Growth differentiation factor 11 (GDF11) and myostatin (or GDF8) are closely related members of the transforming growth factor β superfamily and are often perceived to serve similar or overlapping roles. Yet, despite commonalities in protein sequence, receptor utilization and signaling, accumulating evidence suggests that these 2 ligands can have distinct functions in many situations. GDF11 is essential for mammalian development and has been suggested to regulate aging of multiple tissues, whereas myostatin is a well-described negative regulator of postnatal skeletal and cardiac muscle mass and modulates metabolic processes. In this review, we discuss the biochemical regulation of GDF11 and myostatin and their functions in the heart, skeletal muscle, and brain. We also highlight recent clinical findings with respect to a potential role for GDF11 and/or myostatin in humans with heart disease. Finally, we address key outstanding questions related to GDF11 and myostatin dynamics and signaling during development, growth, and aging.
RATIONALE: Growth differentiation factor 11 (GDF11) and GDF8 are members of the transforming growth factor-β superfamily sharing 89% protein sequence homology. We have previously shown that circulating GDF11 levels decrease with age in mice. However, a recent study by Egerman et al reported that GDF11/8 levels increase with age in mouse serum.
OBJECTIVE: Here, we clarify the direction of change of circulating GDF11/8 levels with age and investigate the effects of GDF11 administration on the murine heart.
METHODS AND RESULTS: We validated our previous finding that circulating levels of GDF11/8 decline with age in mice, rats, horses, and sheep. Furthermore, we showed by Western analysis that the apparent age-dependent increase in GDF11 levels, as reported by Egerman et al, is attributable to cross-reactivity of the anti-GDF11 antibody with immunoglobulin, which is known to increase with age. GDF11 administration in mice rapidly activated SMAD2 and SMAD3 signaling in myocardium in vivo and decreased cardiac mass in both young (2-month-old) and old (22-month-old) mice in a dose-dependent manner after only 9 days.
CONCLUSIONS: Our study confirms an age-dependent decline in serum GDF11/8 levels in multiple mammalian species and that exogenous GDF11 rapidly activates SMAD signaling and reduces cardiomyocyte size. Unraveling the molecular basis for the age-dependent decline in GDF11/8 could yield insight into age-dependent cardiac pathologies.
Fluorescence-activated cell sorting (FACS) strategies to purify distinct cell types from the pool of fetal human myofiber-associated (hMFA) cells were developed. We demonstrate that cells expressing the satellite cell marker PAX7 are highly enriched within the subset of CD45(-)CD11b(-)GlyA(-)CD31(-)CD34(-)CD56(int)ITGA7(hi) hMFA cells. These CD45(-)CD11b(-)GlyA(-)CD31(-)CD34(-)CD56(int)ITGA7(hi) cells lack adipogenic capacity but exhibit robust, bipotent myogenic and osteogenic activity in vitro and engraft myofibers when transplanted into mouse muscle. In contrast, CD45(-)CD11b(-)GlyA(-)CD31(-)CD34(+) fetal hMFA cells represent stromal constituents of muscle that do not express PAX7, lack myogenic function, and exhibit adipogenic and osteogenic capacity in vitro. Adult muscle likewise contains PAX7(+) CD45(-)CD11b(-)GlyA(-)CD31(-)CD34(-)CD56(int)ITGA7(hi) hMFA cells with in vitro myogenic and osteogenic activity, although these cells are present at lower frequency in comparison to their fetal counterparts. The ability to directly isolate functionally distinct progenitor cells from human muscle will enable novel insights into muscle lineage specification and homeostasis.
Ex vivo expansion of satellite cells and directed differentiation of pluripotent cells to mature skeletal muscle have proved difficult challenges for regenerative biology. Using a zebrafish embryo culture system with reporters of early and late skeletal muscle differentiation, we examined the influence of 2,400 chemicals on myogenesis and identified six that expanded muscle progenitors, including three GSK3β inhibitors, two calpain inhibitors, and one adenylyl cyclase activator, forskolin. Forskolin also enhanced proliferation of mouse satellite cells in culture and maintained their ability to engraft muscle in vivo. A combination of bFGF, forskolin, and the GSK3β inhibitor BIO induced skeletal muscle differentiation in human induced pluripotent stem cells (iPSCs) and produced engraftable myogenic progenitors that contributed to muscle repair in vivo. In summary, these studies reveal functionally conserved pathways regulating myogenesis across species and identify chemical compounds that expand mouse satellite cells and differentiate human iPSCs into engraftable muscle.
Calorie restriction (CR) extends life span and ameliorates age-related pathologies in most species studied, yet the mechanisms underlying these effects remain unclear. Using mouse skeletal muscle as a model, we show that CR acts in part by enhancing the function of tissue-specific stem cells. Even short-term CR significantly enhanced stem cell availability and activity in the muscle of young and old animals, in concert with an increase in mitochondrial abundance and induction of conserved metabolic and longevity regulators. Moreover, CR enhanced endogenous muscle repair and CR initiated in either donor or recipient animals improved the contribution of donor cells to regenerating muscle after transplant. These studies indicate that metabolic factors play a critical role in regulating stem cell function and that this regulation can influence the efficacy of recovery from injury and the engraftment of transplanted cells.
Satellite cells reside beneath the basal lamina of skeletal muscle fibers and include cells that act as precursors for muscle growth and repair. Although they share a common anatomical localization and typically are considered a homogeneous population, satellite cells actually exhibit substantial heterogeneity. We used cell-surface marker expression to purify from the satellite cell pool a distinct population of skeletal muscle precursors (SMPs) that function as muscle stem cells. When engrafted into muscle of dystrophin-deficient mdx mice, purified SMPs contributed to up to 94% of myofibers, restoring dystrophin expression and significantly improving muscle histology and contractile function. Transplanted SMPs also entered the satellite cell compartment, renewing the endogenous stem cell pool and participating in subsequent rounds of injury repair. Together, these studies indicate the presence in adult skeletal muscle of prospectively isolatable muscle-forming stem cells and directly demonstrate the efficacy of myogenic stem cell transplant for treating muscle degenerative disease.
Frame-disrupting mutations in the DMD gene, encoding dystrophin, compromise myofiber integrity and drive muscle deterioration in Duchenne muscular dystrophy (DMD). Removing one or more exons from the mutated transcript can produce an in-frame mRNA and a truncated, but still functional, protein. In this study, we developed and tested a direct gene-editing approach to induce exon deletion and recover dystrophin expression in the mdx mouse model of DMD. Delivery by adeno-associated virus (AAV) of clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 endonucleases coupled with paired guide RNAs flanking the mutated Dmd exon23 resulted in excision of intervening DNA and restored the Dmd reading frame in myofibers, cardiomyocytes, and muscle stem cells after local or systemic delivery. AAV-Dmd CRISPR treatment partially recovered muscle functional deficiencies and generated a pool of endogenously corrected myogenic precursors in mdx mouse muscle.
Current therapies for sarcomas are often inadequate. This study sought to identify actionable gene targets by selective targeting of the molecular networks that support sarcoma cell proliferation. Silencing of asparagine synthetase (ASNS), an amidotransferase that converts aspartate into asparagine, produced the strongest inhibitory effect on sarcoma growth in a functional genomic screen of mouse sarcomas generated by oncogenic Kras and disruption of Cdkn2a. ASNS silencing in mouse and human sarcoma cell lines reduced the percentage of S phase cells and impeded new polypeptide synthesis. These effects of ASNS silencing were reversed by exogenous supplementation with asparagine. Also, asparagine depletion via the ASNS inhibitor amino sulfoximine 5 (AS5) or asparaginase inhibited mouse and human sarcoma growth in vitro, and genetic silencing of ASNS in mouse sarcoma cells combined with depletion of plasma asparagine inhibited tumor growth in vivo. Asparagine reliance of sarcoma cells may represent a metabolic vulnerability with potential anti-sarcoma therapeutic value.
The most common form of heart failure occurs with normal systolic function and often involves cardiac hypertrophy in the elderly. To clarify the biological mechanisms that drive cardiac hypertrophy in aging, we tested the influence of circulating factors using heterochronic parabiosis, a surgical technique in which joining of animals of different ages leads to a shared circulation. After 4 weeks of exposure to the circulation of young mice, cardiac hypertrophy in old mice dramatically regressed, accompanied by reduced cardiomyocyte size and molecular remodeling. Reversal of age-related hypertrophy was not attributable to hemodynamic or behavioral effects of parabiosis, implicating a blood-borne factor. Using modified aptamer-based proteomics, we identified the TGF-β superfamily member GDF11 as a circulating factor in young mice that declines with age. Treatment of old mice to restore GDF11 to youthful levels recapitulated the effects of parabiosis and reversed age-related hypertrophy, revealing a therapeutic opportunity for cardiac aging.
Parabiosis experiments indicate that impaired regeneration in aged mice is reversible by exposure to a young circulation, suggesting that young blood contains humoral "rejuvenating" factors that can restore regenerative function. Here, we demonstrate that the circulating protein growth differentiation factor 11 (GDF11) is a rejuvenating factor for skeletal muscle. Supplementation of systemic GDF11 levels, which normally decline with age, by heterochronic parabiosis or systemic delivery of recombinant protein, reversed functional impairments and restored genomic integrity in aged muscle stem cells (satellite cells). Increased GDF11 levels in aged mice also improved muscle structural and functional features and increased strength and endurance exercise capacity. These data indicate that GDF11 systemically regulates muscle aging and may be therapeutically useful for reversing age-related skeletal muscle and stem cell dysfunction.
In the adult central nervous system, the vasculature of the neurogenic niche regulates neural stem cell behavior by providing circulating and secreted factors. Age-related decline of neurogenesis and cognitive function is associated with reduced blood flow and decreased numbers of neural stem cells. Therefore, restoring the functionality of the niche should counteract some of the negative effects of aging. We show that factors found in young blood induce vascular remodeling, culminating in increased neurogenesis and improved olfactory discrimination in aging mice. Further, we show that GDF11 alone can improve the cerebral vasculature and enhance neurogenesis. The identification of factors that slow the age-dependent deterioration of the neurogenic niche in mice may constitute the basis for new methods of treating age-related neurodegenerative and neurovascular diseases.