Simvastatin Inuences Selected Aspects of Duchenne Muscular Dystrophy Pathology in a Mouse Model

Background: Duchenne muscular dystrophy (DMD) is an incurable disease, caused by the mutations in the DMD gene, encoding dystrophin, an actin-binding cytoskeletal protein. Lack of functional dystrophin results in muscle weakness, degeneration, and as an outcome cardiac and respiratory failure. As there is still no cure for affected individuals, the pharmacological compounds with the potential to treat or at least attenuate the symptoms of the disease are under constant evaluation. The pleiotropic agents, 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors, known as statins, have been suggested to exert benecial effects in the mouse model of DMD. On the other hand, they were also reported to induce skeletal-muscle myopathy. Methods: Several methods including functional assessment of muscle function via grip strength measurement and treadmill test, enzymatic assays, histological analysis of muscle damage, gene expression evaluation, and immunouorescence staining were conducted to study simvastatin-related alterations in mdx mice. Results: In our study, simvastatin treatment of mdx mice did not result in improved running performance; however, some benecial effect was observed when grip strength was evaluated. Creatine kinase and lactate dehydrogenase activity, markers of muscle injury, were diminished after simvastatin delivery in mdx mice. Nevertheless, no signicant changes in inammation, brosis, and necrosis were noted. Interestingly, simvastatin mRNA level embryonic myosin heavy chain isoform, a declined percentage of nucleated myobers, and miR-1 upregulation, an alteration in the muscle regeneration. some angiogenic results muscle but not in the diaphragm. Alexa Fluor 568 (for detection of CD31). Pictures of the whole tissue were taken and CD31/α-SMA positive vessels were analyzed quantitatively per muscle area. The results were presented as a number of vessels per area. Necrosis was assessed by the immunouorescent staining of the IgG/IgM/IgA (goat anti-mouse IgG, IgM, IgA Alexa Fluor 488 antibody, Thermo Fisher Scientic) with laminin α2 (rabbit anti-mouse antibody, Abcam, ab11576; secondary antibody: goat anti-rabbit Alexa Fluor 568) and showed as a percentage of necrotic bers in the stained muscle. Evaluation of the muscle cross-sectional area (CSA) and the mean ber area were determined by semi-automatic muscle analysis using segmentation of histology (SMASH) staining of laminin. for The expression of miR-1, and miR-206 (miRCURY LNA™ miRNA PCR Assays) was normalized to the constitutive SNORD68 gene (miRCURY LNA™ PCR Relative quantication of gene expression was calculated based on the comparative C t method (ΔC t = C t gene of interest – C t Eef2/SNORD68 ) and presented as the relative expression in comparison to vehicle-treated animals.

from DMD lose the ability to walk and ultimately die in the 2nd to 3rd decade of life, due to cardiac or respiratory failure (7,8).
Taking into account the diversity of the processes which may affect DMD progression and the constant need for the development of effective therapeutics, new factors are suggested to exhibit bene cial effects on this so far incurable disease. In our previous study, we have found that lack of heme oxygenase-1 (Hmox1, HO-1), a heme-degrading enzyme, exerting anti-oxidant and cytoprotective activities leads to a more severe disease state. Knock-out of Hmox1 resulted in aggravated in ammation and brosis as well as the impaired running capacity of dystrophic animals (9). Hmox1 expression may be modulated by the plethora of compounds, including 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors (10), commonly known as statins, discovered 40 years ago (11) and used as lipid-lowering drugs for the treatment of hypercholesterolemia and reduction of atherosclerosis. Interestingly, in 2015, Whitehead et al. for the rst time described the protective effect of statins in dystrophic mice (12). The authors showed that simvastatin improved muscle health, reduced in ammation, oxidative stress, and increased autophagy in mdx animals. A few years later, the same group suggested long-term improvement in heart functions in response to simvastatin treatment (13). However, other studies performed in animal models did not con rm such favorable properties. Verhaart et al. found no positive outcome of simvastatin treatment on muscle function, histology, or expression of genes involved in several DMD-related processes such as in ammation, brosis, and oxidative stress (14). Moreover, also Finkler et al. did not demonstrate the salutary, anti-dystrophic impact of a different statin, rosuvastatin (15).
What is relevant, even the devastating role of statins in muscle biology has been reported. Several in vitro studies showed the toxic effects of those drugs on muscle cells. Importantly, concentrations of statins required to induce deleterious effects in vitro are far beyond the physiological range, being typically greater than 1 µM. Such concentrations are considerably (100-1000 times) higher than those found in vivo in humans (16). For instance, 24 h stimulation with 10 µM or 50 µM simvastatin, atorvastatin, or rosuvastatin exerted toxic effects on C2C12 myoblast cell line (17) whereas even 100 µM cerivastatin, uvastatin, and atorvastatin were used to induce cell death and mitochondrial toxicity in L6 rat skeletal muscle cell line (18).
There are also discrepant data about the incidence of different kinds of myopathy in humans after statin therapy. Previous studies indicated a high risk of such adverse effects, e.g. showing that > 10% of statin users in the general population can be affected (19,20). Noteworthy, a recent systematic review of clinical trials found adverse muscle symptoms only in < 1% compared with placebo controls (21). Statinrelated muscle symptoms also appear to be exacerbated by several factors, including exercise (22), older age, and female sex (23). Nevertheless, published a few years ago meta-analysis by Iwere and Hewitt clearly showed, that even in aged patients (65 + years), the risk of statin-induced myopathy was comparable to placebo patients (24) which was also con rmed recently by Zhou et al. (25). Moreover, in September 2020, the observational analysis provided data from three large trials on 58 390 peoples treated with simvastatin for a mean of 3.4 years (HPS, SEARCH, and HPS2-THRIVE) reporting the extremely low risk of simvastatin-induced myopathy with speci c criteria for identi cation of the individuals at high and at lower risk (26). These data implicate that the fear of statin-caused myopathy might be in many cases overestimated. Notably, the above-mentioned risk factors for statin-induced myopathy are not relevant to boys with DMD.
Based on the published, contradictory results in the eld of muscular dystrophy (5,9) and our previous expertise in terms of the role of statins, including the angiogenesis process (27,28), we aimed at the evaluation of the effect of simvastatin in mdx animals as well as tried to identify the additional molecular mechanisms responsible for the potential improvement in muscle health and function in DMD. We have found that simvastatin can ameliorate selected aspects of DMD pathology such as the elevated creatine kinase (CK) and lactate dehydrogenase (LDH) levels; however, it does not affect the important processes contributing to dystrophy progression like brosis, in ammation, or angiogenesis. Moreover, the simvastatin effect was, at least in our hands, muscle-speci c. Therefore, we may conclude, that simvastatin treatment could be tested as a potential supportive therapy for DMD; however, it requires further, thorough investigation.

Animals
Animal experiments were conducted in accordance with national and European legislation, after approval by the 2nd Institutional Animal Care and Use Committee (IACUC) in Kraków, Poland (approval number: 323/2018). Mdx mice C57BL/10ScSn-Dmd mdx /J and control mice C57BL/10ScSnJ (WT) were purchased from the Jackson Laboratory. Mice were bred on a mixed C57BL/10ScSn and C57BL/6 × FVB background as described by us previously (9) and were housed in speci c pathogen-free (SPF) conditions with water and food available ad libitum. Genotyping of animals was performed using PCR on the DNA isolated from the tails. Only males were used for the experiments. The rst dose of simvastatin was given to the 6-week-old male littermates or age-matched mice from generation F2 to F5 and the administration was continued for 28 days until the age of 10 weeks.

Simvastatin treatment
An activation procedure was based on the published protocol (29). Brie y, 4 mg of simvastatin (Sigma-Aldrich) was dissolved in 200 µl of ethanol. Then 300 µl of 0.1 N NaOH was added to the solution and subsequently incubated at 50 °C for 2 h. The pH was brought to 7.2 by HCl, and the concentration of the stock solution was adjusted to 2 mg/ml. The stock solution was kept at 4 °C. For 28 days both WT and mdx mice received 10 mg/kg body weight (BW)/day via oral gavage. Mice of both genotypes were randomly separated into the vehicle (solvent)-and simvastatin-treated groups. The administered dose was chosen based on the literature data (12).

Grip strength assay
Forelimb grip strength was assessed one day before the rst dose, at day 15, and one day after the last administration, using a grip strength meter with a triangular pull bar (Ugo Basile) as described earlier (30,31). The measurements were repeated 3 times with a 1 min break in between. The results were calculated as an average from 3 measurements, normalized to BW, and expressed as N/kg BW.

Treadmill test
The treadmill exhaustion test was performed after the last dose of simvastatin using the Exer-3/6 (Columbus Instruments) at 15 degrees downhill by the investigator blind to the mice genotype. We employed the protocol described previously (9) with modi cation. Brie y, after 3 daily acclimation sessions of 15 min at 8 m/min and one day at 20 m/min, 10-week-old male mice were subjected to an exhaustion treadmill test. Mice were warmed up at 5 m/min for 5 min before the test. For the test, mice ran on the treadmill at 5 m/min for 2 min, 7 m/min for 2 min, 8 m/min for 2 min, 10 m/min for 5 min, and 12 m/min for 15 min. Afterward, speed was increased by 1 m/min to a nal speed of 20 m/min. Exhaustion was de ned by the inability of the animal to remain on the treadmill despite stimulation by gentle touching.

Blood cell count
The blood was collected directly from the vena cava to the EDTA-coated tubes and analyzed using scil Vet abc (Horiba). The total number of white blood cells (WBC) and the percentage of granulocytes, monocytes, and lymphocytes among WBC, was calculated in 10-week-old mice treated with vehicle and simvastatin.
Analyses were conducted according to our previous studies (4,9,32) after taking pictures of the whole tissues. The assessment of in ammation and brosis extent was conducted using arbitrary units, respectively: 0 -no signs of in ammation/collagen deposition; 1 -any sign of leukocyte in ltration and myo ber swelling/collagen deposition; 2 -visible in ammation, myo ber swelling, and rhabdomyolysis/collagen deposition; 3 -signs of in ammation, myo ber swelling, and rhabdomyolysis which take around half of a eld of view/collagen deposition takes up around half of the eld of view; 4a substantial part of the muscle in the eld of view is in ltrated and degenerated/collagen deposition takes the substantial part of the eld of view. The analysis of CNF indicating the level of regeneration was performed based on H&E staining; 10-15 pictures/tissue were randomly taken and the percentage of CNF among all bers was calculated.
Immuno uorescent staining of CD31/α-SMA positive vessels was performed as described by us previously with slight modi cations (33). Primary antibodies: rabbit anti-human α-SMA (Abcam, ab5694) and rat anti-mouse CD31 (BD Pharmingen, 550274) were used followed by the incubation with secondary antibodies: goat anti-rat Alexa Fluor 488 (for detection of α-SMA) and goat anti-rabbit Alexa Fluor 568 (for detection of CD31). Pictures of the whole tissue were taken and CD31/α-SMA positive vessels were analyzed quantitatively per muscle area. The results were presented as a number of vessels per area. Necrosis was assessed by the immuno uorescent staining of the IgG/IgM/IgA (goat anti-mouse IgG, IgM, IgA Alexa Fluor 488 antibody, Thermo Fisher Scienti c) with laminin α2 (rabbit anti-mouse antibody, Abcam, ab11576; secondary antibody: goat anti-rabbit Alexa Fluor 568) and showed as a percentage of necrotic bers in the stained muscle. Evaluation of the muscle cross-sectional area (CSA) and the mean ber area were determined by semi-automatic muscle analysis using segmentation of histology (SMASH) (34) based on immuno uorescent staining of laminin.
The stainings were visualized under Nikon Eclipse Ti uorescent microscope. All histological assessments were analyzed by the investigator blind to the mice group using ImageJ software. If necessary, the brightness and/or contrast were adjusted to all of the pictures equally.

Determination of serum CK and LDH concentrations
To estimate the activity of CK and LDH diagnostic Liquick Cor-CK and Liquick Cor-LDH kits were used, respectively, according to the manufacturer protocols (Cormay). Blood was collected from vena cava and was allowed to clot at room temperature for 30 min and then centrifuged at 4 °C for 10 min at 2000 g. The assay was performed using a clear serum, without the signs of hemolysis. The absorbance values were then converted to CK and LDH (U/l). RNA isolation, reverse transcription (RT), and quantitative real-time PCR (qRT-PCR) Collected muscles were protected in RNAlater (Sigma-Aldrich), snap-frozen in liquid nitrogen, and stored at -80 °C for downstream analyses. RNA was isolated as in our previous study (9) using the Chomczynski-Sacchi method (35). Its quality and concentration were determined by NanoDrop Spectrophotometer (Thermo Fisher Scienti c). qRT-PCR was performed as described previously (9) using StepOne Plus Real-Time PCR (Applied Biosystems -Thermo Fisher Scienti c) and SYBR Green PCR Master Mix (Sigma-Aldrich), speci c primers (listed in Table 1), and cDNA obtained in the RT reaction with recombinant M-MuLV reverse transcriptase (Thermo Fisher Scienti c). Eef2 was used as a housekeeping gene. LNA miRCURY RT-PCR Kit and miRCURY LNA SYBR PCR Kit (Qiagen, Hilden, Germany) were applied for miRNAs determination. The expression of miR-1, miR-133a, and miR-206 (miRCURY LNA™ miRNA PCR Assays) was normalized to the constitutive SNORD68 gene (miRCURY LNA™ miRNA PCR Assay). Relative quanti cation of gene expression was calculated based on the comparative C t method (ΔC t = C t gene of interest -C t Eef2/SNORD68 ) and presented as the relative expression in comparison to vehicle-treated animals. The fragments of muscles were snap-frozen in liquid nitrogen, homogenized in 1% Triton X-100 in PBS using TissueLyser (QIAGEN), and centrifuged (7 000 g, 10 min, 4 °C). The protein lysates were collected, and total protein concentration was determined by bicinchoninic acid (BCA, Sigma-Aldrich) assay. 100 µg of protein lysate was used to determine the level of vascular endothelial growth factor (VEGF), broblast growth factor-2 (FGF2), endoglin (CD105), and stromal cell-derived factor-1 (SDF1) according to the vendor's instructions (R&D Systems). To assess the level of osteopontin (OPN), 750 times-diluted mouse serum was subjected to the test and the concentration was quanti ed based on the absorbance values according to the manufacturer's protocol (R&D Systems).

Statistical analyses
Data are presented as mean ± SEM. Differences between groups were tested for statistical signi cance using the one-way ANOVA followed by Tukey's post-hoc test or the unpaired 2-tailed Student's t-test (when statin treatment was evaluated in mdx mice only); p ≤ 0.05 was considered signi cant. The outliers were identi ed based on Grubb's test.

Simvastatin treatment decreases CK and LDH activities in mdx mice
Administration of simvastatin for 28 days in a dose of 10 mg/kg BW/day by oral gavage caused a slight drop in the BW both in WT and mdx mice, the effect visible after the delivery of the rst few doses. No other prominent changes in the mice's behavior were observed. The initial drop in the body weight did not exceed an alarming percentage or aggravated further in the following days. Moreover, animals started to gain weight over time in a similar matter to the vehicle group (Fig. 1A). The blood cell analysis did not reveal any detrimental effect of statin treatment measured at the end of the experiment (Fig. 1B-E). Despite no apparent changes in the number of white blood cells (WBC) (Fig. 1B), a signi cant rise in the percentage of granulocytes (Fig. 1C) and monocytes (Fig. 1D) was noted at the expense of the lymphocytes (Fig. 1E) in the vehicle and simvastatin-treated mdx mice in comparison to appropriate WT.
One of the hallmarks of DMD is the elevated level of LDH and CK, markers of muscle damage (36). Accordingly, the activity of CK (Fig. 1F) and LDH (Fig. 1G) was potently increased in the dystrophic animals, whereas it dropped in mdx mice treated with simvastatin. No disturbing changes as the result of simvastatin were apparent in WT animals, hence for clarity we next focused on the investigation of simvastatin effectiveness predominantly in dystrophic individuals.
Simvastatin treatment fails to improve the exercise capacity of the mdx mice but signi cantly augments forelimb grip strength To assess the functional effect of statin treatment we carried out two types of tests. Conducted after the last dose of statin administration treadmill experiment did not show any difference in the running capacity of mdx mice when compared to the vehicle-treated mice ( Fig. 2A). However, the rate of the increase in forelimb grip strength was higher for dystrophic animals treated with simvastatin in comparison to the vehicle group -the tendency was visible already on day 15 and sustained till day 29, after the last dose of the treatment (Fig. 2B).
Simvastatin treatment does not affect in ammation and necrosis of the muscles in dystrophic animals H&E staining did not reveal any effect of simvastatin on in ammation, regardless of the type of the analyzed muscle (Fig. 3A, B). Accordingly, the expression of an in ammatory gene, heme oxygenase-1 (Hmox1) was affected by the treatment neither in gastrocnemius nor in the diaphragm (Fig. 3C). The muscle necrosis, as assessed by the immuno uorescent staining of the IgG/IgM/IgA, the membraneimpermeable markers, was also not attenuated in mdx mice upon simvastatin administration (Fig. 3D).

Simvastatin does not reduce brosis in dystrophic animals
In dystrophic muscles, collagen deposition was clearly visible; however, the simvastatin treatment did not attenuate brosis as shown by semi-quantitative analysis of trichrome staining both in gastrocnemius (Fig. 4A) and in diaphragm (Fig. 4B) muscles. Of note, the mRNA (Spp1 gene) and protein level of OPN, one of the markers of brosis, was not affected by statin treatment (Fig. 4C -E). Furthermore, the expression of other brotic factors, including transforming growth factor-beta 1 (Tgfb1) and matrix metalloproteinase 11 (Mmp11) was unchanged in both analyzed muscles (Fig. 4F, G). Although there was a signi cant decrease in collagen type I alpha 1 chain (Col1a1) in the gastrocnemius muscle (Fig. 4F) no such effect was found in the diaphragm (Fig. 4G).

Simvastatin treatment in uences muscle regeneration
Based on the laminin staining of the muscle, we were able to evaluate the size of the bers, which appeared to be larger in the gastrocnemius muscle of dystrophic animals upon simvastatin treatment ( Fig. 5A, B). Importantly, also the expression of embryonic myosin heavy chain isoform Myh3, encoding eMyHC especially relevant in the matter of muscle regeneration (37), was perceived to be declined in gastrocnemius muscle (Fig. 5C). Simultaneously, the percentage of CNF was lower when simvastatintreated mdx mice were compared to the vehicle group (Fig. 5D). Interestingly, increased during muscle regeneration, the protein level of FGF2 (38), was reduced in gastrocnemius muscle of simvastatinreceiving animals (Fig. 5E). As microRNAs, especially so-called myomiRs, play an important role in muscle regeneration (39) we decided to check the expression of the three, most commonly used miRNAs: miR-1, miR-133a, and miR-206. Results for simvastatin treated mdx animals shown a signi cant upregulation of miR-1, a prominent rising tendency in miR-133a, and no difference in miR-206 (Fig. 5F).
Notably, no changes in ber size and CNF were noticed in the diaphragm (Fig. 5G-I).

Simvastatin treatment does not affect vascularization in dystrophic muscles
Recent discoveries underline the role of dysregulation of angiogenesis in DMD pathology (4,5,31). In our previous studies, we have found concentration-and cell-type dependent effect of statins on VEGF synthesis and overall angiogenic activity (27,40,41). However, in the C2C12 mouse myoblast cell line simvastatin at the physiologically relevant concentrations (0.1-1 µM) did not affect Vegfa ( Supplementary Fig. 1A). Interestingly, in vivo, the decrease in Vegfa and also kinase insert domain receptor (Kdr) expression after simvastatin delivery was found in gastrocnemius of mdx mice, indicating the possible anti-angiogenic effect (Fig. 6A). Nevertheless, such alterations were not perceived for other angiogenic genes, such as angiopoietin-1 (Ang1) and C-X-C motif chemokine 12 (Cxcl12), also known as gene coding stromal cell-derived factor 1 (SDF1) (Fig. 6A). Importantly, VEGF (Fig. 6B), as well as SDF1 ( Fig. 6C) and endoglin (CD105) (Fig. 6D) protein level was also unaffected. Furthermore, no effect of statin treatment on the analyzed factors was detected in diaphragm muscle, both on mRNA and protein level ( Fig. 6E-H). Moreover, when the number of CD31 + /α-SMA + vessels was evaluated in gastrocnemius muscle, no differences were noted after statin delivery (Fig. 6I). Interestingly, a signi cant rise in the number of CD31 + /α-SMA + vessels was observed in the diaphragm, once again showing discrepancies between various muscles (Fig. 6J).

Discussion
Despite many years of intensive and profound studies, DMD remains an incurable disease. Newest, most promising therapies, using the latest advances in genetic modi cation, namely CRISPR/Cas9 technology, are still far from clinical introduction and acceptance. Thus, glucocorticoids, with prednisolone and de azacort being the most commonly used, still serve as a gold standard therapy for patients suffering from DMD. Unfortunately, except for undoubtful bene cial effects in e.g. prolonging ambulation, their daily administration was shown to exert many side effects leading to, among others, osteoporosis, diabetes, or muscle atrophy (42). As there is a constant need to investigate novel strategies, which could at least attenuate the severity of the disease, many researchers focus not only on new drug discoveries but also repurposing of the already existing ones.
HMG-CoA reductase inhibitors, commonly known as statins, seem to be the perfect choice for such investigation. Despite the still ongoing discussion regarding statin-induced myopathy, myositis, and rhabdomyolysis (16,20,22,43) more and more studies describe that the bene ts of treatment outweigh the possible risks which, of note, are usually not relevant to DMD boys (25,26). In our study, no further deterioration in in ammation, brosis, and necrosis was visible as the consequence of simvastatin delivery to dystrophic animals. Importantly, no elevation in, strongly associated with statin-induced myopathy, CK level (43), indicated no additional damage to the muscle after one month of simvastatin administration. Moreover, no signi cant or alarming systemic changes were observed in regards to the total WBC or distinguished subpopulation e.g. granulocytes, lymphocytes, and monocytes, at the end of the experiment.
In contrast to the previous discussion about the deleterious muscle-related alterations, over recent years, several studies demonstrated the positive effects of statins on overall skeletal muscle health, including their anti-in ammatory and anti-brotic properties (44,45). In the present study we have found that among various processes contributing to dystrophy progressions, simvastatin can ameliorate, in a muscle-type speci c manner, only selected aspects of DMD pathology in uencing regeneration markers without any effect on in ammation, brosis, or angiogenesis. Interestingly, Whitehead et al., already in 2015 showed the protective in uence of simvastatin in dystrophic animals (12). However, the recent publication by Verhaart et al. described the lack of the effect of this statin and even put into question whether presented by Whitehead et al. high level of simvastatin (obtained after administration in the food and drink) in the blood of the animals was possible to be obtained (14). When different statins were investigated by other groups, results were also inconclusive. It was shown that pravastatin, another FDAapproved cholesterol-lowering drug, can be considered in DMD therapy as it can upregulate utrophin A expression via eEF1A2 (46). Utrophin A, which is an autosomal homolog of dystrophin, is suggested to functionally compensate for dystrophin loss in DMD muscles (47). On the other hand, Finkler et al. demonstrated no bene cial effects of rosuvastatin. Moreover, a visible accretion of in ammation was remarked upon treatment (15). Such discrepancies in the obtained results might be related to several divergences in the applied methodology, including age and background of the mice, type and dose of the statin that was used, route of administration, and length of time the drug was given to the animals.
Nevertheless, so diverse strategies give an undoubtful chance to investigate the effects of statins from different perspectives and various stages of mice development and disease progression. In our study, the dose of simvastatin was 10 mg/kg BW. In that matter, the applied approach was similar to the one used by other groups (12,14). Moreover, despite the most promising results were obtained by Whitehead et al. when simvastatin was provided in food and water (12), we strongly believe that oral gavage administration is more relevant, giving us the opportunity to more precisely control the given dose.
Additionally, literature data con rm this method of statin delivery to be more effective in the manner of the obtained in the blood simvastatin concentration (14). Notable, although the effect of simvastatin treatment on mdx mice was not as profound as in Whitehead et al. (12) study, we did remark some interesting changes.
We observed not only typical for dystrophic animals and described in our previous papers (4,5,9), elevation in the most commonly acknowledged muscle damage markers: CK and LDH activity level, but also a signi cant decrease in given parameters in mdx mice upon simvastatin treatment, what is in line with work by Whitehead et al. (12). Such an outcome might suggest lower degeneration of the muscle bers. However, no alterations in typical muscle degeneration markers, namely necrosis, and in ammation (48), allow us to con rm such a hypothesis. In contrast to the results obtained by Whitehead et al., which showed visibly reduced in ammatory cell in ltration and a diminished number of CD68 macrophages (12), we did not observe the anti-in ammatory potential of simvastatin when the histological assessment of gastrocnemius and diaphragm muscles was performed. Moreover, the expression of the Hmox1 gene, coding anti-oxidant, and cytoprotective HO-1 enzyme was also not affected by the treatment. Similar results were obtained by Verhaart et al. (14), who reported no effect on in ammation even with the prolonged by two months, in comparison to us, time of drug administration.
Furthermore, in opposition to the published data suggesting the anti-brotic role of simvastatin in mdx animals (12, 13), we did not observe any effect of the treatment on brosis, neither in histological assessment of collagen deposition nor expression of brosis-related genes. Those observations were con rmed by the evaluation of OPN expression, a recently described biomarker of DMD associated with regeneration, in ammation, and brosis (49,50), which was also not affected by simvastatin.
On the other hand, interesting results were obtained in our study concerning muscle regeneration. When gastrocnemius muscle was investigated we noticed a signi cant increase in the mean myo ber size.
Together with histological assessment of the CNF, a decrease in the expression of Myh3, which is important in muscle repair (51), and downregulation of highly expressed during regeneration, FGF2 protein (38), we might imply normalization of the regenerative process in investigated muscle. We suggest that a decline in the number of CNF and Myh3 mRNA level could be related to faster maturation of the regenerating bers and as a result, loss of the related to that process markers (48). Moreover, a signi cant rise in muscle-speci c miR-1 and increasing tendency in miR-133a, so-called myomiRs, might indicate more e cient muscle regeneration, as it was demonstrated that such upregulation promotes muscle differentiation and therefore, improves muscle repair (52,53). Interestingly, no effect of simvastatin on dystrophic muscle regeneration was demonstrated by Whitehead et al. and Verhaart et al. (12,14). Moreover, it needs to be emphasized that such alterations were noticed only in the gastrocnemius muscle. When we analyzed the diaphragm, we did not observe any changes. It shows that, at least in our hands, simvastatin treatment-related outcomes are strongly muscle type-speci c. In order to better understand the complex mechanisms exerted by statins, their effects on various cell types including muscle satellite cells (mSC), crucial for muscle regeneration (54), should be investigated.
Enlargement in muscle ber size together with improved muscle regeneration could further explain observed by us amelioration in the forelimb grip strength. Interestingly, we described that even though in both vehicle and simvastatin groups strength was augmented, the rate of the improvement was signi cantly higher in drug-treated mdx mice. Interestingly, when a measurement of the speci c force of the muscle was performed by Whitehead et al. (12) and Verhaart et al. (14), they showed signi cant improvement and lack of any effect, respectively. As overall mice performance in a treadmill test, in uenced not only by muscle strength but also by the respiratory and cardiovascular systems, was not altered by the treatment, we might speculate that obtained by us effect might not be strong enough to cause systemic changes in the mice.
To expand already described knowledge we decided to investigate also a different aspect of DMD progression -angiogenesis alterations, especially because our previous studies revealed a decrease in a major pro-angiogenic factor, VEGF, both at mRNA and protein level in skeletal muscles of dystrophic animals in comparison to wild-type counterparts (4,5,31). Importantly, improvement of endothelial function and vasculoprotective action are well-recognized statin effects (55). Our previous experiments clearly showed that statins regulate angiogenesis. We have demonstrated that atorvastatin at the pharmacologically relevant concentration (100 nM) enhanced the expression of endothelial nitric oxide synthase (eNOS) in human microvascular endothelial cells (HMEC-1). Moreover, atorvastatin prevented the hypoxia-induced decline in eNOS expression (28). The regulation of several angiogenic factors was observed by us after statin stimulation in human umbilical vein endothelial cells (HUVEC) but these effects may be also cell-and dose-dependent (27,41). Moreover, accelerated vascularization upon simvastatin treatment was also demonstrated in models of peripheral ischemia and corneal neovascularization (56). In the present study, despite signi cant changes in some of the tested angiogenic genes in gastrocnemius muscle, no complementary effects were noticed on the protein level.
Although a decrease in Vegfa/Kdr signaling might indicate the anti-angiogenic effect of simvastatin, neither VEGF protein nor abundance of CD31/α-SMA double-positive blood vessels were observed.
Despite the elevated number of vessels in the diaphragm, no other investigated factors were affected, rather suggesting no profound effect on angiogenesis as the result of simvastatin administration.
Noteworthy, it again shows that simvastatin might not be in uencing various muscles in the same manner.

Conclusion
In conclusion, we suggest that simvastatin has the potential to positively in uence selected aspects of DMD pathology. Despite muscle-dependent changes and the requirement for further research, improvement in forelimb grip strength with decreased CK and LDH activity and ameliorated muscle regeneration, allow us to consider simvastatin as a potential drug in combined therapies for DMD.

Consent for publication
Not applicable.

Availability of data and materials
The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.

Competing interests
The authors declare that they have no competing interests. Figure 2 The rate of forelimb grip strength increase is elevated in dystrophic animals upon simvastatin treatment.

Figures
(A) Downhill running treadmill test presented as the percentage of the running distance compared to vehicle-treated animals; n=5-6/group, mean ± SEM. (B) Forelimb grip strength analysis shown as a strength increase rate at day 15 and day 29 of statin delivery in comparison to day 0; n=5-7/group, mean ± SEM; * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001.

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