Author contributions: S.L.: collection of data, data analysis and interpretation, and manuscript writing; H.S., B.J., Y.G., Z.C., C.C. and C.H.: collection of data; C.K.: provision of study materials and manuscript writing; W.S.P.: provision of study materials; L.W.: conception and design, data analysis and interpretation, and manuscript writing.
Disclosure of potential conflicts of interest is found at the end of this article.
First published online in STEM CELLSEXPRESS January 10, 2013.
Muscular dystrophies are a group of devastating diseases characterized by progressive muscle weakness and degeneration, with etiologies including muscle gene mutations and regenerative defects of muscle stem cells. Notch signaling is critical for skeletal myogenesis and has important roles in maintaining the muscle stem cell pool and preventing premature muscle differentiation. To investigate the functional impact of Notch signaling blockade in muscle stem cells, we developed a conditional knock-in mouse model in which endogenous Notch signaling is specifically blocked in muscle stem cell compartment. Mice with Notch signaling inhibition in muscle stem cells showed several muscular dystrophic features and impaired muscle regeneration. Analyses of satellite cells and isolated primary myoblasts revealed that Notch signaling blockade in muscle stem cells caused reduced activation and proliferation of satellite cells but enhanced differentiation of myoblasts. Our data thus indicate that Notch signaling controls processes that are critical to regeneration in muscular dystrophy, suggesting that Notch inhibitor therapies could have potential side effects on muscle functions. STEM CELLS2013;31:823–828
Skeletal muscle is capable of initiating a highly regulated myogenic process leading to muscle growth, maintenance, and repair [1, 2]. This process is mainly dependent upon a quiescent pool of stem cells (satellite cells). In response to growth, injury, or increased exercise, satellite cells become activated and proliferate as mononucleated progenitor cells (myoblasts), which then differentiate into multinucleated myotubes and further mature as functional muscles. Importantly, satellite cells are able to self-renew to replenish the stem cell pool for further rounds of muscle regeneration.
Muscular dystrophies are a heterogeneous group of devastating diseases characterized by progressive muscle weakness and degeneration , and currently there are no effective treatments. The development and progression of muscular dystrophies is a result of multiple factors including muscle gene mutations and cell-autonomous failure in muscle stem cells [3–5]. Currently, stem cell-mediated therapeutic strategies such as muscle stem cell transplantation or functional enhancement of endogenous muscle stem cells are being pursued to improve muscle functions and pathology in muscular dystrophy patients [2, 3, 6, 7]. However, the development of effective therapies requires a better understanding of molecular and cellular mechanisms regulating muscle stem cell behaviors.
Notch signaling is a developmental signaling pathway with critical roles in the highly coordinated muscle regenerative process [8–11]. Notch signaling is required for muscle stem cell maintenance as well as their proliferation and activation in response to regenerative cues [8–12]. At a later stage of myogenesis, when sufficient myoblasts are produced, Notch signaling must be switched off to allow myoblast differentiation and subsequent reconstitution of functional muscles, as Notch activation blocks myogenic differentiation . Therefore, Notch signaling is critical in maintaining an appropriate population of muscle progenitor cells and preventing premature differentiation .
However, specific stages of the myogenic process that require Notch signaling have not been rigorously determined, since genetic analysis mainly focused on a CSL-deficient mouse model [8, 11, 13]. CSL is the Notch pathway transcription factor with dual functions as a transcriptional repressor or activator in the absence or presence of Notch receptor activation, respectively ; consequently, CSL deficiency leads to de-repression of, or loss of, Notch target gene transcription depending on cellular Notch status. Differential functional impacts on myogenic differentiation were observed as CSL depletion blocked murine C2C12 myoblast differentiation, whereas inhibition of active Notch signaling via γ-secretase inhibitor promoted differentiation (unpublished data). Therefore, a more rigorous genetic model is needed to investigate Notch-dependent myogenic step(s) and how Notch signaling deregulation might affect muscle functions. Understanding the knowledge gaps regarding the in vivo roles of Notch signaling in muscle stem cells is important because Notch signaling modulation is being widely investigated in the treatment of diseases and tissue regeneration [15, 16]. Here, we developed a mouse model in which a pan-Notch inhibitor dnMAML1 specifically blocks Notch signaling in muscle stem cells. Our data revealed that Notch signaling blockade in muscle stem cells might affect the development and progression of muscular dystrophies.
MATERIALS AND METHODS
Detailed materials and methods were provided as supporting information.
RESULTS AND DISCUSSION
To study the functional impact of blocking endogenous active Notch signaling in muscle stem cells, we crossed ROSA26dnMAML1/dnMAML1 mice  with Pax7-Cre mice  to generate offsprings including Pax7-Cre+/dnMAML1+ (Mutants) and Pax7-Cre−/dnMAML1+ littermate controls (Fig. 1A). dnMAML1 is a 62-amino-acid peptide derived from the Notch coactivator MAML1 that interferes with the ability of MAML coactivators to form Notch transcriptional complexes (Notch/CSL/MAML) [19, 20], thereby blocking transcription induced by all Notch receptors. Therefore, the mutant mice expressed the pan-Notch inhibitor dnMAML1-gfp in muscle stem cells driven by the Pax7 promoter.
We observed that the Pax7-Cre+/dnMAML1+ mutant mice were smaller in size and weight (Fig. 1B, 1C), displayed muscle weakness and poor balance (dragging both hind limbs simultaneously when walking) (supporting information Video S1), showed hair loss on the head (Fig. 1B, 1D), and some developed spinal deformation (Fig. 1D) and hydrocephalus (not shown) after 2–6 months of age. These mice had a shortened life span and the majority died within 8 months after birth (Fig. 1E). The mutant mice had major muscle defects with smaller muscle masses (Fig. 2A, 2B). Histological analysis of the severely abnormally moving mice revealed that the mutants had severe muscle damage and degeneration as indicated by the central nucleated myofibers (Fig. 2C) as well as fibrous tissue replacement in their muscles (Fig. 2D), suggesting that Notch-depleted satellite cells are inefficient to repair muscle damages induced by normal activity. The mutant muscles had a significantly reduced number of Pax7-positive satellite cells in both p15 and p30 mice (Fig. 2E) but enhanced number of MyoD-positive myogenic cells (Fig. 2F, 2G). These data suggest that satellite cell depletion in the mutant mice possibly results from reduced self-renewal and excessive myogenic commitment/differentiation of satellite cells [8, 11, 12].
Moreover, primary myoblasts isolated from the mutant mice showed reduced proliferative capacity (Fig. 3A) compared to the controls, but no change in cellular apoptosis (not shown), which is consistent with previous reports [8, 11]. The mutant myoblasts were prone to differentiation as shown by the generation of larger and longer myofibers (Fig. 3B) and increased expression of muscle differentiation markers Myogenin and Myosin (Fig. 3C). Thus, Notch loss-of-function causes premature differentiation and reduced proliferation of muscle stem cells, suggesting that deregulated Notch signaling contributes to the dystrophic phenotype.
We next determined the effect of Notch signaling inhibition in satellite cells on muscle regenerative capacity in vivo in response to cardiotoxin-induced injury. Muscle regeneration consists of two key steps: the initial activation and proliferation of muscle stem cells, and subsequent myogenic differentiation to restore the functional muscles. At day 1 after injury, Pax7 expression was lower in the mutant mice (Fig. 4A–4C), suggesting that Notch signaling inhibition results in reduced satellite cell activation and proliferation. The defects in stem cells likely affected the later myogenic differentiation, as reduced induction of myogenic transcription factor Myogenin was observed 3 days postinjury (Fig. 4D). At day 7, control muscles reconstituted with numerous newly regenerated centrally nucleated myofibers (Fig. 4E), whereas mutant muscles consisted of the majority of smaller regenerated myofibers, with some showing abnormally large but hollow (Fig. 4F) or absence of the nuclei (Fig. 4G).
Therefore, Notch inhibition in muscle stem cells results in defective regenerative capacity, which is consistent with a role of Notch signaling in maintaining and activation of muscle stem cells [8, 11]. It should be noted that a prenatal patterning and other defects cannot be excluded because of Pax7-cre expression in fetal muscles as well as other tissues . Future work using mouse models with Notch inhibition postnatally will provide conclusive evidence for a role of active Notch signaling in muscle regeneration. Most importantly, we showed that Notch signaling inhibition likely affect the pathogenesis or progression of muscular dystrophy diseases. Therefore, potential side effects of Notch inhibitors on muscle functions for the treatment of cancers and other diseases should be considered, although short-tem Notch inhibition appeared not to affect muscles in a clinically symptomatic way .
We used a specific genetic tool, the pan-Notch inhibitor dnMAML1, to block endogenous active Notch signaling in muscle stem cell compartment and revealed a critical role of Notch signaling in muscle stem cell maintenance and differentiation. The Pax7-Cre+/dnMAML1+ mutant mice exhibit several important muscular dystrophic features and impaired muscle regeneration. Therefore, impaired Notch signaling likely contributes to the pathogenic mechanisms of muscular dystrophies. Long-term clinical applications of Notch inhibitors might have significant side effects on muscle functions.
We thank Dr. Moyi Li for the technical help with muscle injection, Marda Jorgensen for helping with immunohistochemical staining, and Dr. Abigail McElhinny for critical reading of this manuscript and helpful comments. This work is supported in part by the University of Florida Shands Cancer Center Startup fund (L.W) and Muscular Dystrophy Association (L.W).
DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST
The authors indicate no potential conflicts of interest.