Hypochlorous acid exposure impairs skeletal muscle function and Ca2+ signalling: implications for Duchenne muscular dystrophy pathology

Duchenne muscular dystrophy (DMD) is a fatal X‐linked disease characterised by severe muscle wasting. The mechanisms underlying the DMD pathology likely involve the interaction between inflammation, oxidative stress and impaired Ca2+ signalling. Hypochlorous acid (HOCl) is a highly reactive oxidant produced endogenously via myeloperoxidase; an enzyme secreted by neutrophils that is significantly elevated in dystrophic muscle. Oxidation of Ca2+‐handling proteins by HOCl may impair Ca2+ signalling. This study aimed to determine the effects of HOCl on skeletal muscle function and its potential contribution to the dystrophic pathology. Extensor digitorum longus (EDL), soleus and interosseous muscles were surgically isolated from anaesthetised C57 (wild‐type) and mdx (dystrophic) mice for measurement of ex vivo force production and intracellular Ca2+ concentration. In whole EDL muscle, HOCl (200 μM) significantly decreased maximal force and increased resting muscle tension which was only partially reversible by dithiothreitol. The effects of HOCl (200 μM) on maximal force in slow‐twitch soleus were lower than found in the fast‐twitch EDL muscle. In single interosseous myofibres, HOCl (10 μM) significantly increased resting intracellular Ca2+ concentration and decreased Ca2+ transient amplitude. These effects of HOCl were reduced by the application of tetracaine, Gd3+ or streptomycin, implicating involvement of ryanodine receptors and transient receptor potential channels. These results demonstrate the potent effects of HOCl on skeletal muscle function potentially mediated by HOCl‐induced oxidation to Ca2+ signalling proteins. Hence, HOCl may provide a link between chronic inflammation, oxidative stress and impaired Ca2+ handling that is characteristic of DMD and presents a potential therapeutic target for DMD.


Introduction
Duchenne muscular dystrophy (DMD) is an X-linked progressive muscle-wasting disease which affects approximately 1 in 3500-6000 boys worldwide (Emery, 1991).DMD is a fatal disease where death typically results from either respiratory or cardiac failure at an average age of 30 years (Landfeldt et al., 2020).The mechanisms which lead to muscle wasting in DMD remain unresolved but likely involves a complex interaction between chronic inflammation, reactive oxygen species (ROS) and impaired Ca 2+ handling, which leads to muscle necrosis (De Backer et al., 2002;Deconinck & Dan, 2007).Despite numerous preclinical studies reporting the beneficial effects of anti-inflammatory and anti-oxidant based treatments, and the promising developments in gene therapy, available therapies for DMD have limited efficacy and/or are prohibitively expensive (Kawamura et al., 2020;Komaki et al., 2020;Rafael-Fortney et al., 2011;Raman et al., 2019).The mechanisms of DMD are also complicated by the fact that fast-twitch muscles are preferentially affected by the DMD pathology more than the slow-twitch muscles (Webster et al., 1988).A better understanding of the mechanisms underlying the DMD pathology could identify strategies to develop improved therapeutic treatments.Hypochlorous acid (HOCl), a highly reactive oxidant, may provide a unifying explanation for the multiple hallmarks of the DMD pathology and is a potential therapeutic target.
Several studies have demonstrated that HOCl exposure impairs the function of Ca 2+ -handling proteins on the sarcoplasmic reticulum (SR), including the ryanodine receptors (RyR) and sarcoplasmic reticulum Ca 2+ -ATPase (SERCA) pumps (Favero et al., 1998(Favero et al., , 2003;;Lafoux et al., 2010).However, these studies have been conducted almost exclusively in isolated SR vesicles or chemically skinned fibres.The extent to which these effects are manifest in more physiologically relevant preparations such as intact muscle fibres and whole skeletal muscle is unclear.Importantly, these past studies have not indicated whether HOCl affects whole muscle force production.HOCl may also affect other Ca 2+ -handling proteins such as transient receptor potential (TRP) channel proteins on the sarcolemma.
Together, these previous studies suggest that HOCl concentration is likely to be elevated in dystrophic muscle due to the increased activity of MPO as a consequence of chronic inflammation.Furthermore, HOCl has the potential to disrupt Ca 2+ homeostasis by oxidation of multiple Ca 2+ -handling proteins, leading to skeletal muscle dysfunction similar to that seen in DMD.Thus, HOCl may provide a link between the major hallmarks of DMD; chronic inflammation, increased ROS production and impaired Ca 2+ handling.Therefore, this study aimed to investigate the effects of HOCl on whole muscle function and Ca 2+ handling in non-dystrophic (C57) and dystrophic (mdx) muscle.

Materials and methods
All reagents were purchased from Merck (Australia) unless otherwise stated.

Ethical approval
All animal experiments were conducted in accordance with the guidelines of the Australian National Health and Medical Research Council Code of Practice for the care and use of animals for scientific purposes (2013), and the Animal Welfare act of Western Australia (2002).This J Physiol 601.23 study was approved by the Animal Ethics Committee of the University of Western Australia (Approval numbers: RA/3/100/1501 and RA/3/100/1625).Experiments were conducted on 8-12-week-old male control C57Bl/10 (C57) mice or dystrophic C57B1/10ScSn mdx/mdx (mdx) mice sourced from the Animal Resource Centre (Murdoch, Western Australia).Animals were housed in the Preclinical Facility at the University of Western Australia, maintained at 20-22°C on a 12 h light/dark cycle and given free access to a standard chow diet and water.

Surgical isolation of EDL, soleus and interosseous muscles
All whole-muscle experiments were conducted using intact surgically excised extensor digitorum longus (EDL) and soleus muscles.EDL muscles are primarily fast-twitch muscles, while soleus muscles are primarily slow-twitch muscles (Hayes & Williams, 1996).Single intact fibres from the interosseous muscle (predominately fast-twitch) were used for intracellular Ca 2+ measurements (Friedrich et al., 2008).
Mice were anaesthetised via an intraperitoneal injection of sodium pentobarbitone (40 mg/kg body weight, Lethabarb, Virbac, Australia) before removal of EDL and interosseous muscles.Mice were then killed using an overdose of sodium pentobarbitone (>120 mg/kg of body weight, intraperitoneal injection).No mice died before the removal of the EDL/interosseous muscles were completed.
Optimum muscle length (L o ) was identified by manually adjusting the muscle length until peak twitch force was recorded.A series of isometric stabilising contractions were performed at 3 min intervals followed by a control 120 Hz (0.5 s stimulation duration throughout protocol) maximal contraction before the beginning of the timed contractile protocol and any experimental interventions.
Skeletal muscle contractile function was assessed, in the presence or absence of 200 μM HOCl, via a series of 10 maximal (120 Hz) isometric contractions at 2 min intervals followed by a 60 min monitoring period where maximal isometric contractions were recorded every 10 min.The active force and resting tension were recorded for all contractions.
The reversibility of HOCl oxidation was also assessed using the antioxidant, dithiothreitol (DTT).Muscles were exposed to the previously described contractile protocol but the HOCl solution was replaced with either 5 mM DTT exposure or fresh Ringer solution (washout) for the 60 min monitoring period.
In separate experiments, the initial HOCl exposure protocol was preceded by 30 min incubation with tetracaine (150 μM) or streptomycin (300 μM) to investigate the potential involvement of RyRs and TRP channel proteins, respectively.
At the end of the protocol, the muscles were removed from the organ bath, their tendons were then removed and the muscles were blotted on filter paper and weighed.For each muscle, the optimal fibre length (L f ) was calculated from a previously established L f :L o ratio of 0.44 (Burkholder et al., 1994) and the cross-sectional area of each muscle was estimated by dividing muscle wet mass by the product L f and the density of mammalian skeletal muscle (1.056 mg/mm 3 ) (Mendez & Keys, 1960), and used to calculate specific force (N/cm 2 ).

Measurement of contractile function in isolated soleus muscles
The effects of 200 μM HOCl on isolated soleus muscle were assessed using the same protocol described for EDL muscles.Due to the predominately slow fibre type, an 80 Hz stimulation frequency was used to maximally stimulate the muscles, and a higher tetanic stimulation train duration (1 s) was applied.In these experiments, all n values are the number of muscles and are as follows: (1) C57 control (n = 7), (2) mdx control (n = 5), (3) C57 200 μM HOCl (n = 7) and (4) mdx 200 μM HOCl (n = 6).

Measurement of intracellular Ca 2+ in intact interosseous fibres
Isolated interosseous muscles were incubated in 1.5 ml of 0.2% collagenase at 37°C for 90 min, washed in phosphate-buffered saline and placed in a small well filled with physiological rodent saline (PRS; Contains: NaCl (138 mM), KCl (2.7 mM), MgCl 2 (1.06 mM), HEPES (12.4 mM), CaCl 2 (1.8 mM) and glucose (5.6 mM)) and then carefully agitated using a glass pipette to encourage dissociation of the muscle into individual fibres.Single interosseous fibres were placed in PRS solution containing 10 μM FURA 2-AM ester and 10 μM Pluronic F-127 for 30 min in the dark at room temperature.The supernatant was then removed, and the muscle fibres were bathed in a solution containing only PRS for a further 30 min to allow time for the FURA-2 AM de-esterification process.Fura-2 was used because it is sensitive to small changes in resting Ca 2+ concentration, while also able to measure large decreases Ca 2+ transient amplitude.Fibres were then placed in a separate dish containing HEPES-free Ringer solution at room temperature and mounted on an inverted Nikon microscope (Nikon 'S Flour 1.30 oil immersion' , Japan) for fluorescence measurements.Fibres were electrically stimulated via parallel platinum electrodes connected to an isolated pulse stimulator (AM Systems, Model 1600) in series with a power amplifier (Ebony, Dual Channel Power Amplifier EP500B).Cells containing the FURA 2-AM were exposed to UV light at excitation wavelengths of 340 nm and 380 nm.The ratio of the two emission intensities (at 340 nm and 380 nm) was used as an indicator of the relative cytosolic Ca 2+ concentration.Only one fibre per each dish was imaged.Data were subsequently analysed using the Strathclyde Electrophysiology Data Recorder software package (Glasgow, UK).
Fibres from C57 and mdx mice were exposed to either 10 μM HOCl or to the equivalent volume of distilled water as a control to determine the effects of HOCl exposure on resting cytosolic Ca 2+ levels and Ca 2+ transient amplitude.Control twitch contractions were induced by electrical stimulation once every 30 s for 2 min prior to the addition of HOCl.The data were presented relative to the final twitch of this 2 min period.The involvement of RyR and TRP channel proteins in the HOCl-mediated effects on Ca 2+ handling was investigated by the addition of tetracaine (150 μM) and Gd 3+ (300 μM), respectively, prior to the addition of HOCl.

Calculation of Ca 2+ concentrations
Ca 2+ concentrations were calculated using the following equation (Grynkiewicz et al., 1985).R is the ratio of fluorescence emission at 340 nm and 380 nm, K d is the apparent dissociation constant, R max is the maximum ratio at saturating Ca 2+ , R min is the minimum ratio under Ca 2+ -free conditions and β is the ratio of the 380 nm fluorescence under minimum and maximum [Ca 2+ ] conditions.To calculate maximum and minimum values for [Ca 2+ ] tightly Ca 2+ -buffered (EGTA Ca 2+ buffer) solutions were prepared containing either maximal Ca 2+ (CaCO 3 = 47.5 mM, EGTA = 50 mM, MgO = 8.5 mM, HEPES = 90 mM, ATP = 8 mM, creatine phosphate = 10 mM, pH = 7.1) or minimum Ca 2+ (CaCO 3 = 0 mM, EGTA = 50 mM, MgO = 8.5 mM, HEPES = 90 mM, ATP = 8 mM, creatine phosphate = 10 mM, pH = 7.1).Fluorescent measurements were obtained as per the methodology described previously except FURA 2 salt was used rather that FURA 2-AM.

Data analysis
Data were analysed in GraphPad Prism 9 using two-way ANOVAs with Tukey's post hoc analysis.All results are presented as means and standard deviations (SD).N values represent the number of muscles for all whole-muscle experiments in the experiments using isolated intact interosseous muscle fibres; n represents the number of fibres.Due to the volume p-value comparisons in these time course data, some p-values have been reported in supplementary tables S1-S24.

HOCl decreases maximal force and increases resting tension in isolated whole EDL muscle and is only partially reversible by DTT
In intact EDL muscles, exposure to 200 μM HOCl significantly decreased maximal force and increased resting tension in both C57 and mdx muscle (Fig. 1; P values < 0.0001).The effects of HOCl were rapid with significant changes evident within 2-4 min of exposure (P values < 0.05).At the end of the protocol, resting tension in HOCl-exposed C57 muscles was increased by 52% (from 0.781 g (SD: 0.279 g) to 1.14 g (SD: 0.294 g)) which was not significantly larger than the 31% increase found in mdx muscles (from 0.836 g (SD: 0.423 g) to 1.13 g (SD: 0.615 g)).Interestingly, HOCl exposure decreased maximal isometric force by 49% in mdx muscles (from 16 N/cm 2 (SD: 3.15 N/cm 2 ) to 8.21 N/cm 2 (SD: 2.88 N/cm 2 )) which was significantly greater (P < 0.001) than the 26% decrease observed in C57 muscles (from 18.4 N/cm 2 (SD: 3.17 N/cm 2 ) to 13.6 N/cm 2 (SD: 2.85 N/cm 2 )).
The antioxidant, DTT, effectively reversed the HOCl-induced increase in resting tension from the 20 to 40 and 80 min marks in C57 and from the 30 to 80 min marks in mdx (P values < 0.05) but was ineffective in reversing the effects of HOCl on maximal force.Resting tension decreased almost immediately after the addition of DTT (at 20 min in Fig. 1A, B) and by the end of the protocol was not significantly different from the initial resting tension before exposure to HOCl (C57 initial = 0.726 g (SD: 0.118 g), C57 final = 0.823 g (SD: 0.274 g); mdx initial = 0.705 g (SD: 0.119 g), mdx final = 0.707 g (SD: 0.169 g)).In contrast, DTT exposure did not ameliorate the effects of HOCl on maximal force in either C57 (P = 0.708; initial maximal force = 20.7 N/cm 2 (SD: 2.16 N/cm 2 ), final maximal force = 15.9N/cm 2 (SD: 2.4 N/cm 2 )) or mdx groups (P = 0.193; initial maximal force = 15.1 N/cm 2 (SD: 2.66 N/cm 2 ), final maximal force = 10.4N/cm 2 (SD: 1.51 N/cm 2 )).

HOCl decreases maximal force and increases resting tension in isolated slow-twitch soleus whole muscle
In intact soleus muscles, exposure to 200 μM HOCl significantly decreased maximal force and increased resting tension in both C57 and mdx mice (Fig. 2; P values < 0.001).At the end of the protocol, resting tension in HOCl-exposed C57 muscles was increased by 80% (from 0.693 g (SD: 0.161 g) to 1.198 g (SD: 0.16 g)), compared with a 47% increase recorded in mdx muscles (from 0.597 g (SD: 0.142 g) to 0.841 g (SD: 0.242 g)).Similarly, at the end of the protocol, maximal isometric force in HOCl-exposed C57 muscles decreased by 15% (from 16.44 N/cm 2 (SD: 2.69 N/cm 2 g) to 13.75 N/cm 2 (SD: 2.18 N/cm 2 g)), compared with the 13% decrease recorded in mdx muscles (from 16.64 N/cm 2 (SD: 3.78 N/cm 2 g) to 13.92 N/cm 2 (SD: 2.76 N/cm 2 g)).These decreases in maximal force were comparatively lower compared with the fast-twitch EDL muscle which, as detailed in the previous section, decreased by 26% in C57 muscle and 49% in mdx muscle.

HOCl increases resting intracellular Ca 2+ and decreases Ca 2+ transient amplitude
The potential involvement of altered Ca 2+ handling in the HOCl-mediated effects on intact muscles was investigated in single intact interosseous fibres isolated from C57 and mdx mice.Consistent with the effects on intact EDL muscles, HOCl exposure (10 μm) produced a rapid and significant increase in resting Ca 2+ concentration and a decrease in Ca 2+ transient amplitude in both C57 and mdx fibres (Fig. 3A-E).

Effects of HOCl on resting cytosolic Ca 2+ levels and whole muscle resting tension are mediated in part via RyR
Tetracaine was used to determine the contribution of RyRs to the HOCl-induced increase in resting cytosolic Ca 2+ levels in single interosseus fibres and the increase in resting tension in intact EDL muscles.In both C57 and mdx fibres, prior incubation with tetracaine (150 μM) abolished the HOCl-induced rise in resting cytosolic Ca 2+ (C57: P = 0.0045; mdx: P = 0.0017; Fig. 4A, B).In intact EDL muscles, tetracaine significantly delayed the initial increase in resting tension after HOCl exposure (200 μM) (C57: P = 0.0001; mdx: P = 0.0361; Fig. 4C,  D).In EDL muscles from C57 mice, the resting tension was significantly lower (P values < 0.05) in the tetracaine group than the HOCl-only group for the first 20 min, but then progressively increased over time reaching a level similar to the HOCl-only group by the end of the 80 min protocol (P values > 0.05).The resting tension in EDL muscles from mdx mice showed a similar response as for C57 mice, with a notable delay in the HOCl-induced increase in resting tension in the presence of tetracaine.
These results suggest that Ca 2+ release from the SR is responsible for the initial rise in cytosolic Ca 2+ and increase in resting tension.However, the delayed increase in resting tension suggests that HOCl may also affect additional Ca 2+ signalling pathways.

Discussion
Our results indicate that the rapid impairment of muscle function after exposure to HOCl was due, at least in part, to impaired Ca 2+ handling likely mediated by oxidation of RyR and TRP channel proteins.We propose that HOCl may provide a unifying hypothesis in DMD for the link between chronic inflammation, oxidative stress and impaired Ca 2+ handling, and could be a potential target for the development of future DMD therapies.We have also found that the detrimental effects of HOCl on maximal force were reduced in whole soleus muscle compared with whole EDL muscles, which is consistent with the differential effects of DMD on fast-twitch and slow-twitch muscle types (Webster et al., 1988).These results complement previous findings that HOCl levels are already likely to be elevated in mdx muscle compared with C57 muscle due to the fivefold increase in MPO activity (Terrill, Boyatzis et al., 2013a).Although the effects of HOCl on EDL maximal force were more severe in mdx than C57, these effects were not limited to mdx mice.This observation suggests a potential role for HOCl in other inflammatory muscle pathologies which exhibit high neutrophil levels such as autoimmune myositis and myasthenia gravis (Duan et al., 2022;Tidball & Wehling-Henricks, 2005).
Our results are consistent with the effects of HOCl on contractile function and Ca 2+ handling that have been reported in cardiac myocytes.Exposure to HOCl (100 μM) decreased the amplitude of intracellular Ca 2+ transients and increased cytosolic resting Ca 2+ concentration (Eley, Eley et al., 1991a).HOCl also impairs contractility in isolated rat hearts (Eley, Korecky et al., 1991b) which is likely to be a consequence of both reduced Ca 2+ transient amplitude and impaired myofilament force production (MacFarlane & Miller, 1994).The similarities between the effects of HOCl on cardiac and skeletal muscle are important given the role of cardiac and respiratory dysfunction in DMD patients and may reflect the impact of systemic inflammation stemming from skeletal muscle damage.
Our results suggest that HOCl could contribute, with other previously described mechanisms, to the perturbed cytosolic Ca 2+ signalling observed in dystrophin-deficient skeletal muscle in DMD.Reduced expression/activity of SERCA1 in mdx muscle has also been proposed to explain increased cytosolic Ca 2+ (Divet & Huchet-Cadiou, 2002;Mazala et al., 2015).Interestingly, Favero et al. (1998) found that HOCl can inhibit SERCA in isolated SR vesicles, suggesting that oxidation of SERCA1 by HOCl may partially explain the reduced activity of SERCA1 in mdx muscle but probably not the reduced expression.Separately, eccentric contraction-induced force loss may also partially explain the increases in cytosolic Ca 2+ in DMD.Lindsay et al. (2020) found that the force loss in anterior crural muscles undergoing an eccentric protocol could be diminished by the addition of the antioxidant N-acetyl cysteine during the eccentric protocol or the addition of small molecule Ca 2+ modulators which either increased SERCA activity or decreased Ca 2+ leak from RyR.This suggests that acute increases in cytosolic Ca 2+ in mdx muscle may be induced by an eccentric protocol via ROS production.The ensuing damage and chronic inflammation potentially brought about by these damaging contractions in dystrophic muscle may also induce further HOCl generation via chronically increased muscular neutrophil levels (De Luca et al., 2005;Tulangekar & Sztal, 2021).
In this study, we have shown mechanistic detail on the effects of HOCl on intact skeletal muscle function and our results support the likely involvement of RyR in the disruption to Ca 2+ homeostasis.RyRs contain multiple sulfhydryl groups that are subject to reversible oxidation (Salama et al., 2000).Redox modification of RyR can significantly increase RyR-mediated SR Ca 2+ leak (Aracena et al., 2005;Bellinger et al., 2009).Increased Ca 2+ leak from the SR via RyR explains both the increases in resting cytosolic Ca 2+ and increased resting tension found in this study after HOCl exposure.A decrease in the releasable SR Ca 2+ concentration could also contribute to the HOCl-induced reduction in Ca 2+ transient amplitude and decreased maximal force levels reported here.However, the fact that blocking RyR with tetracaine only delayed the rise in resting tension in intact EDL muscles and did not completely abolish it, suggests that additional Ca 2+ handling proteins are likely to be affected by HOCl.
To this end, we provided evidence that TRP channels are also involved in the HOCl-mediated disruption to Ca 2+ homeostasis.Prior incubation with Gd 3+ or streptomycin prevented the increase in resting cytosolic Ca 2+ and the rise in resting tension in single fibres and intact muscles, respectively, suggesting that TRP channels on the sarcolemma are also targets of HOCl oxidation.
TRP channels are sensitive to oxidation and have been implicated in the altered Ca 2+ homeostasis in mdx muscle fibres (Gervasio et al., 2008), although the specific TRP channel protein subtype(s) targeted by HOCl is/are unclear as both streptomycin and Gd 3+ are non-selective blockers (Caldwell et al., 1998;Hamill & Mcbride, 1996).TRPC1 expression is increased in mdx muscle compared with wild-type, and Ca 2+ entry via TRPC1 induces muscle damage in mdx mice (Gervasio et al., 2008), thus implicating this channel in the DMD pathology.Expression of both TRPC1 and TRPC3 is elevated in skeletal muscles of the DMD mdx rat; however, increased TRPC3 expression occurs much earlier than TRPC1 and specific inhibition of TRPC3 restored sarcolemmal Ca 2+ permeability in muscles from DMD mdx rats to levels similar to those of wild-type (Creismeas et al., 2021).This result suggests that TRPC3 may be more relevant to the DMD mdx pathology.TRPV2 has also been implicated in the DMD pathology; knockout of TRPV2 in mdx muscle reduced abnormal Ca 2+ influx and other markers of muscle damage and necrosis (Iwata et al., 2009).Both streptomycin and Gd 3+ can block TRPC1, TRPC3 and TRPV2 (Seth et al., 2009;Siveen et al., 2020;Trebak et al., 2002), thus implicating all three of these channels as potential targets of HOCl oxidation.Further studies are required to determine the specific TRP channels that mediate the effects of HOCl on sarcolemmal Ca 2+ permeability.
Taken together, these results suggest that HOCl acts on both TRP channel proteins and RyRs.This is not surprising considering previous observations that, even at low concentrations (10-20 μM), HOCl oxidises protein thiol groups on plasma membrane proteins and alters trans-membrane ion transport (Schraufstätter et al., 1990).However, HOCl is also highly membrane permeable (Tatsumi & Fliss, 1994) which may explain the capacity of HOCl to oxidise both extracellular and intracellular Ca 2+ handling proteins.Increased TRP-induced Ca 2+ influx would increase SR Ca 2+ sequestration and loading via SERCA, which may further exacerbate the SR Ca 2+ leak via RyR and the resulting increase in cytosolic Ca 2+ and resting tension (denoted pathway 1 in Fig. 7).However, in the presence of tetracaine, Ca 2+ release from the SR via RyR is prevented, and once the SR is maximally loaded with Ca 2+ , any additional Ca 2+ entering the cell via TRP channel proteins will remain in the cytosol (denoted pathway 2 in Fig. 7) (Divet et al., 2007) where it could activate the contractile apparatus resulting in the delayed increase in resting tension that was observed in our tetracaine experiments.It should be acknowledged that SERCA function may also be compromised by HOCl exposure.However, full inhibition of SERCA is only evident at concentrations (3 mM; Favero et al. (1998)) much higher than those used in this study (200 μM); hence this proposed explanation would fit the data despite the possible inhibitory effects of HOCl on SERCA function.
Our results demonstrate the clear functional implications of HOCl-induced oxidation to RyRs and TRPs.However, oxidation to these proteins may also have further impacts such as activation of calpains or opening of the mitochondrial permeability pore.Calpains are Ca 2+ -sensitive proteases activated by elevated cytosolic Ca 2+ .Calpain activity is increased in human DMD muscle and mdx mice (Murphy et al., 2006(Murphy et al., , 2013;;Ono & Sorimachi, 2012;Spencer et al., 1995;Tidball & Spencer, 2000).Involvement of calpain in the mdx pathology seems likely as treatment of mdx mice with the calpain inhibitor, leupeptin, delays necrosis (Badalamente & Stracher, 2000).
Additionally, HOCl oxidation could result in opening of the thiol oxidation-sensitive mitochondrial permeability pore, either directly or via increased cytosolic Ca 2+ levels caused by HOCl oxidation of RyRs and TRPs (Costantini et al., 1996).Therapies that target opening of the mitochondrial permeability pore have been shown to lead to an overall improvement in muscle function in mdx mice (Millay et al., 2008;Reutenauer et al., 2008).
Our results also indicate that HOCl is highly potent oxidant in muscle tissue as acute exposure to HOCl (200 μM) reduced maximal force production by ∼50% in mdx mice, and by a lesser, but still significant level (25%) in non-dystrophic muscle.The potency of HOCl was further demonstrated in the Ca 2+ measurement results as a comparatively low concentration of 10 μM HOCl was sufficient to cause an immediate increase in resting cytosolic Ca 2+ of 87% in C57 and 56% in mdx fibres.Comparative studies using H 2 O 2 , required a 30-fold higher concentration (300 μM H 2 O 2 ) with an exposure of 15 min to induce a 56% increase in resting intracellular Ca 2+ in single interosseous fibres (Andrade et al., 1998).Further to this, increasing HOCl to 25 μM severely impacted the function of intact isolated fibres, such that intracellular Ca 2+ transients were unable to be recorded in half of the fibres tested.These observations highlight the potency of HOCl, relative to H 2 O 2 , in skeletal muscle tissue.The lower concentration of HOCl required to significantly affect single fibres compared with whole muscle is likely explained by the difference in tissue size and surface area-to-volume ratios in single fibres relative to whole muscles.
The more severe effect of HOCl on maximal force production in mdx compared with C57 muscle is an interesting result indicating that HOCl may have a more potent detrimental effect in mdx muscle.The mechanisms which underlie this result are unlikely due to perturbations to Ca 2+ signalling since the effects of HOCl on Ca 2+ signalling were found to be relatively similar across both groups in this study.Differential oxidation to the contractile filaments might explain this result as HOCl could oxidise individual contractile proteins to a greater extent or different proteins in mdx muscle.In support of this idea, contractile proteins including myosin, actin, troponin and titin have been shown to be redox sensitive, thus suggesting that the contractile filaments may also be a target of HOCl (Andrade et al., 1998(Andrade et al., , 2001;;Crowder & Cooke, 1984;Menazza et al., 2010;Prochniewicz et al., 2008;Siddiqui et al., 2016;Watanabe et al., 2020).
Interestingly, we found that HOCl induced a similar increase in resting tension in slow-twitch soleus muscle compared with fast-twitch EDL muscle, whereas, in contrast, HOCl had more severe effects on maximal force in EDL muscle than soleus muscle.This result is notable as it further suggests that HOCl may be directly involved in the DMD pathology as in DMD, fast-twitch fibres are preferentially affected compared with slow-twitch fibres (Webster et al., 1988).
A limitation of our study was the inability to measure intracellular HOCl concentrations in vivo.While the fivefold increased MPO activity in mdx mice suggests that HOCl levels are likely increased, the high reactivity and short half-life of HOCl limit the capacity to directly quantify the intramuscular HOCl concentration (Terrill, Boyatzis et al., 2013a;Weiss et al., 1982).Despite several innovative approaches to directly measure HOCl in vivo (Chen et al., 2022;Gan et al., 2021;Ye et al., 2022), endogenous concentrations of HOCl in rodent skeletal muscle remain unknown.However, in conditions associated with chronic inflammation, such as DMD, the infiltration of neutrophils into the muscle tissue is likely to result in localised elevations in HOCl concentration (Iwasaki et al., 2022).
This study has used the mdx mouse to investigate the effects of HOCl on skeletal muscle function and its potential involvement in the dystrophic pathology.The mdx mouse, while a useful model showing clear differences from the wild-type C57 mouse, displays a less severe dystrophic pathology than that of humans and has a relatively normal lifespan and normal reproductive cycle (Carnwath & Shotton, 1987;Dangain & Vrbova, 1984;Muntoni et al., 1993).It would be valuable to investigate the potential role of HOCl in the pathophysiology of DMD using animal models which more closely reflect human DMD, such as the golden retriever muscular dystrophy model or the newly developed dystrophic rat model (R-DMDdel52) (Barthelemy et al., 2014;Goddard et al., 2018).

Effect of HOCl partially reversible by the antioxidant DTT and potential treatments for HOCl-induced muscle dysfunction
This study also investigated the potential of a thiol reductant (DTT) to reverse the effects of HOCl-induced muscle dysfunction.Although DTT effectively reversed the effects of HOCl on resting tension, it failed to significantly reverse the effects on maximal force, suggesting that HOCl causes both reversible and irreversible protein oxidation in isolated muscle.Eley et al. (1989) reported similar results in isolated rat papillary (cardiac) muscle in which DTT (1 mM) almost fully reversed the effects of HOCl (300 μM) on resting tension but only partially reversed the effects on maximal force.The limited effectiveness of DTT in recovering maximal force production may explain the limited success of clinical trials of antioxidants for DMD and supports the development of treatments that reduce oxidant levels by preventing production or scavenging ROS (e.g.resveratrol, epicatechin) rather than reversing oxidation (Angelini et al., 2022;Shakibaei et al., 2012;Shay et al., 2015).
Taurine has been previously investigated as a putative treatment for DMD (recently reviewed: Merckx & De Paepe (2022)) and has been effective in ameliorating the dystrophic pathology in mdx mice as evidenced through increased muscle force production that was attributed to anti-inflammatory and anti-oxidant effects in addition to directly enhancing SR Ca 2+ and force production in skeletal muscle (Bakker & Berg, 2002;Caprogrosso et al., 2016;Terrill, Grounds et al., 2016a;Terrill, Pinniger et al., 2016b;Terrill et al., 2017;Terrill, Webb et al., 2020).HOCl reacts strongly with taurine to form taurine chloramine, which can inhibit pro-inflammatory mediators including TNFα and IL-1β (Choi et al., 2006).
An alternative treatment option is the use of MPO inhibitors.Although various MPO inhibitors have been investigated in preclinical studies using mouse models of diseases such as multiple system atrophy (Stefanova et al., 2012), Parkinson's disease (Choi et al., 2005;Jucaite et al., 2015) and post-myocardial infarction (Ali et al., 2016), the use of MPO inhibitors as a therapeutic treatment for DMD has not yet been investigated.The development of treatments for DMD which target reduced HOCl may also be important as a secondary treatment to currently available genetic treatments for DMD in instances where genetic treatments are not completely effective (Angelini et al., 2022).

Conclusions and implications for DMD disease pathology
The results from this study suggest a potential involvement of HOCl in the pathophysiology of DMD.A proposed mechanism for HOCl-induced muscle dysfunction is presented in Fig. 7.In this mechanism, the absence of dystrophin leads to a condition of chronic inflammation in which the increased presence of neutrophils results in increased MPO expression and activity (Hodgetts et al., 2006;Terrill, Radley-Crabb, et al., 2013b;Terrill, Pinniger et al., 2016b).The activity of MPO is almost five times higher in skeletal muscles of mdx mice compared with muscles of wild-type C57 mice, and is likely to result in significantly elevated concentrations of HOCl in dystrophic muscle via the actions of MPO on H 2 O 2 (Terrill, Boyatzis et al., 2013a).As this study has shown, in the short term, HOCl can target RyRs and TRP channel proteins leading to increases in cytosolic Ca 2+ and decreases in active force production.The results from this study suggest that HOCl could explain the complex interaction of inflammation, oxidative stress and Ca 2+ dysregulation that result in muscle weakness and wasting in DMD, and might present a potential therapeutic target for the development of future treatments for DMD. and revising of the work.P.P. was involved in the acquisition of all whole soleus muscle data; data analysis and interpretation of the data, and drafting of the revised manuscript.P.A., A.B. and G.P. were involved in conception and design of the work, analysis and interpretation of the data and drafting and revising of the work.

Figure 1 .
Figure 1.Effects of HOCl on resting tension and maximum isometric force and reversibility by dithiothreitol (DTT) in extensor digitorum longus (EDL) muscles from C57 and mdx mice Exposure to HOCl (200 μM) significantly increased resting tension in both C57 (A) and mdx (B) and decreased maximal force in both C57 (C) and mdx (D) muscles.Exposure to DTT (5 mM) significantly reversed increases resting tension in both C57 (A) and mdx (B) but did not significantly reverse the effects of HOCl on maximal force in either C57 (C) or mdx (D) muscles.* Indicates a significant difference between control and HOCl (P < 0.05), îndicates a significant difference between DTT and HOCl.Data presented as means ± SD.Sample size numbers were as follows: C57 control (n = 6), mdx control (n = 12), C57 200 μM HOCl (n = 8), mdx 200 μM HOCl (n = 11), C57 5 mM DTT (n = 6) and mdx 5 mM DTT (n = 6).[Colour figure can be viewed at wileyonlinelibrary.com]

Figure 7 .
Figure 7. Potential cellular mechanism of HOCl-induced muscle damage in Duchenne muscular dystrophy (DMD) HOCl is likely to be present in high concentrations in DMD due to increased neutrophil activity and MPO expression.HOCl impairs Ca 2+ handling by oxidising RyR and TRP channel proteins.Pathway 1 involves extracellular Ca 2+ entry into the SR via TRP channels; pathway 2 for extracellular Ca 2+ entry into the cytosol via TRP channels.HOCl may provide the link between the chronic inflammation, oxidative stress and disrupted Ca 2+ homeostasis seen in DMD.'HOCl' in red indicates oxidised by HOCl.DHPR: dihydropyridine receptor; HOCl: hypochlorous acid; MPO: myeloperoxidase; RyR: ryanodine receptors; TRP: transient receptor potential channel proteins; SERCA: sarcoplasmic reticulum Ca 2+ -ATPase; SR: sarcoplasmic reticulum.Created with BioRender.com.[Colour figure can be viewed at wileyonlinelibrary.com]