Even in adulthood, injured muscles can repair themselves largely because they contain groups of stem cells known as satellite cells. These cells divide to produce progenitor cells that later develop, or differentiate, into new muscle fibers. However as muscles get older, this repair process becomes less effective, in part because the satellite cells do not respond as strongly to injury. It remains obscure precisely why the repair process declines with age.

A protein called TGFβ is part of a signaling pathway that prevents the muscle progenitor cells from differentiating into muscle fibers, and TGFβ signaling is overactive in older muscles. Most TGFβ signaling operates via a protein called Smad4, and Paris et al. now show that older satellite cells and progenitor cells from the muscles of old mice produce less Smad4 when they are regenerating.

Next, the gene for Smad4 was deleted specifically from the satellite cells of mice. By examining the fate of these cells, Paris et al. found that Smad4 normally maintained the population of satellite cells by preventing them from differentiating into muscle fibers too soon. This was the case when both adult and aged muscle was regenerating.

All in all, Smad4 is clearly important for directing satellite cells to regenerate properly; aged cells have less Smad4 and are less able to regenerate. Future studies are now needed to determine how disrupting Smad4 in other resident cell types may influence the regeneration of muscles in mice.

The regenerative capacity of adult skeletal muscle is endowed in a population of Pax7-expressing resident stem cells called satellite cells (SCs) (Brack et al., 2012). Genetic studies utilizing lineage labeling, as well as cell ablation, have established that Pax7-expressing SCs are essential for various aspects of skeletal muscle regeneration (Relaix and Zammit, 2012; Liu et al., 2015). At homeostasis, SCs reside in a quiescent state at the interface between skeletal muscle fibers (myofibers) and the surrounding basal lamina (Brack et al., 2012). In response to degenerative stimuli, SCs activate and undergo proliferative expansion, providing myogenic progenitors (MPs) necessary for myofiber regeneration (Brack et al., 2012). Skeletal muscle regeneration requires a balance between SC/MP amplification and terminal myogenic commitment in order to efficiently form multinucleated myofibers (Brack et al., 2012). Moreover, there is evidence that this balance is compromised in aging skeletal muscle, where changes in SC function and their surrounding environment occur, yielding defective progenitors and stem cells (Chakkalakal et al., 2012; Sousa-Victor et al., 2015; Almada and Wagers, 2016; Blau et al., 2015). Therefore, elucidating factors that regulate SC and derived MP fate is critical in order to develop interventions to combat aged skeletal muscle regenerative decline, in which these cells are lost.

Transforming growth factor beta (TGFβ) superfamily signaling is crucial for the renewal and maintenance of various tissue-specific stem cell populations (Oshimori and Fuchs, 2012). Superfamily ligands include TGFβs, bone morphogenetic proteins (BMPs), growth and differentiation factors (GDFs), Activins, and Myostatin. These ligands bind to transmembrane type II receptors with differing specificities. This association leads to the eventual formation of transmembrane complexes composed of type I (activin-like kinases, ALKs) and type II receptor homodimers (Massagué, 2008). The formation of these complexes triggers the phosphorylation of receptor SMADs (R-SMADs). The R-SMADs associated with TGFβ/Activin/Inhibin and BMP ligands are SMAD2/3 and SMAD1/5/8 respectively. Phosphorylated R-SMADs, associated with the cofactor SMAD4, accumulate in the nucleus, where together with chromatin modifiers and other transcriptional co-factors they promote the expression of target genes. Depending on cell type and context, the TGFβ superfamily pathways regulate many cellular processes including differentiation, renewal, quiescence, and apoptosis (Oshimori and Fuchs, 2012; Massagué, 2012). In aged regenerating skeletal muscle, TGFβ superfamily signaling is widely considered to be abnormally elevated, and is thought to inhibit SC activation and terminal myogenic differentiation (Kollias and McDermott, 2008; Trendelenburg et al., 2012, 2009). However, it has also been proposed that during regeneration TGFβ superfamily ligands may enable SC and MP expansion (Ono et al., 2011; Sinha et al., 2014).

Smad4 is recognized as the canonical cofactor for TGFβ superfamily signaling and was initially identified as a tumor suppressor in pancreatic cancer (Malkoski and Wang, 2012; Miyaki and Kuroki, 2003; Hussein et al., 2003; Nishita et al., 2000). Loss-of-function SMAD4 mutations lead to familial juvenile polyposis, which can be associated with hereditary hemorrhagic telangiectasia, and aggressive forms of various cancers (Malkoski and Wang, 2012; Akhurst, 2004). In myogenic culture systems derived from immortalized cell lines or using sequential pre-plate techniques, knockdown of Smad4 promotes myogenic differentiation (Dey et al., 2012; Ono et al., 2011). Furthermore, global reduction of expression through direct intramuscular injection of Smad4 siRNAs or viral vectors with Smad4 shRNAs into injured mouse skeletal muscle can promote the formation of larger regenerated muscle fibers relative to controls (Dey et al., 2012; Lee et al., 2015). However, given that multiple non-myogenic cell types, such as inflammatory cells and fibro/adipogenic progenitors, also contribute to SC and MP fate decisions during skeletal muscle regeneration; It is unclear which cellular mechanisms promote hypertrophy of regenerated myofibers with non-targeted Smad4 loss. In contrast, specific loss of Smad4 in MPs compromises myogenic differentiation during embryonic skeletal muscle development (Han et al., 2012). Additionally, consistent with the critical role for Smad4 in stem and progenitor cell function, targeted deletion of Smad4 in hematopoietic, hair follicle, and neural stem and derived progenitor cell populations leads to their depletion during homeostasis and regeneration (Karlsson et al., 2007; Yang et al., 2009; Mira et al., 2010). Moreover, targeted loss of Smad4 in myofibers leads to modest deterioration during growth and aggravation of denervation-induced atrophy in adults (Sartori et al., 2013). Recently, gain-of-function SMAD4 mutations that prevent ubiquitination and subsequent degradation have been identified as the cause of the rare developmental disorder Myhre syndrome in humans (Caputo et al., 2014; Le Goff et al., 2012). Patients with Myhre syndrome are characterized by short stature, various musculoskeletal abnormalities, and hypertrophied musculature (Caputo et al., 2014; Le Goff et al., 2012). Although Smad4 clearly has crucial roles in skeletal muscle and tissue-specific stem and progenitor cell biology, to date no studies have explicitly examined whether or not there is a cell-autonomous requirement for Smad4 in SCs and derived MPs during skeletal muscle regeneration.

In this study we show, in comparison to adult, evidence of failure to induce Smad4 expression in aged SCs and MPs during skeletal muscle regeneration. In order to examine the consequences of cell-specific Smad4 loss, we utilized transgenic mice expressing tamoxifen-inducible Cre recombinase under the control of Pax7 regulatory elements to perform targeted deletion of Smad4 in SCs. We found that specific disruption of Smad4 in adult SCs resulted in insufficient SC and derived MP amplification, which was accompanied by severe deficiencies in adult skeletal muscle regeneration. Unexpectedly, with specific loss of Smad4 in aged SCs in an environment of presumably high TGFβ activity, aged skeletal muscle regeneration was not improved.

Here we find Smad4 to be a factor that is normally induced in adult, but lost in aged, SCs and MPs in regenerating muscles. Furthermore, we demonstrate that in an inducible mouse model driving specific loss of Smad4 in SCs, a decline in the number of proliferating Pax7+ SCs and MPs, and consequently severe deficits in skeletal muscle regeneration are experienced regardless of age. Several studies have specifically manipulated components of TGFβ superfamily signaling in myogenic progenitors and SCs (Guardiola et al., 2012; Han et al., 2012; Huang et al., 2014). However, until now, no reports have examined the cell-autonomous roles of Smad4, the common cofactor for all branches of canonical TGFβ superfamily signaling, in adult or aged Pax7+ SCs and determined the subsequent consequences on skeletal muscle regeneration.

Consistent with previous reports, Smad4 disruption did promote myogenic terminal commitment (Dey et al., 2012; Ono et al., 2011; Lee et al., 2015). However, declines in proliferating SCs and MPs during regeneration were also observed. Myofibers are multinucleated cells formed by the fusion of many terminally committed myogenic cells. Therefore, the progression to the formation of mature multinucleated myofibers can be impeded at multiple levels, including impaired myogenic differentiation and/or reduced MP amplification through a variety of mechanisms: inhibition of cell cycle entry, cell death, and/or premature terminal commitment (Sousa-Victor et al., 2015; Brack et al., 2012). Given that we have detected differences in myogenic cell proliferation with no increased cell death or cell cycle inhibitor expression, we suspect that the reduced number of MPs in regenerating skeletal muscles following acute SC-specific Smad4 loss is likely due to a heightened propensity for terminal commitment. In contrast, specific disruption of Smad4 in embryonic mouse MPs, driven by Myf5-Cre, altered terminal myogenic commitment and myofiber formation in the tongue (Han et al., 2012). Intuitively, these divergent results could reflect differences in the intrinsic mechanisms that regulate SCs and MPs in response to the environments of embryonic tongue growth versus adult TA regeneration. Another possibility is that the Myf5-Cre, in addition to targeting the myogenic lineage, drives recombination in further resident cell populations with the capacity to directly or indirectly influence skeletal muscle growth and regeneration (Huang et al., 2014). It will be of interest to determine in what manner specific disruption of Smad4 in other resident non-myogenic cell populations may influence adult or aged skeletal muscle regeneration. Although in adult Smad4 null SC cultures we observed reductions in Fgf6 and Fgfr4 expression similar to embryonic Smad4 deleted muscle, we also detected a robust induction of Fgf2. We have previously found that the presence of elevated levels of FGF2 in aged skeletal muscle is associated with a propensity for aged SCs to progress toward terminal fates (Chakkalakal et al., 2012). Therefore, compensatory induction of FGF2 resulting from Smad4 loss may be a unique feature of adult SCs that drives terminal myogenic commitment, the mechanisms of which will require further investigation.

Previous work has shown that loss of BMP signaling induces premature terminal myogenic commitment, preventing SC and MP amplification (Ono et al., 2011). Furthermore, Id1, which we found to be reduced in aged wildtype and adult P7:S4KO SCs and derived MPs, was identified as a SMAD target required for SC and MP amplification (Ono et al., 2011). Indeed, through direct interactions Id1 can also promote Myogenin degradation (Vinals and Ventura, 2004). Therefore, loss of Id1 in aged and P7:S4KO SCs and MPs during regeneration could potentially stabilize Myogenin and thus promote excessive terminal commitment that impedes myogenic cell amplification. Although we observe loss of Smad4 and Id1 expression, some reports have described the presence of elevated TGFβ and phospho-Smad activity in aged SCs and MPs from regenerating skeletal muscle (Carlson et al., 2008; Pessina et al., 2015). Although less characterized, TGFβ can function through non-canonical Smad4-independent pathways (Derynck and Zhang, 2003; Massagué, 2012). For instance, pSmad3, which is elevated in aged SCs and MPs, also associates with Drosha in a Smad4-independent complex required for microRNA processing (Davis et al., 2008). Studies examining keratinocyte differentiation have shown Iκβ kinase to be a critical regulator of signaling through Smad2/3 that is Smad4-independent (Descargues et al., 2008). Non-canonical TGFβ mediators such as TAK1 (TGFβ activated kinase 1) and the downstream target p38 are both shown to be elevated in aged myogenic cells (Cosgrove et al., 2014; Bernet et al., 2014; Trendelenburg et al., 2012). Notably, heightened p38 activity in aged SCs hinders myogenic cell amplification through mechanisms that include promotion of terminal myogenic commitment (Cosgrove et al., 2014; Bernet et al., 2014). Collectively, it will be critical to determine if loss of Smad4 in aged and P7:S4KO SCs and MPs could impair myogenic cell amplification through promotion of multiple Smad4-independent pathways.

Although the TGFβ superfamily pathways are capable of influencing multiple cell types that contribute to the regeneration of skeletal muscle, few studies have examined specific inhibition of these pathways in adult or aged SCs and their derived progenitors. For example, it has been shown that SC-specific loss of Cripto, an inhibitor of Activin, Myostatin, and TGFβ signaling in myogenic cells, leads to smaller regenerated myofibers (Guardiola et al., 2012). Additionally, genetic ablation of the BMP receptor Alk3 in embryonic myogenic cells, utilizing MyoD-Cre and Myf5-Cre mice, was found to result in ineffective adult skeletal muscle regeneration, however this occurs primarily through mechanisms other than major dysfunction within the SC pool (Huang et al., 2014). This conclusion was based primarily on the lack of observable depletion or dysfunction of Alk3 null SCs in vivo or in culture, respectively, which is thought to occur due to stimulation of the expression of the related Alk6 as a compensatory mechanism to maintain SC function (Huang et al., 2014). In this study, we determined that inducible Pax7-driven loss of Smad4 impedes SC-derived myogenic proliferation and proper skeletal muscle regeneration. Further studies will be needed to determine in what way targeted disruption of Alk3, induced later in life, in adult or aged SCs may affect skeletal muscle regeneration at these ages, with potential compensation possibly being eliminated.

Although not specifically targeting SCs or MPs, intramuscular delivery of Smad4 siRNA or shRNA at later time points during regeneration has been shown to stimulate myofiber hypertrophy (Dey et al., 2012; Lee et al., 2015). Furthermore, utilizing miR-26 disruption to increase, and miR-431 mimics to decrease, Smad4 expression at later timeponts during regeneration promotes myofiber atrophy and hypertrophy respectively (Dey et al., 2012; Lee et al., 2015). However, it is intuitive that since microRNAs often target many genes, the aforementioned manipulations likely do not alter Smad4 expression alone. Therefore, it remains unclear whether the balance between myogenic progenitor expansion and terminal fate commitment could be manipulated to be beneficial to adult and aged muscle if cell-specific Smad4 loss was induced at different timepoints during regeneration or by alternative, more transient, means.

Here, we demonstrate that induction of Smad4 is compromised in aged SCs and myogenic cells during skeletal muscle regeneration. Disruption of Smad4 specifically in adult SCs leads to phenotypes observed in aged regenerating skeletal muscle including compromised proliferation and amplification of SC-derived MPs. Collectively, these data indicate that Smad4 function in SCs is essential for adult and aged skeletal muscle regeneration in the mouse. Moving forward, careful dissection of Smad4-mediated pathways and targets may yield novel factors that promote skeletal muscle genesis in the contexts of aging and disease.

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Author details

1. Nicole D Paris

   Center for Musculoskeletal Research, Department of Orthopaedics and Rehabilitation, University of Rochester Medical Center, Rochester, United States

   Contribution

   NDP, Conception and design, Acquisition of data, Analysis and interpretation of data, Drafting or revising the article

   Competing interests

   The authors declare that no competing interests exist.

   "This ORCID iD identifies the author of this article:" 0000-0003-0654-0983

2. Andrew Soroka

   1. Center for Musculoskeletal Research, Department of Orthopaedics and Rehabilitation, University of Rochester Medical Center, Rochester, United States
   2. Department of Pathology, University of Rochester Medical Center, Rochester, United States

   Contribution

   AS, Designed and performed myogenic differentiation experiment and wrote some portions of the manuscript

   Competing interests

   The authors declare that no competing interests exist.

3. Alanna Klose

   Center for Musculoskeletal Research, Department of Orthopaedics and Rehabilitation, University of Rochester Medical Center, Rochester, United States

   Contribution

   AK, Performed all RNA isolations and RT-qPCR experiments, Analysis and interpretation of data

   Competing interests

   The authors declare that no competing interests exist.

4. Wenxuan Liu

   1. Center for Musculoskeletal Research, Department of Orthopaedics and Rehabilitation, University of Rochester Medical Center, Rochester, United States
   2. Department of Biomedical Genetics, University of Rochester Medical Center, Rochester, United States

   Contribution

   WL, Designed and performed assessment of SC number experiments, Analysis and interpretation of data

   Competing interests

   The authors declare that no competing interests exist.

5. Joe V Chakkalakal

   1. Center for Musculoskeletal Research, Department of Orthopaedics and Rehabilitation, University of Rochester Medical Center, Rochester, United States
   2. Stem Cell and Regenerative Medicine Institute, University of Rochester Medical Center, Rochester, United States
   3. The Rochester Aging Research Center, University of Rochester Medical Center, Rochester, United States

   Contribution

   JVC, Conception and design, Analysis and interpretation of data, Drafting or revising the article

   For correspondence

   joe_chakkalakal@urmc.rochester.edu

   Competing interests

   The authors declare that no competing interests exist.

   "This ORCID iD identifies the author of this article:" 0000-0002-8440-7312

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