (Fig. 4 C) and CK (Fig. 4 D) activity was changed between dystrophic mice additionally lacking Nrf2 and mdx animals, indicating a comparable level of muscle injury. As suspected, plasma LDH and CK levels of mdx mice were elevated compared with those of WT mice (Fig. 4. C, D, respectively).
Lack of Nrf2 transcriptional activity does not aggravate the inflammatory reaction in dystrophic skeletal muscles
Since Nrf2 has been reported as a master regulator of antioxidative responses that contributes to the anti-inflammatory process (30,31), we have assessed whether it can affect the inflammatory reaction in skeletal muscle in our experimental conditions. Analysis of H&E staining demonstrated that mdx mice lacking transcriptional activity of Nrf2 had a similar inflammation score to mdx mice in both, GM (Fig. 5 A, B) and diaphragm (Fig. 5 C, D). Moreover, the expression of Hmox1, an anti-inflammatory factor shown by us to be up-regulated in dystrophic muscles (9), was again potently elevated in GM of mdx mice. However, it was the same in Nrf2tKOmdx mice, indicating that Nrf2 transcriptional activity is dispensable for Hmox1 induction in the muscles (Fig. 5 E).
To shed more light on the inflammatory status, we have performed a comprehensive analysis of different leukocyte populations within hind limb muscles using flow cytometry. Mdx mice demonstrated an abundance of macrophages defined as CD45+F4/80+CD11b+ cells. However, no further changes in the infiltration of this population into skeletal muscle were caused by Nrf2 transcriptional deficiency (Fig. 5 F, G). Due to the diverse functions of different subpopulations of macrophages (4), in the next step, we have investigated M1-like and M2-like macrophages, based on the gating strategy discriminating between MHCII and CD206 expression by CD45+F4/80+CD11b+ cells. The subsets of both M1-like (CD45+F4/80+CD11b+MHCIIhiCD206low) and M2-like (CD45+F4/80+CD11b+MHCIIlow CD206hi) macrophages were much more elevated in dystrophic mice in comparison to WT but the lack of transcriptionally active Nrf2 did not further change their number (Fig. 5 H, I, J).
The number of NK cells (CD45+SSClowCD3-NK1.1+) was significantly higher in mdx mice in comparison to WT. Conversely, dystrophic mice additionally lacking Nrf2 transcriptional activity exhibited decreased NK number reaching a similar number to the one observed in WT animals (Fig. 6 A, C). The number of lymphocytes T (CD45+SSClowCD3+) (Fig. 6 B, C), T helper (Th; CD45+SSClowCD3+CD4+CD8-) (Fig. 6 D, F), and T cytotoxic (Tc; CD45+SSClowCD3+CD4-CD8+) (Fig. 6 E, F) were the same among all four genotypes.
The role of Nrf2 transcriptional deficiency on muscle fibrosis
We have found that transforming growth factor beta-1 (Tgfb1) and collagen type I alpha 1 (Col1a1) were upregulated in mdx vs. WT animals and were further elevated in mdx mice lacking additionally transcriptionally active Nrf2 (Fig. 7 A, B), suggesting that the transcriptional deficiency of Nrf2 could enhance fibrosis. To confirm those results a histological analysis of collagen deposition based on Masson’s trichrome staining was performed. Accordingly, endomysial collagen content was significantly elevated in both GM (Fig. 7 C, D) and diaphragm (Fig. 7 E, F) of dystrophic mice; however, it was not further exacerbated in mdx mice lacking transcriptionally active Nrf2. Moreover, as FAPs are also involved in the progression of DMD (32), we checked their numbers using flow cytometry. Our results showed that FAPs, defined as CD45-CD31-Sca1+a7i-CD34+ cells, were upregulated by dystrophin deficiency, but their numbers were not further affected by the lack of transcriptionally active Nrf2 (Fig. 7 G, H).
A decrease in the expression of angiogenic mediators in mdx mice is not affected by the lack of transcriptionally active Nrf2
Dysregulation of angiogenesis may greatly contribute to DMD progression (33). Moreover, Nrf2 was shown to regulate neovascularization and to exert a pivotal role in angiogenic signal transduction and angiogenic potential of not only endothelial cells itself but also bone marrow-derived proangiogenic cells (34). Therefore, we aimed to investigate the angiogenic signaling in our model. Firstly, we have checked the mRNA and protein level of the major proangiogenic factor, VEGF, in GM of mice of all genotypes. The mRNA expression was diminished in mdx mice but no further changes were observed in double knockouts (Fig. 8 A). Concomitantly, the level of VEGF protein was potently down-regulated in dystrophic GM; however, the lack of transcriptionally active Nrf2 did not affect this production in both healthy and dystrophic mice (Fig. 8 B). A similar trend of changes was found when the expression of Kdr, a receptor for VEGF was evaluated (Fig. 8 C).
Nrf2 transcriptional deficiency does not affect the number and proliferation of muscle SCs but it may influence muscle regeneration
The number of SCs evaluated based on IHF staining and calculation of the ratio of Pax7-positive nuclei to myofibers revealed an increased number of Pax7+ cells in dystrophin-deficient mice in comparison to healthy ones, however, the additional effect of the lack of transcriptionally active Nrf2 was not observed (Fig. 9 A, B). Furthermore, flow cytometry analysis demonstrated a considerable increase in the number of MyoD-positive SCs (CD45-CD31-Sca1-α7integrin+MyoD+) in mdx mice, but it was not further changed by Nrf2 transcriptional deficiency (Fig. 9 C). We have also checked the proliferation of MyoD-positive SCs by cytofluorimetric analysis of cells expressing Ki67. Significantly enhanced proliferation of MyoD+ SCs in dystrophic mice compared to healthy animals was observed. This was not further potentiated in Nrf2tKOmdx mice (Fig. 9 D, E).
Although there was no effect of the lack of transcriptionally active Nrf2 on the number and proliferation of SCs, we have shown that the regeneration process is affected in the course of chronic injury in dystrophic mice, and what is more – it is additionally altered by the Nrf2 status. Accordingly, dystrophic mice showed higher expression of myogenic regulatory factors such as myogenic differentiation 1 (Myod1) and myogenin (Myog) than their healthy counterparts, and the expression of those factors was further enhanced by Nrf2 transcriptional deficiency (Fig. 10 A, B).
Additionally, we have checked the mRNA level of muscle-specific microRNAs which also contribute to the process of muscle regeneration (35). Expression of miR-206 (Fig. 10 C) was elevated in mdx mice in comparison to age-matched WT animals whereas miR-1 (Fig. 10 D) and miR-133a/b (Fig. 10 E) showed the opposite pattern. However, none of them were affected by Nrf2 transcriptional deficiency.
Finally, the number of myofibers expressing eMyHC, the marker of regeneration, was diminished in mdx mice additionally lacking transcriptionally active Nrf2 in comparison to mdx counterparts (Fig. 10 F, G). However, histological analysis of centrally nucleated fibers did not show differences between mdx and Nrf2tKOmdx animals (Fig. 10 H, I).
Chronic treadmill exercise aggravates skeletal muscle degeneration and inflammation in dystrophic animals lacking transcriptional activity of Nrf2
In order to identify the role of Nrf2 under aggravated dystrophic conditions, we have analysed muscle functionality, degeneration, and inflammation after 4 weeks of chronic treadmill exercises. Firstly, reduced muscle function measured by the grip strength test in mdx mice in comparison to healthy counterparts was shown, however, it was not additionally altered by the transcriptional deficiency of Nrf2 (Fig. 11 A). Moreover, the activity of LDH and CK in plasma did not differ in Nrf2tKOmdx vs. mdx animals (Fig.11 B, C, respectively).
The number of necrotic fibers (Fig. 11 D, E) was significantly higher in GM of mdx mice in comparison to WT, and interestingly, it was further increased in Nrf2tKOmdx animals in comparison to mdx (Fig. 11 D, E). However, this was not observed in the diaphragm (Fig. 11 F, G). Furthermore, the same pattern was noticed in the case of inflammatory infiltration evaluated through H&E staining – higher inflammatory extent in Nrf2tKOmdx animals in comparison to mdx in GM (Fig. 11 H, I), but not in the diaphragm (Fig. 11 J, K).
As muscle fibrosis might be enhanced by chronic treadmill exercises (36), we have checked if it is altered by an additional lack of Nrf2. Collagen deposition analysis based on Masson’s trichrome staining revealed increased collagen content in mdx mice, however, it was not further altered by the transcriptional deficiency of Nrf2, neither in GM (Fig. 12 A, B) nor the diaphragm (Fig. 12 C, D). Moreover, the plasma level of osteopontin, a biomarker of DMD associated with fibrosis (37), was comparable between mdx and Nrf2tKOmdx mice (Fig. S1 A).
Inflammation and fibrosis are not affected in 24-week-old dystrophic mice by the lack of transcriptional activity of Nrf2
To assess the role of Nrf2 transcriptional deficiency on inflammation and fibrosis processes in old mice, we have performed H&E and Masson’s trichrome staining on GM and diaphragm of 24-week-old animals. We have observed increased inflammatory infiltration based on H&E staining in both gastrocnemius (Fig. 13 A, B) and diaphragm (Fig. 13 C, D) muscles in mdx mice in comparison to their healthy counterparts, however, it was not further changed by the lack of transcriptionally active Nrf2. Similarly, the same trend was observed in the case of fibrosis content, analysed based on Masson’s trichrome staining. Increased collagen deposition in GM (Fig. 13 E, F) and diaphragm (Fig. 13 G, H) of dystrophic mice was demonstrated, but it was not altered by the lack of Nrf2 transcriptional activity in both muscles.