Prox1 gene transfer combined with voluntary exercise improves dystrophic muscle fragility in Mdx mice Running head: Prox1 improves fragility of exercised Mdx muscle

Background. Voluntary exercise can improve skeletal muscle fragility, i.e. higher susceptibility to contraction induced-injury, as shown by a greater force drop following lengthening contractions, in the dystrophic Mdx mice as compared to healthy mice with dystrophin. This beneficial effect is related to the activation of the calcineurin activation. Unfortunately, voluntary running only partly rescued fragility, so it would be interesting to combined the effects of exercise, for example, with those of others treatments activating the calcineurin pathway and promoting slow and more oxidative fibres. This is of particular interest because slow muscle fibres are apparently less affected and genetic or pharmacological treatments promoting slow and more oxidative fibres are been shown to be beneficial in the Mdx mice . Methods. Here, we tested whether voluntary exercise (1 month of running in a wheel) combined with Prospero-related homeobox factor 1 gene ( Prox1) transfer would better improve functional dystrophic features in Mdx mice as compared to the voluntary exercise single approach. Prox1 is known to promote the promotion of slow contractile gene program in healthy muscle . We slower molecular and functional contractile features transfer absolute maximal force production Mdx mice. Conclusion. Our the beneficial effects of voluntary exercise and Prox1 transfer on fragility Mdx mice.


Backround
Dystrophin deficiency results in Duchenne muscular dystrophy (DMD), the most common lethal inherited muscle disease in boys. Dystrophin is a costameric protein that plays a role in force transmission and sarcolemma stability in skeletal muscle (1)(2)(3). Muscle of dystrophin deficient mice exhibits two important functional dystrophic features. The first is weakness, i.e. reduced specific maximal force (absolute maximal force generated relative to muscle cross-sectional area or weight), whereas absolute maximal force was unchanged because muscle is hypertrophied in the "classic" Mdx DMD model (C57BL/10ScSn-Dmd Mdx/J)(4-6), or decreased in the "new" D2.B10 DMD model (D2-Mdx) (7). The second is fragility, i.e., fast and low oxidative muscle in Mdx mice, but not slow and high oxidative muscle, is susceptible to damage caused by lengthening (eccentric) contractions, which cause an immediate marked force drop following lengthening contractions (7)(8)(9)(10)(11)(12).
Interestingly, chronic muscular exercise can improve these functional dystrophic features in Mdx mice (13)(14)(15). In particular, voluntary wheel running (1 week to 1 year) can increase specific or absolute maximal forces in hindlimb muscle of Mdx mice (5,(16)(17)(18)(19)(20)(21). It was also reported that voluntary running improves (reduces) fragility in Mdx mouse fast muscle (21,22), whereas physical inactivity (disuse) aggravates it (21). Different signalling pathways are activated by exercise in healthy muscle, leading to beneficial muscle remodelling, i.e. cellular and molecular muscle adaptations, such as the promotion of slower and more oxidative muscle fibres (23)(24)(25)(26). Numerous studies support the notion that the muscle remodelling induced by chronic exercise is partly dependent on the activation of the calcineurin pathway in healthy muscle (25)(26)(27)(28)(29)(30). Recently, we found that the improved fragility induces by voluntary running in Mdx mice was related to calcineurin pathway activation, and changes in the program of genes involved in slower contractile features of muscle fibre (22). Unfortunately, voluntary running only partly rescued fragility (21,22), so it would be interesting to combined the effects of exercise, for example, with those of others treatments targeting the calcineurin pathway.
While voluntary exercise offers potential therapeutic benefit, additional adjunct therapies could further improve functional dystrophic features. In the recent years, genetic or pharmacological treatments promoting slower and more oxidative fibres are been shown to be beneficial in the Mdx mice. In fact, several studies support the idea that activation of the AMPK, calcineurin, E2F1, ERRγ, IGF1, SIRT1 and PGC1 signalling pathways alleviates some of the dystrophic features in Mdx muscle (31)(32)(33)(34)(35)(36)(37)(38)(39)(40)(41)(42)(43)(44)(45)(46)(47)(48)(49)(50). For example, genetic activation of calcineurin pathway improves fragility in fast muscle of the Mdx mouse, but decreases maximal force production, thus, aggravating weakness (47). Recently, it was demonstrated that the loss of Prospero-related homeobox factor 1 (Prox1) represses the expression of slow contractile genes in healthy fast muscle, whereas its overexpression via Prox1 transfer has the opposite effect and downregulates the fast contractile genes (51,52). In particular, the inactivation of Prox1 reduces the expression of the slowest myosin heavy chain Myh7 in fast healthy muscle, without affecting oxidative capacity (succinate dehydrogenase staining) and absolute maximal force (52). It was found that Prox1, that is more expressed in slow fibres, is involved in the activation of the NFAT/calcineurin pathway and several of its downstream genes involved in the promotion of slow gene program, and is a regulator of satellite cell differentiation (51). However, it is not yet known (i) whether Prox1 transfer is also beneficial for functional dystrophic features, and in particular (ii) whether voluntary running and Prox1 transfer have additive beneficial effects.
The purpose of the present study was to analyse the effect of Prox1 transfer in voluntary exercised Mdx mice on hindlimb muscle weakness and fragility. We demonstrate that voluntary running and Prox1 transfer have additive beneficial effects on fragility, without affecting specific maximal force. However, we also found that Prox1 transfer reduced muscle weight and consequently reduced absolute maximal force in voluntary exercised Mdx mice.

Animals and voluntary running
All procedures were performed in accordance with national and European legislations and were approved by our institutional Ethics Committee "Charles Darwin" (Project # 01362.02). Mdx mice (C57BL/10ScSc-DMDMdx/J)(Mdx) and sex and age-matched wildtype control mice (C57BL/10)(C57) were used. Mice were randomly divided into different control and experimental groups. In the first set of experiment, Mdx mice at 2.5 months of age were placed (Mdx+wheel) or not (Mdx) in separate cages containing a wheel and were allowed to run 1 month ad libitum. Mdx mouse runners received Prox1 transfer into the muscle 3 days before the initiation of voluntary exercise (Mdx+wheel+Prox1). The running distances were collected and daily running distance was 4.2 ± 0.1 km/day. In the second set of experiments, the muscle of sedentary Mdx mice received (Mdx+Prox1) or not (Mdx) Prox1 transfer. Muscles were measured and collected 4 weeks after Prox1 transfer.

Muscle weakness and fragility
Muscle weakness (reduced specific maximal force) and fragility (susceptibility to contraction-induced injury) were evaluated by measuring the in situ TA muscle contraction in response to nerve stimulation, as described previously (53,54). Mice were anesthetized using pentobarbital (60 mg/kg, ip). Body temperature was maintained at 37°C using radiant heat. The knee and foot were fixed with pins and clamps and the distal tendon of the muscle was attached to a lever arm of a servomotor system (305B, Dual-Mode Lever, Aurora Scientific) using a silk ligature. The sciatic nerve was proximally crushed and distally stimulated by a bipolar silver electrode using supramaximal square wave pulses of 0.1 ms duration. We measured the absolute maximal force that was generated during isometric contractions in response to electrical stimulation (frequency of 75-150 Hz, train of stimulation of 500 ms). Absolute maximal force was determined at L0 (length at which maximal tension was obtained during the tetanus). Absolute maximal force was normalized to the muscle mass as an estimate of specific maximal force, an index of muscle weakness.
The rate of force development (RFD), a marker of fibre-type, was measured when the force increased from 5 to 50% of P0. Fragility, i.e., susceptibility to contraction-induced injury in Mdx mice was estimated from the force drop resulting from lengthening contraction-induced injury. The sciatic nerve was stimulated for 700 ms (frequency of 150 Hz). A maximal isometric contraction of the TA muscle was initiated during the first 500 ms. Then, muscle lengthening (10% L0) at a velocity All sequences of primers used are presented in Table 1.

Histology
Transverse serial sections (8 µm) of TA muscles were obtained using a cryostat, in the midbelly region. Some of sections were processed for histological analysis according to standard protocols (succinic dehydrogenase, SDH). Other sections were processed for immunohistochemistry as described previously (55).

Statistical analysis
Groups were statistically compared using Student T-test, and variance analysis (1 or 2 ways).
If necessary, subsequent Bonferroni post-hoc test was also performed. For groups that did not pass tests of normality and equal variance, non-parametric tests were used (Kruskal Wallis and Wilcoxon). Values are means ± SEM.

1-Prox1 transfer in voluntary exercised Mdx mice promotes slower contractile features
We first determined whether Prox1 transfer increased slower contractile features in  Figure   1C). Moreover, there was no difference between Mdx+wheel+Prox1 and Mdx+wheel muscles in the expression of a marker of oxidative capacity, Sdha, a gene encoding a complex of the mitochondrial respiratory chain ( Figure 1B). In line, histological analyses revealed that the percentage of the cross-sectional muscle area occupied by weak succinic dehydrogenase staining (SDH) staining was not different between Mdx+wheel+Prox1 muscle and Mdx+wheel muscle ( Figure 1D). In situ isometric TA muscle force production in response to nerve stimulation was also performed and indicated that the rate of force development, a marker of the contractile functional phenotype, was decreased (x 0.5) in Mdx+wheel+Prox1 muscle as compared to Mdx+wheel muscle (p < 0.05)( Figure 1E). These data demonstrate that intramuscular delivery of AAV-Prox1 induced a substantial fast to slower contractile transition in the TA muscle of voluntary exercised Mdx mice, in line with previous studies using not-exercised healthy muscle (51,52).  Figure   1G).

2-Prox1 transfer in voluntary exercised Mdx mice further improves muscle fragility
An immediate force drop was observed following lengthening contractions in Mdx muscle There was no such force drop following 9 lengthening contractions in C57 muscle with dystrophin (22,54). This result indicated the higher susceptibility to contraction-induced injury in Mdx muscle, i.e. fragility. In line with previous published results (21,22), the force drop following lengthening contractions in Mdx+wheel muscle was reduced as compared to Mdx muscle (p < 0.05) (Figure 2A The increased expression of slow contractile genes (Myh7 and Tnni1) and the reduced expression of fast contractile gene (Myh4) described above can explained at least in part, the improved fragility in Mdx+wheel+Prox1 muscle since in the recent years, genetic or pharmacological treatments promoting slow fibre gene program are been shown to improve fragility in the Mdx mice. In addition, we tested the possibility that Prox1 transfer also improved fragility via the modifications of the expression of genes coding membrane ions channels since fragility in Mdx mice was also related to excitability and membrane ions channels (22,54). Several genes coding ion membrane channels interacting with dystrophin are involved in muscle excitability, such as Scn4a, Cacna1s, Slc8a1, Trpc1 and chrna1 (57)(58)(59). We found that the expression of Trpc1 encoding for transient receptor potential cation channel subfamily C member 1 (x 2.1) was increased in Mdx+wheel+Prox1 muscle as Increased NADPH oxidase 2 (NOX2) activity was also related to fragility in Mdx mice (60)(61)(62). Thus, we determined whether the reduced force drop following lengthening contractions induced by Prox1 transfer was associated to change in NOX2 pathway. We found no change in the expression of PrxII, Gp91phox, P47phox and Rac1 ( Figure 2D

Prox1 transfer only improves fragility in voluntary exercised Mdx mice
The present study confirms our previous studies (21,22) showing that voluntary exercise alleviates the fragility, a major dystrophic functional feature, in fast anterior crural muscles (TA and extensor digitorum longus) of Mdx mice, such as Dmd based preclinical therapy (54). We found that this beneficial effect of Prox1 transfer in exercised Mdx mice is related to the promotion of slower contractile features, but not to a more oxidative phenotype. This observed relationship between improved fragility and slower contractile features was supported by the 2 following points. First, slow muscle is less fragile than fast muscle in Mdx mice (9)(10)(11). Second, exercise and pharmacological or genetic activation of signalling pathways, such as calcineurin, PPAR-β, PGC1-α, and AMPK, promoting a slower and more oxidative gene program, improve fragility in Mdx mice (21,22,38,46,47,(72)(73)(74). It was previously demonstrated that Prox1 promotes slower fibres by activating the NFATcalcineurin pathway (51), a signalling pathway playing an important role in fibre type specification (27,75). In particular, Prox1 induces nuclear translocation of NFATC1 (51), reflecting the activation of the NFAT-calcineurin pathway (75).
In addition, it is possible that Prox1 transfer improves fragility in voluntary exercised Mdx mice by a preserved excitability, as voluntary exercise and Dmd based therapy (22,54). In our experiments, reduced excitability, i.e. plasmalemma electrical dysfunction leading to defective generation and propagation of muscle potential action, largely contributes to the immediate force drop following lengthening contractions in Mdx mice (22,54). It remains to be determined whether the upregulation of the membrane ion stretch-activated channel Trpc1 induced by Prox1 transfer in voluntary exercised Mdx mice contributes to this improvement. Higher level of TRPC1 or activity of stretch-activated channels are generally associated with a worst dystrophic phenotype and fragility (8,76,77). In line with the present study, it was previously reported that the improved TA muscle excitability and fragility induced by voluntary exercise and calcineurin pathway activation were also related to change in the expression of genes encoding membrane ion channels (22).
Previous studies suggest that increased NOX2 activity is related to fragility in Mdx mice (60)(61)(62). However, our results show that Prox1 transfer in exercised Mdx muscle does not reduce the expression of Nox2 subunits (Gp91phox, P47phox and Rac1), which are shown to produce an elevated level of ROS in Mdx mice (62). Moreover, we found no increased expression of the gene encoding the antioxidant enzyme PrxII, whose overexpression improves fragility in Mdx mice (61). Concerning Des it is not impossible that the increased expression of Des in exercised Mdx mice in response to Prox1 transfer contributes to the improvement of the fragility, since desmin plays a structural role and participate to lateral force transmission (2). This beneficial effect of Prox1 overexpression on fragilty in exercised Mdx mice does not appear to be related to the reduced muscle weight that we observed, since it can be associated with an aggravation of fragility (21)

Prox1 transfer reduces maximal force production in both exercised and sedentary Mdx mice
Although Prox1 transfer improves fragility in voluntary exercised Mdx mice, we found that it has a detrimental effect on maximal force production (-33%). This latter effect was caused by a reduced muscle weight that was likely explained by decreased muscle fibre diameter since we found no increased numbers of fibres expressing MHC-1, MHC-2a and MHC-2x, that are smaller than the fibres expressing MHC-2b (56) whereas the total number of fibres was likely not altered by Prox1 transfer. Our result suggesting that Prox1 is a factor influencing muscle growth and maintenance in Mdx mice is another interesting finding. In line, several genetic or pharmacological treatments promoting slower and more oxidative fibres are been shown to induce atrophy in Mdx mice (46,47,50). Unexpectedly, we found that the atrophic state in response to Prox1 transfer is associated to downregulation of Mstn, a negative regulator of muscle growth (79,80), without other transcriptional changes regarding several well-known atrophic processes.

Conclusion
Our results indicate that Prox1 transfer promotes a slower molecular and functional

Consent for publication
Not applicable.

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

Gene Forward Reverse
House keeping gene