The role of Nrf2 in acute and chronic muscle injury

The nuclear factor erythroid 2-related factor 2 (Nrf2) is considered as a master cytoprotective factor regulating the expression of genes encoding anti-oxidant, anti-inflammatory, and detoxifying proteins. The role of Nrf2 in the pathophysiology of skeletal muscles has been evaluated in different experimental models, however, due to inconsistent data, we aimed to investigate how Nrf2 transcriptional deficiency (Nrf2tKO) affects muscle functions both in an acute and chronic injury. The acute muscle damage was induced in mice of two genotypes—WT and Nrf2tKO mice by cardiotoxin (CTX) injection. To investigate the role of Nrf2 in chronic muscle pathology, mdx mice that share genetic, biochemical, and histopathological features with Duchenne muscular dystrophy (DMD) were crossed with mice lacking transcriptionally active Nrf2 and double knockouts (mdx/Nrf2tKO) were generated. To worsen the dystrophic phenotype, the analysis of disease pathology was also performed in aggravated conditions, by applying a long-term treadmill test. We have observed slightly increased muscle damage in Nrf2tKO mice after CTX injection. Nevertheless, transcriptional ablation of Nrf2 in mdx mice did not significantly aggravate the most deleterious, pathological hallmarks of DMD related to degeneration, inflammation, fibrotic scar formation, angiogenesis, and the number and proliferation of satellite cells in non-exercised conditions. On the other hand, upon chronic exercises, the degeneration and inflammatory infiltration of the gastrocnemius muscle, but not the diaphragm, turned to be increased in Nrf2tKOmdx in comparison to mdx mice. In conclusion, the lack of transcriptionally active Nrf2 influences moderately muscle pathology in acute CTX-induced muscle injury and chronic DMD mouse model, without affecting muscle functionality. Hence, in general, we demonstrated that the deficiency of Nrf2 transcriptional activity has no profound impact on muscle pathology in various models of muscle injury.


Background
Duchenne muscular dystrophy (DMD) is the most common form of muscular dystrophies [1], which affects one in 5000-6000 male births [2]. DMD is the lethal Xchromosome linked recessive genetic neuromuscular disorder, caused by mutations in the gene encoding dystrophin [1]. Dystrophin deficiency leads to progressive muscle weakness, severe muscular atrophy, cardiomyopathy, and respiratory impairments, the two latter being the leading causes of mortality among patients with DMD [2]. Dystrophin, a cytoskeletal protein, is a major structural element of the dystrophin-glycoprotein complex (DGC), which is responsible for maintaining cellular integrity by linking the sarcolemmal and actin cytoskeleton to the extracellular matrix component laminin [3]. The loss of dystrophin disrupts the complex resulting in sarcolemmal instability that makes cells more susceptible to damage and leads to necrosis of muscle fibers [3]. Consequently, it results in the activation of the innate immune system, excessive inflammatory response, and increased oxidative stress [4].
In the early stage of the inflammatory response, muscles are infiltrated by neutrophils and pro-inflammatory, phagocytic M1-like macrophages which are a rich source of Th1 cytokines, that promote the activation and chemotaxis of myeloid cells to damaged tissue. Moreover, cytokines affect proliferation, migration, and differentiation of muscle satellite cells (SCs), progenitors of mature skeletal muscle. Subsequently, the recruitment of anti-inflammatory and pro-regenerative subpopulation of M2-like macrophages is observed [5]. In addition to macrophages and neutrophils, other inflammatory cells, including T-lymphocytes (cytotoxic, helper) may contribute to disease progression [6].
Subsequently, the injury leads to muscle regeneration, a process that depends on activation and proliferation of SCs and their differentiation into myotubes and later on regenerating myofibres that are centrally nucleated and exhibit expression of embryonic myosin heavy chain (eMyHC) isoform. Eventually, in chronic injury, the continuous cycles of myofiber degeneration and regeneration induce exhaustion of SCs and substitution of muscle with fibroadipose tissue [4]. Additionally, oxidative stress with elevated production of reactive oxygen species (ROS) has been proposed as important contributors in the pathogenesis of the DMD in humans [7] and mdx mice (murine model of DMD) [8].
Recently, we have shown that expression of heme oxygenase-1 (HO-1, encoded by Hmox1), antiinflammatory, and cytoprotective enzyme, is strongly elevated in muscles of mdx mice and muscle biopsies of DMD patients [9]. Genetic loss of HO-1 not only exacerbates dystrophic phenotype and inflammation in mdx mice [9] but also aggravates skeletal muscle injury in acute muscle damage model, i.e., following cardiotoxin (CTX) injection [10]. Expression of Hmox1 is regulated, among others, by the redox-sensitive nuclear factor erythroid 2-related factor 2 (Nrf2, encoded by Nfe2l2 gene) belonging to Cap "n" collar (Cnc)-bZIP (basic leucine zipper) family of transcription factors [11]. Nrf2 plays a cytoprotective role as a master regulator of genes encoding oxidative stress response and phase II detoxifying proteins by interacting with the anti-oxidant response element (ARE) sequence. Under normal circumstances, Nrf2 is sequestered in the cytoplasm by Kelch-like ECH-associating protein 1 (Keap1) through the Nrf2-Keap1 complex, which suppresses Nrf2 activity by targeting it for ubiquitination and degradation. In stressful conditions, Nrf2 dissociates from Keap1, translocates into the nucleus, and induces the expression of target genes [12]. The role of Nrf2 in skeletal muscle aging and adaptations to exercise through the regulation of mitochondrial function, maintaining the cellular redox balance, control of oxidative stress, influencing apoptotic signaling, and providing proper contractile properties has been demonstrated [13,14]. Moreover, although the involvement of Nrf2 in DMD progression has been suggested [15][16][17][18], the possible protective mechanisms were not fully discovered. Therefore, we aimed to evaluate the impact of Nrf2 transcriptional deficiency on the acute muscle damage caused by CTX injection and chronic injury using a murine model of DMD-mdx mice.

Animal models
All animal procedures and experiments were performed in accordance with national and European legislation, after approval by the 1st and 2nd Institutional Animal Care and Use Committee (IACUC) in Kraków, Poland (approval numbers: 66/2013, 199/2018, 148/2020). Mice were kept in specific-pathogen-free (SPF) conditions with water and food available ad libitum under controlled temperature and humidity and 14 h/10 h (light/ dark) cycles.
In the experiments, if not stated otherwise, 10-12week-old male littermates or age-matched mice from generation F2 to F5 were used. Accordingly, the double knockout animals lacking both dystrophin and Nrf2 expression (Nrf2 tKO Dmd mdx/Y ) were compared to their mdx littermates (Nrf2 +/+ Dmd mdx/Y ). Additionally, mdx mice were analyzed vs. WT (Nrf2 +/+ Dmd +/Y ) mice and the comparison of Nrf2 tKO (Nrf2 tKO Dmd +/Y ) mice vs. WT mice was studied as well.
The generation of double knockouts was hence done accordingly to other studies in which mdx mice were crossed with relevant knockouts [9,[22][23][24][25]. Genotyping of animals was performed by PCR on the DNA isolated from the tails.

CTX-induced injury
Male and female C57BL/6 J (WT and Nrf2 tKO ) mice at 8-15 weeks of age were used for the myoinjury experiment. Hind limbs of mice were shaved and gastrocnemius muscles (GM) were injected with 25 μl of 20 μmol/ l cardiotoxin from Mozambique spitting cobra (Naja mossambica mossambica; Sigma-Aldrich), while control mice were injected with saline. Animals were provided with analgesia (50 μl, 0.03 mg/ml buprenorphine) after injection and in the next 2 days. Mice were euthanized on the 1st, 3rd, 7th, 14th, and 28th day after CTX injury. Subsequently, plasma was taken and GM were harvested for further analyses.

Treadmill test
To assess muscle functionality and performance, the treadmill test was performed as described previously [9,24]. Additionally, to worsen the dystrophic phenotype, mice were subjected to chronic treadmill exercises. The training protocol started at the age of 4 weeks. After 3 days of acclimatization, mice underwent a run on a horizontal treadmill at 12 m/min for 30 min twice a week for 4 weeks, according to published protocols [26,27]. Experiments were performed using the Exer-3/6 treadmill (Columbus Instruments).

Forelimb grip strength test
Forelimb grip strength was assessed using a grip strength meter (GSM) with a triangular pull bar (Ugo Basile) according to the published protocol with modifications [28]. Briefly, the animals were gently held by the tail allowing them to grasp the grid using forelimbs. Afterward, mice were moved horizontally toward the bar and pulled back until the grip was released. The measurements were repeated 5 times with a 1-min break in between. The results were calculated as an average from 5 measurements, normalized to body weight, and expressed as N/kg BW.

Plasma creatine kinase and lactate dehydrogenase measurement
Plasma was obtained by blood collection from vena cava to heparin-coated tubes followed by centrifugation at 1000×g for 10 min at 4°C just before the terminal procedure and collection of GM. The activity of CK and LDH were measured using diagnostic Liquick Cor-CK and Liquick Cor-LDH kit, respectively (P.Z. CORMAY) following the manufacturer's instruction using plasma diluted 10 times for measurements.

Histological analysis
GM were dissected, immediately fixed in 10% formalin, dehydrated, embedded in paraffin, and cut into 4 μm sections. Subsequently, sections were deparaffinized, rehydrated, and subjected to histological stainings. Hematoxylin and eosin staining (H&E, Sigma-Aldrich) was performed to visualize inflammation and regenerating myofibers according to standard protocols. For Masson's trichrome staining assessing collagen content (fibrosis evaluation), sections were fixed overnight in Bouin's solution and sequentially treated with Biebrich scarlet-acid fuchsin, phosphotungstic acid/phosphomolybdic acid, and aniline blue (Sigma-Aldrich, according to the vendor's instructions). Inflammation, muscle damage, fibrosis, and regeneration scoring was done by a blinded experimenter using the following description of arbitrary scale: 0-no signs of inflammation/collagen deposition/ regenerating fibers 1-any sign of inflammation, myofibre swelling/ collagen deposition/regenerating fibers 2-signs of inflammation, myofibre swelling, and rhabdomyolysis/collagen deposition/regenerating fibers are well visible and significant but occupy less than half of the field of view 3-signs of inflammation, myofibre swelling, and rhabdomyolysis/collagen deposition/regenerating fibers take up more than half of the field of view 4-signs of inflammation, myofibre swelling, and rhabdomyolysis/collagen deposition/regenerating fibers take up all field of view Immunohistofluorescent stainings GM were harvested and snap-frozen in optimal cutting temperature compound (OCT, Leica) in liquid nitrogenchilled isopentane and stored at −80°C until processed. Frozen tissues were cryosectioned (10 μm) using a cryostat (Leica) and placed on glass slides coated previously with poly-L-lysine (Sigma-Aldrich).
Gastrocnemius muscle cross-sectional area (CSA) and the mean fiber area were determined by semi-automatic muscle analysis using segmentation of histology (SMAS H) [29] based on immunofluorescent staining of laminin described above. Only the injured area of the muscle was examined by the experimenter blinded to the mouse genotype.

Gene expression analysis by quantitative real-time PCR
Harvested skeletal muscle tissues were stored in RNAlater RNA Stabilization Solution (Invitrogen) at −80°C until processed. GM from 12-week-old mice were used to isolate RNA by homogenization in 1 ml of Qiazol Total RNA Isolation Reagent (Qiagen) using TissueLyser (Qiagen), following the manufacturer's instructions. The concentration and quality of RNA were determined spectrophotometrically (NanoDrop, Thermo Fisher Scientific). To synthesize cDNA, the reverse transcription reaction was performed on 1 μg RNA using RevertAid reverse transcriptase (Thermo Fisher Scientific) or Mys-tiCq® microRNA cDNA Synthesis Mix (Sigma-Aldrich). qPCR was performed with Applied Biosystems TM Ste-pOnePlus Real-Time PCR (Thermo Fisher Scientific) in a mixture containing cDNA, SYBR Green PCR Master Mix (SYBR Green qPCR Kit, Sigma-Aldrich), forward and reverse primers recognizing murine genes (Table 1), and muscle-specific murine microRNAs (Table 2). A universal reverse primer for miRNAs qPCR was supplied by a vendor. The relative quantification of gene expression was quantified based on the comparative Ct (threshold cycle value) method. Gene expression levels were calculated by normalizing to the level of housekeeping gene elongation factor 2 (Eef2) or constitutive small nuclear RNA U6 in the case of microRNA.

Analysis of mononucleated cell populations in skeletal muscles by flow cytometry
Samples for flow cytometry were prepared as described previously [9,10]. Briefly, mice were euthanized and Table 1 Sequences of primers used for qPCR analysis

Gene name
Forward primer Reverse primer perfused immediately with saline containing 0.5 U/ml heparin through the left ventricle. Then, hind limbs muscles were excised, weighted, minced, and digested at 37°C for 45 min in a solution containing collagenase IV (5 mg/ ml; Gibco; Invitrogen) and dispase (1.2 U/ml; Gibco; Invitrogen). Digested muscles were passed through 100 μm cell strainer, washed with PBS, pelleted after centrifugation, and resuspended in PBS + 2% fetal bovine serum. Subsequently, the number of cells per milliliter was calculated using the Bürker chamber. Samples were stained with the following antibodies: rat anti-mouse CD45-APC-eFluor780 (

Protein analysis
To assess monocyte chemoattractant protein 1 (MCP-1) and vascular endothelial growth factor A (VEGF) protein level, GM lysates were subjected to Luminex TM measurement according to the manufacturer's instructions (Life Technologies) whereas osteopontin concentration in plasma was determined by ELISA following the vendor's protocol (R&D System). The results from MCP-1 and VEGF measurement were calculated as pg/mg of total protein and the level of osteopontin was shown in ng/ml.

Statistics
Data are presented as mean ± SEM and analyzed with the unpaired two-tailed Student's t test to determine differences between two groups or one-way ANOVA followed by Tukey's post hoc test for multiple groups. p ≤ 0.05 was considered as significant. Grubb's test was used to identify significant outliers, GraphPad Prism for graphs and statistical analyses.

Lack of transcriptionally active Nrf2 enhances skeletal muscle damage after CTX-induced injury
To analyze 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. The level of muscle damage and inflammatory infiltration evaluated based on H&E staining (Fig. 1a, b) was significantly higher in Nrf2 tKO mice on the 3rd day after muscle damage. Although the activity of CK (Fig. 1c) was increased in Nrf2 tKO animals on day 1 after injection, a statistically significant difference between WT and Nrf2 tKO mice was not evident. On the other hand, the level of LDH (Fig. 1d) was significantly elevated in mice of both genotypes, and additionally, it was much higher in Nrf2 tKO 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. 1e) and mRNA level of Hmox1 (Fig. 1f), Il1b (Fig. 1g) and Il6 (Fig. 1h) 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. 1i, 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 Nrf2 tKO animals on the 7th day after injury, however, there were no differences among genotypes in all analyzed timepoints (Fig. 2a). Accordingly, the number of eMyHC + fibers (Fig. 2b, c), CSA  (Fig. 2d), and mean fiber area (Fig. 2e) 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 (Nrf2 tKO mdx). 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. 3a). However, we did not observe an effect of the transcriptional deficiency of Nrf2 on the running pattern. The exercise capacity of Nrf2 tKO animals was comparable to WT mice and Nrf2 tKO mdx mice run similar distance as mdx counterparts (Fig. 3a). Body weight (Fig. 3b) and GM mass (Fig. 3c) were significantly increased in mdx mice in comparison to healthy animals, while in Nrf2 tKO mdx, no striking differences compared with age-matched mdx mice were found.
Transcriptional knockout of Nrf2 does not exacerbate muscle degeneration in non-exercised 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. 4a, b) nor LDH (Fig. 4c) and CK (Fig. 4d) 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. 4c, d, respectively).
Lack of Nrf2 transcriptional activity does not aggravate the inflammatory reaction in non-exercised dystrophic skeletal muscles Since Nrf2 has been reported as a master regulator of anti-oxidative responses that contributes to the antiinflammatory 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. 5a, b) and diaphragm (Fig. 5c, d).
Moreover, the expression of Hmox1, an antiinflammatory factor shown by us to be upregulated in dystrophic muscles [9], was again potently elevated in GM of mdx mice. However, it was the same in Nrf2 tKO mdx mice, indicating that Nrf2 transcriptional activity is dispensable for Hmox1 induction in the muscles (Fig. 5e).
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 + MHCII hi CD206 low ) and M2-like (CD45 + F4/80 + CD11b + MHCII low CD206 hi ) 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. 5h, i, j).
The role of Nrf2 transcriptional deficiency on muscle fibrosis in non-exercised mdx mice 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. 7a, 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. 7c, d) and diaphragm (Fig. 7e, 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. 7g, h).
A decrease in the expression of angiogenic mediators in non-exercised 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 marrowderived 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. 8a). Concomitantly, the level of VEGF protein was potently downregulated in dystrophic GM; however, the lack of transcriptionally active Nrf2 did not affect this production in both healthy and dystrophic mice (Fig. 8b). A similar trend of changes was found when the expression of Kdr, a receptor for VEGF was evaluated (Fig. 8c).

Nrf2 transcriptional deficiency does not affect the number and proliferation of muscle SCs but it may influence muscle regeneration in non-exercised dystrophic animals
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. 9a, 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. 9c). 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 Nrf2 tKO mdx mice (Fig. 9d, 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. 10a, 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. 10c) was elevated in mdx mice in comparison to age-matched WT animals whereas miR-1 (Fig. 10d) and miR-133a/b (Fig. 10e) 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. 10f, g). However, histological analysis of centrally nucleated fibers did not show differences between mdx and Nrf2 tKO mdx animals (Fig. 10h, 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 analyzed 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. 11a). Moreover, the activity of LDH and CK in plasma did not differ in Nrf2 tKO mdx vs. mdx animals (Fig. 11b, c, respectively).
The number of necrotic fibers (Fig. 11d, e) was significantly higher in GM of mdx mice in comparison to WT, and interestingly, it was further increased in Nrf2 tKO mdx animals in comparison to mdx (Fig. 11d, e). However, this was not observed in the diaphragm (Fig. 11f, g). Furthermore, the same pattern was noticed in the case of inflammatory infiltration evaluated through H&E staining-higher inflammatory extent in Nrf2 tKO mdx animals in comparison to mdx in GM (Fig. 11h, i), but not in the diaphragm (Fig. 11j, 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. 12a, b) nor the diaphragm (Fig. 12c,  d). Moreover, the plasma level of osteopontin, a biomarker of DMD associated with fibrosis [37], was comparable between mdx and Nrf2 tKO mdx 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. 13a, b) and diaphragm (Fig. 13c, 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, and analyzed based on Masson's trichrome staining. Increased collagen deposition in GM (Fig. 13e, f) and diaphragm (Fig. 13g, h) of dystrophic mice was demonstrated, but it was not altered by the lack of Nrf2 transcriptional activity in both muscles.

Discussion
DMD is still an incurable disease with very limited treatment possibilities, including corticosteroids [38]. However, despite having a beneficial effect on muscle function, these drugs cause many side effects [39]. Therefore, identifying new targets for anti-inflammatory treatment may contribute to the development of novel therapeutic strategies. Taking into consideration the pleiotropic activity of Nrf2, which drives the expression of anti-inflammatory, anti-oxidant, and cytoprotective genes, we aimed to investigate its role in the acute muscle injury induced by CTX injection and in the progression of DMD using two different mouse modelsmdx and exacerbated dystrophic phenotype-mdx mice subjected to the long-term treadmill exercise. In our study, we found that the absence of transcriptionally active Nrf2 may be associated with increased muscle damage after acute muscle injury following CTX injection and under aggravated dystrophic conditions, however, it did not significantly affect the pathophysiological hallmarks of DMD progression.
Due to the disruption of the DGC complex and increased sarcolemmal permeability in DMD [40], serum proteins such as IgG and IgM, which are typically found only in circulation, are accumulated in damaged, necrotic myofibers [41]. We have shown that the number of such necrotic fibers does not differ between nonexercised mdx and Nrf2 tKO mdx mice. Although we have demonstrated higher levels of CK and LDH in the serum of non-exercised mdx mice in comparison to healthy counterparts, their levels were not further elevated in We have also performed such analysis after a single CTX injury, a model where the kinetics of degeneration and inflammation processes are more stable than during DMD development. Although a similar experimental model was previously utilized by Shelar et al. [42], the degeneration has not been checked. In our hand, Nrf2 transcriptional deficiency was associated with the aggravation of LDH activity in plasma on 1st day after injury and enhanced muscle damage on 3rd day following CTX injury. Hence, it might be hypothesized that at the stage of massive macrophages infiltration after CTX injury occurring just on day 3 [43], the lack of Nrf2 manifests more profoundly, impairing the proper inflammatory response. However, this hypothesis would require further verification.
Inflammation is one of the most prominent features of dystrophic muscles. In our studies, contrary to the expectations, inflammation was not exacerbated in dystrophic muscles lacking transcriptionally active Nrf2 both when total inflammatory extent was evaluated on histological sections and by detailed analysis of immune   subtypes by flow cytometry. Of note, despite the fact T h and T c lymphocytes contribute to the pathogenesis of DMD [6] and their functions may be regulated by Nrf2 [44], their numbers were changed neither by dystrophin nor Nrf2 deficiency in non-exercised animals. An interesting finding of our study is related to the NK cells' abundance-their number was significantly decreased in Nrf2 tKO mdx mice in comparison to mdx, in which they are elevated in the muscles [45]. Hence, our observations are consistent with previous reports showing an increased percentage of NK cells following activation of Nrf2 [46]. Petrillo et al. demonstrated a higher expression of Nrf2 and its target, HO-1, in muscle biopsies from DMD patients in comparison to healthy individuals [15]. In our model, we observed an elevated level of HO-1 in the CTX-injured muscles on day 1 after injection, as well as in the dystrophic, non-exercised mice, consistent with our previous results [9,10]. However, the effect of Nrf2 deficiency on the level of this important inflammatory mediator has been noticed neither in Nrf2 tKO following CTX injury nor in non-exercised Nrf2 tKO mdx indicating that HO-1 may be regulated independently of Nrf2. Indeed, such an Nrf2-independent regulation of HO-1, relying on, e.g., Foxo1 transcription factor, has been demonstrated in the muscles and SCs [9,47].
Concomitantly, we have revealed that Nrf2 plays a dispensable role in endomysial fibrosis in dystrophic muscles, the process of accumulation of connective tissue, which is the characteristic attribute of DMD pathology [4], is associated with poor outcome [48] and linked to the increased expression of TGF-β1 and collagen [49][50][51]. We have observed elevated mRNA level of Tgfb1 and Col1a1 not only in non-exercised mdx animals but even at a higher degree in dystrophic mice additionally lacking transcriptionally active Nrf2, what is consistent with studies showing that Nrf2 acts as a protective agent against fibrosis [52][53][54]. However, although the lack of transcriptional activity of Nrf2 in dystrophic mice affected mRNA of fibrotic genes, it was not reflected in the collagen deposition in muscle's histological sections. Moreover, the number of FAPs remained comparable between those two groups of animals. The observed differences may be caused by the fact that the mRNA level points to a particular time-point, whereas collagen deposition showed changes arising during disease progression.
Additionally, to fully understand the potential role of Nrf2 in DMD progression, we have studied the inflammation and fibrosis in older-24-week-old animals. Accordingly, differences between non-exercised mdx and Nrf2 tKO mdx were observed neither in GM nor in the diaphragm. Importantly, our data are in line with the work by Takemoto et al. [55] what might indicate that older mdx mice are not a good model to study DMD progression due to disease stabilization at later stages [56][57][58]. Therefore, we have also looked at the role of Nrf2 in exacerbated dystrophic phenotype-mice subjected to long-term treadmill exercises. Interestingly, we have observed an increased number of necrotic fibers in GM of dystrophic mice in comparison to the diaphragm what is in line with the previous findings [59]. However, again, we did not find any changes in dystrophic muscle functionality and other pathological aspects of DMD driven by the lack of Nrf2 transcriptional activity except for the higher level of degeneration and inflammation in GM of Nrf2 tKO mdx mice, but not in the diaphragm. The latter could be explained by the applied experimental protocol of chronic exercises with relatively low speed and maintaining horizontal orientation, which possibly did not influence the diaphragm, contrary to the application of more acute protocols [60].
Effective muscle regeneration is achieved by SCs [61]. In DMD, repeated cycles of muscle damage and regeneration disturbed the balance between self-renewal and differentiation, leading to premature depletion of the SCs pool [62]. We have shown that the absolute number of Pax7-positive SCs, as well as MyoD-positive SCs, was notably increased in dystrophic animals as in the previous studies performed on mdx mice [63]. Nevertheless, regardless of the method used, the number, as well as the proliferation of dystrophic SCs, was not additionally altered by Nrf2 transcriptional deficiency. It is well established that muscle regeneration is controlled by myogenic regulatory factors (MRFs) such as MyoD and myogenin. Their elevated level was observed by us in mdx mice and was further upregulated by additional Nrf2 transcriptional deficiency, the mechanism of which is currently investigated. Newly formed myofibers are characterized by the expression of unique myosin isoforms such as eMyHC. The number of eMyHC-positive myofibers was lower in mdx mice additionally lacking transcriptional activity of Nrf2 in comparison to mdx. However, because there are many repeated cycles of muscle damage and regeneration in DMD, it is difficult to conclude how Nrf2 affects this process based on transiently expressed eMyHC [64]. Therefore, we also checked muscle regeneration after acute muscle injury induced by CTX injection, a method that allows studying muscle regeneration in more controlled and reproducible conditions [65]. In the model of CTX-induced muscle injury, no effect of the lack of Nrf2 transcriptional deficiency was observed. This finding is consistent with previous studies where a similar level of regeneration in transcriptionally deficient Nrf2 and control mice was demonstrated [55]. Contrary, Shelar et al. presented delayed regeneration of tibialis anterior muscle in Nrf2-deficient mice [42], however, a few experimental differences (e.g., the age of animals and divergent muscles injected with various doses and types of CTX) may potentially explain discrepancies in the results obtained in both studies.
Overall, our results did not show the prominent effects of Nrf2 deficiency both in acute and in chronic muscle injury. However, it is still possible that beneficial effects could be observed through Nrf2 induction or overexpression. Sun et al. have shown that dystrophic phenotype may be markedly alleviated in mdx mice by treatment with sulforaphane (SFN) [16,18], an isothiocyanate that activates Nrf2 by modifying Keap1 cysteines [66]. Moreover, several studies have outlined that some features of DMD might be improved by other antioxidant compounds like resveratrol [67] or curcumin [68], but their effect was not as strong as SFN [16,18]. However, it is important to remember that such compounds might work not solely through activation of Nrf2, but they may also involve other mechanisms [69][70][71], and in the above studies the involvement of the Nrf2 pathway was not investigated in details.

Conclusion
Lack of transcriptionally active Nrf2 is associated with slightly increased muscle damage after acute muscle injury caused by CTX injection as well as degeneration and inflammatory infiltration of the gastrocnemius muscle in dystrophic mice upon chronic treadmill exercises. Nevertheless, Nrf2 ablation does not significantly aggravate the most deleterious pathological events such as degeneration, inflammation, angiogenesis, and fibrotic scar formation as well as the number and proliferation of SCs in the non-exercised mdx mice model. Therefore, we conclude that the deficiency of Nrf2 transcriptional activity has no profound impact on muscle pathology in various models of muscle injury.