APE1/Ref-1 inhibition via APX3330 in young dystrophic mdx mice

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Goodman, Nicole Stupka, Nicholas Giourmas, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-9507119/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Aims Chronic inflammation and oxidative stress are key components in Duchenne muscular dystrophy (DMD) pathology. Apurinic/apyrimidinic endonuclease 1/redox factor-1 (APE1/Ref-1) is a multifunctional protein involved in inflammatory and oxidative stress pathways through its redox domain and is an emerging therapeutic target for inflammatory conditions. This study aimed to investigate the effects of APX3330, a small molecular inhibitor of APE1’s Ref-1 function on mdx mouse pathology, a model of DMD. Methods Three-week-old mdx mice and wildtype (WT) C57Bl/10 mice were randomised into four groups: 1) WT untreated control; 2) WT treated with APX3330 (25 mg·kg -1 ); 3) mdx untreated control; or 4) mdx treated with APX3330 (25 mg·kg -1 ). After three weeks, ex vivo contractile function and histological analysis were performed in extensor digitorum longus (EDL) and soleus muscles. Results APX3330 treated WT mice displayed a lower absolute and specific force in EDL (p < 0.01, p < 0.001, respectively) and soleus (p < 0.05) muscles, despite having a higher muscle mass to body mass ratio than untreated mice (p < 0.05). In APX3330 treated dystrophic mice, soleus muscles exhibited larger fibres (p < 0.0001), while EDL muscles showed smaller fibres (p < 0.01), longer time to peak tension (p < 0.05), and higher twitch to tetanic force ratio (p < 0.01). Dystrophic EDL muscles also demonstrated a higher amount of CD68 positive monocytes/macrophages compared to untreated mdx mice (p < 0.05). APX3330 treatment did not alter muscle histopathology. Discussion APX3330 treatment demonstrated deleterious effects in both WT and dystrophic mice. Despite literature indicating anti-inflammatory effects, APX3330 was unable to ameliorate the dystrophic phenotype in any meaningful capacity. Duchenne muscular dystrophy inflammation oxidative stress APE1/Ref-1 APX3330 Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction Duchenne muscular dystrophy (DMD) is one of the most severe forms of childhood muscular dystrophy which affects 1 in every 5,000 boys ( 1 ). DMD is caused by mutations in the DMD gene resulting in the loss of the cytoskeletal protein, dystrophin, subsequently disrupting the formation of the dystrophin-glycoprotein complex (DGC) ( 2 , 3 ). DGC destabilisation exposes muscle fibres to mechanical stress, with continuous cycles of muscle damage and insufficient repair eventually leading to contractile tissue being replaced by fibroadipose tissue, which is then heightened by inflammation and oxidative stress ( 4 , 5 ). The current standard of care for DMD patients are corticosteroids (prednisolone/deflazacort), potent anti-inflammatory drugs that delay the loss of ambulation and improve respiratory capacity by moderately improving muscle function ( 6 ). However, adverse side effects are associated with corticosteroids, including weight gain, bone loss, and delayed growth, limiting their effectiveness ( 7 ). Therefore, investigation into alternative therapies that mitigate inflammation while improving dystrophic muscle function and pathology with a more favourable side-effect profile is necessary. Apurinic/apyrimidinic endonuclease 1/redox factor 1(APE1/Ref-1) is a multifunctional protein initially involved in the base excision repair (BER) pathway that targets DNA lesions that increase oxidative stress ( 8 ). Another major role of APE/Ref-1 is the redox regulation of transcription factors, in which APE1/Ref-1 reduces transcription factors, including nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB), to promote DNA binding and activation of genes involved in pro-inflammatory and oxidative stress signaling cascades ( 9 ). Repression of APE1/Ref-1 has shown to induce protective, antioxidant effects by enhancing the activity and abundance of nuclear factor erythroid 2-related factor 2 (NRF2) target proteins ( 10 ). However, the effects of APE1/Ref-1 inhibition on inflammation and oxidative stress appear to be dependent on biological context with some in vitro studies reporting protective effects of APE1/Ref-1 overexpression in acute models of inflammation ( 11 – 15 ). Alternatively, in chronic inflammation and oxidative stress, constant activation of APE1/Ref-1 may lead to a pathological response. Indeed, chronic inflammatory conditions that involve heightened inflammation and oxidative stress, such as myocarditis and chronic colitis, show an elevated expression of APE1/Ref-1 ( 16 – 18 ). APX3330 is a small molecular inhibitor of APE1/Ref-1 which targets the redox domain, rendering the protein unable to reduce transcription factors, keeping transcription factors in an oxidized and inactive state ( 19 ). This then inhibits downstream activity of these transcription factors, including those involved in inflammation and oxidative stress pathways. Indeed, APX3330 has shown to reduce inflammatory cytokine expression in RAW264.7 cells ( 20 ) and the infiltration of CD45 positive immune cells in the colon of Winnie mice, a model of chronic colitis ( 21 ). Furthermore, in the Winnie mouse, APX3330 decreased mitochondrial superoxide production in the myenteric plexus, alleviating inflammation-induced oxidative stress, which led to neuroprotective effects, reduction in disease severity and restoration of GI function ( 21 ). The significant reduction in markers of inflammation and oxidative stress in these models indicate potential application in other disease states with similar pathology, including muscular dystrophy (reviewed in ( 22 )). To date, few studies have evaluated the role of APE1/Ref-1 in skeletal muscle ( 23 – 26 ). Our lab has previously investigated the inhibition of APE1/Ref-1 via APX3330 in the mdx mouse model, the most commonly used mouse model for DMD, during a period of elevated, yet stable, inflammation (6–12 weeks of age); however, this did not result in any meaningful benefit beyond reduction in inflammatory markers ( 27 ). Although APX3330 was unable to alter dystrophic pathology during 6–12 weeks of age, the pathology is quite established during this time ( 28 ). To investigate the potential of APX3330 as a preventative, treatment must be administered prior to the onset of the dystrophic phenotype (3–6 weeks of age) ( 28 ). Beginning at 3 weeks of age covers a peak degeneration/regeneration period as seen during childhood in DMD patients. Therefore, we hypothesise that APE1/Ref-1 inhibition with APX3330 treatment could delay dystrophic hindlimb muscle pathology in mdx mice if administered within the juvenile time point prior to peak damage and inflammation (3–6 weeks of age). Methods Ethics approval and animals This study was approved by the Animal Ethics Committee at Victoria University (AEC 22/005). All experiments conformed to the Australian Code for the care and use of animals for scientific purposes (8th ed., 2013). Clinical trial number: not applicable Dystrophic female mdx mice and C57Bl/10 wildtype controls were bred and housed at the Western Centre for Health, Research and Education (WCHRE, Victoria, Australia) in a light- and temperature-controlled room (12h light/dark cycle, 21°C) with ad libitum access to food and water. At 3 weeks of age, wildtype (WT) and mdx mice were randomly allocated into treated (n = 12) or untreated groups (n = 8). Treatment mice received an intraperitoneal (IP) injection of APX3330 (25 mg·kg − 1 ) dissolved in Cremophor (2%), ethanol (2%) and sterile water (96%) twice a day for 3 weeks. APX3330 was received as a generous gift by Dr. Mark R Kelley (Indiana University School of Medicine, Indiana, USA). We utilised the same treatment regime used by Fishel et al., 2011 ( 29 ) and Sahakian et al., 2020 ( 21 ), where, in models of pancreatic cancer and ulcerative colitis, respectively, APX3330 decreased inflammatory cell infiltration and the activation of inflammatory cell signalling cascades, including NF-κB ( 21 , 29 ). Additionally, the doses were kept the same as our previous study, ( 27 ), in which APX3330 was effective in lowering macrophages/monocytes, allowing direct comparison, and determine whether APX3330 could be more effective as a preventative treatment during a younger age point, prior to the establishment of the dystrophic phenotype. Tissue extraction Mice were deeply anaesthetised with 2–4% isoflurane with oxygen set to a flow rate of 0.6 mL·min − 1 , and non-survival surgery was performed. Extensor digitorum longus (EDL) and soleus muscles were then tied tendon to tendon with 4.0 suture thread and immediately excised for muscle contractile function analysis. Following the completion of the contractile protocol, muscles were embedded with optimal cutting temperature (OCT) compound (Tissue-Tek OCT Compound; Sakura Finetek, California, USA) in isopentane pre-cooled in liquid nitrogen for histological analysis. Contractile function Ex vivo evaluation of muscle contractile properties was performed as previously described ( 30 ). Excised EDL and soleus muscles were placed into a contractile chamber (Danish Myo Technology (DMT) A/S, Hinnerup, Denmark) containing Krebs-Heinseleit Ringer’s solution (118 mM NaCl, 4.75 mM KCl, 1 mM Na 2 HPO 4 , 1.18 mM MgSO 4 ·7H 2 O, 2.5 mM CaCl 2 , 24.8 mM NaHCO 3 , 11 mM D-Glucose; pH 7.4). Individual baths were bubbled with 5% CO 2 in O 2 (BOC gases, Melbourne, Australia) and maintained at 30°C. The proximal end of the muscle was clamped onto a previously calibrated force transducer and the distal end was secured to a micromanipulator with stimulating electrodes flanking the muscle belly. Optimal length (L O ) was determined for each muscle via a series of twitch contractions at increasing lengths to ensure optimal overlap of sarcomeres, and muscle length was measured with callipers. A force-frequency relationship was established by delivering frequencies of 10, 20, 30, 40, 50, 60, 80, 100, 120 and 150 Hz. Absolute force (maximum isometric contraction; P O ) was recorded as the highest force obtained in the force-frequency protocol, with other forces reported as a percentage of that maximum. To normalise force produced for different muscle sizes, specific force (sP O ) was calculated as maximal force produced per cross-sectional area (CSA), using L O and muscle mass, assuming the muscle density is 1.06 g/cm 3 and fibre-muscle length ratios are 0.44 for the EDL and 0.71 for the soleus ( 31 , 32 ). Following the force-frequency protocol, each muscle was stimulated with a single pulse three times in 5 second intervals for the analysis of basic contractile properties (peak twitch force (P t ), time to peak (TTP), and relaxation time (½RT)). Muscle fatiguability was assessed by sending repeated intermittent electrical stimuli for 3 minutes. To obtain comparable levels of fatigue, the EDL was stimulated every 4 seconds at 100 Hz for 350 ms and the soleus every 2 seconds at 80 Hz for 500 ms. Data was collected and analysed using LabChart Pro version 8.0 software, customised for this experiment (ADInstruments, Dunedin, New Zealand). Histology Haematoxylin & Eosin (H&E) – To assess muscle morphology, including damaged areas and regenerating fibres, 10 µM cryosections from EDL and soleus muscles were stained with haematoxylin and eosin (H&E) as previously described ( 30 ). A quantitative estimate of degeneration within each section was determined by classifying areas of inflammatory cell infiltrate and centrally nucleated fibres as “unhealthy tissue” ( 33 ). The proportion of centrally nucleated fibres and fibre size and distribution were also assessed. Succinate Dehydrogenase (SDH) – To assess changes in oxidative capacity, a succinate dehydrogenase (SDH) stain was performed on EDL and soleus muscle sections as previously described ( 30 ). Briefly, sections were incubated in SDH solution (0.05% nitro blue tetrazolium; 0.2 M sodium succinate; 0.2 M phosphate buffer, pH 7.6) for 1hr at 37°C, fixed in formal saline (0.9% NaCl; 10% formaldehyde) and mounted in glycerol. Images were converted to 8bit, threshold adjusted to exclude non-specific staining and SDH intensity was measured in full cross sections of the muscle. Picro-Sirius Red (PSR) – To assess changes in collagen, a Picro-sirius red (PSR) stain was performed on EDL and soleus muscles. Briefly, sections were airdried then fixed in 10% neutral buffered formalin (NBF). Sections were then incubated in Bouins fixative (formaldehyde; picric acid; acetic acid) overnight, washed under running water and incubated in 0.1% PSR solution (Sirius Red F3B; picric acid) for 1hr and dipped in water. Sections were dehydrated in 3 changes of ethanol and 3 changes of xylene to ensure no further colour changes and mounted in xylene. Images were converted to 8bit, threshold adjusted to exclude non-specific staining and PSR intensity was measured in full cross sections of the muscle. Imaging – Following staining, images of whole muscle sections at 200x magnification were captured with a Zeiss Axio Imager Microscope (Zeiss, Germany), running the Metafer 4 V3.13.3 imaging software (MetaSystems, Germany) and stitched together by a VSlide V1.1.120 software (MetaSystems, Germany). All images were analysed using ImageJ software (NIH, Maryland, USA). Immunohistochemistry for CD68 positive cells Immunohistochemical analysis was performed as previously described ( 34 ). Briefly, muscle sections were fixed in 4% paraformaldehyde (PFA) and permeabilised in TBST (Tris-buffered saline with 0.05% Tween-20). Sections were then incubated in a serum free blocking solution (Dako, #X0909) before incubation in primary antibody (anti-CD68, ab125212, Abcam, 1:500) prepared in antibody diluent (Dako, #S3022) at room temperature. The anti-CD68 antibody (ab125212, Abcam, 1:500) was used for the detection of monocyte and macrophage infiltration. Slides were then incubated with fluorophore-conjugated secondary Alexa Fluro 594 goat anti-rabbit antibody (A11012, Thermo Fisher Scientific, 1:1000 in antibody diluent) and counterstained with wheat germ agglutinin (WGA; W11262, Thermo Fisher Scientific, 1:50 in phosphate buffered saline (PBS)), and mounted in mounting medium with DAPI (ab104139, Abcam). Additional sections from untreated mdx mice stained without the primary antibody were used as a negative control. Images of whole sections (100x) and at 200x magnification were taken using the confocal microscope (STELLARIS 5 confocal microscope, Leica Microsystems). Whole muscle sections were analysed using ImageJ software (NIH, Bethsda, MD, USA) and result expressed as cells/mm 2 . Statistical analysis All values are expressed as means ± SEM. An unpaired t-test was used to evaluate differences in CD68 positive cells between treated and untreated mdx mice. In every other instance, four group comparisons were analysed using a two-way ANOVA with strain and treatment as factors, followed by a Tukey post hoc analysis test where there was a significant interaction. When data was not normally distributed, a Kruskal-Wallis test was performed for four-group comparisons, followed by a Dunn post-hoc analysis test. Differences between groups were considered significant if p ≤ 0.05. All data analysis was performed using GraphPad Prism 9.0 (GraphPad Software Inc., California, USA). Results APX3330 appears to be detrimental to muscle contractile properties Body weights were significantly lower in the APX-treated animals (Table I), potentially suggesting impaired growth, although the effect was primarily seen in the wildtype animals. Interestingly, the muscle masses of both muscles in both strains were not different, resulting in higher muscle mass to body mass ratios (MM:BM) in the APX3330-treated wildtype EDL and soleus of both strains. Dystrophic hindlimbs muscles exhibited various indications of muscle weakness compared to their wildtype counterparts. Although specific force was lower in both muscles, weakness was mostly observed in the soleus muscles, including reductions in peak force, time to peak, and ½ relaxation time, as only optimal length was increased in mdx EDL muscle compared to wildtypes (Table I). When muscle force at submaximal frequencies was normalised to maximal force output, no change was observed between wildtype and mdx EDL muscles (Fig. 1 A), yet force was significantly reduced at 40–100 Hz in mdx soleus muscles compared to wildtype (Fig. 1 B). No strain differences were observed in the EDL fatigue/recovery results (Fig. 1 C), however, mdx soleus muscles were more fatigue resistant between 90–120 sec of the fatigue protocol (Fig. 1 D). With the addition of APX3330, wildtype mice displayed decreases in peak force and specific force in both EDL and soleus muscles, despite increases in the MM:BM (Table I), suggesting increased non-contractile material. Force development at higher frequencies were also reduced in the EDL (80 and 120 Hz; Fig. 1 A) and soleus (80 and 100 Hz; Fig. 1 B) with treatment. Interestingly, fatigue resistance was enhanced in the EDL and soleus (Fig. 1 C & 1 D), yet the overall rate of recovery was lowered in the EDL and unchanged in the soleus (Fig. 1 E & 1 F). These findings suggest that the treatment, and/or stress response induced by the treatment regimen, may hinder growth, increase muscle infiltrate and/or have detrimental effects on muscle contractile properties in wildtype mice. In dystrophic EDL muscle, APX3330 increased the time to peak, indicating a delay in the rate of force development, and an increased P t /P O ratio, suggesting increased muscle stiffness and/or shift to a slower contractile phenotype (Table I). The soleus of mdx mice showed an increased MM:BM, enhanced force at 40–100 Hz, similar to wildtype levels, while increasing fatigue resistance between 120 and 180 sec of the fatigue protocol (Fig. 1 C & 1 D). However, the soleus muscles displayed a reduction in absolute and specific force (Table I), thus, it is possible that the muscle is just consistently producing low forces. Table I. Body mass and muscle morphology characteristics Measure WT VEH n=8 WT APX n=12 mdx VEH n=8 mdx APX n=12 Body Mass (g) 19.2 ± 0.3 15.6 ± 0.3 #### 18.1 ± 0.3 17.8 ± 0.3 #### Muscle Measure EDL SOL WT CTL n=8 WT APX n=12 mdx CTL n=8 mdx APX n=12 WT CTL n=8 WT APX n=12 mdx CTL n=8 mdx APX n=12 Mass (mg) 6.4 ± 0.2 8.7 ± 1.7 7.3 ± 0.3 8.0 ± 0.5 6.5 ± 0.3 7.2 ± 0.9 ^ 6.7 ± 0.5 7.3 ± 0.4 MM/BM 0.33 ± 0.01 0.50 ± 0.05 ## 0.40 ± 0.02 0.42 ± 0.02 0.34 ± 0.01 0.46 ± 0.05 # 0.37 ± 0.03 0.41 ± 0.03 # L O (mm) 9.3 ± 0.3 8.6 ± 0.2 9.6 ± 0.3 * 9.7 ± 0.1 * 8.8 ± 0.2 8.7 ± 0.1 ^ 8.6 ± 0.2 ^ 8.5 ± 0.2 P t (mN) 28.7 ± 3.9 20.9 ± 2.5 20.0 ± 1.5 23.1 ± 2.7 16.6 ± 2.2 15.5 ± 1.2 12.7 ± 1.3 * 12.2 ± 0.8 * TTP (ms) 26.0 ± 0.3 27.2 ± 0.3 # 26.5 ± 0.5 27.2 ± 0.3 # 32.7 ± 0.9 33.4 ± 0.5 ^ 31.8 ± 0.5 * 32.0 ± 0.4 ^* ½ RT (ms) 16.6 ± 0.7 18.3 ± 0.3 18.6 ± 1.6 19.6 ± 0.6 27.5 ± 1.4 28.8 ± 0.9 24.4 ± 0.8 ** 25.4 ± 0.8 ** P O (mN) 150.1 ± 14.3 96.0 ± 5.0 ^## 113.7 ± 9.4 104.1 ± 11.6 136.5 ± 11.8 120.2 ± 6.4 ^# 109.8 ± 7.5 ** 89.8 ± 6.8 **# P t /P O 0.19 ± 0.01 0.20 ± 0.01 ## 0.18 ± 0.01 0.22 ± 0.01 ^## 0.12 ± 0.01 0.13 ± 0.00 ^ 0.11 ± 0.01 0.21 ± 0.07 CSA (mm 2 ) 0.15 ± 0.00 0.21 ± 0.03 0.16 ± 0.01 0.17 ± 0.01 ^ 0.16 ± 0.01 0.20 ± 0.03 0.16 ± 0.01 0.19 ± 0.01 sP O (N/cm 2 ) 10.3 ± 1.0 5.9 ± 0.6 ### 6.5 ± 0.4 ^* 6.3 ± 0.8 8.7 ± 0.6 6.7 ± 0.9 # 7.0 ± 0.6 * 5.2 ± 0.6 *# Abbreviations: WT = wildtype mice; MDX = mdx mice; CTL = control; APX = APX3330 treated; EDL = extensor digitorum longus; SOL = soleus; MM = muscle mass; BM = body mass; L O = optimal length; P t = peak twitch; TTP = time to peak; ½ RT = half relaxation time; P O = peak tetanic absolute force; CSA = cross-sectional area; sP O = specific force. Symbols indicate: ^ n = n-1. *p < 0.05, **p < 0.01, strain effect. # p < 0.05, ## p < 0.01, ### p < 0.001, #### p < 0.0001, treatment effect. APX3330 increases mean fibre size in mdx muscles, yet does not alter oxidative capacity or fibrosis in hindlimbs As anticipated, dystrophic mdx mice exhibited a lower percentage of healthy tissue area and a higher proportion of centralised nuclei in both EDL (Figure 2A & 2B) and soleus (Figure 3A & 3B) muscles, compared to wildtype mice. In the wildtype mice, APX3330 treatment resulted in a decrease in the average fibre area in the EDL muscles (Figure 2C), indicating a leftward shift in fibre size distribution (Figure 2D). Conversely, treatment had the opposite effect on the WT soleus muscles, showing an increase in the mean fibre area and a rightward shift in fibre distribution (Figure 3C & 3D). When treated with APX3330, the mean fibre area increased in both the EDL (Figure 2C & 2E) and soleus (Figure 3C & 2E) muscles of mdx mice. Initially, this might suggest larger fibres and, therefore, increased strength. However, since the treatment also exacerbated muscle weakness, it may be characteristic of oedema-induced swelling or displaying a pseudo-hypertrophic phenotype, such that the quality of the contractile tissue is diminished despite the increase in size. Additionally, mdx mice displayed a lower oxidative capacity in the EDL (Figure 4A), with no changes in the soleus (Figure 4B), and increased fibrosis in the soleus (p<0.01; Figure 5B). No increase in fibrosis was detected in the EDL (Figure 5A), possibly due to variability in values obtained in the WT control group, as the picrosirius red staining was also observed within the fibres, which was not the case in the other groups, and would have inflated the percentage area values. APX3330 treatment increased monocyte and macrophage infiltration in the EDL Infiltration of CD68 positive monocytes and macrophages were elevated in mdx EDL muscle compared to WT mice (Figure 6). This elevation was further increased significantly in APX3330 treated mdx mice. This is contrary to our previous results in older mice in which the number of CD68 positive cells was lowered in APX3330 treated mdx mice compared to vehicle treated mdx mice (27). This could indicate that the physical action of vehicle and/or drug administration could contribute to cell infiltration, as the control group in this study were not treated with a vehicle. However, it is important to note that the CD68 results of the mdx control group in this study are also similar to the mdx vehicle group in our previous study, indicating that the addition of vehicle had little effect on the number of CD68 positive cells and, instead, that APX3330 may not be effective at this dosage and/or age point. Discussion Although, in our previous study, APX3330 was able to reduce inflammatory cell infiltration in mdx mice during the relatively stable phase of dystrophic pathology (6-12 wks), it did not induce profound changes in muscle pathophysiology (27). Given that APX3330 has previously demonstrated the ability to decrease inflammation and improve disease outcomes in models characterised by chronic, heightened inflammation, it was hypothesised that APX3330 would be more beneficial during the peak of damage and repair in mdx dystrophic pathology, when inflammation is at its highest (3-6 wks). However, treatment with APX3330 resulted in stunted growth and impaired muscle force in wildtype mice. Similar observations were made in mdx mice treated with APX3330, although force development and fatigue resistance in the soleus appeared to be improved. Interestingly, APX3330 treatment in mdx EDL muscle increased the number of CD68 positive cells, indicating that the treatment caused more inflammation than it resolved. Overall, these findings suggest that APX3330 may not be a suitable treatment for dystrophic pathology, with the current dose and/or mechanism of action insufficient to ameliorate muscle dystrophy, during a peak damage/repair period. Within wildtype mice, the administration of APX3330 resulted in a decrease in body weight but an increase in the ratio of muscle mass to body mass. This initially suggests that muscle mass is maintained despite the reduction in overall body mass. However, the hindlimb muscles of the treated mice exhibited lower absolute and specific force compared to the control group, as well as reduced force development at higher frequencies. These findings indicate that the treatment and/or the associated stress response from the treatment regimen may hinder growth, cause infiltration of non-contractile material and/or negatively affect muscle contractile properties. Interestingly, APX3330 treatment enhanced fatigue resistance and coupled with the decrease in rate of recovery, implies that muscle force is preserved and a possible shift to slower fibres, particularly in the EDL muscles. However, it is also important to note that this could be attributed to already low maximal forces, making further reduction difficult. When evaluating fibre area, the treatment led to a decrease fibre area in the EDL, while an increase was observed in the soleus. This observation further suggests the preservation of slow twitch fibre phenotype and/or the loss of fast twitch fibres. Overall, the treatment in wildtype mice appears to the weaken muscles and inhibit growth, thus presenting a negative impact on these mice. Despite the detrimental effects of APX3330 on wildtype muscle, the objective of this study was to investigate potential therapeutic effects on dystrophic mdx muscle. DMD pathology is typically characterised by preferential damage toward fast-twitch fibres which is also observed in mdx mice (35). However, this study found that mdx mice exhibited various signs of muscle weakness in the slow-twitch soleus muscle, including reductions in peak twitch, peak force, specific force, time to peak and ½ relaxation time. Additionally, the mdx soleus muscles showed a diminished force frequency response, further indicating muscle weakness. Conversely, the EDL muscles of mdx mice only demonstrated a lower specific force and an increase in optimal length which could be attributed to multiple factors, such as increased fibrosis, altered muscle architecture or a compensatory mechanism aiming to optimise force production (36, 37). However, this alteration was the only functional measure that displayed a change. As anticipated, mdx mice exhibited a decrease in healthy tissue area and an increase in the proportion of centrally located nuclei in both the EDL and soleus muscles. EDL mdx muscles showed a decrease in oxidative capacity while no change was seen in the soleus. Fibrosis was increased in the soleus muscles, yet this change was not significant in the EDL, possibly due to variation in the wildtype control group that displayed red staining within the fibre compared to the other groups. The changes observed in the histopathology are all indicative of the typical dystrophic phenotype (38-40). Treatment in mdx mice resulted in various changes in muscle contractile properties. Specifically, in the EDL, there was an increase in MM:BM, peak twitch to peak tetanic force ratio, time to peak, and mean fibre area. These findings suggest a delayed rate of force development and reduced elasticity. These changes could indicate a shift to a slower phenotype or a possible increase in calcium influx, which may be associated with increase twitch tension. Indeed, studies have shown calcium regulatory dysfunction in mdx mice and subsequent amelioration with sarco/endoplasmic reticulum calcium ATPase (SERCA) activation (41, 42), however, further investigation involving calcium uptake and SERCA activity would be necessary to determine the exact cause of these changes. Alternatively, muscle weakness could also be resultant of the stress response as the treatment regimen is quite invasive. Decreases in body weight, skeletal muscle mass and grip strength in mice after being subjected to repetitive water-immersion restraint stress has been observed (43). Interestingly, in the soleus muscle, treatment led to an increase in force at lower frequencies of activation, seemingly returning to wildtype levels. Furthermore, the treatment enhanced fatigue resistance. At face value, these changes appear to be beneficial, however, it is important to note that these changes could also result from overall low maximal forces, as observed by decreases in absolute and specific force, similar to the effects seen in wildtype mice. This suggests that the treated muscles are less efficient at generating force and exhibit impaired contractile function, which negatively impacts motor function. In addition, APX3330 treatment was not able to ameliorate dystrophic pathology as there were no improvements in either healthy tissue area or the proportion of centrally nucleated fibres. However, treatment was able to increase mean fibre area. Usually, increases in fibre area are often associated with improved muscle strength, yet as lower peak and specific forces were observed, it does not appear to be as a result of increased contractile material. Further work would be needed to investigate the cause. Ultimately, due to reductions in maximal force-generating capacity, it appears that APX3330 treatment is not effectively addressing the underlying muscle weakness that is associated with dystrophic muscles. While interpreting the results of this study, it is important to note that treated mice were compared to untreated control mice. Our previous study did not indicate any treatment-based differences between vehicle treated and APX3330 treated groups within the wildtype mice (27), indicating the relative safety of the APX3330 drug. However, due to the rigorous treatment regime of twice daily injections, it is also important to consider the effects of the entire regimen independent of vehicle-related effects. Previous studies have also reported that mice receiving just two intraperitoneal injections of saline 24 hours apart, was enough to significantly increase corticosterone levels compared to mice that were not handled and mice that received only one saline injection (44). Minimising confounding factors, such as injection stress, would reveal if treatment effects were benefiting the dystrophic pathology or if the vehicle and/or treatment regime caused more damage which could then be ameliorated by the drug. Although a vehicle group was ultimately omitted from this study, the current study revealed that APX3330 treatment had a more detrimental impact on muscle in both wildtype and dystrophic mice. This important finding suggests the need for a more potent drug that would require less injections, or an alternative mode of administration including orally or slow-release pellets. Higher doses of APX3330 may also be required to further inhibit inflammation and have a positive impact on muscle pathology and function in mdx mice. Additional dosage studies may be required to determine the efficacy of APX3330 in skeletal muscle and whether these changes are due to direct action in the muscle or indirect action via alternative pathways. Given the ambiguous nature of APE1, it may be upregulated in dystrophy, as observed previously (27), in attempts to resolve inflammation and oxidative stress. Therefore, stimulation studies should also be considered to determine its role in skeletal muscle. In this study, the administration of APX3330 treatment, coupled with a treatment regimen administered twice daily, demonstrated deleterious effects in wildtype mice and limited benefits in dystrophic mice. To evaluate the separate impacts of the treatment regimen and the treatment more effectively itself, it is crucial to include a control group receiving a vehicle. Furthermore, the investigation of alternative methods for drug administration may result in more advantageous outcomes. The exploration of different delivery routes or dosing schedules could mitigate the stress response and provide guidance for more efficacious treatment strategies. This study further corroborates the notion that solely targeting inflammation is insufficient to improve dystrophic pathology. Abbreviations DMD Duchenne muscular dystrophy APE1/Ref 1–Apurinic/apyrimidinic endonuclease 1/redox factor 1 WT wildtype EDL extensor digitorum longus DGC dystrophin–glycoprotein complex BER base excision repair NF κB–nuclear factor kappa–light–chain–enhancer of activated B cells NRF2 nuclear factor erythroid 2–related factor 2 AEC Animal Ethics Committee WCHRE Western Centre for Health, Research and Education IP intraperitoneal OCT optimal cutting temperature DMT Danish Myo Technology NaCl sodium chloride KCl potassium chloride Na 2 HPO 4 sodium phosphate dibasic MgSO 4 ·7H 2 O magnesium sulphate heptahydrate CaCl 2 calcium chloride NaHCO 3 sodium bicarbonate CO 2 carbon dioxide O 2 oxygen L O optimal length P O peak tetanic force sP O specific force CSA cross sectional area P t peak twitch force TTP time to peak ½RT half relaxation time H&E haematoxylin and eosin SDH succinate dehydrogenase PSR picro–sirius red NBF neutral buffered formalin PFA paraformaldehyde TBST tris–buffered saline with Tween–20 PBS phosphate buffered saline SEM standard error of mean ANOVA analysis of variance SOL soleus MM muscle mass BM body mass MDX mdx mice CTL control APX APX3330 SERCA sarco/endoplasmic reticulum calcium ATPase Declarations Ethical Publication Statement We confirm that we have read the Journal’s position on issues involved in ethical publication and affirm that this report is consistent with those guidelines. Disclosure of Conflicts of Interest None of the authors has any conflict of interest to disclose. Authorship Contribution Statement H.L: Investigation, data collection and analysis, writing – original draft, review and editing. C.A.G: Conceptualization, supervision, writing – review and editing. N.S: Resources, visualization, writing – review and editing. N.G: Data collection, writing – review and editing. L.S: Resources, writing – review and editing. K.N: Resources, writing – review and editing. A.H: Conceptualization, supervision, writing – review and editing. Data Availability Statement The authors confirm that the data supporting the findings of this study are available within the article and/or its supplementary materials. Funding Statement No financial support for the research, authorship, and/or publication of this article was received. References Kariyawasam D, D’Silva A, Mowat D, Russell J, Sampaio H, Jones K, et al. Incidence of Duchenne muscular dystrophy in the modern era; an Australian study. European Journal of Human Genetics. 2022;30(12):1398-404. Hoffman EP, Brown RH, Jr., Kunkel LM. Dystrophin: the protein product of the Duchenne muscular dystrophy locus. Cell. 1987;51(6):919-28. Allen DG, Whitehead NP, Froehner SC. Absence of Dystrophin Disrupts Skeletal Muscle Signaling: Roles of Ca2+, Reactive Oxygen Species, and Nitric Oxide in the Development of Muscular Dystrophy. Physiol Rev. 2016;96(1):253-305. Li W, Zheng Y, Zhang W, Wang Z, Xiao J, Yuan Y. Progression and variation of fatty infiltration of the thigh muscles in Duchenne muscular dystrophy, a muscle magnetic resonance imaging study. Neuromuscul Disord. 2015;25(5):375-80. Desguerre I, Mayer M, Leturcq F, Barbet JP, Gherardi RK, Christov C. Endomysial fibrosis in Duchenne muscular dystrophy: a marker of poor outcome associated with macrophage alternative activation. J Neuropathol Exp Neurol. 2009;68(7):762-73. Mah JK. An Overview of Recent Therapeutics Advances for Duchenne Muscular Dystrophy. Methods Mol Biol. 2018;1687:3-17. Buchman AL. Side effects of corticosteroid therapy. J Clin Gastroenterol. 2001;33(4):289-94. Fung H, Demple B. A vital role for Ape1/Ref1 protein in repairing spontaneous DNA damage in human cells. Mol Cell. 2005;17(3):463-70. Kelley MR, Georgiadis MM, Fishel ML. APE1/Ref-1 role in redox signaling: translational applications of targeting the redox function of the DNA repair/redox protein APE1/Ref-1. Curr Mol Pharmacol. 2012;5(1):36-53. Fishel ML, Wu X, Devlin CM, Logsdon DP, Jiang Y, Luo M, et al. Apurinic/Apyrimidinic Endonuclease/Redox Factor-1 (APE1/Ref-1) Redox Function Negatively Regulates NRF2. Journal of Biological Chemistry. 2015;290(5):3057-68. Baek H, Lim CS, Byun HS, Cho HS, Lee YR, Shin YS, et al. The anti-inflammatory role of extranuclear apurinic/apyrimidinic endonuclease 1/redox effector factor-1 in reactive astrocytes. Mol Brain. 2016;9(1):99. Angkeow P, Deshpande SS, Qi B, Liu YX, Park YC, Jeon BH, et al. Redox factor-1: an extra-nuclear role in the regulation of endothelial oxidative stress and apoptosis. Cell Death & Differentiation. 2002;9(7):717-25. Kim CS, Son SJ, Kim EK, Kim SN, Yoo DG, Kim HS, et al. Apurinic/apyrimidinic endonuclease1/redox factor-1 inhibits monocyte adhesion in endothelial cells. Cardiovasc Res. 2006;69(2):520-6. Tang W, Lin D, Chen M, Li Z, Zhang W, Hu W, et al. PTEN-mediated mitophagy and APE1 overexpression protects against cardiac hypoxia/reoxygenation injury. In Vitro Cell Dev Biol Anim. 2019;55(9):741-8. Hao J, Du H, Liu F, Lu JC, Yang XC, Cui W. Apurinic/apyrimidinic endonuclease/redox factor 1 (APE1) alleviates myocardial hypoxia-reoxygenation injury by inhibiting oxidative stress and ameliorating mitochondrial dysfunction. Exp Ther Med. 2019;17(3):2143-51. Shin JH, Choi S, Lee YR, Park MS, Na YG, Irani K, et al. APE1/Ref-1 as a Serological Biomarker for the Detection of Bladder Cancer. Cancer Res Treat. 2015;47(4):823-33. Jin SA, Lim BK, Seo HJ, Kim SK, Ahn KT, Jeon BH, et al. Elevation of Serum APE1/Ref-1 in Experimental Murine Myocarditis. Int J Mol Sci. 2017;18(12). Hofseth LJ, Khan MA, Ambrose M, Nikolayeva O, Xu-Welliver M, Kartalou M, et al. The adaptive imbalance in base excision–repair enzymes generates microsatellite instability in chronic inflammation. Journal of Clinical Investigation. 2003;112(12):1887-94. Hartman GD, Lambert-Cheatham NA, Kelley MR, Corson TW. Inhibition of APE1/Ref-1 for Neovascular Eye Diseases: From Biology to Therapy. Int J Mol Sci. 2021;22(19). Jedinak A, Dudhgaonkar S, Kelley MR, Sliva D. Apurinic/Apyrimidinic endonuclease 1 regulates inflammatory response in macrophages. Anticancer Res. 2011;31(2):379-85. Sahakian L, Filippone RT, Stavely R, Robinson AM, Yan XS, Abalo R, et al. Inhibition of APE1/Ref-1 Redox Signaling Alleviates Intestinal Dysfunction and Damage to Myenteric Neurons in a Mouse Model of Spontaneous Chronic Colitis. Inflamm Bowel Dis. 2020. Lalunio H, Stupka N, Goodman CA, Hayes A. The Potential of Targeting APE1/Ref-1 as a Therapeutic Intervention for Duchenne Muscular Dystrophy. Antioxid Redox Signal. 2025;42(13-15):641-54. Szczesny B, Tann AW, Mitra S. Age- and tissue-specific changes in mitochondrial and nuclear DNA base excision repair activity in mice: Susceptibility of skeletal muscles to oxidative injury. Mech Ageing Dev. 2010;131(5):330-7. Wang P, Li CG, Qi Z, Cui D, Ding S. Acute exercise stress promotes Ref1/Nrf2 signalling and increases mitochondrial antioxidant activity in skeletal muscle. Exp Physiol. 2016;101(3):410-20. Buck M, Chojkier M. Muscle wasting and dedifferentiation induced by oxidative stress in a murine model of cachexia is prevented by inhibitors of nitric oxide synthesis and antioxidants. The EMBO Journal. 1996;15(8):1753-65. Yuzefovych LV, Musiyenko SI, Wilson GL, Rachek LI. Mitochondrial DNA damage and dysfunction, and oxidative stress are associated with endoplasmic reticulum stress, protein degradation and apoptosis in high fat diet-induced insulin resistance mice. PLoS One. 2013;8(1):e54059. Lalunio H, Goodman CA, Stupka N, Giourmas N, Debruin DA, Sahakian L, et al. APE1/Ref-1 inhibition via APX3330 lowers monocyte/macrophage infiltration without ameliorating the structure and function of dystrophic mdx hindlimb muscles. Physiol Rep. 2025;13(15):e70494. Radley-Crabb HG, Marini JC, Sosa HA, Castillo LI, Grounds MD, Fiorotto ML. Dystropathology increases energy expenditure and protein turnover in the mdx mouse model of duchenne muscular dystrophy. PLoS One. 2014;9(2):e89277. Fishel ML, Jiang Y, Rajeshkumar NV, Scandura G, Sinn AL, He Y, et al. Impact of APE1/Ref-1 redox inhibition on pancreatic tumor growth. Mol Cancer Ther. 2011;10(9):1698-708. Debruin DA, Timpani CA, Lalunio H, Rybalka E, Goodman CA, Hayes A. Exercise May Ameliorate the Detrimental Side Effects of High Vitamin D Supplementation on Muscle Function in Mice. J Bone Miner Res. 2020;35(6):1092-106. Brooks SV, Faulkner JA. Contractile properties of skeletal muscles from young, adult and aged mice. J Physiol. 1988;404:71-82. Close RI. Dynamic properties of mammalian skeletal muscles. Physiol Rev. 1972;52(1):129-97. Grounds M. Quantification of histopathology in haemotoxylin and eosin stained muscle sections. Treat NMD, Neuromuscular Network; DMD_M. 2014;1:1-13. McRae NL, Addinsall AB, Howlett KF, McNeill B, McCulloch DR, Stupka N. Genetic reduction of the extracellular matrix protein versican attenuates inflammatory cell infiltration and improves contractile function in dystrophic mdx diaphragm muscles. Sci Rep. 2020;10(1):11080. Head SI, Williams DA, Stephenson DG. Abnormalities in Structure and Function of Limb Skeletal Muscle Fibres of Dystrophic mdx Mice. Proceedings: Biological Sciences. 1992;248(1322):163-9. Konno RN, Nigam N, Wakeling JM, Ross SA. The Contributions of Extracellular Matrix and Sarcomere Properties to Passive Muscle Stiffness in Cerebral Palsy. Front Physiol. 2021;12:804188. Zollner AM, Abilez OJ, Bol M, Kuhl E. Stretching skeletal muscle: chronic muscle lengthening through sarcomerogenesis. PLoS One. 2012;7(10):e45661. Carnwath JW, Shotton DM. Muscular dystrophy in the mdx mouse: histopathology of the soleus and extensor digitorum longus muscles. J Neurol Sci. 1987;80(1):39-54. Gehrig SM, Koopman R, Naim T, Tjoakarfa C, Lynch GS. Making fast-twitch dystrophic muscles bigger protects them from contraction injury and attenuates the dystrophic pathology. Am J Pathol. 2010;176(1):29-33. Marshall PA, Williams PE, Goldspink G. Accumulation of collagen and altered fiber-type ratios as indicators of abnormal muscle gene expression in the mdx dystrophic mouse. Muscle Nerve. 1989;12(7):528-37. Nogami K, Maruyama Y, Sakai-Takemura F, Motohashi N, Elhussieny A, Imamura M, et al. Pharmacological activation of SERCA ameliorates dystrophic phenotypes in dystrophin-deficient mdx mice. Hum Mol Genet. 2021;30(11):1006-19. Wasala NB, Yue Y, Lostal W, Wasala LP, Niranjan N, Hajjar RJ, et al. Single SERCA2a Therapy Ameliorated Dilated Cardiomyopathy for 18 Months in a Mouse Model of Duchenne Muscular Dystrophy. Molecular Therapy. 2020;28(3):845-54. Fushimi S, Nohno T, Katsuyama H. Chronic Stress Induces Type 2b Skeletal Muscle Atrophy via the Inhibition of mTORC1 Signaling in Mice. Med Sci (Basel). 2023;11(1). Drude S, Geissler A, Olfe J, Starke A, Domanska G, Schuett C, et al. Side effects of control treatment can conceal experimental data when studying stress responses to injection and psychological stress in mice. Lab Anim (NY). 2011;40(4):119-28. Additional Declarations No competing interests reported. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-9507119","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":637797572,"identity":"9282e5c0-b2ce-409e-9e3e-a8a1b1f0af61","order_by":0,"name":"Hannah Lalunio","email":"","orcid":"","institution":"The University of Melbourne","correspondingAuthor":false,"prefix":"","firstName":"Hannah","middleName":"","lastName":"Lalunio","suffix":""},{"id":637797573,"identity":"9709159e-909a-42fe-a27b-be0043158533","order_by":1,"name":"Craig A. Goodman","email":"","orcid":"","institution":"The University of Melbourne","correspondingAuthor":false,"prefix":"","firstName":"Craig","middleName":"A.","lastName":"Goodman","suffix":""},{"id":637797574,"identity":"f7b603ae-bed9-42bf-a45f-ee78dead36bd","order_by":2,"name":"Nicole Stupka","email":"","orcid":"","institution":"Victoria University","correspondingAuthor":false,"prefix":"","firstName":"Nicole","middleName":"","lastName":"Stupka","suffix":""},{"id":637797575,"identity":"d7ad69d7-dced-42d5-83ae-0ce58e7ed39d","order_by":3,"name":"Nicholas Giourmas","email":"","orcid":"","institution":"Victoria University","correspondingAuthor":false,"prefix":"","firstName":"Nicholas","middleName":"","lastName":"Giourmas","suffix":""},{"id":637797576,"identity":"860900a3-1420-4b16-b391-071df5a5c29b","order_by":4,"name":"Lauren Sahakian","email":"","orcid":"","institution":"Victoria University","correspondingAuthor":false,"prefix":"","firstName":"Lauren","middleName":"","lastName":"Sahakian","suffix":""},{"id":637797577,"identity":"8bc499fe-1a4a-482f-a5b1-ed3734609b60","order_by":5,"name":"Kulmira Nurgali","email":"","orcid":"","institution":"Victoria University","correspondingAuthor":false,"prefix":"","firstName":"Kulmira","middleName":"","lastName":"Nurgali","suffix":""},{"id":637797578,"identity":"9c3d0f67-a37b-4e61-a0a2-7f537f88026b","order_by":6,"name":"Alan Hayes","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA60lEQVRIiWNgGAWjYDACdiBmbACxmA8wwNl4ATNcGVsCyVp4DIjTwt/M/OwB4w67xH7+M9+keRhsZDccYH4mgU+LxGE2cwPGM8mJM2fkbgNqSTPecIDNDK8WhsMMZhKMbcyJG27wbjbmYTicuOEAA34t8ofZvwG11CfuP3/mMVDLf6AWoAg+LQaHeUC2AA1nyGF8zMNwAKiFB78thod5yiQS244bz7iRZvhwjkGy8czDPMUW+LTIHW/fJvGxrVq2v//wgwNvKuxk+463b7yBTwsYJDAwODZA3MkAiSligD2R6kbBKBgFo2AkAgBMRUeOspwndwAAAABJRU5ErkJggg==","orcid":"","institution":"Victoria University","correspondingAuthor":true,"prefix":"","firstName":"Alan","middleName":"","lastName":"Hayes","suffix":""}],"badges":[],"createdAt":"2026-04-23 13:08:33","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-9507119/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-9507119/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":109099835,"identity":"d3b00f68-7126-42c3-be9d-b6ffbb614d18","added_by":"auto","created_at":"2026-05-12 14:18:50","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":68281,"visible":true,"origin":"","legend":"\u003cp\u003eForce-frequency relationship, fatigue and recovery. \u003cem\u003eForce-frequency relationship of the \u003c/em\u003e\u003cem\u003e\u003cstrong\u003e(A)\u003c/strong\u003e\u003c/em\u003e\u003cem\u003e EDL and \u003c/em\u003e\u003cem\u003e\u003cstrong\u003e(B)\u003c/strong\u003e\u003c/em\u003e\u003cem\u003e soleus were determined and expressed as a percentage of absolute maximum force (P\u003c/em\u003e\u003csub\u003e\u003cem\u003eO\u003c/em\u003e\u003c/sub\u003e\u003cem\u003e). Muscle fatiguability and recovery for the \u003c/em\u003e\u003cem\u003e\u003cstrong\u003e(C) \u003c/strong\u003e\u003c/em\u003e\u003cem\u003eEDL and \u003c/em\u003e\u003cem\u003e\u003cstrong\u003e(D)\u003c/strong\u003e\u003c/em\u003e\u003cem\u003e soleus were determined and expressed as a percentage of initial force produced. Recovery rate for 0-5mins, 0-1min and 1-5mins were calculated for the \u003c/em\u003e\u003cem\u003e\u003cstrong\u003e(E) \u003c/strong\u003e\u003c/em\u003e\u003cem\u003eEDL and \u003c/em\u003e\u003cem\u003e\u003cstrong\u003e(F) \u003c/strong\u003e\u003c/em\u003e\u003cem\u003esoleus. *p \u0026lt; 0.05, **p \u0026lt; 0.01, ***p \u0026lt; 0.001, ****p \u0026lt; 0.0001, strain effect. \u003c/em\u003e\u003csup\u003e\u003cem\u003e#\u003c/em\u003e\u003c/sup\u003e\u003cem\u003ep \u0026lt; 0.05, \u003c/em\u003e\u003csup\u003e\u003cem\u003e##\u003c/em\u003e\u003c/sup\u003e\u003cem\u003ep \u0026lt; 0.01, \u003c/em\u003e\u003csup\u003e\u003cem\u003e###\u003c/em\u003e\u003c/sup\u003e\u003cem\u003ep \u0026lt; 0.001, \u003c/em\u003e\u003csup\u003e\u003cem\u003e####\u003c/em\u003e\u003c/sup\u003e\u003cem\u003ep \u0026lt; 0.0001, treatment effect. \u003c/em\u003e\u003csup\u003e\u003cem\u003ea\u003c/em\u003e\u003c/sup\u003e\u003cem\u003ep \u0026lt; 0.05, strain difference between control treated groups. \u003c/em\u003e\u003csup\u003e\u003cem\u003eb\u003c/em\u003e\u003c/sup\u003e\u003cem\u003ep \u0026lt; 0.05, \u003c/em\u003e\u003csup\u003e\u003cem\u003ebb\u003c/em\u003e\u003c/sup\u003e\u003cem\u003ep \u0026lt; 0.01, treatment difference between wildtype groups. \u003c/em\u003e\u003csup\u003e\u003cem\u003ec\u003c/em\u003e\u003c/sup\u003e\u003cem\u003ep \u0026lt; 0.05, \u003c/em\u003e\u003csup\u003e\u003cem\u003ecc\u003c/em\u003e\u003c/sup\u003e\u003cem\u003ep \u0026lt; 0.01, treatment difference between mdx groups. n = 8-12. WT = wildtype mice; MDX = mdx mice; CTL = control; APX = APX3330 treated; EDL = extensor digitorum longus; SOL = soleus.\u003c/em\u003e\u003c/p\u003e","description":"","filename":"Picture1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-9507119/v1/568533b7c8f23889f675ebc1.jpg"},{"id":109100009,"identity":"7200d871-f0b0-4120-a0f6-bf40e0f8f028","added_by":"auto","created_at":"2026-05-12 14:19:44","extension":"jpeg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":456828,"visible":true,"origin":"","legend":"\u003cp\u003eHaematoxylin \u0026amp; Eosin (H\u0026amp;E) histological analysis of the extensor digitorum longus (EDL) in wildtype and mdx mice. \u003cem\u003e\u003cstrong\u003e(A)\u003c/strong\u003e\u003c/em\u003e\u003cem\u003e Healthy tissue area expressed as a percentage of total area. \u003c/em\u003e\u003cem\u003e\u003cstrong\u003e(B)\u003c/strong\u003e\u003c/em\u003e\u003cem\u003e Centralised nuclei displayed as a percentage of total fibres counted. \u003c/em\u003e\u003cem\u003e\u003cstrong\u003e(C)\u003c/strong\u003e\u003c/em\u003e\u003cem\u003e Mean fibre size and fibre size distribution in the \u003c/em\u003e\u003cem\u003e\u003cstrong\u003e(D)\u003c/strong\u003e\u003c/em\u003e\u003cem\u003e wildtype and \u003c/em\u003e\u003cem\u003e\u003cstrong\u003e(E)\u003c/strong\u003e\u003c/em\u003e\u003cem\u003e mdx groups. Representative images are depicted in panel \u003c/em\u003e\u003cem\u003e\u003cstrong\u003e(F)\u003c/strong\u003e\u003c/em\u003e\u003cem\u003e. ****p \u0026lt; 0.0001, strain effect. \u003c/em\u003e\u003csup\u003e\u003cem\u003e####\u003c/em\u003e\u003c/sup\u003e\u003cem\u003ep \u0026lt; 0.0001, treatment effect. n = 3-5 per group. WT = wildtype; MDX = mdx mice; CTL = control; APX = APX3330 treated; EDL = extensor digitorum longus.\u003c/em\u003e\u003c/p\u003e","description":"","filename":"floatimage2.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-9507119/v1/10fc2da48eb14fbed35f2c53.jpeg"},{"id":109099998,"identity":"f3bed7df-bbec-42a5-bd33-d0f3a90ca1b1","added_by":"auto","created_at":"2026-05-12 14:19:40","extension":"jpeg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":480017,"visible":true,"origin":"","legend":"\u003cp\u003eHaematoxylin \u0026amp; Eosin (H\u0026amp;E) histological analysis of the soleus (SOL) in wildtype and mdx mice. \u003cem\u003e\u003cstrong\u003e(A)\u003c/strong\u003e\u003c/em\u003e\u003cem\u003e Healthy tissue area expressed as a percentage of total area. \u003c/em\u003e\u003cem\u003e\u003cstrong\u003e(B)\u003c/strong\u003e\u003c/em\u003e\u003cem\u003e Centralised nuclei displayed as a percentage of total fibres counted. \u003c/em\u003e\u003cem\u003e\u003cstrong\u003e(C)\u003c/strong\u003e\u003c/em\u003e\u003cem\u003e Mean fibre size and fibre size distribution in the \u003c/em\u003e\u003cem\u003e\u003cstrong\u003e(D)\u003c/strong\u003e\u003c/em\u003e\u003cem\u003e wildtype and \u003c/em\u003e\u003cem\u003e\u003cstrong\u003e(E)\u003c/strong\u003e\u003c/em\u003e\u003cem\u003e mdx groups. Representative images are depicted in panel \u003c/em\u003e\u003cem\u003e\u003cstrong\u003e(F)\u003c/strong\u003e\u003c/em\u003e\u003cem\u003e. ****p \u0026lt; 0.0001, strain effect. \u003c/em\u003e\u003csup\u003e\u003cem\u003e####\u003c/em\u003e\u003c/sup\u003e\u003cem\u003ep \u0026lt; 0.0001, treatment effect. n = 4-5 per group. WT = wildtype; MDX = mdx mice; CTL = control; APX = APX3330 treated; SOL = soleus.\u003c/em\u003e\u003c/p\u003e","description":"","filename":"floatimage3.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-9507119/v1/2fbfcad88decc4d2f9815ecc.jpeg"},{"id":109099939,"identity":"8bcd5dd6-5c47-45bb-a849-6b7257ec9c27","added_by":"auto","created_at":"2026-05-12 14:19:26","extension":"jpeg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":370601,"visible":true,"origin":"","legend":"\u003cp\u003eSuccinate Dehydrogenase (SDH) histological analysis of the extensor digitorum longus (EDL) and soleus (SOL) in wildtype and mdx mice. \u003cem\u003e\u003cstrong\u003e(A)\u003c/strong\u003e\u003c/em\u003e\u003cem\u003e SDH positive fibres as a percentage of total area in the EDL. \u003c/em\u003e\u003cem\u003e\u003cstrong\u003e(B)\u003c/strong\u003e\u003c/em\u003e\u003cem\u003e SDH positive fibres as a percentage of total area in the SOL. Representative images are depicted in panel \u003c/em\u003e\u003cem\u003e\u003cstrong\u003e(C)\u003c/strong\u003e\u003c/em\u003e\u003cem\u003e. *p ≤ 0.05 strain effect, analysed using a two-way ANOVA, n=3-6 per group. WT = wildtype mice; MDX = mdx mice; CTL = control; APX = APX3330 treated; EDL = extensor digitorum longus; SOL = soleus.\u003c/em\u003e\u003c/p\u003e","description":"","filename":"floatimage4.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-9507119/v1/076952c1579c9d5a4723bebc.jpeg"},{"id":109100007,"identity":"74a26afc-7b38-4049-9c22-071daf16f8a4","added_by":"auto","created_at":"2026-05-12 14:19:43","extension":"jpeg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":443369,"visible":true,"origin":"","legend":"\u003cp\u003ePicrosirius Red (PSR) histological analysis of the extensor digitorum longus (EDL) and soleus (SOL) in wildtype and mdx mice. \u003cem\u003e\u003cstrong\u003e(A)\u003c/strong\u003e\u003c/em\u003e\u003cem\u003e Picrosirius red staining as a percentage of total area in the EDL. \u003c/em\u003e\u003cem\u003e\u003cstrong\u003e(B)\u003c/strong\u003e\u003c/em\u003e\u003cem\u003e Picrosirius red staining as a percentage of total area in the SOL. Representative images are depicted in panel \u003c/em\u003e\u003cem\u003e\u003cstrong\u003e(C)\u003c/strong\u003e\u003c/em\u003e\u003cem\u003e. **p ≤ 0.01 strain effect, analysed using a two-way ANOVA, n=5-6 per group. WT = wildtype mice; MDX = mdx mice; CTL = control; APX = APX3330 treated; EDL = extensor digitorum longus; SOL = soleus.\u003c/em\u003e\u003c/p\u003e","description":"","filename":"floatimage5.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-9507119/v1/be4b22723aba7271be5bba22.jpeg"},{"id":109099892,"identity":"171b4d1d-a23f-48a2-83cf-36428cfbff42","added_by":"auto","created_at":"2026-05-12 14:19:12","extension":"jpeg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":333395,"visible":true,"origin":"","legend":"\u003cp\u003eMonocyte and macrophage infiltration in the extensor digitorum longus (EDL) of wildtype and mdx mice. \u003cem\u003e\u003cstrong\u003e(A) \u003c/strong\u003e\u003c/em\u003e\u003cem\u003eCD68+ monocytes and macrophages were labelled using anti-CD68+ (red) antibody in EDL cross-sections. Connective tissue to visualise muscle architecture is labelled with WGA (green) and nuclei are labelled with nuclei marker DAPI (blue).\u003c/em\u003e\u003cem\u003e\u003cstrong\u003e (B) \u003c/strong\u003e\u003c/em\u003e\u003cem\u003eQuantification of CD68+ cells in EDL cross-sections.\u003c/em\u003e\u003cem\u003e\u003cstrong\u003e \u003c/strong\u003e\u003c/em\u003e\u003cem\u003eScale bar = 50µm, x20 magnification, **p ≤ 0.01 strain effect, analysed using a student’s t-test, n = 2 mice for wildtype groups and n = 6 for mdx groups. WT = wildtype mice; MDX = mdx mice; CTL = control; APX = APX3330 treated; EDL = extensor digitorum longus; SOL = soleus.\u003c/em\u003e\u003c/p\u003e","description":"","filename":"floatimage6.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-9507119/v1/5ca65ce76212e20007348034.jpeg"},{"id":109523604,"identity":"24e15de5-eefc-46aa-96d5-1b02a28a587a","added_by":"auto","created_at":"2026-05-19 06:41:42","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2499019,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-9507119/v1/4a1fc099-f817-47ac-a338-e53567bdf9d9.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"APE1/Ref-1 inhibition via APX3330 in young dystrophic mdx mice","fulltext":[{"header":"Introduction","content":"\u003cp\u003eDuchenne muscular dystrophy (DMD) is one of the most severe forms of childhood muscular dystrophy which affects 1 in every 5,000 boys (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e). DMD is caused by mutations in the \u003cem\u003eDMD\u003c/em\u003e gene resulting in the loss of the cytoskeletal protein, dystrophin, subsequently disrupting the formation of the dystrophin-glycoprotein complex (DGC) (\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e). DGC destabilisation exposes muscle fibres to mechanical stress, with continuous cycles of muscle damage and insufficient repair eventually leading to contractile tissue being replaced by fibroadipose tissue, which is then heightened by inflammation and oxidative stress (\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e). The current standard of care for DMD patients are corticosteroids (prednisolone/deflazacort), potent anti-inflammatory drugs that delay the loss of ambulation and improve respiratory capacity by moderately improving muscle function (\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e). However, adverse side effects are associated with corticosteroids, including weight gain, bone loss, and delayed growth, limiting their effectiveness (\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e). Therefore, investigation into alternative therapies that mitigate inflammation while improving dystrophic muscle function and pathology with a more favourable side-effect profile is necessary.\u003c/p\u003e \u003cp\u003eApurinic/apyrimidinic endonuclease 1/redox factor 1(APE1/Ref-1) is a multifunctional protein initially involved in the base excision repair (BER) pathway that targets DNA lesions that increase oxidative stress (\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e). Another major role of APE/Ref-1 is the redox regulation of transcription factors, in which APE1/Ref-1 reduces transcription factors, including nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB), to promote DNA binding and activation of genes involved in pro-inflammatory and oxidative stress signaling cascades (\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e). Repression of APE1/Ref-1 has shown to induce protective, antioxidant effects by enhancing the activity and abundance of nuclear factor erythroid 2-related factor 2 (NRF2) target proteins (\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e). However, the effects of APE1/Ref-1 inhibition on inflammation and oxidative stress appear to be dependent on biological context with some \u003cem\u003ein vitro\u003c/em\u003e studies reporting protective effects of APE1/Ref-1 overexpression in acute models of inflammation (\u003cspan additionalcitationids=\"CR12 CR13 CR14\" citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e). Alternatively, in chronic inflammation and oxidative stress, constant activation of APE1/Ref-1 may lead to a pathological response. Indeed, chronic inflammatory conditions that involve heightened inflammation and oxidative stress, such as myocarditis and chronic colitis, show an elevated expression of APE1/Ref-1 (\u003cspan additionalcitationids=\"CR17\" citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eAPX3330 is a small molecular inhibitor of APE1/Ref-1 which targets the redox domain, rendering the protein unable to reduce transcription factors, keeping transcription factors in an oxidized and inactive state (\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e). This then inhibits downstream activity of these transcription factors, including those involved in inflammation and oxidative stress pathways. Indeed, APX3330 has shown to reduce inflammatory cytokine expression in RAW264.7 cells (\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e) and the infiltration of CD45 positive immune cells in the colon of Winnie mice, a model of chronic colitis (\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e). Furthermore, in the Winnie mouse, APX3330 decreased mitochondrial superoxide production in the myenteric plexus, alleviating inflammation-induced oxidative stress, which led to neuroprotective effects, reduction in disease severity and restoration of GI function (\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e). The significant reduction in markers of inflammation and oxidative stress in these models indicate potential application in other disease states with similar pathology, including muscular dystrophy (reviewed in (\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e)).\u003c/p\u003e \u003cp\u003eTo date, few studies have evaluated the role of APE1/Ref-1 in skeletal muscle (\u003cspan additionalcitationids=\"CR24 CR25\" citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e). Our lab has previously investigated the inhibition of APE1/Ref-1 via APX3330 in the \u003cem\u003emdx\u003c/em\u003e mouse model, the most commonly used mouse model for DMD, during a period of elevated, yet stable, inflammation (6\u0026ndash;12 weeks of age); however, this did not result in any meaningful benefit beyond reduction in inflammatory markers (\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e). Although APX3330 was unable to alter dystrophic pathology during 6\u0026ndash;12 weeks of age, the pathology is quite established during this time (\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e). To investigate the potential of APX3330 as a preventative, treatment must be administered prior to the onset of the dystrophic phenotype (3\u0026ndash;6 weeks of age) (\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e). Beginning at 3 weeks of age covers a peak degeneration/regeneration period as seen during childhood in DMD patients. Therefore, we hypothesise that APE1/Ref-1 inhibition with APX3330 treatment could delay dystrophic hindlimb muscle pathology in \u003cem\u003emdx\u003c/em\u003e mice if administered within the juvenile time point prior to peak damage and inflammation (3\u0026ndash;6 weeks of age).\u003c/p\u003e"},{"header":"Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eEthics approval and animals\u003c/h2\u003e \u003cp\u003e This study was approved by the Animal Ethics Committee at Victoria University (AEC 22/005). All experiments conformed to the Australian Code for the care and use of animals for scientific purposes (8th ed., 2013). Clinical trial number: not applicable\u003c/p\u003e \u003cp\u003eDystrophic female \u003cem\u003emdx\u003c/em\u003e mice and C57Bl/10 wildtype controls were bred and housed at the Western Centre for Health, Research and Education (WCHRE, Victoria, Australia) in a light- and temperature-controlled room (12h light/dark cycle, 21\u0026deg;C) with \u003cem\u003ead libitum\u003c/em\u003e access to food and water. At 3 weeks of age, wildtype (WT) and \u003cem\u003emdx\u003c/em\u003e mice were randomly allocated into treated (n\u0026thinsp;=\u0026thinsp;12) or untreated groups (n\u0026thinsp;=\u0026thinsp;8). Treatment mice received an intraperitoneal (IP) injection of APX3330 (25 mg\u0026middot;kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) dissolved in Cremophor (2%), ethanol (2%) and sterile water (96%) twice a day for 3 weeks. APX3330 was received as a generous gift by Dr. Mark R Kelley (Indiana University School of Medicine, Indiana, USA). We utilised the same treatment regime used by Fishel et al., 2011 (\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e) and Sahakian et al., 2020 (\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e), where, in models of pancreatic cancer and ulcerative colitis, respectively, APX3330 decreased inflammatory cell infiltration and the activation of inflammatory cell signalling cascades, including NF-κB (\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e). Additionally, the doses were kept the same as our previous study, (\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e), in which APX3330 was effective in lowering macrophages/monocytes, allowing direct comparison, and determine whether APX3330 could be more effective as a preventative treatment during a younger age point, prior to the establishment of the dystrophic phenotype.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eTissue extraction\u003c/h3\u003e\n\u003cp\u003eMice were deeply anaesthetised with 2\u0026ndash;4% isoflurane with oxygen set to a flow rate of 0.6 mL\u0026middot;min\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, and non-survival surgery was performed. Extensor digitorum longus (EDL) and soleus muscles were then tied tendon to tendon with 4.0 suture thread and immediately excised for muscle contractile function analysis. Following the completion of the contractile protocol, muscles were embedded with optimal cutting temperature (OCT) compound (Tissue-Tek OCT Compound; Sakura Finetek, California, USA) in isopentane pre-cooled in liquid nitrogen for histological analysis.\u003c/p\u003e\n\u003ch3\u003eContractile function\u003c/h3\u003e\n\u003cp\u003e \u003cem\u003eEx vivo\u003c/em\u003e evaluation of muscle contractile properties was performed as previously described (\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e). Excised EDL and soleus muscles were placed into a contractile chamber (Danish Myo Technology (DMT) A/S, Hinnerup, Denmark) containing Krebs-Heinseleit Ringer\u0026rsquo;s solution (118 mM NaCl, 4.75 mM KCl, 1 mM Na\u003csub\u003e2\u003c/sub\u003eHPO\u003csub\u003e4\u003c/sub\u003e, 1.18 mM MgSO\u003csub\u003e4\u003c/sub\u003e\u0026middot;7H\u003csub\u003e2\u003c/sub\u003eO, 2.5 mM CaCl\u003csub\u003e2\u003c/sub\u003e, 24.8 mM NaHCO\u003csub\u003e3\u003c/sub\u003e, 11 mM D-Glucose; pH 7.4). Individual baths were bubbled with 5% CO\u003csub\u003e2\u003c/sub\u003e in O\u003csub\u003e2\u003c/sub\u003e (BOC gases, Melbourne, Australia) and maintained at 30\u0026deg;C.\u003c/p\u003e \u003cp\u003eThe proximal end of the muscle was clamped onto a previously calibrated force transducer and the distal end was secured to a micromanipulator with stimulating electrodes flanking the muscle belly. Optimal length (L\u003csub\u003eO\u003c/sub\u003e) was determined for each muscle via a series of twitch contractions at increasing lengths to ensure optimal overlap of sarcomeres, and muscle length was measured with callipers. A force-frequency relationship was established by delivering frequencies of 10, 20, 30, 40, 50, 60, 80, 100, 120 and 150 Hz. Absolute force (maximum isometric contraction; P\u003csub\u003eO\u003c/sub\u003e) was recorded as the highest force obtained in the force-frequency protocol, with other forces reported as a percentage of that maximum. To normalise force produced for different muscle sizes, specific force (sP\u003csub\u003eO\u003c/sub\u003e) was calculated as maximal force produced per cross-sectional area (CSA), using L\u003csub\u003eO\u003c/sub\u003e and muscle mass, assuming the muscle density is 1.06 g/cm\u003csup\u003e3\u003c/sup\u003e and fibre-muscle length ratios are 0.44 for the EDL and 0.71 for the soleus (\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eFollowing the force-frequency protocol, each muscle was stimulated with a single pulse three times in 5 second intervals for the analysis of basic contractile properties (peak twitch force (P\u003csub\u003et\u003c/sub\u003e), time to peak (TTP), and relaxation time (\u0026frac12;RT)). Muscle fatiguability was assessed by sending repeated intermittent electrical stimuli for 3 minutes. To obtain comparable levels of fatigue, the EDL was stimulated every 4 seconds at 100 Hz for 350 ms and the soleus every 2 seconds at 80 Hz for 500 ms. Data was collected and analysed using LabChart Pro version 8.0 software, customised for this experiment (ADInstruments, Dunedin, New Zealand).\u003c/p\u003e\n\u003ch3\u003eHistology\u003c/h3\u003e\n\u003cp\u003e \u003cem\u003eHaematoxylin \u0026amp; Eosin (H\u0026amp;E)\u003c/em\u003e \u0026ndash; To assess muscle morphology, including damaged areas and regenerating fibres, 10 \u0026micro;M cryosections from EDL and soleus muscles were stained with haematoxylin and eosin (H\u0026amp;E) as previously described (\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e). A quantitative estimate of degeneration within each section was determined by classifying areas of inflammatory cell infiltrate and centrally nucleated fibres as \u0026ldquo;unhealthy tissue\u0026rdquo; (\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e). The proportion of centrally nucleated fibres and fibre size and distribution were also assessed.\u003c/p\u003e \u003cp\u003e \u003cem\u003eSuccinate Dehydrogenase (SDH)\u003c/em\u003e \u0026ndash; To assess changes in oxidative capacity, a succinate dehydrogenase (SDH) stain was performed on EDL and soleus muscle sections as previously described (\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e). Briefly, sections were incubated in SDH solution (0.05% nitro blue tetrazolium; 0.2 M sodium succinate; 0.2 M phosphate buffer, pH 7.6) for 1hr at 37\u0026deg;C, fixed in formal saline (0.9% NaCl; 10% formaldehyde) and mounted in glycerol. Images were converted to 8bit, threshold adjusted to exclude non-specific staining and SDH intensity was measured in full cross sections of the muscle.\u003c/p\u003e \u003cp\u003e \u003cem\u003ePicro-Sirius Red (PSR)\u003c/em\u003e \u0026ndash; To assess changes in collagen, a Picro-sirius red (PSR) stain was performed on EDL and soleus muscles. Briefly, sections were airdried then fixed in 10% neutral buffered formalin (NBF). Sections were then incubated in Bouins fixative (formaldehyde; picric acid; acetic acid) overnight, washed under running water and incubated in 0.1% PSR solution (Sirius Red F3B; picric acid) for 1hr and dipped in water. Sections were dehydrated in 3 changes of ethanol and 3 changes of xylene to ensure no further colour changes and mounted in xylene. Images were converted to 8bit, threshold adjusted to exclude non-specific staining and PSR intensity was measured in full cross sections of the muscle.\u003c/p\u003e \u003cp\u003e \u003cem\u003eImaging\u003c/em\u003e \u0026ndash; Following staining, images of whole muscle sections at 200x magnification were captured with a Zeiss Axio Imager Microscope (Zeiss, Germany), running the Metafer 4 V3.13.3 imaging software (MetaSystems, Germany) and stitched together by a VSlide V1.1.120 software (MetaSystems, Germany). All images were analysed using ImageJ software (NIH, Maryland, USA).\u003c/p\u003e\n\u003ch3\u003eImmunohistochemistry for CD68 positive cells\u003c/h3\u003e\n\u003cp\u003eImmunohistochemical analysis was performed as previously described (\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e). Briefly, muscle sections were fixed in 4% paraformaldehyde (PFA) and permeabilised in TBST (Tris-buffered saline with 0.05% Tween-20). Sections were then incubated in a serum free blocking solution (Dako, #X0909) before incubation in primary antibody (anti-CD68, ab125212, Abcam, 1:500) prepared in antibody diluent (Dako, #S3022) at room temperature. The anti-CD68 antibody (ab125212, Abcam, 1:500) was used for the detection of monocyte and macrophage infiltration. Slides were then incubated with fluorophore-conjugated secondary Alexa Fluro 594 goat anti-rabbit antibody (A11012, Thermo Fisher Scientific, 1:1000 in antibody diluent) and counterstained with wheat germ agglutinin (WGA; W11262, Thermo Fisher Scientific, 1:50 in phosphate buffered saline (PBS)), and mounted in mounting medium with DAPI (ab104139, Abcam). Additional sections from untreated \u003cem\u003emdx\u003c/em\u003e mice stained without the primary antibody were used as a negative control. Images of whole sections (100x) and at 200x magnification were taken using the confocal microscope (STELLARIS 5 confocal microscope, Leica Microsystems). Whole muscle sections were analysed using ImageJ software (NIH, Bethsda, MD, USA) and result expressed as cells/mm\u003csup\u003e2\u003c/sup\u003e.\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eAll values are expressed as means\u0026thinsp;\u0026plusmn;\u0026thinsp;SEM. An unpaired t-test was used to evaluate differences in CD68 positive cells between treated and untreated \u003cem\u003emdx\u003c/em\u003e mice. In every other instance, four group comparisons were analysed using a two-way ANOVA with strain and treatment as factors, followed by a Tukey post hoc analysis test where there was a significant interaction. When data was not normally distributed, a Kruskal-Wallis test was performed for four-group comparisons, followed by a Dunn post-hoc analysis test. Differences between groups were considered significant if p\u0026thinsp;\u0026le;\u0026thinsp;0.05. All data analysis was performed using GraphPad Prism 9.0 (GraphPad Software Inc., California, USA).\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eAPX3330 appears to be detrimental to muscle contractile properties\u003c/h2\u003e \u003cp\u003eBody weights were significantly lower in the APX-treated animals (Table I), potentially suggesting impaired growth, although the effect was primarily seen in the wildtype animals. Interestingly, the muscle masses of both muscles in both strains were not different, resulting in higher muscle mass to body mass ratios (MM:BM) in the APX3330-treated wildtype EDL and soleus of both strains.\u003c/p\u003e \u003cp\u003eDystrophic hindlimbs muscles exhibited various indications of muscle weakness compared to their wildtype counterparts. Although specific force was lower in both muscles, weakness was mostly observed in the soleus muscles, including reductions in peak force, time to peak, and \u0026frac12; relaxation time, as only optimal length was increased in \u003cem\u003emdx\u003c/em\u003e EDL muscle compared to wildtypes (Table I). When muscle force at submaximal frequencies was normalised to maximal force output, no change was observed between wildtype and \u003cem\u003emdx\u003c/em\u003e EDL muscles (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA), yet force was significantly reduced at 40\u0026ndash;100 Hz in \u003cem\u003emdx\u003c/em\u003e soleus muscles compared to wildtype (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). No strain differences were observed in the EDL fatigue/recovery results (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC), however, \u003cem\u003emdx\u003c/em\u003e soleus muscles were more fatigue resistant between 90\u0026ndash;120 sec of the fatigue protocol (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eWith the addition of APX3330, wildtype mice displayed decreases in peak force and specific force in both EDL and soleus muscles, despite increases in the MM:BM (Table I), suggesting increased non-contractile material. Force development at higher frequencies were also reduced in the EDL (80 and 120 Hz; Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA) and soleus (80 and 100 Hz; Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB) with treatment. Interestingly, fatigue resistance was enhanced in the EDL and soleus (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC \u0026amp; \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD), yet the overall rate of recovery was lowered in the EDL and unchanged in the soleus (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE \u0026amp; \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eF). These findings suggest that the treatment, and/or stress response induced by the treatment regimen, may hinder growth, increase muscle infiltrate and/or have detrimental effects on muscle contractile properties in wildtype mice.\u003c/p\u003e \u003cp\u003eIn dystrophic EDL muscle, APX3330 increased the time to peak, indicating a delay in the rate of force development, and an increased P\u003csub\u003et\u003c/sub\u003e/P\u003csub\u003eO\u003c/sub\u003e ratio, suggesting increased muscle stiffness and/or shift to a slower contractile phenotype (Table I). The soleus of \u003cem\u003emdx\u003c/em\u003e mice showed an increased MM:BM, enhanced force at 40\u0026ndash;100 Hz, similar to wildtype levels, while increasing fatigue resistance between 120 and 180 sec of the fatigue protocol (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC \u0026amp; \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD). However, the soleus muscles displayed a reduction in absolute and specific force (Table I), thus, it is possible that the muscle is just consistently producing low forces.\u003c/p\u003e \u003cp\u003e \u003cb\u003eTable I. Body mass and muscle morphology characteristics\u003c/b\u003e \u003c/p\u003e\u003ctable border=\"0\" cellspacing=\"0\" cellpadding=\"0\" width=\"99%\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 12px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u003cem\u003e\u003cu\u003eMeasure\u003c/u\u003e\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" style=\"width: 21px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eWT VEH n=8\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" style=\"width: 21px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eWT APX n=12\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" style=\"width: 21px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u003cem\u003emdx\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;VEH\u003cem\u003e\u0026nbsp;\u003c/em\u003en=8\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" style=\"width: 21px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u003cem\u003emdx\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;APX\u003cem\u003e\u0026nbsp;\u003c/em\u003en=12\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 12px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u003cem\u003eBody Mass (g)\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" style=\"width: 21px;\"\u003e\n \u003cp\u003e19.2\u0026nbsp;\u0026plusmn;\u0026nbsp;0.3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" style=\"width: 21px;\"\u003e\n \u003cp\u003e15.6\u0026nbsp;\u0026plusmn;\u0026nbsp;0.3\u003csup\u003e####\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" style=\"width: 21px;\"\u003e\n \u003cp\u003e18.1\u0026nbsp;\u0026plusmn;\u0026nbsp;0.3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" style=\"width: 21px;\"\u003e\n \u003cp\u003e17.8\u0026nbsp;\u0026plusmn;\u0026nbsp;0.3\u003csup\u003e####\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"2\" style=\"width: 12px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u003cem\u003e\u003cu\u003eMuscle Measure\u003c/u\u003e\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"4\" style=\"width: 43px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u003cu\u003eEDL\u003c/u\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"4\" style=\"width: 43px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u003cu\u003eSOL\u003c/u\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 10px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eWT CTL\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003en=8\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 10px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eWT APX\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003en=12\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 10px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u003cem\u003emdx\u003c/em\u003e CTL\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003en=8\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 11px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u003cem\u003emdx\u003c/em\u003e APX\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003en=12\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 10px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eWT CTL\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003en=8\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 10px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eWT APX\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003en=12\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 10px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u003cem\u003emdx\u003c/em\u003e CTL\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003en=8\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 10px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u003cem\u003emdx\u003c/em\u003e APX\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003en=12\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 12px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u003cem\u003eMass (mg)\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 10px;\"\u003e\n \u003cp\u003e6.4 \u0026plusmn; 0.2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 10px;\"\u003e\n \u003cp\u003e8.7 \u0026plusmn; 1.7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 10px;\"\u003e\n \u003cp\u003e7.3 \u0026plusmn; 0.3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 11px;\"\u003e\n \u003cp\u003e8.0 \u0026plusmn; 0.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 10px;\"\u003e\n \u003cp\u003e6.5 \u0026plusmn; 0.3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 10px;\"\u003e\n \u003cp\u003e7.2 \u0026plusmn; 0.9\u003csup\u003e^\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 10px;\"\u003e\n \u003cp\u003e6.7 \u0026plusmn; 0.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 10px;\"\u003e\n \u003cp\u003e7.3 \u0026plusmn; 0.4\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 12px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u003cem\u003eMM/BM\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 10px;\"\u003e\n \u003cp\u003e0.33 \u0026plusmn; 0.01\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 10px;\"\u003e\n \u003cp\u003e0.50 \u0026plusmn; 0.05\u003csup\u003e##\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 10px;\"\u003e\n \u003cp\u003e0.40 \u0026plusmn; 0.02\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 11px;\"\u003e\n \u003cp\u003e0.42 \u0026plusmn; 0.02\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 10px;\"\u003e\n \u003cp\u003e0.34 \u0026plusmn; 0.01\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 10px;\"\u003e\n \u003cp\u003e0.46 \u0026plusmn; 0.05\u003csup\u003e#\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 10px;\"\u003e\n \u003cp\u003e0.37 \u0026plusmn; 0.03\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 10px;\"\u003e\n \u003cp\u003e0.41 \u0026plusmn; 0.03\u003csup\u003e#\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 12px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u003cem\u003eL\u003csub\u003eO\u003c/sub\u003e (mm)\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 10px;\"\u003e\n \u003cp\u003e9.3 \u0026plusmn; 0.3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 10px;\"\u003e\n \u003cp\u003e8.6 \u0026plusmn; 0.2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 10px;\"\u003e\n \u003cp\u003e9.6 \u0026plusmn; 0.3\u003csup\u003e*\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 11px;\"\u003e\n \u003cp\u003e9.7 \u0026plusmn; 0.1\u003csup\u003e*\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 10px;\"\u003e\n \u003cp\u003e8.8 \u0026plusmn; 0.2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 10px;\"\u003e\n \u003cp\u003e8.7 \u0026plusmn; 0.1\u003csup\u003e^\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 10px;\"\u003e\n \u003cp\u003e8.6 \u0026plusmn; 0.2\u003csup\u003e^\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 10px;\"\u003e\n \u003cp\u003e8.5 \u0026plusmn; 0.2\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 12px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u003cem\u003eP\u003csub\u003et\u003c/sub\u003e (mN)\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 10px;\"\u003e\n \u003cp\u003e28.7 \u0026plusmn; 3.9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 10px;\"\u003e\n \u003cp\u003e20.9 \u0026plusmn; 2.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 10px;\"\u003e\n \u003cp\u003e20.0 \u0026plusmn; 1.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 11px;\"\u003e\n \u003cp\u003e23.1 \u0026plusmn; 2.7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 10px;\"\u003e\n \u003cp\u003e16.6 \u0026plusmn; 2.2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 10px;\"\u003e\n \u003cp\u003e15.5 \u0026plusmn; 1.2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 10px;\"\u003e\n \u003cp\u003e12.7 \u0026plusmn; 1.3\u003csup\u003e*\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 10px;\"\u003e\n \u003cp\u003e12.2 \u0026plusmn; 0.8\u003csup\u003e*\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 12px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u003cem\u003eTTP (ms)\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 10px;\"\u003e\n \u003cp\u003e26.0 \u0026plusmn; 0.3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 10px;\"\u003e\n \u003cp\u003e27.2 \u0026plusmn; 0.3\u003csup\u003e#\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 10px;\"\u003e\n \u003cp\u003e26.5 \u0026plusmn; 0.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 11px;\"\u003e\n \u003cp\u003e27.2 \u0026plusmn; 0.3\u003csup\u003e#\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 10px;\"\u003e\n \u003cp\u003e32.7 \u0026plusmn; 0.9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 10px;\"\u003e\n \u003cp\u003e33.4 \u0026plusmn; 0.5\u003csup\u003e^\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 10px;\"\u003e\n \u003cp\u003e31.8 \u0026plusmn; 0.5\u003csup\u003e*\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 10px;\"\u003e\n \u003cp\u003e32.0 \u0026plusmn; 0.4\u003csup\u003e^*\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 12px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u003cem\u003e\u0026frac12; RT (ms)\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 10px;\"\u003e\n \u003cp\u003e16.6 \u0026plusmn; 0.7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 10px;\"\u003e\n \u003cp\u003e18.3 \u0026plusmn; 0.3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 10px;\"\u003e\n \u003cp\u003e18.6 \u0026plusmn; 1.6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 11px;\"\u003e\n \u003cp\u003e19.6 \u0026plusmn; 0.6\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 10px;\"\u003e\n \u003cp\u003e27.5 \u0026plusmn; 1.4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 10px;\"\u003e\n \u003cp\u003e28.8 \u0026plusmn; 0.9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 10px;\"\u003e\n \u003cp\u003e24.4 \u0026plusmn; 0.8\u003csup\u003e**\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 10px;\"\u003e\n \u003cp\u003e25.4 \u0026plusmn; 0.8\u003csup\u003e**\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 12px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u003cem\u003eP\u003csub\u003eO\u003c/sub\u003e (mN)\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 10px;\"\u003e\n \u003cp\u003e150.1 \u0026plusmn; 14.3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 10px;\"\u003e\n \u003cp\u003e96.0 \u0026plusmn; 5.0\u003csup\u003e^##\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 10px;\"\u003e\n \u003cp\u003e113.7 \u0026plusmn; 9.4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 11px;\"\u003e\n \u003cp\u003e104.1 \u0026plusmn; 11.6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 10px;\"\u003e\n \u003cp\u003e136.5 \u0026plusmn; 11.8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 10px;\"\u003e\n \u003cp\u003e120.2 \u0026plusmn; 6.4\u003csup\u003e^#\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 10px;\"\u003e\n \u003cp\u003e109.8 \u0026plusmn; 7.5\u003csup\u003e**\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 10px;\"\u003e\n \u003cp\u003e89.8 \u0026plusmn; 6.8\u003csup\u003e**#\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 12px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u003cem\u003eP\u003csub\u003et\u0026nbsp;\u003c/sub\u003e/P\u003csub\u003eO\u003c/sub\u003e\u0026nbsp;\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 10px;\"\u003e\n \u003cp\u003e0.19 \u0026plusmn; 0.01\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 10px;\"\u003e\n \u003cp\u003e0.20 \u0026plusmn; 0.01\u003csup\u003e##\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 10px;\"\u003e\n \u003cp\u003e0.18 \u0026plusmn; 0.01\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 11px;\"\u003e\n \u003cp\u003e0.22 \u0026plusmn; 0.01\u003csup\u003e^##\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 10px;\"\u003e\n \u003cp\u003e0.12 \u0026plusmn; 0.01\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 10px;\"\u003e\n \u003cp\u003e0.13 \u0026plusmn; 0.00\u003csup\u003e^\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 10px;\"\u003e\n \u003cp\u003e0.11 \u0026plusmn; 0.01\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 10px;\"\u003e\n \u003cp\u003e0.21 \u0026plusmn; 0.07\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 12px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u003cem\u003eCSA (mm\u003csup\u003e2\u003c/sup\u003e)\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 10px;\"\u003e\n \u003cp\u003e0.15 \u0026plusmn; 0.00\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 10px;\"\u003e\n \u003cp\u003e0.21 \u0026plusmn; 0.03\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 10px;\"\u003e\n \u003cp\u003e0.16 \u0026plusmn; 0.01\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 11px;\"\u003e\n \u003cp\u003e0.17 \u0026plusmn; 0.01\u003csup\u003e^\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 10px;\"\u003e\n \u003cp\u003e0.16 \u0026plusmn; 0.01\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 10px;\"\u003e\n \u003cp\u003e0.20 \u0026plusmn; 0.03\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 10px;\"\u003e\n \u003cp\u003e0.16 \u0026plusmn; 0.01\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 10px;\"\u003e\n \u003cp\u003e0.19 \u0026plusmn; 0.01\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 12px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u003cem\u003esP\u003csub\u003eO\u003c/sub\u003e (N/cm\u003csup\u003e2\u003c/sup\u003e)\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 10px;\"\u003e\n \u003cp\u003e10.3 \u0026plusmn; 1.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 10px;\"\u003e\n \u003cp\u003e5.9 \u0026plusmn; 0.6\u003csup\u003e###\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 10px;\"\u003e\n \u003cp\u003e6.5 \u0026plusmn; 0.4\u003csup\u003e^*\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 11px;\"\u003e\n \u003cp\u003e6.3 \u0026plusmn; 0.8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 10px;\"\u003e\n \u003cp\u003e8.7 \u0026plusmn; 0.6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 10px;\"\u003e\n \u003cp\u003e6.7 \u0026plusmn; 0.9\u003csup\u003e#\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 10px;\"\u003e\n \u003cp\u003e7.0 \u0026plusmn; 0.6\u003csup\u003e*\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 10px;\"\u003e\n \u003cp\u003e5.2 \u0026plusmn; 0.6\u003csup\u003e*#\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u003cem\u003eAbbreviations: WT = wildtype mice; MDX = mdx mice; CTL = control; APX = APX3330 treated; EDL = extensor digitorum longus; SOL = soleus; MM = muscle mass; BM = body mass; L\u003csub\u003eO\u003c/sub\u003e = optimal length; P\u003csub\u003et\u003c/sub\u003e = peak twitch; TTP = time to peak; \u0026frac12; RT = half relaxation time; P\u003csub\u003eO\u003c/sub\u003e = peak tetanic absolute force; CSA = cross-sectional area; sP\u003csub\u003eO\u003c/sub\u003e = specific force. Symbols indicate: ^ n = n-1. *p \u0026lt; 0.05, **p \u0026lt; 0.01, strain effect. \u003csup\u003e#\u003c/sup\u003ep \u0026lt; 0.05, \u003csup\u003e##\u003c/sup\u003ep \u0026lt; 0.01, \u003csup\u003e###\u003c/sup\u003ep \u0026lt; 0.001, \u003csup\u003e####\u003c/sup\u003ep \u0026lt; 0.0001, treatment effect.\u003c/em\u003e\u003csup\u003e\u0026nbsp;\u003c/sup\u003e\u003c/p\u003e\n\u003cp\u003eAPX3330 increases mean fibre size in mdx muscles, yet does not alter oxidative capacity or fibrosis in hindlimbs\u003c/p\u003e\n\u003cp\u003eAs anticipated, dystrophic \u003cem\u003emdx\u003c/em\u003e mice exhibited a lower percentage of healthy tissue area and a higher proportion of centralised nuclei in both EDL (Figure 2A \u0026amp; 2B) and soleus (Figure 3A \u0026amp; 3B) muscles, compared to wildtype mice.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eIn the wildtype mice, APX3330 treatment resulted in a decrease in the average fibre area in the EDL muscles (Figure 2C), indicating a leftward shift in fibre size distribution (Figure 2D). Conversely, treatment had the opposite effect on the WT soleus muscles, showing an increase in the mean fibre area and a rightward shift in fibre distribution (Figure 3C \u0026amp; 3D). \u0026nbsp;\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eWhen treated with APX3330, the mean fibre area increased in both the EDL (Figure 2C \u0026amp; 2E) and soleus (Figure 3C \u0026amp; 2E) muscles of \u003cem\u003emdx\u003c/em\u003e mice. Initially, this might suggest larger fibres and, therefore, increased strength. However, since the treatment also exacerbated muscle weakness, it may be characteristic of oedema-induced swelling or displaying a pseudo-hypertrophic phenotype, such that the quality of the contractile tissue is diminished despite the increase in size.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAdditionally, \u003cem\u003emdx\u003c/em\u003e mice displayed a lower oxidative capacity in the EDL (Figure 4A), with no changes in the soleus (Figure 4B), and increased fibrosis in the soleus (p\u0026lt;0.01; Figure 5B). No increase in fibrosis was detected in the EDL (Figure 5A), possibly due to variability in values obtained in the WT control group, as the picrosirius red staining was also observed within the fibres, which was not the case in the other groups, and would have inflated the percentage area values.\u003c/p\u003e\n\u003ch2\u003eAPX3330 treatment increased monocyte and macrophage infiltration in the EDL\u003c/h2\u003e\n\u003cp\u003eInfiltration of CD68 positive monocytes and macrophages were elevated in \u003cem\u003emdx\u003c/em\u003e EDL muscle compared to WT mice (Figure 6). This elevation was further increased significantly in APX3330 treated \u003cem\u003emdx\u003c/em\u003e mice. This is contrary to our previous results in older mice in which the number of CD68 positive cells was lowered in APX3330 treated \u003cem\u003emdx\u003c/em\u003e mice compared to vehicle treated \u003cem\u003emdx\u003c/em\u003e mice (27). This could indicate that the physical action of vehicle and/or drug administration could contribute to cell infiltration, as the control group in this study were not treated with a vehicle. However, it is important to note that the CD68 results of the \u003cem\u003emdx\u003c/em\u003e control group in this study are also similar to the \u003cem\u003emdx\u003c/em\u003e vehicle group in our previous study, indicating that the addition of vehicle had little effect on the number of CD68 positive cells and, instead, that APX3330 may not be effective at this dosage and/or age point.\u0026nbsp;\u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eAlthough, in our previous study, APX3330 was able to reduce inflammatory cell infiltration in \u003cem\u003emdx\u0026nbsp;\u003c/em\u003emice during the relatively stable phase of dystrophic pathology (6-12 wks), it did not induce profound changes in muscle pathophysiology (27). Given that APX3330 has previously demonstrated the ability to decrease inflammation and improve disease outcomes in models characterised by chronic, heightened inflammation, it was hypothesised that APX3330 would be more beneficial during the peak of damage and repair in \u003cem\u003emdx\u003c/em\u003e dystrophic pathology, when inflammation is at its highest (3-6 wks). However, treatment with APX3330 resulted in stunted growth and impaired muscle force in wildtype mice. Similar observations were made in \u003cem\u003emdx\u003c/em\u003e mice treated with APX3330, although force development and fatigue resistance in the soleus appeared to be improved. Interestingly, APX3330 treatment in \u003cem\u003emdx\u003c/em\u003e EDL muscle increased the number of CD68 positive cells, indicating that the treatment caused more inflammation than it resolved. Overall, these findings suggest that APX3330 may not be a suitable treatment for dystrophic pathology, with the current dose and/or mechanism of action insufficient to ameliorate muscle dystrophy, during a peak damage/repair period.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;Within wildtype mice, the administration of APX3330 resulted in a decrease in body weight but an increase in the ratio of muscle mass to body mass. This initially suggests that muscle mass is maintained despite the reduction in overall body mass. However, the hindlimb muscles of the treated mice exhibited lower absolute and specific force compared to the control group, as well as reduced force development at higher frequencies. These findings indicate that the treatment and/or the associated stress response from the treatment regimen may hinder growth, cause infiltration of non-contractile material and/or negatively affect muscle contractile properties. Interestingly, APX3330 treatment enhanced fatigue resistance and coupled with the decrease in rate of recovery, implies that muscle force is preserved and a possible shift to slower fibres, particularly in the EDL muscles. However, it is also important to note that this could be attributed to already low maximal forces, making further reduction difficult. When evaluating fibre area, the treatment led to a decrease fibre area in the EDL, while an increase was observed in the soleus. This observation further suggests the preservation of slow twitch fibre phenotype and/or the loss of fast twitch fibres. Overall, the treatment in wildtype mice appears to the weaken muscles and inhibit growth, thus presenting a negative impact on these mice. \u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;Despite the detrimental effects of APX3330 on wildtype muscle, the objective of this study was to investigate potential therapeutic effects on dystrophic \u003cem\u003emdx\u003c/em\u003e muscle. DMD pathology is typically characterised by preferential damage toward fast-twitch fibres which is also observed in \u003cem\u003emdx\u003c/em\u003e mice (35). However, this study found that \u003cem\u003emdx\u003c/em\u003e mice exhibited various signs of muscle weakness in the slow-twitch soleus muscle, including reductions in peak twitch, peak force, specific force, time to peak and \u0026frac12; relaxation time. Additionally, the \u003cem\u003emdx\u003c/em\u003e soleus muscles showed a diminished force frequency response, further indicating muscle weakness. Conversely, the EDL muscles of \u003cem\u003emdx\u003c/em\u003e mice only demonstrated a lower specific force and an increase in optimal length which could be attributed to multiple factors, such as increased fibrosis, altered muscle architecture or a compensatory mechanism aiming to optimise force production (36, 37). However, this alteration was the only functional measure that displayed a change. As anticipated, \u003cem\u003emdx\u003c/em\u003e mice exhibited a decrease in healthy tissue area and an increase in the proportion of centrally located nuclei in both the EDL and soleus muscles. EDL \u003cem\u003emdx\u003c/em\u003e muscles showed a decrease in oxidative capacity while no change was seen in the soleus. Fibrosis was increased in the soleus muscles, yet this change was not significant in the EDL, possibly due to variation in the wildtype control group that displayed red staining within the fibre compared to the other groups. The changes observed in the histopathology are all indicative of the typical dystrophic phenotype (38-40).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eTreatment in \u003cem\u003emdx\u003c/em\u003e mice resulted in various changes in muscle contractile properties. Specifically, in the EDL, there was an increase in MM:BM, peak twitch to peak tetanic force ratio, time to peak, and mean fibre area. These findings suggest a delayed rate of force development and reduced elasticity. These changes could indicate a shift to a slower phenotype or a possible increase in calcium influx, which may be associated with increase twitch tension. Indeed, studies have shown calcium regulatory dysfunction in \u003cem\u003emdx\u003c/em\u003e mice and subsequent amelioration with sarco/endoplasmic reticulum calcium ATPase (SERCA) activation (41, 42), however, further investigation involving calcium uptake and SERCA activity would be necessary to determine the exact cause of these changes. Alternatively, muscle weakness could also be resultant of the stress response as the treatment regimen is quite invasive. Decreases in body weight, skeletal muscle mass and grip strength in mice after being subjected to repetitive water-immersion restraint stress has been observed (43). Interestingly, in the soleus muscle, treatment led to an increase in force at lower frequencies of activation, seemingly returning to wildtype levels. Furthermore, the treatment enhanced fatigue resistance. At face value, these changes appear to be beneficial, however, it is important to note that these changes could also result from overall low maximal forces, as observed by decreases in absolute and specific force, similar to the effects seen in wildtype mice. This suggests that the treated muscles are less efficient at generating force and exhibit impaired contractile function, which negatively impacts motor function. In addition, APX3330 treatment was not able to ameliorate dystrophic pathology as there were no improvements in either healthy tissue area or the proportion of centrally nucleated fibres. However, treatment was able to increase mean fibre area. Usually, increases in fibre area are often associated with improved muscle strength, yet as lower peak and specific forces were observed, it does not appear to be as a result of increased contractile material. Further work would be needed to investigate the cause. Ultimately, due to reductions in maximal force-generating capacity, it appears that APX3330 treatment is not effectively addressing the underlying muscle weakness that is associated with dystrophic muscles.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eWhile interpreting the results of this study, it is important to note that treated mice were compared to untreated control mice. Our previous study did not indicate any treatment-based differences between vehicle treated and APX3330 treated groups within the wildtype mice (27), indicating the relative safety of the APX3330 drug. However, due to the rigorous treatment regime of twice daily injections, it is also important to consider the effects of the entire regimen independent of vehicle-related effects. Previous studies have also reported that mice receiving just two intraperitoneal injections of saline 24 hours apart, was enough to significantly increase corticosterone levels compared to mice that were not handled and mice that received only one saline injection (44). Minimising confounding factors, such as injection stress, would reveal if treatment effects were benefiting the dystrophic pathology or if the vehicle and/or treatment regime caused more damage which could then be ameliorated by the drug. Although a vehicle group was ultimately omitted from this study, the current study revealed that APX3330 treatment had a more detrimental impact on muscle in both wildtype and dystrophic mice. This important finding suggests the need for a more potent drug that would require less injections, or an alternative mode of administration including orally or slow-release pellets. Higher doses of APX3330 may also be required to further inhibit inflammation and have a positive impact on muscle pathology and function in \u003cem\u003emdx\u003c/em\u003e mice. Additional dosage studies may be required to determine the efficacy of APX3330 in skeletal muscle and whether these changes are due to direct action in the muscle or indirect action via alternative pathways. Given the ambiguous nature of APE1, it may be upregulated in dystrophy, as observed previously (27), in attempts to resolve inflammation and oxidative stress. Therefore, stimulation studies should also be considered to determine its role in skeletal muscle.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eIn this study, the administration of APX3330 treatment, coupled with a treatment regimen administered twice daily, demonstrated deleterious effects in wildtype mice and limited benefits in dystrophic mice. To evaluate the separate impacts of the treatment regimen and the treatment more effectively itself, it is crucial to include a control group receiving a vehicle. Furthermore, the investigation of alternative methods for drug administration may result in more advantageous outcomes. The exploration of different delivery routes or dosing schedules could mitigate the stress response and provide guidance for more efficacious treatment strategies. This study further corroborates the notion that solely targeting inflammation is insufficient to improve dystrophic pathology.\u0026nbsp;\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cdiv class=\"DefinitionList\"\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eDMD\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eDuchenne muscular dystrophy\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eAPE1/Ref\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003e1\u0026ndash;Apurinic/apyrimidinic endonuclease 1/redox factor 1\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eWT\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ewildtype\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eEDL\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eextensor digitorum longus\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eDGC\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003edystrophin\u0026ndash;glycoprotein complex\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eBER\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ebase excision repair\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eNF\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eκB\u0026ndash;nuclear factor kappa\u0026ndash;light\u0026ndash;chain\u0026ndash;enhancer of activated B cells\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eNRF2\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003enuclear factor erythroid 2\u0026ndash;related factor 2\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eAEC\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eAnimal Ethics Committee\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eWCHRE\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eWestern Centre for Health, Research and Education\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eIP\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eintraperitoneal\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eOCT\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eoptimal cutting temperature\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eDMT\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eDanish Myo Technology\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eNaCl\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003esodium chloride\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eKCl\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003epotassium chloride\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eNa\u003csub\u003e2\u003c/sub\u003eHPO\u003csub\u003e4\u003c/sub\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003esodium phosphate dibasic\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eMgSO\u003csub\u003e4\u003c/sub\u003e\u0026middot;7H\u003csub\u003e2\u003c/sub\u003eO\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003emagnesium sulphate heptahydrate\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eCaCl\u003csub\u003e2\u003c/sub\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ecalcium chloride\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eNaHCO\u003csub\u003e3\u003c/sub\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003esodium bicarbonate\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eCO\u003csub\u003e2\u003c/sub\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ecarbon dioxide\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eO\u003csub\u003e2\u003c/sub\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eoxygen\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eL\u003csub\u003eO\u003c/sub\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eoptimal length\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eP\u003csub\u003eO\u003c/sub\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003epeak tetanic force\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003esP\u003csub\u003eO\u003c/sub\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003especific force\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eCSA\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ecross sectional area\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eP\u003csub\u003et\u003c/sub\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003epeak twitch force\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eTTP\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003etime to peak\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u0026frac12;RT\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ehalf relaxation time\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eH\u0026amp;E\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ehaematoxylin and eosin\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eSDH\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003esuccinate dehydrogenase\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003ePSR\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003epicro\u0026ndash;sirius red\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eNBF\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eneutral buffered formalin\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003ePFA\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eparaformaldehyde\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eTBST\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003etris\u0026ndash;buffered saline with Tween\u0026ndash;20\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003ePBS\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ephosphate buffered saline\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eSEM\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003estandard error of mean\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eANOVA\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eanalysis of variance\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eSOL\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003esoleus\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eMM\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003emuscle mass\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eBM\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ebody mass\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eMDX\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003e \u003cem\u003emdx\u003c/em\u003e mice\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eCTL\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003econtrol\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eAPX\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eAPX3330\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eSERCA\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003esarco/endoplasmic reticulum calcium ATPase\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthical Publication Statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe confirm that we have read the Journal\u0026rsquo;s position on issues involved in ethical publication and affirm that this report is consistent with those guidelines.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDisclosure of Conflicts of Interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNone of the authors has any conflict of interest to disclose.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthorship Contribution Statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eH.L: Investigation, data collection and analysis, writing \u0026ndash; original draft, review and editing. C.A.G: Conceptualization, supervision, writing \u0026ndash; review and editing. N.S: Resources, visualization, writing \u0026ndash; review and editing. N.G: Data collection, writing \u0026ndash; review and editing. L.S: Resources, writing \u0026ndash; review and editing. K.N: Resources, writing \u0026ndash; review and editing. A.H: Conceptualization, supervision, writing \u0026ndash; review and editing.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Availability Statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors confirm that the data supporting the findings of this study are available within the article and/or its supplementary materials.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding Statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNo financial support for the research, authorship, and/or publication of this article was received.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eKariyawasam D, D\u0026rsquo;Silva A, Mowat D, Russell J, Sampaio H, Jones K, et al. Incidence of Duchenne muscular dystrophy in the modern era; an Australian study. European Journal of Human Genetics. 2022;30(12):1398-404.\u003c/li\u003e\n\u003cli\u003eHoffman EP, Brown RH, Jr., Kunkel LM. Dystrophin: the protein product of the Duchenne muscular dystrophy locus. Cell. 1987;51(6):919-28.\u003c/li\u003e\n\u003cli\u003eAllen DG, Whitehead NP, Froehner SC. Absence of Dystrophin Disrupts Skeletal Muscle Signaling: Roles of Ca2+, Reactive Oxygen Species, and Nitric Oxide in the Development of Muscular Dystrophy. Physiol Rev. 2016;96(1):253-305.\u003c/li\u003e\n\u003cli\u003eLi W, Zheng Y, Zhang W, Wang Z, Xiao J, Yuan Y. Progression and variation of fatty infiltration of the thigh muscles in Duchenne muscular dystrophy, a muscle magnetic resonance imaging study. Neuromuscul Disord. 2015;25(5):375-80.\u003c/li\u003e\n\u003cli\u003eDesguerre I, Mayer M, Leturcq F, Barbet JP, Gherardi RK, Christov C. Endomysial fibrosis in Duchenne muscular dystrophy: a marker of poor outcome associated with macrophage alternative activation. J Neuropathol Exp Neurol. 2009;68(7):762-73.\u003c/li\u003e\n\u003cli\u003eMah JK. An Overview of Recent Therapeutics Advances for Duchenne Muscular Dystrophy. Methods Mol Biol. 2018;1687:3-17.\u003c/li\u003e\n\u003cli\u003eBuchman AL. Side effects of corticosteroid therapy. J Clin Gastroenterol. 2001;33(4):289-94.\u003c/li\u003e\n\u003cli\u003eFung H, Demple B. A vital role for Ape1/Ref1 protein in repairing spontaneous DNA damage in human cells. Mol Cell. 2005;17(3):463-70.\u003c/li\u003e\n\u003cli\u003eKelley MR, Georgiadis MM, Fishel ML. APE1/Ref-1 role in redox signaling: translational applications of targeting the redox function of the DNA repair/redox protein APE1/Ref-1. Curr Mol Pharmacol. 2012;5(1):36-53.\u003c/li\u003e\n\u003cli\u003eFishel ML, Wu X, Devlin CM, Logsdon DP, Jiang Y, Luo M, et al. Apurinic/Apyrimidinic Endonuclease/Redox Factor-1 (APE1/Ref-1) Redox Function Negatively Regulates NRF2. Journal of Biological Chemistry. 2015;290(5):3057-68.\u003c/li\u003e\n\u003cli\u003eBaek H, Lim CS, Byun HS, Cho HS, Lee YR, Shin YS, et al. The anti-inflammatory role of extranuclear apurinic/apyrimidinic endonuclease 1/redox effector factor-1 in reactive astrocytes. Mol Brain. 2016;9(1):99.\u003c/li\u003e\n\u003cli\u003eAngkeow P, Deshpande SS, Qi B, Liu YX, Park YC, Jeon BH, et al. Redox factor-1: an extra-nuclear role in the regulation of endothelial oxidative stress and apoptosis. Cell Death \u0026amp; Differentiation. 2002;9(7):717-25.\u003c/li\u003e\n\u003cli\u003eKim CS, Son SJ, Kim EK, Kim SN, Yoo DG, Kim HS, et al. Apurinic/apyrimidinic endonuclease1/redox factor-1 inhibits monocyte adhesion in endothelial cells. Cardiovasc Res. 2006;69(2):520-6.\u003c/li\u003e\n\u003cli\u003eTang W, Lin D, Chen M, Li Z, Zhang W, Hu W, et al. PTEN-mediated mitophagy and APE1 overexpression protects against cardiac hypoxia/reoxygenation injury. In Vitro Cell Dev Biol Anim. 2019;55(9):741-8.\u003c/li\u003e\n\u003cli\u003eHao J, Du H, Liu F, Lu JC, Yang XC, Cui W. Apurinic/apyrimidinic endonuclease/redox factor 1 (APE1) alleviates myocardial hypoxia-reoxygenation injury by inhibiting oxidative stress and ameliorating mitochondrial dysfunction. Exp Ther Med. 2019;17(3):2143-51.\u003c/li\u003e\n\u003cli\u003eShin JH, Choi S, Lee YR, Park MS, Na YG, Irani K, et al. APE1/Ref-1 as a Serological Biomarker for the Detection of Bladder Cancer. Cancer Res Treat. 2015;47(4):823-33.\u003c/li\u003e\n\u003cli\u003eJin SA, Lim BK, Seo HJ, Kim SK, Ahn KT, Jeon BH, et al. Elevation of Serum APE1/Ref-1 in Experimental Murine Myocarditis. Int J Mol Sci. 2017;18(12).\u003c/li\u003e\n\u003cli\u003eHofseth LJ, Khan MA, Ambrose M, Nikolayeva O, Xu-Welliver M, Kartalou M, et al. The adaptive imbalance in base excision\u0026ndash;repair enzymes generates microsatellite instability in chronic inflammation. Journal of Clinical Investigation. 2003;112(12):1887-94.\u003c/li\u003e\n\u003cli\u003eHartman GD, Lambert-Cheatham NA, Kelley MR, Corson TW. Inhibition of APE1/Ref-1 for Neovascular Eye Diseases: From Biology to Therapy. Int J Mol Sci. 2021;22(19).\u003c/li\u003e\n\u003cli\u003eJedinak A, Dudhgaonkar S, Kelley MR, Sliva D. Apurinic/Apyrimidinic endonuclease 1 regulates inflammatory response in macrophages. Anticancer Res. 2011;31(2):379-85.\u003c/li\u003e\n\u003cli\u003eSahakian L, Filippone RT, Stavely R, Robinson AM, Yan XS, Abalo R, et al. Inhibition of APE1/Ref-1 Redox Signaling Alleviates Intestinal Dysfunction and Damage to Myenteric Neurons in a Mouse Model of Spontaneous Chronic Colitis. Inflamm Bowel Dis. 2020.\u003c/li\u003e\n\u003cli\u003eLalunio H, Stupka N, Goodman CA, Hayes A. The Potential of Targeting APE1/Ref-1 as a Therapeutic Intervention for Duchenne Muscular Dystrophy. Antioxid Redox Signal. 2025;42(13-15):641-54.\u003c/li\u003e\n\u003cli\u003eSzczesny B, Tann AW, Mitra S. Age- and tissue-specific changes in mitochondrial and nuclear DNA base excision repair activity in mice: Susceptibility of skeletal muscles to oxidative injury. Mech Ageing Dev. 2010;131(5):330-7.\u003c/li\u003e\n\u003cli\u003eWang P, Li CG, Qi Z, Cui D, Ding S. Acute exercise stress promotes Ref1/Nrf2 signalling and increases mitochondrial antioxidant activity in skeletal muscle. Exp Physiol. 2016;101(3):410-20.\u003c/li\u003e\n\u003cli\u003eBuck M, Chojkier M. Muscle wasting and dedifferentiation induced by oxidative stress in a murine model of cachexia is prevented by inhibitors of nitric oxide synthesis and antioxidants. The EMBO Journal. 1996;15(8):1753-65.\u003c/li\u003e\n\u003cli\u003eYuzefovych LV, Musiyenko SI, Wilson GL, Rachek LI. Mitochondrial DNA damage and dysfunction, and oxidative stress are associated with endoplasmic reticulum stress, protein degradation and apoptosis in high fat diet-induced insulin resistance mice. PLoS One. 2013;8(1):e54059.\u003c/li\u003e\n\u003cli\u003eLalunio H, Goodman CA, Stupka N, Giourmas N, Debruin DA, Sahakian L, et al. APE1/Ref-1 inhibition via APX3330 lowers monocyte/macrophage infiltration without ameliorating the structure and function of dystrophic mdx hindlimb muscles. Physiol Rep. 2025;13(15):e70494.\u003c/li\u003e\n\u003cli\u003eRadley-Crabb HG, Marini JC, Sosa HA, Castillo LI, Grounds MD, Fiorotto ML. Dystropathology increases energy expenditure and protein turnover in the mdx mouse model of duchenne muscular dystrophy. PLoS One. 2014;9(2):e89277.\u003c/li\u003e\n\u003cli\u003eFishel ML, Jiang Y, Rajeshkumar NV, Scandura G, Sinn AL, He Y, et al. Impact of APE1/Ref-1 redox inhibition on pancreatic tumor growth. Mol Cancer Ther. 2011;10(9):1698-708.\u003c/li\u003e\n\u003cli\u003eDebruin DA, Timpani CA, Lalunio H, Rybalka E, Goodman CA, Hayes A. Exercise May Ameliorate the Detrimental Side Effects of High Vitamin D Supplementation on Muscle Function in Mice. J Bone Miner Res. 2020;35(6):1092-106.\u003c/li\u003e\n\u003cli\u003eBrooks SV, Faulkner JA. Contractile properties of skeletal muscles from young, adult and aged mice. J Physiol. 1988;404:71-82.\u003c/li\u003e\n\u003cli\u003eClose RI. Dynamic properties of mammalian skeletal muscles. Physiol Rev. 1972;52(1):129-97.\u003c/li\u003e\n\u003cli\u003eGrounds M. Quantification of histopathology in haemotoxylin and eosin stained muscle sections. Treat NMD, Neuromuscular Network; DMD_M. 2014;1:1-13.\u003c/li\u003e\n\u003cli\u003eMcRae NL, Addinsall AB, Howlett KF, McNeill B, McCulloch DR, Stupka N. Genetic reduction of the extracellular matrix protein versican attenuates inflammatory cell infiltration and improves contractile function in dystrophic mdx diaphragm muscles. Sci Rep. 2020;10(1):11080.\u003c/li\u003e\n\u003cli\u003eHead SI, Williams DA, Stephenson DG. Abnormalities in Structure and Function of Limb Skeletal Muscle Fibres of Dystrophic mdx Mice. Proceedings: Biological Sciences. 1992;248(1322):163-9.\u003c/li\u003e\n\u003cli\u003eKonno RN, Nigam N, Wakeling JM, Ross SA. The Contributions of Extracellular Matrix and Sarcomere Properties to Passive Muscle Stiffness in Cerebral Palsy. Front Physiol. 2021;12:804188.\u003c/li\u003e\n\u003cli\u003eZollner AM, Abilez OJ, Bol M, Kuhl E. Stretching skeletal muscle: chronic muscle lengthening through sarcomerogenesis. PLoS One. 2012;7(10):e45661.\u003c/li\u003e\n\u003cli\u003eCarnwath JW, Shotton DM. Muscular dystrophy in the mdx mouse: histopathology of the soleus and extensor digitorum longus muscles. J Neurol Sci. 1987;80(1):39-54.\u003c/li\u003e\n\u003cli\u003eGehrig SM, Koopman R, Naim T, Tjoakarfa C, Lynch GS. Making fast-twitch dystrophic muscles bigger protects them from contraction injury and attenuates the dystrophic pathology. Am J Pathol. 2010;176(1):29-33.\u003c/li\u003e\n\u003cli\u003eMarshall PA, Williams PE, Goldspink G. Accumulation of collagen and altered fiber-type ratios as indicators of abnormal muscle gene expression in the mdx dystrophic mouse. Muscle Nerve. 1989;12(7):528-37.\u003c/li\u003e\n\u003cli\u003eNogami K, Maruyama Y, Sakai-Takemura F, Motohashi N, Elhussieny A, Imamura M, et al. Pharmacological activation of SERCA ameliorates dystrophic phenotypes in dystrophin-deficient mdx mice. Hum Mol Genet. 2021;30(11):1006-19.\u003c/li\u003e\n\u003cli\u003eWasala NB, Yue Y, Lostal W, Wasala LP, Niranjan N, Hajjar RJ, et al. Single SERCA2a Therapy Ameliorated Dilated Cardiomyopathy for 18 Months in a Mouse Model of Duchenne Muscular Dystrophy. Molecular Therapy. 2020;28(3):845-54.\u003c/li\u003e\n\u003cli\u003eFushimi S, Nohno T, Katsuyama H. Chronic Stress Induces Type 2b Skeletal Muscle Atrophy via the Inhibition of mTORC1 Signaling in Mice. Med Sci (Basel). 2023;11(1).\u003c/li\u003e\n\u003cli\u003eDrude S, Geissler A, Olfe J, Starke A, Domanska G, Schuett C, et al. Side effects of control treatment can conceal experimental data when studying stress responses to injection and psychological stress in mice. Lab Anim (NY). 2011;40(4):119-28.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Duchenne muscular dystrophy, inflammation, oxidative stress, APE1/Ref-1, APX3330","lastPublishedDoi":"10.21203/rs.3.rs-9507119/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-9507119/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eAims\u003c/h2\u003e \u003cp\u003eChronic inflammation and oxidative stress are key components in Duchenne muscular dystrophy (DMD) pathology. Apurinic/apyrimidinic endonuclease 1/redox factor-1 (APE1/Ref-1) is a multifunctional protein involved in inflammatory and oxidative stress pathways through its redox domain and is an emerging therapeutic target for inflammatory conditions. This study aimed to investigate the effects of APX3330, a small molecular inhibitor of APE1\u0026rsquo;s Ref-1 function on \u003cem\u003emdx\u003c/em\u003e mouse pathology, a model of DMD.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e \u003cp\u003eThree-week-old \u003cem\u003emdx\u003c/em\u003e mice and wildtype (WT) C57Bl/10 mice were randomised into four groups: 1) WT untreated control; 2) WT treated with APX3330 (25 mg\u0026middot;kg\u003csup\u003e-1\u003c/sup\u003e); 3) \u003cem\u003emdx\u003c/em\u003e untreated control; or 4) \u003cem\u003emdx\u003c/em\u003e treated with APX3330 (25 mg\u0026middot;kg\u003csup\u003e-1\u003c/sup\u003e). After three weeks, \u003cem\u003eex vivo\u003c/em\u003e contractile function and histological analysis were performed in extensor digitorum longus (EDL) and soleus muscles.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eAPX3330 treated WT mice displayed a lower absolute and specific force in EDL (p\u0026thinsp;\u0026lt;\u0026thinsp;0.01, p\u0026thinsp;\u0026lt;\u0026thinsp;0.001, respectively) and soleus (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) muscles, despite having a higher muscle mass to body mass ratio than untreated mice (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). In APX3330 treated dystrophic mice, soleus muscles exhibited larger fibres (p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001), while EDL muscles showed smaller fibres (p\u0026thinsp;\u0026lt;\u0026thinsp;0.01), longer time to peak tension (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05), and higher twitch to tetanic force ratio (p\u0026thinsp;\u0026lt;\u0026thinsp;0.01). Dystrophic EDL muscles also demonstrated a higher amount of CD68 positive monocytes/macrophages compared to untreated \u003cem\u003emdx\u003c/em\u003e mice (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). APX3330 treatment did not alter muscle histopathology.\u003c/p\u003e\u003ch2\u003eDiscussion\u003c/h2\u003e \u003cp\u003eAPX3330 treatment demonstrated deleterious effects in both WT and dystrophic mice. Despite literature indicating anti-inflammatory effects, APX3330 was unable to ameliorate the dystrophic phenotype in any meaningful capacity.\u003c/p\u003e","manuscriptTitle":"APE1/Ref-1 inhibition via APX3330 in young dystrophic mdx mice","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-05-12 14:18:01","doi":"10.21203/rs.3.rs-9507119/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"0b855e09-4a2d-494d-8c71-822c14148a65","owner":[],"postedDate":"May 12th, 2026","published":true,"recentEditorialEvents":[{"type":"decision","content":"Rejected","date":"2026-05-19T06:28:01+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-05-19T04:45:24+00:00","index":38,"fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-05-18T05:29:45+00:00","index":37,"fulltext":""},{"type":"reviewerAgreed","content":"108629733520897121747941034672068606636","date":"2026-05-10T05:36:31+00:00","index":35,"fulltext":""},{"type":"reviewerAgreed","content":"118611475698504040568286464539119094431","date":"2026-05-06T07:21:54+00:00","index":30,"fulltext":""},{"type":"reviewersInvited","content":"21","date":"2026-05-04T16:05:12+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-04-29T22:50:54+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-04-29T22:50:36+00:00","index":"","fulltext":""}],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2026-05-19T06:40:39+00:00","versionOfRecord":[],"versionCreatedAt":"2026-05-12 14:18:01","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-9507119","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-9507119","identity":"rs-9507119","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}