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. 8 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 are no differences among genotypes in all time-points (Fig. 2 A). Accordingly, the number of eMyHC+ fibers was similar at the time of the peak of regeneration (7th day after injury) (Fig. 2 B, C).
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 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 test. As shown by us (10) and others (25) previously, and confirmed in the present study, mdx mice were able to run a shorter distance than WT. However, we did not see any effect of 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 and GM mass significantly increased in mdx mice in comparison to healthy animals, while in Nrf2tKOmdx no striking differences in body/GM mass compared with age-matched mdx mice were observed (Fig. 3 B, C).
Transcriptional knock-out of Nrf2 does not exacerbate 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 lacking additionally Nrf2 and mdx animals, indicating a comparable level of muscle injury. As suspected, serum LDH and CK levels of mdx mice were elevated compared with those of WT mice (Fig. 4. C, D).
Lack of Nrf2 transcriptional activity does not aggravate the inflammatory reaction in dystrophic skeletal muscles
Since Nrf2 has been reported to be a master regulator of antioxidative responses and contributes to the anti-inflammatory process (27,28), 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 (Fig. 5 A, B). Moreover, the expression of Hmox1, an anti-inflammatory factor shown by us to be up-regulated in dystrophic muscles (10), 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 C).
For a broader view of inflammatory status, we have performed a comprehensive analysis of different leukocyte populations within skeletal muscles of hind limbs of mice of 4 genotypes using flow cytometry. Mdx mice demonstrated an elevated proportion of monocytes defined as CD45+F4/80-CD11b+Ly6C+Ly6G- cells. However, no further changes in the infiltration of this population into skeletal muscle were caused by Nrf2 transcriptional deficiency (Fig. 5 D, E). Similarly, the percentage of macrophages (CD45+F4/80+CD11b+) was significantly higher in mdx mice in comparison to WT, however, it was not changed by the additional lack of Nrf2 transcriptional activity (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+MHCIIlowCD206hi) macrophages were much more abundant in dystrophic mice in comparison to WT but the lack of transcriptionally active Nrf2 did not change further their percentage (Fig. 5 H, I, J).
Next, we have studied the population of NK cells and various populations of lymphocytes. The percentage of NK cells (CD45+SSClowCD3-NK1.1+) (Fig. 6 A, C), lymphocytes T (CD45+SSClowCD3+) (Fig. 6 B, C), T helper (Th; CD45+SSClowCD3+CD8-CD4+) (Fig. 6 D, F), and T cytotoxic (Tc; CD45+SSClowCD3+CD8+ CD4-) (Fig. 6 E, F) were the same among four genotypes. Interestingly, despite the lack of differences in the percentage of T regulatory (Treg; CD45+SSClowCD3+CD8-CD4+FoxP3+CD25+) cells between dystrophic mice and their healthy counterparts, their level was elevated in Nrf2tKOmdx compared to mdx (Fig. 6 G, H).
The role of Nrf2 transcriptional deficiency on muscle fibrosis
We have found that mRNAs encoding 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 dystrophic mice in comparison to WT animals. However, it was not further exacerbated in mdx mice lacking transcriptionally active Nrf2 (Fig. 7 C, D).
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 (29). Moreover, Nrf2 was shown to regulate neovascularization and exert a pivotal role in angiogenic signal transduction and angiogenic potential of endothelial cells and bone marrow-derived proangiogenic cells (30). 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 in 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 gastrocnemius muscle, however, the lack of transcriptionally active Nrf2 did affect this production neither in healthy and dystrophic mice (Fig. 8 B). A similar trend of changes was found when the expression of Kdr, receptor for VEGF were evaluated (Fig. 8 C).
Nrf2 transcriptional deficiency does not affect the number and proliferation of muscle SCs but it may influence muscle regeneration
To elucidate whether Nrf2 affects SCs functions, we analysed the number and proliferation of SCs isolated from skeletal muscles of four genotypes: WT, Nrf2tKO, mdx, Nrf2tKOmdx. Flow cytometry analysis of SCs (CD45-CD31-Sca1-α7integrin+) percentage among nucleated cells revealed a considerable decrease in dystrophin-deficient mice in comparison to WT counterparts, but it was not further changed by Nrf2 transcriptional deficiency (Fig. 9 A, B). Accordingly, the percentage of quiescent SCs (CD45-CD31-Sca1-α7integrin+CD34+) was decreased in mdx and Nrf2tKOmdx, but it was not additionally affected by the lack of transcriptionally active Nrf2 (Fig. 9 C). Whereas, the percentage of activated SCs (CD45-CD31-Sca1-α7integrin+CD34-) did not differ between four genotypes (Fig. 9 D).
Moreover, we have checked the proliferation of CD34+ and CD34- SCs by cytofluorimetric analysis of cells in S+G2M phase. We have noticed a significant enhancement in the proliferation of both, SCs CD34+ and CD34- in dystrophic muscles compared to normal mice, but additional influence of Nrf2 transcriptional deficiency was not observed (Fig. 9 E, F).
Importantly, the percentage of SCs analysed among all nucleated cells by flow cytometry can be misleading due to the excessive inflammation in the muscles of mdx animals. Therefore, we have additionally analysed SCs number based on IHF staining by counting the ratio of Pax7-positive nuclei to myofibers. According to performed staining, the number of Pax7+ cells was increased in mdx mice in comparison to healthy ones (Fig. 9 G, H). However, none of the methods showed the additional effect of the lack of transcriptionally active Nrf2 on SCs number.
Although there is no effect of 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. 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 (31). Expression of miR-206 was elevated in mdx mice in comparison to age-matched WT animals whereas miR-1 and miR-133a/b showed the opposite pattern. However, none of them were affected by Nrf2 transcriptional deficiency (Fig. 10 C-E).
Finally, the number of myofibers expressing the marker of regeneration – eMyHC was diminished in mdx mice lacking additionally 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).