Halofuginone promotes satellite cell activation and survival in muscular dystrophies

Abstract

Halofuginone is a leading agent in preventing fibrosis and inflammation in various muscular dystrophies. We hypothesized that in addition to these actions, halofuginone directly promotes the cell-cycle events of satellite cells in the mdx and dysf(-/-) mouse models of early-onset Duchenne muscular dystrophy and late-onset dysferlinopathy, respectively. In both models, addition of halofuginone to freshly prepared single gastrocnemius myofibers derived from 6-week-old mice increased BrdU incorporation at as early as 18h of incubation, as well as phospho-histone H3 (PHH3) and MyoD protein expression in the attached satellite cells, while having no apparent effect on myofibers derived from wild-type mice. BrdU incorporation was abolished by an inhibitor of mitogen-activated protein kinase/extracellular signal-regulated protein kinase, suggesting involvement of this pathway in mediating halofuginone's effects on cell-cycle events. In cultures of myofibers and myoblasts isolated from dysf(-/-) mice, halofuginone reduced Bax and induced Bcl2 expression levels and induced Akt phosphorylation in a time-dependent manner. Addition of an inhibitor of the phosphinositide-3-kinase/Akt pathway reversed the halofuginone-induced cell survival, suggesting this pathway's involvement in mediating halofuginone's effects on survival. Thus, in addition to its known role in inhibiting fibrosis and inflammation, halofuginone plays a direct role in satellite cell activity and survival in muscular dystrophies, regardless of the mutation. These actions are of the utmost importance for improving muscle pathology and function in muscular dystrophies.

Pharmacological therapeutics targeting the secondary defects and downstream pathology of Duchenne muscular dystrophy

“…Halofuginone is a low molecular weight plant alkaloid that plays dual roles – it inhibits TFG-β-mediated collagen synthesis signaling by impeding Smad 3 binding to DNA 102 , but also enhances Akt and MAPK/ERK signaling to promote myotube fusion in mdx and wild type primary myoblasts 103 . Ten weeks of halofuginone treatment in 4-week old mdx mice decreased diaphragm fibrosis, increased the number of revertant fibers 104 , and promoted satellite cell activation and survival 105 . The pro-proliferative features of halofuginone, in addition to its anti-fibrotic action, could have positive implications for muscle regeneration in DMD patients.…”

“…NO stimulates the conversion of guanosine triphosphate (GTP) to cyclic guanosine monophosphate (cGMP), stimulating vasodilation. In healthy muscle, nNOS is enriched at the sarcolemma in fast twitch muscle fibers, but in mdx and DMD muscle, nNOS is mislocalized and its activity decreases by 80% 105, 190, 191 , implicating aberrant NO signaling in contributing to dystrophic pathology. Accordingly, mouse studies validate negative effects of low NO-induced ischemia on skeletal muscle contraction and fatigue 192, 193 .…”

Early pathological signs in youngdysf−/−mice are improved by halofuginone

“…In MDs, halofuginone has been reported to inhibit muscle fibrosis and pathology in several mouse models [25,26], including dysferlinopathies post-disease initiation, and to prevent the late outcome of the disease [27]. Moreover, halofuginone increased myofiber-attached satellite cell proliferation and myotube fusion in vitro, and reduced apoptosis in mouse models for DMD and dysferlinopathies, demonstrating a direct effect of halofuginone on muscle cells [28][29][30].Halofuginone has recently been shown to ameliorate sarcolemmal integrity and lysosome trafficking in dysf -/myotubes and single myofibers. Halofuginone increased synaptotagmin-7 levels and its spread across the cytosol in dysf -/myofibers and muscle tissue, and increased its colocalization with lysosomes [31].The mechanism of action of halofuginone is not fully understood.…”

“…">Cell preparation and fusion-index assayPrimary myoblasts from the hindlimb muscles of 5-week-old Wt or dysf -/mice were prepared as described previously [29,30]. Cells were plated at 5 × 10 4 cell/cm 2 on 60-mm diameter Petri dishes and grown as described previously [30]. Growing myoblasts were induced to differentiate with DMEM containing 2% (v/v) horse serum for 24 h [28,30], then the medium was switched to 20% (v/v) fetal bovine serum (FBS) in DMEM for another 24 h (day 2), 96 h (day 5) and 192 h (day 8).…”

“…Cells were plated at 5 × 10 4 cell/cm 2 on 60-mm diameter Petri dishes and grown as described previously [30]. Growing myoblasts were induced to differentiate with DMEM containing 2% (v/v) horse serum for 24 h [28,30], then the medium was switched to 20% (v/v) fetal bovine serum (FBS) in DMEM for another 24 h (day 2), 96 h (day 5) and 192 h (day 8). Cells were fixed and stained for myosin heavy chain (MyHC) antibody (MF-20, diluted 1:10; Hybridoma Bank, Iowa City, IA).…”