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Epigenetic Regulators Modulate Muscle Damage in Duchenne Muscular Dystrophy Model

Histone acetyl transferases (HATs) and histone deacetylases (HDAC) control transcription during myogenesis. HDACs promote chromatin condensation, inhibiting gene transcription in muscle progenitor cells until myoblast differentiation is triggered and HDACs are released. HATs, namely CBP/p300, activate myogenic regulatory and elongation factors promoting myogenesis. HDAC inhibitors are known to improve regeneration in dystrophic muscles through follistatin upregulation. However, the potential of directly modulating HATs remains unexplored. We tested this possibility in a well-known zebrafish model of Duchenne muscular dystrophy. Interestingly, CBP/p300 transcripts were found downregulated in the absence of Dystrophin. While investigating CBP rescuing potential we observed that dystrophin-null embryos overexpressing CBP actually never show significant muscle damage, even before a first regeneration cycle could occur. We found that the pan-HDAC inhibitor trichostatin A (TSA) also prevents early muscle damage, however the single HAT CBP is as efficient even in low doses. The HAT domain of CBP is required for its full rescuing ability. Importantly, both CBP and TSA prevent early muscle damage without restoring endogenous CBP/p300 neither increasing follistatin transcripts. This suggests a new mechanism of action of epigenetic regulators protecting dystrophin-null muscle fibres from detaching, independent from the known improvement of regeneration upon damage of HDACs inhibitors. This study builds supporting evidence that epigenetic modulators may play a role in determining the severity of muscle dystrophy, controlling the ability to resist muscle damage. Determining the mode of action leading to muscle protection can potentially lead to new treatment options for muscular dystrophies in the future.


INTRODUCTION
There is no cure to date for muscular dystrophies caused by absent or malfunctioning Dystrophin, be it lethal Duchenne or milder Becker type. Current therapies aim at alleviating the symptoms and delaying the progression of a disease that can be life threatening. One promising pharmacological treatment is to inhibit histone deacetylases (HDACs) (Consalvi et al., 2011). HDACs can regulate gene transcription in muscle progenitor cells, by controlling the activity of myogenic regulatory factors and MEF2 family proteins (McKinsey et al., 2000;Palacios and Puri, 2006). HDACs promote chromatin condensation, inhibiting gene transcription until myoblast differentiation is triggered and HDACs are released. Similarly, blocking HDACs leads to hyper acetylated chromatin, inhibiting condensation and therefore facilitating transcription. HDAC inhibitors ameliorate the dystrophic phenotype by promoting myogenesis and improving regeneration in dystrophic muscles (Consalvi et al., 2011;Giordani and Puri, 2013;Iezzi et al., 2002). Several studies show that follistatin upregulation by HDAC inhibitors is responsible for boosting regeneration in dystrophic muscles (Iezzi et al., 2004;Mozzetta et al., 2013). While studies have been focusing on blocking HDACs to promote hyper acetylated chromatin and transcription, the potential of directly modulating histone acetyl transferases (HATs) on a muscle dystrophy context remains unexplored. CBP (CREB Binding Protein) is a nuclear transcriptional co-activator with HAT activity, belonging to the p300/CBP family (Bedford et al., 2010). It is ubiquitously expressed and acetylates both histone and nonhistone proteins to regulate transcription. CBP was shown to functionally activate MyoD by acetylation and to directly interact with MEF2C (Giordani and Puri, 2013). CBP expression in zebrafish muscle was reported recently (Batut et al., 2015). We used the zebrafish model, extensively characterised and widely used for studying muscular dystrophy (Bajanca et al., 2015), to explore the effects of overexpressing CBP in a Dystrophin-null background. We have found that overexpressing this single HAT rescues the dystrophic phenotype as efficiently as blocking HDACs. Moreover, we observed that both treatments inhibit the early appearance of dystrophic fibres, prior to any effect on regeneration could take place. We report a follistatinindependent mechanism protecting against fibre damage.

RESULTS
To test whether Dystrophin absence affects HAT's expression we performed quantitative realtime PCR (qPCR) for p300 and/or CBP (crebbp) transcripts ( Figure 1A). Two-way ANOVA shows highly significant differences [F(1,24)=31.5, p<0.0001] between HAT genes expression in controls and embryos injected with well described Dystrophin morpholino cocktail (also see Supplementary Methods, Supplementary Figure 1A). Multiple comparisons indicate strong downregulation of crebbpb and p300b and milder but significant decrease of p300a expression (p values: crebbpb = 0.0010, p300a = 0.0247, p300b = 0.0045). Therefore, the absence of Dystrophin significantly affects HAT expression.
Taking these results, we set to determine whether the dystrophic phenotype could be rescued by overexpressing RFP-tagged murine CBP in dystrophin-null embryos (dmd ta222a/ta222a ).
Nuclear and dose dependent expression of the transgene was confirmed (Supplementary Figure   1B,C). To test the hypothesis that CBP overexpression improves regeneration, embryos were analysed at 2 dpf, as fibre tips start detaching from somitic borders (Bajanca et al., 2015), and 6 dpf, when regeneration upon damage is underway (Gurevich et al., 2016;Pipalia et al., 2016).
While dystrophin-null embryos show characteristic dystrophic muscles from early stages, overexpressing CBP clearly reduced damage not only at later stages but, unexpectedly, also at 2 dpf ( Figure 1B). Fast swimming was also restored, a characteristic failure of dystrophic embryos. Absence of damage as early as 2 dpf cannot be explained by stimulating regeneration, which takes several days (Gurevich et al., 2016;Pipalia et al., 2016), suggesting that CBP overexpression may act at an earlier phase preventing muscle degeneration in the first place.  Figure 1D). On average, 83% of dystrophin-null (dmd ta222a/ta222a ) embryos showed clear dystrophic muscles at 2 dpf ( Figure   1C, dmd -/-). The prevalence of a dystrophic signature was drastically reduced to 28% upon treatment with TSA and 27% when a low dose of CBP (+CBPlow) was used. A 10-fold increase of the CBP dose (+CBPhigh) improves the rescue significance (p<0.0001). Therefore, CBP overexpression is at least as efficient as HDACs inhibition in protecting dystrophin-null muscles from damage. Similar results were obtained in the Dystrophin morphant background for both HDACs inhibitor and CBP ( Figure 1D,E). Immunostaining for RFP-CBP shows that rescue is obtained in morphants even when detectable levels are seen in few muscle nuclei (Supplementary Figure 1D). Overexpression of a CBP form lacking active HAT domain leads to mild rescue indicating that the acetylase activity of CBP is essential for full rescue (+HAT; Figure 1D,E).
Since CBP/p300 expression is generally downregulated in Dystrophin-depleted embryos ( Figure 1A), we tested whether CBP overexpression or TSA act though increasing endogenous crebbp/p300 levels. Dystrophin-depleted embryos were co-injected with CBP (10 7 molecules) or treated with TSA and qPCR was performed at 2 dpf ( Figure 1F; Supplementary Figure 1E).
One-way ANOVA revealed statistically significant differences between treatment groups for  Figure 1F). Therefore, rescue is not due to restoring endogenous CBP/p300 transcripts levels.
While the mechanism of action of HDAC inhibition leading to improved regeneration is not fully understood, follistatin upregulation is a key event (Iezzi et al., 2004;Mozzetta et al., 2013).
We questioned whether the follistatin pathway could also be involved in the early protection against muscle damage observed in TSA treatment and CBP overexpression. Quantitative analysis of the two zebrafish follistatin transcripts, fsta and fstb, expression was performed at 2 dpf ( Figure 1G). Dystrophin depletion does not significantly affect fsta or fstb levels. There were no statistically significant differences in the expression of fsta between groups as Therefore, the protection against muscle damage granted by CBP overexpression or TSA treatment does not rely on stimulating follistatin transcription but likely on a follistatinindependent pathway.

FINAL REMARKS
This study shows for the first time that either CBP overexpression or pan-HDAC inhibition are able to prevent, or at least delay, early stages of muscle damage in zebrafish dystrophic muscles.
This observation adds up to the recognised positive effect of HDAC inhibitors on muscle regeneration upon damage (Consalvi et al., 2011). Other authors observed that treating mouse dystrophic muscles (mdx) with deacetylase inhibitors was able to confer resistance to contraction-coupled degeneration (Minetti et al., 2006). However, this was suggested to be mediated by follistatin through satellite cell number increase. We show that damage protection occurs independently from regeneration. Follistatin upregulation is not involved in protecting early zebrafish muscle from damage, supporting the idea that the two rescuing mechanisms triggered by HDAC inhibition are independent. Interestingly, overexpression of a single HAT, CBP, achieves a similar level of protection as pan-HDAC inhibition. The strong downregulation of fstb by TSA but not CBP should be investigated in future work to determine whether these agents may act through different mechanisms of action. Our results prompt for investigating similar mechanisms in other animal models and identifying new therapeutic targets, possibly with more restricted and controlled effects than HDAC inhibitors. This study strengthen the idea that epigenetics plays a role in the progression of symptoms in some patients with Dystrophin mutations. This could explain the variation in severity in Becker muscular dystrophy patients with identical mutation and the variable progression of DMD. Doubt persists on the mechanism leading to protection. To further understand the mechanisms of action triggered by either HDAC inhibition or CBP a high throughput analysis needs to be employed in future studies.

Animals
Fish used were wild-type Danio rerio, dmd ta222a/+ and dmd ta222a/+ crossed to Tg(actc1b:mCherry) pc4 to facilitate identifying dystrophic muscles. All animals were handled in a facility certified by the French Ministry of Agriculture (approval ID B-31-555-10) and in accordance with the guidelines from the European directive on the protection of animals used for scientific purposes (2010/63/UE), French Decret 2013-118. Staging and rearing was performed as described (Westerfield, 1995). Plasmids and morpholinos were injected into 1cell stage embryos at the indicated concentrations ((Johnson et al., 2013); see text).
Phenolthiourea (0.003%) was added to inhibit pigmentation and facilitate muscle analysis. Dystrophic muscles were characterised by abnormal birefringence, supported by observing disruption of the actin reporter pattern when Tg(actc1b:mCherry) pc4 fish were used, and impaired movement (Bajanca et al., 2015;Berger et al., 2012). The absence of Dystrophin expression in rescued embryos was confirmed by immunohistochemistry.

Plasmids, morpholinos and HDACs inhibition
pCS2+mRFP-CBP and pCS2+mRFP-HAT plasmids were created by digesting by BamH1 full length CBP of mouse origin, or CBP deleted for amino acids 1430 to 1570 from pCMV-HA-CBP and pCMV-HA-CBPHAT (provided by A. Harel-Bellan) and inserting them into pCS2+mRFP-N1 vector (Addgene) digested by BamH1. Injections were performed at one-cell stage with the indicated amounts of RFP-CBP and RFP-CBPHAT RNA. Mouse CBP shares 85% protein identity to both zebrafish CBP-A (the product of crebbpb) and zebrafish CBP-B (the product of crebbpa), according to the standard NCBI BLAST tools. Zebrafish protein CREBBPA (also called CREBBPb or CBP-B) corresponds to protein ID ENSDARP00000081684, while CREBBPB protein ID (also called CREBBPa or CBP-A) corresponds to ENSDARP00000086306.
Morpholinos were from Gene-tools and were used as a cocktail as described previously (dmd-MO1 at 4 ng per embryo and dmd-MO6 at 7.5ng/embryo; (Johnson et al., 2013). Embryos were exposed to Trichostatin A (TSA, Sigma) for 24 hours, from 24 to 48 hpf. TSA was added to fish water for a final concentration of 200 nM as described previously (He et al., 2014;Johnson et al., 2013).