*4.5. Pharmacological Approaches to Enhance Satellite Cell Myogenic Capacity*

Besides cell-based therapies and gene replacement strategies aimed at satellite cell reprogramming in DMD, new pharmacological interventions have been recently explored to target muscle stem cell microenvironment and stimulating intrinsic repair (see Table 4). Asymmetric cell division plays a pivotal role in the maintenance of the satellite cell pool. The granulocyte colony-stimulating factor receptor (G-CSFR) is asymmetrically segregated during muscle stem cell division and the G-CSF/G-CSFR axis supports long-term muscle regeneration in mice. Filgrastim, a G-CSF analogue currently being tested for efficacy and safety in a Phase I study on DMD patients (NCT02814110), increased myocytes and improved regeneration in *mdx* mice [128].

A treatment with the secreted factor Wnt7a, which drives the symmetric expansion of satellite cells [129,130], resulted in increased specific muscle force and reduced contractile damage in *mdx* mice. In parallel, it induced hypertrophy and a shift toward slow-twitch in human primary myotubes [19].

β1-integrin is another essential niche molecule that maintains satellite cell homeostasis, sustaining the expansion and self-renewal of the stem cell pool during regeneration. The exogenous administration of β1-integrin enhanced regeneration in vitro and also muscle function in vivo in *mdx* mice [131].

Interesting preclinical results were also obtained via pharmacological inhibition of p38MAPK, which is aberrantly regulated in regenerating dystrophic muscles, although the exact mechanism and the link with regeneration and myogenesis remains to be better determined. Treatment with the p38MAPK-inhibitor SB731445 in the *Sgcd*−/<sup>−</sup> mouse model was able to ameliorate the dystrophic phenotype and to improve the self-renewal of satellite cells [132].

Finally, unacylated ghrelin (UnAG) is a circulating hormone that protects muscle from atrophy, promotes myoblast differentiation, and enhances ischemia-induced muscle regeneration. UnAG was found to reduce muscle degeneration, improve muscle function, and increase dystrophin-null SC self-renewal in *mdx* mice, maintaining the satellite cell pool [133]. These first preclinical observations support the use of drugs aiming to directly restore polarity and proper mitotic division of satellite cells, as part of DMD therapy in the future, although a larger body of evidence regarding the mechanisms underlying their effects in dystrophic muscles will be needed to improve data translatability.

**Table 4.** Pharmacological approaches targeting niche homeostasis in DMD. Synthetic overview of drugs, new or repurposed, targeting the muscle stem cell niche microenvironment in dystrophic muscles by acting on inflammation, fibrosis, or self-renewal, and of their effects on regenerative biomarkers in *mdx* mice muscles. For drugs translated into clinical settings, the stage of development in DMD patients is indicated, and for repurposed drugs, the approval for other pathological conditions. \* ClinicalTrials.Gov identifiers.



#### **Table 4.** *Cont.*


#### **Table 4.** *Cont.*


#### **Table 4.** *Cont.*

Abbreviations: ANG II, angiotensin II; AMPK, AMP-dependent protein kinase; Bax, Bcl-2-associated X protein; Bcl2, B-cell lymphoma 2; CNF, centronucleated fibers; COX1, COX2, cyclooxygenase 1 and 2; COXIBs, cyclooxygenase 2 inhibitors; CTGF, connective tissue growth factor; EMSA, electrophoretic mobility shift assay; Fgf2, fibroblast growth factor 2; G-CSFR, granulocyte colony stimulating factor; GHS-R, growth hormone secretagogue receptor; IL-1Ra, interleukin 1 receptor antagonist; NSAIDs, nonsteroidal anti-inflammatory drugs; PG, prostaglandin; ROS, reactive oxygen species; Wnt7a, wingless-type MMTV integration site family, member 7A.

#### **5. Discussion**

Satellite cells are muscle-committed stem cells resident in skeletal muscle, importantly contributing to muscle growth and differentiation, and allowing an efficient repair process after damage. Multiple intrinsic and extrinsic factors are involved in the orchestration of this complex process, thereby fascinating scientists for potential applications in the field of regenerative medicine, and for developing drugs able to counter progressive muscle wasting disorders, by enhancing an efficient repairing process in inherited or acquired conditions. DMD is a prototype of the efforts in this field, due to the intense research aimed at identifying potential therapies. DMD in fact has no cure; the progressive muscle degeneration is directly related to the events following the primary defect, which are related in a complex cross-talk, to the inefficient regeneration process. Accordingly, satellite cells are believed to be pivotal in determining disease outcome, since the exhaustion of the satellite cell pool causes the absence of a turning point of this muscle disorder. Intrinsic defects of satellite cells, due to the absence of dystrophin, have been described, and the role of the niche in the exhaustion of the satellite cell pool is still debated. As a matter of fact, the niche environment seems to be paramount in the ability of satellite cells to repair the continuous cycles of degeneration/regeneration happening in DMD [15,17,20]. Satellite cells' niche is disrupted over time by the inflammation and fibrosis occurring as the disease progress. Therefore, while the effort to treat the primary defect is still the main target of the scientific community, drugs able to act on muscle environment deserve to be taken into consideration. This approach concerns both novel drugs and repurposed ones, the latter having the additional advantage of a more rapid translational potential from bench to clinic. Drugs with the best clinical potential would ideally target main pathogenic events, reducing damage in parallel, making repair efficient. Knowing all that, we took advantage of the large amount of preclinical data obtained in our and other laboratories with the main aim of summarizing the available evidence of a potential drug action on regenerative efficiency via a direct or indirect action on stem cell niche and satellite cells.

As reviewed here, there are many promising compounds able to improve muscle regeneration, and even glucocorticoids have a positive outcome in improving myofiber regeneration and enhancing the maturation process, mostly in relation to the regimen approach, which may in part account for their clinical efficacy. Importantly, the complex and not fully clear action of glucocorticoids in dystrophic muscle, and mostly in the satellite cell niche, deserves to be better investigated, as these drugs are able to counter degeneration while sustaining the regeneration process.

In particular, drugs acting on different ILs and other inflammatory cytokines are promising [90, 100,104] and should be investigated in more depth. These drugs can take advantage of studies performed in DMD patients, and possibly gain better insight by looking at biomarkers of regeneration. Importantly, great progress has been made to identify reliable non-invasive biomarkers of both pathology progression and regeneration and drug efficacy. These will greatly help the translational assessment of therapy efficacy in the stem cell niche [42,66–83]. In a general situation, even if it is not clear yet whether is the exhaustion of the satellite cell pool or niche disruption drives the pathological progression, we know that drugs acting either on the amelioration of the niche environment or satellite cells' asymmetric division could be good candidates to slow down DMD.

At the same time, what we have learned from a complex disease such as DMD is that the outcome of drug action on regeneration efficiency is not always straightforward in spite of robust hypotheses and a clear mechanism of action. Interestingly, among novel potential therapies, our analysis of literature data enlightened that drugs purely directed against inflammatory molecules (e.g., TNF-α, NF-κB) are mostly able to reduce muscle damage but not improve regeneration, whilst several therapeutic interventions inhibiting or modulating molecules with a pleiotropic action seem to positively impact regenerative biomarkers, in parallel with controlling damage. These molecules, i.e., myokine IL-6, pro-fibrotic TGF-β1, c-AMP dependent pathways and estrogen receptors, and all self-renewal mediators (e.g., G-CSF, Wnt7a, β1-integrin, p38MAPK, ghrelin derivatives), represent potential therapeutic targets of new/repurposed drugs for DMD to specifically support regeneration efficiency, via a direct action on satellite cells or by improving niche environment. In parallel, the effects on regeneration via

the pharmacological modulation of other promising targets, e.g., HDACs and ANGII pathways, need to be further explored. In particular, this topic will grant further insight into the specific roles in the regeneration process of different HDAC isoenzymes, and roles in alternative pathways of the RAS system, such as those mediated by ANG 1-7 via MAS receptor [86,87,118].

Then, we still need to understand key aspects of this multi-actor process that in turn have to be approached at different levels. In fact, there are major key players in disease progression and homeostasis maintenance, and it is reductive to assess that the disease progression is caused by the exhaustion of the satellite cells' niche. Then, combined strategies may work in synergy and such synergy may also occur with innovative molecular or cell therapies able to restore dystrophin expression. In fact, drugs able to address regeneration efficiency, although they will not cure, create ideal circumstances to sustain therapies aimed at correcting primary DMD defects, by creating a healthier environment. This has not been done yet, and the possible outcomes are not predictable, although they would ideally be of high clinical relevance.

In addition, the possibility to enhance our understanding of drug targetable events in myogenesis may also help to improve the in vitro approach of tissue engineering for building up both experimental platforms for drug discovery and simulation of reparative medicine.

**Funding:** This research was funded by Fondazione CON IL SUD "Brains to South" project n. 2018-PDR-00351 granted to O.C. entitled "Optogenetic engineered artificial muscle", by PRIN-MIUR (Research Projects of National Interest Ministry of Education, University and Research) project n. 2017FJSM9S\_005 entitled "New pharmacological strategies modulating PGC1alpha signalling and mitochondrial biogenesis to restore skeletal and cardiac muscle functionality in Duchenne Muscular Dystrophy", granted to A.D.L., and by The Dutch Duchenne Parent Project NL (DPP NL) Small Project entitled "Optogenetic engineered artificial muscle to enhance preclinical studies in DMD", granted to O.C.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**


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