Towards frailty biomarkers: Candidates from genes and pathways regulated in aging and age-related diseases.

OBJECTIVE: Use of the frailty index to measure an accumulation of deficits has been proven a valuable method for identifying elderly people at risk for increased vulnerability, disease, injury, and mortality. However, complementary molecular frailty biomarkers or ideally biomarker panels have not yet been identified. We conducted a systematic search to identify biomarker candidates for a frailty biomarker panel. METHODS: Gene expression databases were searched (http://genomics.senescence.info/genes including GenAge, AnAge, LongevityMap, CellAge, DrugAge, Digital Aging Atlas) to identify genes regulated in aging, longevity, and age-related diseases with a focus on secreted factors or molecules detectable in body fluids as potential frailty biomarkers. Factors broadly expressed, related to several "hallmark of aging" pathways as well as used or predicted as biomarkers in other disease settings, particularly age-related pathologies, were identified. This set of biomarkers was further expanded according to the expertise and experience of the authors. In the next step, biomarkers were assigned to six "hallmark of aging" pathways, namely (1) inflammation, (2) mitochondria and apoptosis, (3) calcium homeostasis, (4) fibrosis, (5) NMJ (neuromuscular junction) and neurons, (6) cytoskeleton and hormones, or (7) other principles and an extensive literature search was performed for each candidate to explore their potential and priority as frailty biomarkers. RESULTS: A total of 44 markers were evaluated in the seven categories listed above, and 19 were awarded a high priority score, 22 identified as medium priority and three were low priority. In each category high and medium priority markers were identified. CONCLUSION: Biomarker panels for frailty would be of high value and better than single markers. Based on our search we would propose a core panel of frailty biomarkers consisting of (1) CXCL10 (C-X-C motif chemokine ligand 10), IL-6 (interleukin 6), CX3CL1 (C-X3-C motif chemokine ligand 1), (2) GDF15 (growth differentiation factor 15), FNDC5 (fibronectin type III domain containing 5), vimentin (VIM), (3) regucalcin (RGN/SMP30), calreticulin, (4) PLAU (plasminogen activator, urokinase), AGT (angiotensinogen), (5) BDNF (brain derived neurotrophic factor), progranulin (PGRN), (6) α-klotho (KL), FGF23 (fibroblast growth factor 23), FGF21, leptin (LEP), (7) miRNA (micro Ribonucleic acid) panel (to be further defined), AHCY (adenosylhomocysteinase) and KRT18 (keratin 18). An expanded panel would also include (1) pentraxin (PTX3), sVCAM/ICAM (soluble vascular cell adhesion molecule 1/Intercellular adhesion molecule 1), defensin α, (2) APP (amyloid beta precursor protein), LDH (lactate dehydrogenase), (3) S100B (S100 calcium binding protein B), (4) TGFß (transforming growth factor beta), PAI-1 (plasminogen activator inhibitor 1), TGM2 (transglutaminase 2), (5) sRAGE (soluble receptor for advanced glycosylation end products), HMGB1 (high mobility group box 1), C3/C1Q (complement factor 3/1Q), ST2 (Interleukin 1 receptor like 1), agrin (AGRN), (6) IGF-1 (insulin-like growth factor 1), resistin (RETN), adiponectin (ADIPOQ), ghrelin (GHRL), growth hormone (GH), (7) microparticle panel (to be further defined), GpnmB (glycoprotein nonmetastatic melanoma protein B) and lactoferrin (LTF). We believe that these predicted panels need to be experimentally explored in animal models and frail cohorts in order to ascertain their diagnostic, prognostic and therapeutic potential.

Protein kinase A activation inhibits DUX4 gene expression in myotubes from patients with facioscapulohumeral muscular dystrophy.

Facioscapulohumeral muscular dystrophy (FSHD) is among the most prevalent of the adult-onset muscular dystrophies. FSHD causes a loss of muscle mass and function, resulting in severe debilitation and reduction in quality of life. Currently, only the symptoms of FSHD can be treated, and such treatments have minimal benefit. The available options are not curative, and none of the treatments address the underlying cause of FSHD. The genetic, epigenetic, and molecular mechanisms triggering FSHD are now quite well-understood, and it has been shown that expression of the transcriptional regulator double homeobox 4 (DUX4) is necessary for disease onset and is largely thought to be the causative factor in FSHD. Therefore, we sought to identify compounds suppressing DUX4 expression in a phenotypic screen using FSHD patient-derived muscle cells, a zinc finger and SCAN domain-containing 4 (ZSCAN4)-based reporter gene assay for measuring DUX4 activity, and ∼3,000 small molecules. This effort identified molecules that reduce DUX4 gene expression and hence DUX4 activity. Among those, ß2-adrenergic receptor agonists and phosphodiesterase inhibitors, both leading to increased cellular cAMP, effectively decreased DUX4 expression by >75% in cells from individuals with FSHD. Of note, we found that cAMP production reduces DUX4 expression through a protein kinase A-dependent mode of action in FSHD patient myotubes. These findings increase our understanding of how DUX4 expression is regulated in FSHD and point to potential areas of therapeutic intervention.

TAK-1/p38/nNFκB signaling inhibits myoblast differentiation by increasing levels of Activin A.

BACKGROUND: Skeletal-muscle differentiation is required for the regeneration of myofibers after injury. The differentiation capacity of satellite cells is impaired in settings of old age, which is at least one factor in the onset of sarcopenia, the age-related loss of skeletal-muscle mass and major cause of frailty. One important cause of impaired regeneration is increased levels of transforming growth factor (TGF)-ß accompanied by reduced Notch signaling. Pro-inflammatory cytokines are also upregulated in aging, which led us hypothesize that they might potentially contribute to impaired regeneration in sarcopenia. Thus, in this study, we further analyzed the muscle differentiation-inhibition pathway mediated by pro-inflammatory cytokines in human skeletal muscle cells (HuSKMCs). METHODS: We studied the modulation of HuSKMC differentiation by the pro-inflammatory cytokines interleukin (IL)-1α and tumor necrosis factor (TNF)-α The grade of differentiation was determined by either imaging (fusion index) or creatine kinase (CK) activity, a marker of muscle differentiation. Secretion of TGF-ß proteins during differentiation was assessed by using a TGF-ß-responsive reporter-gene assay and further identified by means of pharmacological and genetic inhibitors. In addition, signaling events were monitored by western blotting and reverse transcription PCR, both in HuSKMC cultures and in samples from a rat sarcopenia study. RESULTS: The pro-inflammatory cytokines IL-1α and TNF-α block differentiation of human myoblasts into myotubes. This anti-differentiation effect requires activation of TGF-ß-activated kinase (TAK)-1. Using pharmacological and genetic inhibitors, the TAK-1 pathway could be traced to p38 and NFκB. Surprisingly, the anti-differentiation effect of the cytokines required the transcriptional upregulation of Activin A, which in turn acted through its established signaling pathway: ActRII/ALK/SMAD. Inhibition of Activin A signaling was able to rescue human myoblasts treated with IL-1ß or TNF-α, resulting in normal differentiation into myotubes. Studies in aged rats as a model of sarcopenia confirmed that this pro-inflammatory cytokine pathway identified is activated during aging. CONCLUSIONS: In this study, we found an unexpected connection between cytokine and Activin signaling, revealing a new mechanism by which cytokines affect skeletal muscle, and establishing the physiologic relevance of this pathway in the impaired regeneration seen in sarcopenia.

Myostatin reduces Akt/TORC1/p70S6K signaling, inhibiting myoblast differentiation and myotube size.

Myostatin is a negative regulator of skeletal muscle size, previously shown to inhibit muscle cell differentiation. Myostatin requires both Smad2 and Smad3 downstream of the activin receptor II (ActRII)/activin receptor-like kinase (ALK) receptor complex. Other transforming growth factor-beta (TGF-beta)-like molecules can also block differentiation, including TGF-beta(1), growth differentiation factor 11 (GDF-11), activins, bone morphogenetic protein 2 (BMP-2) and BMP-7. Myostatin inhibits activation of the Akt/mammalian target of rapamycin (mTOR)/p70S6 protein synthesis pathway, which mediates both differentiation in myoblasts and hypertrophy in myotubes. Blockade of the Akt/mTOR pathway, using small interfering RNA to regulatory-associated protein of mTOR (RAPTOR), a component of TOR signaling complex 1 (TORC1), increases myostatin-induced phosphorylation of Smad2, establishing a myostatin signaling-amplification role for blockade of Akt. Blockade of RAPTOR also facilitates myostatin's inhibition of muscle differentiation. Inhibition of TORC2, via rapamycin-insensitive companion of mTOR (RICTOR), is sufficient to inhibit differentiation on its own. Furthermore, myostatin decreases the diameter of postdifferentiated myotubes. However, rather than causing upregulation of the E3 ubiquitin ligases muscle RING-finger 1 (MuRF1) and muscle atrophy F-box (MAFbx), previously shown to mediate skeletal muscle atrophy, myostatin decreases expression of these atrophy markers in differentiated myotubes, as well as other genes normally upregulated during differentiation. These findings demonstrate that myostatin signaling acts by blocking genes induced during differentiation, even in a myotube, as opposed to activating the distinct "atrophy program." In vivo, inhibition of myostatin increases muscle creatine kinase activity, coincident with an increase in muscle size, demonstrating that this in vitro differentiation measure is also upregulated in vivo.