Digital Biomarkers Enable Automated, Longitudinal Monitoring in a Mouse Model of Aging.

To understand the growing needs of an aging human population, there is demand for scalable and reproducible approaches to study animal models of aging and to test novel therapeutic interventions. We investigated the sensitivity and utility of a continuous monitoring platform and its digital biomarkers (motion, breathing rate, and wheel running) to evaluate behavioral and physiological differences between "young" (12 weeks) and "old" (23 months) male C57BL/6J mice with or without running wheels in the home cage. Compared to young mice, old mice showed marked reductions in motion and breathing rate, as well as altered circadian rhythms. Mice without running wheels possessed lower breathing rates compared to their counterparts with running wheels. Digital biomarkers showed age-dependent changes in response to routine procedures (cage changes and blood sampling) and alterations in subjects that unexpectedly reached endpoint. Continuous collection of digital biomarkers in the home cage can enhance current approaches by providing unbiased longitudinal monitoring for large-scale aging studies.

Validation of a New Rodent Experimental System to Investigate Consequences of Long Duration Space Habitation.

Animal models are useful for exploring the health consequences of prolonged spaceflight. Capabilities were developed to perform experiments in low earth orbit with on-board sample recovery, thereby avoiding complications caused by return to Earth. For NASA's Rodent Research-1 mission, female mice (ten 32 wk C57BL/6NTac; ten 16 wk C57BL/6J) were launched on an unmanned vehicle, then resided on the International Space Station for 21/22d or 37d in microgravity. Mice were euthanized on-orbit, livers and spleens dissected, and remaining tissues frozen in situ for later analyses. Mice appeared healthy by daily video health checks and body, adrenal, and spleen weights of 37d-flight (FLT) mice did not differ from ground controls housed in flight hardware (GC), while thymus weights were 35% greater in FLT than GC. Mice exposed to 37d of spaceflight displayed elevated liver mass (33%) and select enzyme activities compared to GC, whereas 21/22d-FLT mice did not. FLT mice appeared more physically active than respective GC while soleus muscle showed expected atrophy. RNA and enzyme activity levels in tissues recovered on-orbit were of acceptable quality. Thus, this system establishes a new capability for conducting long-duration experiments in space, enables sample recovery on-orbit, and avoids triggering standard indices of chronic stress.

HDAC4 Controls Muscle Homeostasis through Deacetylation of Myosin Heavy Chain, PGC-1α, and Hsc70.

HDAC4, a class IIa histone deacetylase, is upregulated in skeletal muscle in response to denervation-induced atrophy. When HDAC4 is deleted postnatally, mice are partially protected from denervation. Despite the name "histone" deacetylase, HDAC4 demonstrably deacetylates cytosolic and non-histone nuclear proteins. We developed potent and selective class IIa HDAC inhibitors. Using these tools and genetic knockdown, we identified three previously unidentified substrates of HDAC4: myosin heavy chain, peroxisome proliferator-activated receptor gamma co-activator 1alpha (PGC-1α), and heat shock cognate 71 kDa protein (Hsc70). HDAC4 inhibition almost completely prevented denervation-induced loss of myosin heavy chain isoforms and blocked the action of their E3 ligase, MuRF1. PGC-1α directly interacts with class IIa HDACs; selective inhibitors increased PGC-1α protein in muscles. Hsc70 deacetylation by HDAC4 affects its chaperone activity. Through these endogenous HDAC4 substrates, we identified several muscle metabolic pathways that are regulated by class IIa HDACs, opening up new therapeutic options to treat skeletal muscle disorders and potentially other disease where these specific pathways are affected.

αKlotho Regulates Age-Associated Vascular Calcification and Lifespan in Zebrafish.

The hormone αKlotho regulates lifespan in mice, as knockouts die early of what appears to be accelerated aging due to hyperphosphatemia and soft tissue calcification. In contrast, the overexpression of αKlotho increases lifespan. Given the severe mouse phenotype, we generated zebrafish mutants for αklotho as well as its binding partner fibroblast growth factor-23 (fgf23). Both mutations cause shortened lifespan in zebrafish, with abrupt onset of behavioral and degenerative physical changes at around 5 months of age. There is a calcification of vessels throughout the body, most dramatically in the outflow tract of the heart, the bulbus arteriosus (BA). This calcification is associated with an ectopic activation of osteoclast differentiation pathways. These findings suggest that the gradual loss of αKlotho found in normal aging might give rise to ectopic calcification.

Author Correction: Behavior of mice aboard the International Space Station.

A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has been fixed in the paper.

Skeletal muscle in MuRF1 null mice is not spared in low-gravity conditions, indicating atrophy proceeds by unique mechanisms in space.

Microgravity exposure is associated with loss of muscle mass and strength. The E3 ubiquitin ligase MuRF1 plays an integral role in degrading the contractile apparatus of skeletal muscle; MuRF1 null (KO) mice have shown protection in ground-based models of muscle atrophy. In contrast, MuRF1 KO mice subjected to 21 days of microgravity on the International Space Station (ISS) were not protected from muscle atrophy. In a time course experiment microgravity-induced muscle loss on the ISS showed MuRF1 gene expression was not upregulated. A comparison of the soleus transcriptome profiles between spaceflight and a publicly available data set for hindlimb suspension, a claimed surrogate model of microgravity, showed only marginal commonalities between the models. These findings demonstrate spaceflight induced atrophy is unique, and that understanding of effects of space requires study situated beyond the Earth's mesosphere.

Behavior of mice aboard the International Space Station.

Interest in space habitation has grown dramatically with planning underway for the first human transit to Mars. Despite a robust history of domestic and international spaceflight research, understanding behavioral adaptation to the space environment for extended durations is scant. Here we report the first detailed behavioral analysis of mice flown in the NASA Rodent Habitat on the International Space Station (ISS). Following 4-day transit from Earth to ISS, video images were acquired on orbit from 16- and 32-week-old female mice. Spaceflown mice engaged in a full range of species-typical behaviors. Physical activity was greater in younger flight mice as compared to identically-housed ground controls, and followed the circadian cycle. Within 7-10 days after launch, younger (but not older), mice began to exhibit distinctive circling or 'race-tracking' behavior that evolved into coordinated group activity. Organized group circling behavior unique to spaceflight may represent stereotyped motor behavior, rewarding effects of physical exercise, or vestibular sensation produced via self-motion. Affording mice the opportunity to grab and run in the RH resembles physical activities that the crew participate in routinely. Our approach yields a useful analog for better understanding human responses to spaceflight, providing the opportunity to assess how physical movement influences responses to microgravity.

Supraphysiologic Administration of GDF11 Induces Cachexia in Part by Upregulating GDF15.

The age-related effects of GDF11 have been a subject of controversy. Here, we find that elevated GDF11 causes signs of cachexia in mice: reduced food intake, body weight, and muscle mass. GDF11 also elicited a significant elevation in plasma Activin A, previously shown to contribute to the loss of skeletal muscle. The effects of GDF11 on skeletal muscle could be reversed by administration of antibodies to the Activin type II receptors. In addition to the effects on muscle, GDF11 increased plasma GDF15, an anorectic agent. The anorexia, but not the muscle loss, could be reversed with a GDF15-neutralizing antibody. GDF15 upregulation is due to GDF11-induced recruitment of SMAD2/3 to the GDF15 promoter. Inhibition of GDF15 can restore appetite but cannot restore the GDF11-induced loss of muscle mass, which requires blockade of ActRII signaling. These findings are relevant for treatment of cachexia.

GDF11 Increases with Age and Inhibits Skeletal Muscle Regeneration.

Age-related frailty may be due to decreased skeletal muscle regeneration. The role of TGF-ß molecules myostatin and GDF11 in regeneration is unclear. Recent studies showed an age-related decrease in GDF11 and that GDF11 treatment improves muscle regeneration, which were contrary to prior studies. We now show that these recent claims are not reproducible and the reagents previously used to detect GDF11 are not GDF11 specific. We develop a GDF11-specific immunoassay and show a trend toward increased GDF11 levels in sera of aged rats and humans. GDF11 mRNA increases in rat muscle with age. Mechanistically, GDF11 and myostatin both induce SMAD2/3 phosphorylation, inhibit myoblast differentiation, and regulate identical downstream signaling. GDF11 significantly inhibited muscle regeneration and decreased satellite cell expansion in mice. Given early data in humans showing a trend for an age-related increase, GDF11 could be a target for pharmacologic blockade to treat age-related sarcopenia.

Influence of exercise on the metabolic profile caused by 28 days of bed rest with energy deficit and amino acid supplementation in healthy men.

OBJECTIVE: Muscle loss and metabolic changes occur with disuse [i.e. bed rest (BR)]. We hypothesized that BR would lead to a metabolically unhealthy profile defined by: increased circulating tumor necrosis factor (TNF)-α, decreased circulating insulin-like-growth-factor (IGF)-1, decreased HDL-cholesterol, and decreased muscle density (MD; measured by mid-thigh computerized tomography). METHODS: We investigated the metabolic profile after 28 days of BR with 8 ± 6% energy deficit in male individuals (30-55 years) randomized to resistance exercise with amino acid supplementation (RT, n=24) or amino acid supplementation alone (EAA, n=7). Upper and lower body exercises were performed in the horizontal position. Blood samples were taken at baseline, after 28 days of BR and 14 days of recovery. RESULTS: We found a shift toward a metabolically unfavourable profile after BR [compared to baseline (BLN)] in both groups as shown by decreased HDL-cholesterol levels (EAA: BLN: 39 ± 4 vs. BR: 32 ± 2 mg/dL, RT: BLN: 39 ± 1 vs. BR: 32 ± 1 mg/dL; p<0.001) and Low MD (EAA: BLN: 27 ± 4 vs. BR: 22 ± 3 cm(2), RT: BLN: 28 ± 2 vs. BR: 23 ± 2 cm(2); p<0.001). A healthier metabolic profile was maintained with exercise, including NormalMD (EAA: BLN: 124 ± 6 vs. BR: 110 ± 5 cm(2), RT: BLN: 132 ± 3 vs. BR: 131 ± 4 cm(2); p<0.001, time-by-group); although, exercise did not completely alleviate the unfavourable metabolic changes seen with BR. Interestingly, both groups had increased plasma IGF-1 levels (EAA: BLN:168 ± 22 vs. BR 213 ± 20 ng/mL, RT: BLN:180 ± 10 vs. BR: 219 ± 13 ng/mL; p<0.001) and neither group showed TNFα changes (p>0.05). CONCLUSIONS: We conclude that RT can be incorporated to potentially offset the metabolic complications of BR.

Transforming growth factor-ß superfamily ligand trap ACE-536 corrects anemia by promoting late-stage erythropoiesis.

Erythropoietin (EPO) stimulates proliferation of early-stage erythrocyte precursors and is widely used for the treatment of chronic anemia. However, several types of EPO-resistant anemia are characterized by defects in late-stage erythropoiesis, which is EPO independent. Here we investigated regulation of erythropoiesis using a ligand-trapping fusion protein (ACE-536) containing the extracellular domain of human activin receptor type IIB (ActRIIB) modified to reduce activin binding. ACE-536, or its mouse version RAP-536, produced rapid and robust increases in erythrocyte numbers in multiple species under basal conditions and reduced or prevented anemia in murine models. Unlike EPO, RAP-536 promoted maturation of late-stage erythroid precursors in vivo. Cotreatment with ACE-536 and EPO produced a synergistic erythropoietic response. ACE-536 bound growth differentiation factor-11 (GDF11) and potently inhibited GDF11-mediated Smad2/3 signaling. GDF11 inhibited erythroid maturation in mice in vivo and ex vivo. Expression of GDF11 and ActRIIB in erythroid precursors decreased progressively with maturation, suggesting an inhibitory role for GDF11 in late-stage erythroid differentiation. RAP-536 treatment also reduced Smad2/3 activation, anemia, erythroid hyperplasia and ineffective erythropoiesis in a mouse model of myelodysplastic syndromes (MDS). These findings implicate transforming growth factor-ß (TGF-ß) superfamily signaling in erythroid maturation and identify ACE-536 as a new potential treatment for anemia, including that caused by ineffective erythropoiesis.

Effects of resistance exercise combined with essential amino acid supplementation and energy deficit on markers of skeletal muscle atrophy and regeneration during bed rest and active recovery.

Spaceflight and bed rest (BR) lead to muscle atrophy. This study assessed the effect of essential amino acid (EAA) supplementation and resistance training with decreased energy intake on molecular changes in skeletal muscle after 28-day BR and 14-day recovery. Thirty-one men (31-55 years) subjected to an 8 ± 6% energy deficit were randomized to receive EAA without resistance training (AA, n = 7), or EAA 3 h after (RT, n = 12) or 5 min before (AART, n = 12) resistance training. During BR, myostatin transcript levels increased twofold in the AA group. During recovery, insulin-like growth factor-1 (IGF-1) mRNA increased in all groups, whereas Pax7, MyoD, myogenin, and MRF4 transcripts increased in AA only (all P < 0.05). MAFbx transcripts decreased twofold with AA and RT. Satellite cells did not change during BR or recovery. This suggests that EAA alone is the least protective countermeasure to muscle loss, and several molecular mechanisms are proposed by which exercise attenuates muscle atrophy during BR with energy deficit.

Administration of a soluble activin type IIB receptor promotes skeletal muscle growth independent of fiber type.

This is the first report that inhibition of negative regulators of skeletal muscle by a soluble form of activin type IIB receptor (ACE-031) increases muscle mass independent of fiber-type expression. This finding is distinct from the effects of selective pharmacological inhibition of myostatin (GDF-8), which predominantly targets type II fibers. In our study 8-wk-old C57BL/6 mice were treated with ACE-031 or vehicle control for 28 days. By the end of treatment, mean body weight of the ACE-031 group was 16% greater than that of the control group, and wet weights of soleus, plantaris, gastrocnemius, and extensor digitorum longus muscles increased by 33, 44, 46 and 26%, respectively (P<0.05). Soleus fiber-type distribution was unchanged with ACE-031 administration, and mean fiber cross-sectional area increased by 22 and 28% (P<0.05) in type I and II fibers, respectively. In the plantaris, a predominantly type II fiber muscle, mean fiber cross-sectional area increased by 57% with ACE-031 treatment. Analysis of myosin heavy chain (MHC) isoform transcripts by real-time PCR indicated no change in transcript levels in the soleus, but a decline in MHC I and IIa in the plantaris. In contrast, electrophoretic separation of total soleus and plantaris protein indicated that there was no change in the proportion of MHC isoforms in either muscle. Thus these data provide optimism that ACE-031 may be a viable therapeutic in the treatment of musculoskeletal diseases. Future studies should be undertaken to confirm that the observed effects are not age dependent or due to the relatively short study duration.

Resistance training and timed essential amino acids protect against the loss of muscle mass and strength during 28 days of bed rest and energy deficit.

Spaceflight and bed rest (BR) result in losses of muscle mass and strength. Resistance training (RT) and amino acid (AA) supplementation are potential countermeasures to minimize these losses. However, it is unknown if timing of supplementation with exercise can optimize benefits, particularly with energy deficit. We examined the effect of these countermeasures on body composition, strength, and insulin levels in 31 men (ages 31-55 yr) during BR (28 days) followed by active recovery (14 days). Subjects were randomly assigned to essential AA supplementation (AA group, n = 7); RT with AA given 3 h after training (RT group, n = 12); or RT with AA given 5 min before training (AART group, n = 12). Energy intake was reduced by 8 +/- 6%. Midthigh muscle area declined with BR for the AA > RT > AART groups: -11%, -3%, -4% (P = 0.05). Similarly, greatest losses in lower body muscle strength were seen in the AA group (-22%). These were attenuated in the exercising groups [RT (-8%) and AART (-6%; P < 0.05)]. Fat mass and midthigh intramuscular fat increased after BR in the AA group (+3% and +14%, respectively), and decreased in the RT (-5% and -4%) and AART groups (-1 and -5%; P = 0.05). Muscle mass and strength returned toward baseline after recovery, but the AA group showed the lowest regains. Combined resistance training with AA supplementation pre- or postexercise attenuated the losses in muscle mass and strength by approximately two-thirds compared with AA supplement alone during BR and energy deficit. These data support the efficacy of combined AA and RT as a countermeasure against muscle wasting due to low gravity.