Compression of morbidity in a progeroid mouse model through the attenuation of myostatin/activin signalling

Animal maintenance

Control (Ercc1 ^+/+) and transgenic (Ercc1 ^Δ/−) mice were bred as previously described20, 25 and maintained in accordance to the Animals (Scientific Procedures) Act 1986 (UK) and approved by the Biological Resource Unit of Reading University or the Dutch Ethical Committee at Erasmus MC. Mice were housed in individual ventilated cages under specific pathogen‐free conditions (20–22°C, 12–12 hr light–dark cycle) and provided food and water ad libitum. Because the Ercc1 ^Δ/− mice were smaller, food was administered within the cages, and water bottles with long nozzles were used from around 2 weeks of age. Animals were bred and maintained (for the lifespan cohort) on AIN93G synthetic pellets (Research Diet Services B.V.; gross energy content 4.9 kcal/g dry mass, digestible energy 3.97 kcal/g). Post‐natal myostatin/activin block was induced in 7‐week‐old male mice, through intraperitoneal (IP) injection with 10 mg/kg of sActRIIB‐Fc every week, two times till week 16.26 Each experimental group consisted of a minimum of five male mice. The University of Reading experiments were performed on 12 controls, 9 Ercc1 ^Δ/−, and 14 sActRIIB‐treated Ercc1 ^Δ/− mice (all male mice). Lifespan experiments were performed on both genders, with five male and five female Ercc1 ^Δ/− mice per treatment condition and four males and four female littermate wild‐type controls. End‐of‐life Ercc1 ^Δ/− animals, both sActRIIB and mock treated, were post‐mortem investigated and scored negative for visible tumours, signs of internal bleedings, enlarged spleen size, or abnormally coloured heart or enlarged heart size.

Phenotype scoring

The mice were weighed and visually inspected at least weekly and were scored in a blinded manner by experienced research technicians for the onset of various phenotypical parameters. The onset of body weight was counted as the first week. A decline in body weight was noted after their maximal body weight was reached. Whole‐body tremor was scored if mice were trembling for a combined total of at least 10 s when put on a flat surface for 20 s. Impaired balance was determined by observing the mice walking on a flat surface for 20 s. Mice that had difficulties in maintaining an upright orientation during this period were scored as having imbalance. If mice showed a partial loss of function of the hind limbs, they were scored as having paresis.

Open‐field activity cages monitoring system

Open‐field cages (Linton Instrumentation AM548) with an array of invisible infrared light beams and multiple photocell receptors were used. Beams scan activity at two levels from front to back and left to right was performed to determine movement with data captured using AMON software, running on Windows PCs. The lower grid measured normal X, Y movement, whilst the upper grid measured rearing movement. Mice (14 weeks of age) were acclimatized for 30 min before recording. Data were measured on three occasions at 1 day intervals.

Muscle tension measurements

Dissection of the hind limb was carried out under oxygenated Krebs solution (95% O₂ and 5% CO₂). Under circulating oxygenated Krebs solution, one end of a silk suture was attached to the distal tendon of the extensor digitorum longus (EDL) and the other to a force transducer (FT03). The proximal tendon remained attached to the tibial bone. The leg was secured in the experimental chamber. Silver electrodes were positioned on either side of the EDL. A constant voltage stimulator was used to directly stimulate the EDL, which was stretched to attain the optimal muscle length to produce maximum twitch tension (P _t). Tetanic contractions were invoked by stimulus trains of 500 ms duration at 20, 50, 100, and 200 Hz. The maximum tetanic tension (P _o) was determined from the plateau of the frequency–tension curve.

Protein synthesis measure

The relative rate of protein synthesis was measured using the surface sensing of translation method (SUnSET).27 Briefly, mice were injected exactly 30 min before tissue collection with 0.04 μmol/g body mass puromycin into the peritoneal cavity and then returned to their cages. After tissue collection, muscles were solubilized as for western blotting and then pulled through a slot blotting chamber facilitating the transfer of protein onto a nylon membrane. Thereafter, the membrane was processed identically to a western blot.

Histological analysis and immunohistochemistry

Following dissection, the muscle was immediately frozen in liquid nitrogen‐cooled isopentane and mounted in optimal cutting temperature compound (TAAB O023) cooled by dry ice/ethanol. Immunohistochemistry was performed on 10 μm cryosections that were air‐dried at room temperature (RT) for 30 min before the application of block wash buffer [PBS with 5% foetal calf serum (v/v), 0.05% Triton X‐100]. Antibodies were diluted in wash buffer 30 min before using. Fluorescence‐based secondary antibodies were used to detect all primary antibodies except for CD‐31 where the Vectastain ABC‐HRP kit was deployed (Vector PK‐6100) with an avidin/biotin‐based peroxidase system and DAB peroxidase (HRP) substrate (Vector SK‐4100). Morphometric analysis of muscle fibre size was performed as previously described.28 Details of primary and secondary antibodies are given in Table ​11.

Dihydroethidium staining

Sectioned slides were dried for 30 min at RT. The sections were rehydrated with PBS then incubated with dihydroethidium (DHE) (50 μmol/L in PBS Sigma D7008) for 30 min at 37°C in the dark. Counterstain for nuclei was DAPI‐containing fluorescent mounting medium.

Haematoxylin and eosin

Muscle and liver sections were dewaxed in xylene and rehydration in ethanol prior to incubation with Harris' haematoxylin solution (Sigma HHS16) for 30 s and thereafter in eosin solution (Sigma‐Aldrich 318906) for 2 min.

Succinate dehydrogenase staining

Muscle cyro‐sections were incubated for 3 min at RT in a sodium phosphate buffer containing 75 mM sodium succinate, 1.1 mM Nitroblue Tetrazolium (Sigma‐Aldrich), and 1.03 mM Phenazine Methosulphate (Sigma‐Aldrich). Samples were then fixed in 10% formal‐calcium, dehydrated and cleared in xylene prior to mounting with DPX mounting medium (Fisher).

Transmission electron microscopy

Biceps muscle and the kidney were briefly fixed with 4% paraformaldehyde and 2.5% glutaraldehyde in 0.1 M cacodylate buffer pH 7.4 in situ at RT then dissected, removed, and cut into pieces of 1 mm³ and fixed for 48 h in same solution at 4°C. Tissue blocks were contrasted using 0.5% OsO₄ (Roth, Germany; RT, 1.5 hr) and 1% uranyl acetate (Polysciences, Germany) in 70% ethanol (RT, 1 hr). After dehydration, tissue blocks were embedded in epoxy resin (Durcopan, Roth, Germany), and ultrathin sections of 40 nm thickness were cut using a Leica UC6 ultramicrotome (Leica, Wetzlar, Germany). Sections were imaged using a Zeiss 906 TEM (Zeiss, Oberkochen, Germany) and analysed using ITEM software (Olympus, Germany).26

Blood glucose, growth hormone, insulin, and insulin‐like growth factor‐1 levels

Glucose levels were measured using a freestyle mini blood glucose metre. GH, insulin, and IGF‐1 levels were measured in serum using a rat/mouse growth hormone ELISA (Merck Millipore), ultrasensitive mouse insulin ELISA (Mercodia), or mouse IGF‐1 ELISA (Abcam), respectively.

Micro‐computed tomography imaging

Computed tomography imaging was performed using a high‐speed in vivo micro‐computed tomography (μCT) scanner (Quantum FX, PerkinElmer, Hopkinton, MA, USA). The X‐ray source was set to a current of 160 μA and a voltage of 90 kVp. The field of view was 30 mm × 30 mm for muscle with a voxel size of 60 μm and 20 mm × 20 mm, and voxel size was 40 μm, for bone. The animals received isoflurane anaesthesia (2.5%) to immobilize them during scanning. Following scanning, image segmentation was performed semi‐automatically using the Volume Edit tools within the analysis software package (AnalyzeDirect, Overland Park, KS, USA). Briefly, segmentation masks (object maps) were created using a combination of semi‐automatic and manual techniques (object extraction, region growing, and thresholding tools). These segmentation results were then manually modified if necessary and quantified using the ROI tools.

Protein expression by immunoblotting

Frozen muscles were pulverized with pestle and mortar and solubilized in 50 mM Tris, pH 7.5, 150 mM NaCl, 5 mM MgCl₂, 1 mM DTT, 10% glycerol, 1% SDS, 1% Triton X‐100, 1X Roche Complete Protease Inhibitor Cocktail, and 1X Sigma‐Aldrich Phosphatase Inhibitor Cocktails 1 and 3. Proteins were denatured in Laemmli buffer and resolved on 10% SDS‐PAGEs prior to immunoblotting and probing with antibodies and the SuperSignal West Pico Chemiluminescent substrate (Pierce). Details of antibodies are given in Table ​11.

Quantitative polymerase chain reaction

Fifty to 100 mg of tissue was solubilized in TRIzol (Fisher) using a tissue homogenizer. Total RNA was prepared using the RNeasy Mini Kit (Qiagen, Manchester, UK). Five micrograms of RNA were reverse‐transcribed to cDNA with SuperScript II Reverse Transcriptase (Invitrogen) and analysed by quantitative real‐time RT‐PCR on a StepOne Plus cycler, using the Applied Biosystems SYBR‐Green PCR Master Mix. Primers were designed using the software Primer Express 3.0 (Applied Biosystems). Relative expression was calculated using the ΔΔCt method and normalized to cyclophilin‐B and hypoxanthine‐guanine phosphoribosyltransferase. Primer sequences are given in Table ​11.

Satellite cell culture

Single fibres from EDL were isolated using 0.2% collagenase I in Dulbecco's modified Eagle's medium and either fixed in 2% paraformaldehyde or cultured for 24, 48, and 72 hr as previously described.29

Bone scanning

Tibial samples were scanned and analysed by μCT; 180° scans were performed on a Skyscan 1172F μCT scanner (Skyscan, Kontich, Belgium); the X‐ray source was operated at 50 kV and 200 uA, a 0.5 aluminium filter was used with a 1650 ms exposure time and a pixel size of 5 μm. Projection images were reconstructed into tomograms using NRecon (Skyscan, Kontich, Belgium), and regions of interest were analysed using CTAn (Skyscan, Kontich, Belgium).

The activin ligand trap increases body organismal activity and strength

We determined whether the age‐related reduced muscle mass in Ercc1 ^Δ/− mutants could be prevented by the sActRIIB protein, which we have shown to antagonize signalling mediated by myostatin and related proteins.26 To that end, male Ercc1 ^Δ/− mice were IP injected twice a week with sActRIIB from 7 weeks of age till week 16. Mock‐treated Ercc1 ^Δ/− mutants showed no overall body mass gain in 8 weeks, whereas both control and Ercc1 ^Δ/− animals treated with sActRIIB displayed weight increases of 37% and 18%, respectively (Figures ​1A,1A, S2A, and S2B).

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Figure 1

sActRIIB treatment mitigates body, whole animal activity, grip strength, losses, and specific force loss in Ercc1 ^Δ/− mice. (A) Relative changes in body mass over time. Intraperitoneal injection of Ercc1 ^Δ/− with sActRIIB started at week 7 and tissues collected at the end of week 15. Organismal activity measurements through activity cages. Measurements in (B–E) made at the end of week 14. (F) Rotarod activity. (G) Muscle contraction measurement through assessment of grip strength. (H) Ex vivo assessment of EDL‐specific force. (I) Half relaxation time for the EDL. Levels of (J) growth hormone, (K) glucose, (L) insulin, and (M) insulin‐like growth factor‐1 at beginning of week 15. (N) Food intake and (O) relative food intake at the end of week 15. n = 6 control male mice, n = 5 Ercc1 ^Δ/− untreated male mice, and n = 5 Ercc1 ^Δ/− treated male mice. All analysis performed using non‐parametric Kruskal–Wallis test followed by the Dunn's multiple comparisons except (J) where one‐way analysis of variance followed by Bonferroni's multiple comparison tests was used. *P < 0.05, **P < 0.01, ***P < 0.001. EDL, extensor digitorum longus; IGF‐1, insulin‐like growth factor‐1; sActRIIB, soluble activin receptor type IIB.

Using activity cages, we found that sActRIIB‐treated Ercc1 ^Δ/− mice were more active than both their mock‐treated counterparts and control mice (Figure ​1B1B and Movie S1). Treatment of Ercc1 ^Δ/− mice with sActRIIB increased the distance travelled compared not only with untreated mice but also with control animals (Figure ​1C1C and Movie S1). Total rearing counts and rearing time, measures of locomotor activity as well as exploration and anxiety, were highest in control mice and significantly reduced in Ercc1 ^Δ/− mice. sActRIIB treatment increased these values compared with Ercc1 ^Δ/− but not to normal levels (Figure ​11D–1E). Motor coordination, measured using the Rotarod, showed that Ercc1 ^Δ/− mice at the age of 16 weeks have significant deficit in this skill, which was improved, albeit not to normal levels, by sActRIIB (Figure ​1F).1F). Muscle function, as assessed using a grip metre, revealed that progeric mice had reduced strength compared with control mice. This parameter was significantly improved in Ercc1 ^Δ/− mutants by sActRIIB (Figure ​1G).1G). Ex vivo measure of specific force revealed a significant deficit in this parameter in Ercc1 ^Δ/− mutants that was significantly increased by sActRIIB treatment (Figure ​1H).1H). Half‐relaxation time was increased in Ercc1 ^Δ/− mutants compared with controls but reduced by sActRIIB treatment (Figure ​11I).

We determined the circulatory levels of molecules known to regulate organismal growth and found elevated levels of GH in both untreated and sActRIIB treated Ercc1 ^Δ/− mutants (Figure ​1J),1J), likely as previously noted feedback mechanism in response to prolonged low IGF‐1.2 Indeed, levels of blood glucose, serum insulin, and IGF‐1 were decreased in Ercc1 ^Δ/− mutants as compared with controls, and none of these factors were changed in response to sActRIIB treatment (Figure ​11K–1M). Food intake of Ercc1 ^Δ/− mutants, relative to body weight, was higher than control mice, but unaffected by sActRIIB treatment, excluding indirect effects of diet restriction for which Ercc1 ^Δ/− mice are very sensitive (Figure ​11N–1O).23 Water intake was not affected by the treatment (data not shown).

Quantitative and qualitative improvements to skeletal muscle through soluble activin receptor type IIB treatment

Previous work has shown that sActRIIB treatment increases muscle mass. The increased body weight and grip strength of Ercc1 ^Δ/− mice subjected to sActRIIB prompted us to further examine individual muscles. Treated Ercc1 ^Δ/− mice revealed that all five groups showed significant greater mass compared with those from mock‐treated Ercc1 ^Δ/− animals with a range of 30–62% (TA and plantaris, respectively; Figure ​22A–2C). Activation of signalling pathways initiated through ActRIIB and relevant to this study was found to be elevated in the muscle of Ercc1 ^Δ/− mice and decreased by sActRIIB treatment (Figure S3A). Importantly, the abundance of DNA breaks was not changed by sActRIIB treatment (Figure S3B). Furthermore, sActRIIB failed to increase the mass of any other organ examined including the heart, kidney, and liver (Figure S2C and S2D). We explored the mechanisms underlying the increase in muscle mass following sActRIIB treatment of Ercc1 ^Δ/− mice. Introduction of sActRIIB induced fibre hypertrophy irrespective of MHC expression (Figure ​22D–2G). Of particular note was the finding that some types of fibres in the sActRIIB‐treated Ercc1 ^Δ/− muscles were significantly larger than even in controls (see MHCI and IIA in soleus; Figure ​2E).2E). The total fibre number in EDL was elevated in Ercc1 ^Δ/− mutants and maintained by sActRIIB (Figure ​2H).2H). A similar trend was found in the soleus (Figure ​2H).2H). Of particular note was the observation of a large proportion of fibres with micro‐lesions (including tears to the membrane) isolated from the EDL muscle from Ercc1 ^Δ/− animals, which appeared largely normalized by sActRIIB (Figure ​2I2I and ​and2J).2J). Caspase‐3 activity as a gauge of apoptosis was significantly elevated in muscle of Ercc1 ^Δ/− mice and largely normalized by treatment with sActRIIB (Figures ​2K2K and S3C). The number of fibres displaying centrally located nuclei was elevated in both the EDL and soleus muscles from Ercc1 ^Δ/− mice compared with controls and became even more abundant following sActRIIB treatment (Figures ​2L2L and S3D). The fibres showing supra‐normal levels of SDH activity, indicative of abnormal mitochondrial activity that leads to apoptosis,30 were significantly more frequent in both the EDL and soleus of Ercc1 ^Δ/− mice compared with treated mutants (Figure ​22M–2O). Assessment of ROS activity through DHE intensity showed elevated levels of superoxide in muscle of Ercc1 ^Δ/− animals, which was lowered by sActRIIB treatment although it did not reach the level of control mice (Figure ​22P–2S).31, 32

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Figure 2

Quantitative and qualitative improvements to Ercc1 ^Δ/− skeletal muscle through sActRIIB treatment. (A) Muscle weight at end of week 15. (B) Muscle mass normalized to tibial length. (C) Micro‐computed tomography scan of hind limb to visualize the increase in muscle upon sActRIIB treatment in Ercc1 ^Δ/− mice. (D–G) Cross‐sectional fibre areas assigned to specific myosin heavy chain isoforms. (H) Fibre number increased in EDL and soleus of Ercc1 ^Δ/− mice and further increased following treatment. (I) Incidence of damaged fibres following single fibre isolation. (J) Example of micro‐tear (arrows) in an Ercc1 ^Δ/− EDL fibre. (K) Fibres containing caspase 3 epitope as a percentage of all EDL and soleus fibres. (L) Percentage of fibres with centrally located nuclei in the EDL and soleus. (M) Quantification of hyper‐stained SDH fibres. (N) SDH in control muscle and (O) Ercc1 ^Δ/− muscle showing hyper‐stained fibres (arrows). (P) Quantification of DHE fluorescence in TA muscle fibres. (Q) Control TA fibres with little DHE fluorescence in the body of control fibres. (R) Ercc1 ^Δ/− TA fibres with elevated DHE fluorescence in the body of control fibres. (S) Treated Ercc1 ^Δ/− TA fibres with little DHE fluorescence in the body of fibres. n = 9 control male mice, n = 8 Ercc1 ^Δ/− untreated male mice, and n = 8 Ercc1 ^Δ/− treated male mice. Scale for single fibre 50 μm, SDH 100 μm and DHE 20 μm. One‐way analysis of variance followed by Bonferroni's multiple comparison tests. *P < 0.05, **P < 0.01, ***P < 0.001. DHE, dihydroethidium; EDL, extensor digitorum longus; sActRIIB, soluble activin receptor type IIB; SDH, succinate dehydrogenase; TA, tibialis anterior.

Myosin heavy chain, oxidative fibre profiling, vascular organization, and molecular metabolic analysis of skeletal muscle

Myosin heavy chain analysis revealed that the progeric muscle displayed a faster profile compared with control muscles (Figure S1H–S1J). Treatment of progeric animals with sActRIIB resulted in a shift towards an even faster MHC profile. This was particularly pronounced in the EDL, with an increase in the proportion of type IIB fibres at the expense of both types IIA and IIX (Figure ​33A–3D).

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Figure 3

sActRIIB induces fast and glycolytic transformation of Ercc1 ^Δ/− muscle. (A) MHC profile of EDL muscle. (B–D) EDL MHCIIA/IIB fibre distribution in the three cohorts, controls, Ercc1 ^Δ/−, and Ercc1 ^Δ/− treated with sActRIIB. (E) SDH‐positive and ‐negative fibre profile of EDL muscle. (F–H) SDH stain in the three cohorts. (I) Quantification of EDL capillary density. (J–L) Identification of EDL capillaries with CD‐31 in the three cohorts. Quantitative PCR profiling of (M) angiogenic genes, (N) PGC1α, (O) mitochondrial genes, and (P) regulators of fat metabolism. n = 8 for all cohorts. Scale for SDH 100 μm and CD31 50 μm. One‐way analysis of variance followed by Bonferroni's multiple comparison tests used in all data sets except (E) where non‐parametric Kruskal–Wallis test followed by the Dunn's multiple comparison was used. *P < 0.05, **P < 0.01, ***P < 0.001. EDL, extensor digitorum longus; MHC, myosin heavy chain; SDH, succinate dehydrogenase; sActRIIB, soluble activin receptor type IIB.

To examine the metabolic status of the sActRIIB‐treated muscle, we determined the SDH activity. In both the EDL and the soleus, the number of SDH‐positive fibres was decreased in the progeric mice compared with controls (Figures ​33E–3H and S3E). Introduction of sActRIIB treatment further decreased the number of SDH⁺ fibres and, at the same time, increased the number of SDH⁻ entities in the EDL (Figure ​33E–3H). Similar changes were also recorded in the soleus (Figure S3E). Therefore, the sActRIIB treatment further reduces the status of the already diminished oxidative character of Ercc1 ^Δ/− muscles. Subsequently, we investigated whether changes in the muscle metabolic profile wrought by sActRIIB also induced a remodelling of the vasculature. The capillary density profile indeed showed that the number of blood vessels serving each fibre was lower in Ercc1 ^Δ/− mice (albeit non‐significantly) and further decreased following sActRIIB treatment (Figure ​33I–3L). These changes were underpinned by decreases in the expression of three genes examined that control the development of blood vessels (Figure ​3M).3M). Expression of PGC1α, a key regulator of oxidative properties in muscle, was slightly lower in muscle from Ercc1 ^Δ/− mice compared with controls and was even more suppressed following sActRIIB treatment (Figure ​3N).3N). The changes in genes supporting the development of blood vessels were mirrored by mitochondrial transcript levels. qPCR analysis of eight genes important for the mitochondrial metabolism revealed that seven had decreased expression in Ercc1 ^Δ/− muscles induced by sActRIIB treatment (Figure ​3O).3O). We also investigated genes that control fat metabolism. All seven genes examined were significantly reduced in expression by sActRIIB (Figure ​33P).

Therefore, the attenuation of signalling through sActRIIB results in the patterning of muscle towards a fast contracting status, which has a paucity of oxidative fibres and supporting blood vessels underpinned by changes in the expression of genes that control capillary development and sustain aerobic metabolism.

Ultrastructure and mitochondrial characterization in muscle

The ultrastructure of skeletal muscle in the three cohorts was examined using transmission electron microscopy. Numerous abnormalities were evident in the muscle from Ercc1 ^Δ/− mice including heterogeneous Z‐line lengths, missing Z‐lines, misaligned Z‐lines, split sarcomeres, and large inter‐sarcomeric spaces compared with controls (Figure ​4A,4A, ​A,4B,4B, ​B,4D,4D, ​D,4E,4E, ​E,4G,4G, and ​and4H).4H). These abnormalities were largely absent in muscle from Ercc1 ^Δ/− mice treated with sActRIIB (Figure ​4C,4C, ​C,4F,4F, and ​and4I).4I). Quantification of mitochondria density revealed a decrease in this parameter both within the fibre (sarcomeric region) and directly under the sarcolemma (Figure ​4J4J and ​and4K).4K). Of special note was the alteration (swelling) of mitochondria both within the fibre and immediately under the sarcolemma (Figure ​4H).4H). Quantification of mitochondrial size showed enlargement in the muscle from Ercc1 ^Δ/− mutants, which was reduced by the treatment with sActRIIB (Figure ​4L4L and ​and4M).4M). Mitochondrial hypertrophy has been shown to be a protective response to a decrease in mitochondrial function or number or an indicative excessive fusion.33, 34, 35 It is thought to promote mitochondrial survival by up‐regulating a stress response programme. Indeed, we found that there was an increase in the expression of key genes involved in the mitochondrial unfolded protein response (UPR^MT) pathway in the muscle from Ercc1 ^Δ/− mice (Figure ​4N).4N). We also examined the levels of inflammatory and prohibitin genes, which support mitochondrial function of ensuring correct folding of the cristae.36 Expression of IL6 and IL18 as well as two key prohibitin genes (Phb and Phb2) appeared slightly elevated in the muscle of Ercc1 ^Δ/− mice (Figure ​4O4O and ​and4P).4P). Treatment of Ercc1 ^Δ/− mice with sActRIIB generally prevented these changes (Figure ​44A–4P). Lastly, we examined whether muscle harboured epigenetic modifications involved in the maintenance of heterochromatin that change with age.37, 38 The ageing process causes a decrease in the level of H3K9me3 but an increase in H4K20me3.39 H3K9me3 was decreased, and H4K20me3 increased in Ercc1 ^Δ/− animals in keeping with an age‐related change (Figure ​4Q4Q and ​and4R).4R). Both features were normalized following sActRIIB treatment (Figure ​4Q4Q and ​and44R).

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Figure 4

sActRIIB prevents Ercc1 ^Δ/− muscle ultrastructural abnormalities and supports normal levels of expression of key stress indicators. All Electron microscopy (EM) longitudinal image and quantitative measurements are from the bicep muscle. (A) Low‐power image of control muscle. (B) Low‐power image of Ercc1 ^Δ/− muscle. Note large spaces (black arrowheads), non‐uniform sarcomere width (red arrows), dilated sarcomeric mitochondria (red arrowheads), split sarcomere (black arrow), and disrupted M‐Line (blue arrow). (C) Low‐power image of sActRIIB‐treated Ercc1 ^Δ/− muscle. (D) Higher magnification of sarcomeric region of control muscle showing uniformly sized mitochondria (black arrows). (E) Enlarged mitochondria in sarcomeric region of Ercc1 ^Δ/− muscle (blue arrowhead) and absent (blue arrow) or faint Z‐line (black arrow). (F) Higher magnification of sarcomeric region of treated Ercc1 ^Δ/− mice showing smaller sarcomeric mitochondria (black arrows). (G) Sarcolemma region of control muscle showing compact mitochondria (red arrowhead). (H) Dilated (blue arrowhead) and aberrant mitochondria (blue arrow) in sub‐sarcolemma region of Ercc1 ^Δ/− muscle. (I) Sarcolemma region of treated Ercc1 ^Δ/− mice showing compact mitochondria (red arrowhead). (J, K) Sarcomeric (intrafusal) and sub‐membrane mitochondrial density measurements. (L, M) Sub‐membrane and sarcomeric (intrafusal) mitochondrial size measurements. (N) Expression of mitochondria unfolded protein response gene in gastrocnemius muscle. (O) Expression of inflammatory genes in gastrocnemius muscle. (P) Expression of prohibitin genes in gastrocnemius muscle. (Q) Quantification of EDL fibres expressing H3K9me3 and (R) H4K20me3. EM studies n = 6–7 for all cohorts. All other measures n = 8–9 for all cohorts. Non‐parametric Kruskal–Wallis test followed by the Dunn's multiple comparisons used in (N, O) and the rest with one‐way analysis of variance followed by Bonferroni's multiple comparison tests. *P < 0.05, **P < 0.01. EDL, extensor digitorum longus; sActRIIB, soluble activin receptor type IIB.

These results demonstrate subcellular defects in the Ercc1 ^Δ/− muscle and the expression of genes indicative of ongoing stress. sActRIIB treatment prevented the development of many of these abnormal features.

Connective tissue profiling

Skeletal muscle force transmission relies on proteins that link the contractile apparatus to the extra cellular matrix (ECM). We examined two of its components and determined how they were modified by the Ercc1 ^Δ/− genotype and thereafter by sActRIIB treatment. First, we examined the expression of dystrophin, a key intercellular component that links the cytoskeleton to the ECM. Its RNA expression was decreased in the Ercc1 ^Δ/− muscle, which was subsequently increased to levels greater than controls by sActRIIB (Figure ​5A).5A). We measured the amount of dystrophin specifically located between fibres using quantitative immuno‐fluorescence and confirmed its reduction specifically at this site in the Ercc1 ^Δ/− muscle compared with controls and was significantly increased by sActRIIB (Figure ​5B5B and ​and5D).5D). Thereafter, we examined expression of collagen IV as basement membrane component important for force transmission. Its expression was slightly decreased albeit not reaching statistical significance in Ercc1 ^Δ/− muscle (Figure ​5C).5C). However, sActRIIB caused its level to increase over both untreated Ercc1 ^Δ/− and control levels (Figure ​5C).5C). Collagen IV gene expression levels were reflected at the protein level at the myofibre surface (Figure ​55E).

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Figure 5

Normalization of Ercc1 ^Δ/− extracellular components by sActRIIB and differentiation and self‐renewal of its satellite cells. (A) Dystrophin gene expression measured by quantitative PCR (qPCR). (B) Measure of dystrophin in fibre‐type‐specific manner using quantitative immunofluorescence. (C) Measure of collagen IV expression profiling by qPCR. (D) Immunofluorescence image for dystrophin expression in EDL muscle. (E) Immunofluorescence image for collagen IV expression in EDL muscle. n = 7 for all cohorts. (F) EDL myonuclei count. (G) Quantification of satellite cells on freshly isolated EDL fibres. (H) Quantification of cells on EDL fibres after 72 h culture. (I) Control, mock‐treated Ercc1 ^Δ/−, and sActRIIB‐treated Ercc1 ^Δ/− fibre examined at 72 h for expression of Myogenin (red) and Pax7 (green). Arrows indicated satellite cell progeny. (J) Quantification of EDL differentiated (Pax7⁻/Myogenin⁺) vs. stem cell (Pax7⁺/Myogenin⁻) after 72 h in culture. Fibres collected from three mice from each cohort and minimum of 25 fibres examined. Scale 50 μm. Non‐parametric Kruskal–Wallis test followed by the Dunn's multiple comparisons used for (A–C). Rest of data was analysed using one‐way analysis of variance followed by Bonferroni's multiple comparison tests. *P < 0.05, **P < 0.01, ***P < 0.001. EDL, extensor digitorum longus; sActRIIB, soluble activin receptor type IIB.

Mechanisms underlying fibre size changes

To explore mechanisms regulating muscle mass, we investigated changes in anabolic and catabolic programmes. Surprisingly, levels of phosphorylated Akt (an inducer of anabolism) appeared elevated in the muscle from 16‐week‐old mock‐treated Ercc1 ^Δ/− mice (Figure S4A). Next, we examined downstream targets of pAkt and found that there was a slight decrease in the phosphorylation of 4EBP1 at Thr37/46 but none at Ser65 (Figure S4B). However, there was an elevated level of phosphorylation at another pAkt target, S6 (Figure S4C). The effect of sActRIIB on the anabolic programme of Ercc1 ^Δ/− muscle showed a general increase in the level of pAkt, as well as its two downstream targets, 4EBP1 and S6, relative to both mock‐treated Ercc1 ^Δ/− and control groups (Figure S4A–S4C). Thereafter, we probed the catabolic programme and found that activity of FoxO1 and FoxO3a, key regulators of both ubiquitin‐mediated protein breakdown (FoxO1 significantly, FoxO3a not so), was generally decreased in the muscle from Ercc1 ^Δ/− mice (Figure S4D and S4E), even to a level exceeding controls. Expression of both MuRF1 and Atrogin‐1 downstream targets of FoxO1 and FoxO3a were elevated at the RNA level in the muscle of Ercc1 ^Δ/− mice (Figure S4I and S4J). The LC3 autophagy activity was suppressed compared with controls (Figure S4F). Treatment with sActRIIB caused an elevation in the levels of inactive FoxO1 and FoxO3a (Figure S4D and S4E) and a decrease in the expression of MuRF1 but, surprisingly, not Atrogin‐1 (Figure S4I and S4J). Expression of Mul1, a key regulator of mitophagy,40 did not differ in the three groups (Figure S4K). Significantly, we found an increase in the level of autophagy gauged by the LC3II/I ratio and levels of p62 following sActRIIB treatment (Figure S4F and S4G). We quantified the presence of p62 puncta, which has been used as an indicator of autophagic flux, with an increase in the numbers of p62 puncta implying a decrease in autophagic activity.41 The number of p62 puncta per given area were higher in Ercc1 ^Δ/− EDL muscle compared with controls, and their levels were reduced by sActRIIB treatment (Figure S4L and S4M). Treatment of Ercc1 ^Δ/− mice with sActRIIB resulted in a non‐significant increase in the amount of active eIF2a, a key regulator of the endoplasmic reticulum UPR (UPR^ER) programme (Figure S4H). At the organismal level, we found that the rate of protein synthesis was elevated (but not to significant levels) in Ercc1 ^Δ/− mice and further elevated by sActRIIB treatment (Figure S4N). The abundance of ubiquitinated proteins was elevated in the muscle of Ercc1 ^Δ/− mice but reduced by sActRIIB treatment (Figure S4O).

These results reveal novel characteristics considering the changes in muscle mass in the progeric mice. The muscle of Ercc1 ^Δ/− mice activates both its protein synthesis pathway and has elevated gene expression of molecules that control protein breakdown. However, autophagy is blunted. Treatment of Ercc1 ^Δ/− with sActRIIB results in an increase in the activity of molecules controlling protein synthesis as well as overall rate of protein synthesis, a decrease in the abundance of ubiquitinated proteins E3 as well as an increase in key regulators of autophagy.

Myonuclei and satellite cell profiling

We examined features of individual myofibres to determine the effect of sActRIIB treatment. The number of myonuclei in the fibres from the EDL or the number of SC on them from either PBS‐ or sActRIIB‐treated Ercc1 ^Δ/− mutants was significantly lower than the number in control mice (Figure ​5F5F and ​and5G).5G). We then investigated the proliferative capacity of the SC from the three cohorts and found that, after 72 h of culture, the population from control fibres had undergone a three‐fold increase compared with initial numbers. In sharp contrast, the SC from PBS‐treated Ercc1 ^Δ/− mice failed to undergo any significant proliferation. Importantly, sActRIIB treatment of Ercc1 ^Δ/− mice resulted in SC being able to undergo a 2.3‐fold increase in number (Figure ​5H).5H). Finally, we found that the attenuated differentiation programme of SC from Ercc1 ^Δ/− mice was normalized by sActRIIB treatment (Figure ​55I–5J).

Therefore, sActRIIB treatment mitigates abnormalities in SC proliferation, differentiation and self‐renewal programmes in Ercc1 ^Δ/− animals. However, it did not normalize the low SC number found in mock‐treated Ercc1 ^Δ/− mice.

Inhibition of glomerular anomalies in Ercc1 ^Δ/− mice by soluble activin receptor type IIB

Kidney pathology due to mutations in ERCC1 has been reported in both human and mice.2, 20 Here, we investigated the impact of sActRIIB on kidney function and structure. Proteinuria analysis showed a 12‐fold elevation in the albumin/creatinine ratio in urine from Ercc1 ^Δ/− mutants compared with controls at 16 weeks of age. This measure was reduced to an elevation of 3.7‐fold in the urine of sActRIIB‐treated Ercc1 ^Δ/− mice (Figure ​6A).6A). We investigated the mechanism underlying the proteinuria in Ercc1 ^Δ/− mice and how it is influenced by sActRIIB by examining the ultrastructure of the kidney filtration apparatus. Transmission electron microscopy showed the Ercc1 ^Δ/− podocytes hypertrophic, but additionally, they contained numerous abnormalities, including enlarged mitochondria as well as accumulation of autophagosomes (Figure ​6B,6B, ​B,6C,6C, ​C,6F,6F, and ​and6G,6G, autophagosomes shown in detail in Figure S5A). The most prominent feature was the degree of foot process effacement in the Ercc1 ^Δ/− sample, which contrasted the regular structures found in control samples (Figure ​66E–6G). When foot processes were present, they are significantly broader in Ercc1 ^Δ/− animals compared with controls (Figure ​66E–6G). Glomerular basement membrane was also significantly thicker in Ercc1 ^Δ/− kidneys compared with controls (Figure ​6F,6F, ​F,6G,6G, and I​I6).6). All these features were to a greater degree normalized following the treatment with sActRIIB (Figure ​66B–6I). At the ultrastructural level, enlarged mitochondria area and accumulation of autophagosomes were completely prevented (Figure ​6D6D and ​and6H).6H). Foot processes were evident (Figure ​6H).6H). It should be noted that in some regions, they appeared normal, whereas in other regions, they are still broader compared with controls (Figure ​6D6D and ​and6H).6H). The thickness of the glomerular basement membrane was significantly reduced by sActRIIB treatment compared with mock‐treated progeroid mice but not to normal levels (Figure ​6I).6I). Nuclear size that was enlarged in the glomeruli of Ercc1 ^Δ/− specimens was maintained at normal dimensions by sActRIIB (Figure ​6J).6J). Finally, we examined whether the impact of sActRIIB could be through direct antagonism of myostatin/activin signalling by investigating the distribution of pSmad2/3 in podocytes. There was very little pSmad2/3 in control glomeruli (Figure ​6K).6K). In contrast, abundant pSmad2/3 was found in nuclei of Ercc1 ^Δ/− podocytes (Figure ​6L).6L). Following sActRIIB treatment, the abundance of pSmad2/3 in Ercc1 ^Δ/− podocytes was reduced compared with untreated progeroid mice (Figure ​6M).6M). However, it was still more prominent than controls.

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Figure 6

The prevention of kidney function abnormalities through the maintenance of the filtration barriers by sActRIIB treatment of Ercc1 ^Δ/− mice. (A) Urine protein measurements at the end of week 14. (B–D) Low and (F–H) high magnification of electron microscopy images of podocytes from control, mock‐treated Ercc1 ^Δ/−, and sActRIIB‐treated Ercc1 ^Δ/− mice. Pod indicates the podocyte. (C) Ercc1 ^Δ/− tissue contains autophagosomes (yellow arrow) and enlarged mitochondria (yellow arrowhead). (D) sActRIIB‐treated Ercc1 ^Δ/− mice show some foot process effacement (red arrowheads) but significant number of mature foot processes (red arrow). (E) Quantification of foot process width. (F) Numerous mature foot processes in control sample (red arrows). (G) Very few foot processes in Ercc1 ^Δ/− sample but thickened glomerular basement membrane (red arrowheads). (H) Treated Ercc1 ^Δ/− sample showing numerous mature foot processes (red arrows). (I) Quantification of glomerular basement membrane thickness. (J) Nuclear size measurements in Nephrin‐positive domain. (K) pSmad2/3 profile in control mice (red) in relation to podocytes, identified through Nephrin expression. (L) Abundant levels of pSmad2/3 (red arrows) in Ercc1 ^Δ/− podocytes. (M) Few pSmad2/3 puncta in sActRIIB‐treated Ercc1 ^Δ/− podocytes (red arrow). n = 8 mice examined for each cohort for (A) and n = 5 mice examined for each cohort for (EM). Analysis performed using non‐parametric Kruskal–Wallis test followed by the Dunn's multiple comparisons. *P < 0.05, **P < 0.01. sActRIIB, soluble activin receptor type IIB.

These results show that foot process effacement is an underlying cause of proteinuria in Ercc1 ^Δ/kidneys. sActRIIB treatment not only improved the ultrastructure of the filtration barrier but significantly also reduced proteinuria.

Impact of soluble activin receptor type IIB on ageing‐related liver Ercc1 ^Δ/− abnormalities

The liver undergoes age‐related changes both in humans and rodent models.21, 42, 43 The nuclei in the livers of Ercc1 ^Δ/− mice undergo progressive ageing‐related changes including enlargement, invaginations, and polyploidy. These features have been interpreted to indicate incomplete cytokinesis.44 We found that both liver nuclear size and the number of liver multi‐nucleated cells were increased in tissue from Ercc1 ^Δ/− mice compared with control tissue (Figure ​7A7A and ​and7B).7B). Treatment of Ercc1 ^Δ/− mice with sActRIIB significantly decreased both measures (Figure ​7A7A and ​and7B).7B). Having shown that age‐associated changes in the liver nuclei of Ercc1 ^Δ/− mice were reduced following treatment with sActRIIB, we examined whether this was reflected by changes in epigenetic modification involved in the maintenance of heterochromatin.37, 38 Previous work has shown that levels of H3K9me3 are down‐regulated during ageing,39 and here too, we found such a relationship (Figure ​7C).7C). In contrast, ageing results in an increase in H4K20me3 marks. Here, we saw extensive levels of H4K20me3 in the liver of Ercc1 ^Δ/− and surprisingly of control mice (Figure ​7D7D and ​and7G).7G). Strikingly, the H4K20me3 marks were essentially absent in livers of Ercc1 ^Δ/− mice treated with sActRIIB (Figure ​7D7D and ​and7G).7G). Oxidative stress is one of the key drivers that induce age‐related changes in the liver.45 Again, we deployed the DHE dye to gauge the level of superoxide.31, 32 Superoxide levels were elevated in the liver samples of both Ercc1 ^Δ/− and control mice, compared with treated Ercc1 ^Δ/− mice (Figure ​7E7E and ​and7H).7H). Next, we profiled the metabolic activity of the liver as it is known to undergo a decrease in the level of oxidative phosphorylation with ageing.21, 46 In agreement with the work by Gregg et al. on Ercc1 ^Δ/− livers, we found a decrease in four of the six genes linked to oxidative phosphorylation (Figure ​77F).21 Expression of five genes was significantly increased by sActRIIB treatment relative to their levels in mock Ercc1 ^Δ/− animals (Figure ​7F).7F). Lastly, we determined whether the effects of sActRIIB on the livers of Ercc1 ^Δ/− mice were mediated by direct antagonism of myostatin/activin signalling. Profiling of pSmad2/3 showed that there was no activity in the parenchyma of the livers of the three cohorts (Figure S5B). Only a few pSmad2/3‐expressing cells were found adjacent to smooth muscle in all three cohorts (Figure S5B).

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Figure 7

sActRIIB prevents the development age‐related liver abnormalities and osteoporotic phenotype in Ercc1 ^Δ/−. (A) Measure of liver nuclear size. (B) Profile frequency of multinucleated liver cells. (C) Frequency of H3K9me3‐positive liver cells. (D) Frequency of H4K20me3‐positive liver cells. (E) Quantification of DHE fluorescence to gauge superoxide levels. (F) Quantitative PCR profiling of mitochondrial gene expression. (G) Immunofluorescence images for H4K20me3 distribution in the three cohorts. (H) DHE intensity levels in the three cohorts. (I) Trabecular bone volume measurements. (J) Trabecular tissue volume measurements. (K) Trabecular bone to tissue volume ratios. (L) Trabecular separation indices. (M) Enumeration of trabeculae. (N) Degrees of trabecular anisotrophy. (O) Trabecular pattern factor as a quantification of bone architecture. (P) Structure model index. (Q) Measure of cortical bone volume. (R) Cortical tissue volume measure. Trabecular bone volume measurements. n = 8 for all animals in (A–H) and n = 6 control male mice, five Ercc1 ^Δ/−‐untreated male mice, and six Ercc1 ^Δ/−‐treated male mice in other experiments. One‐way analysis of variance followed by Bonferroni's multiple comparison tests used for (A–F) and non‐parametric Kruskal–Wallis test followed by the Dunn's multiple comparisons for (I–R). *P < 0.05, **P < 0.01, ***P < 0.001. DHE, dihydroethidium; sActRIIB, soluble activin receptor type IIB.

These results show that antagonism of myostatin/activin signalling leads to profound normalization of Ercc1 ^Δ/− liver cell nuclear structure, selective epigenetic modification of DNA and changes in gene expression indicative of increased oxidative phosphorylation and a reduction in superoxide levels.

Prevention of the osteoporotic phenotype in Ercc1 ^Δ/− mice by soluble activin receptor type IIB

Micro‐CT analyses revealed that Ercc1 ^Δ/− mice exhibit a premature ageing‐related osteoporotic phenotype with extreme differences in trabecular and cortical bone mass and architecture compared with control mice. In the trabecular compartment, there was a significant reduction in bone volume, tissue volume, bone volume/tissue volume, trabecular separation, trabecular number, and degree of anisotropy, a measure of how highly oriented substructures are within a volume (Figure ​77I–7N). Significantly higher trabecular pattern factor indicating trabecular connectivity and structure model index a measure of surface convex curvature and an important parameter in measuring the transition of osteoporotic trabecular bone from a plate‐like to rod‐like architecture were also observed in Ercc1 ^Δ/− mice compared with controls animals (Figure ​7O).7O). Cortical bone volume and tissue volume were significantly lower, further demonstrating that Ercc1 ^Δ/− mice have an osteoporotic bone phenotype (Figure ​7Q7Q and ​and77R).

Analysis of trabecular bone revealed significant increase in bone and tissue volume, bone/tissue volume, and trabecular number in Ercc1 ^Δ/− sActRIIB‐treated mice compared with mock‐treated animals, indicating treatment prevents a decrease in the size of the trabecular compartment and the amount of bone present (Figure ​77I–7K). In addition, trabecular pattern factor was significantly lower in the treated group compared with mock‐treated with levels close to the control group, showing trabecular connectivity improved upon treatment (Figure ​7O).7O). Furthermore, the structure model index was significantly lower in the treated group, again with results close to the control group (Figure ​7P).7P). Cortical bone volume and tissue volume were significantly lower in Ercc1 ^Δ/− mice compared with the control groups, with treatment significantly lessened the tissue volume and bone volume decline (Figure ​7Q7Q and ​and77R).

Together, these analyses reveal that sActRIIB treatment produces tibial architecture changes and prevents a decrease in trabecular and cortical bone mass in Ercc1 ^Δ/− mice, mitigating the premature ageing‐related osteoporotic phenotype observed in this and previous studies.

Effects of soluble activin receptor type IIB on Ercc1 ^Δ/− muscle phenotype

Our study documents the profound effect of antagonizing myostatin/activin signalling on body and muscle mass as well as function of Ercc1 ^Δ/− mice. Age‐related decreases in all three parameters were significantly attenuated in the Ercc1 ^Δ/− mutant by the soluble activin receptor ligand trap. The difference in the muscle mass between treated vs. untreated animals ranged between 30% (TA) and 62% (plantaris). The changes in muscle mass are extremely impressive and worthy of comparison with the Mstn ^−/− mutant. The EDL of the Mstn ^−/− was 60% heavier than the wild‐type counterpart.58 Here, after only 8 weeks of sActRIIB treatment, the EDL was 45% heavier. The differences in muscle mass between treated and untreated mice are of note when compared with outcomes that have previously deployed anti‐myostatin/activin, non‐genetic approaches in aged mice. One such study showed that the TA of aged mice underwent an increase of 6%, whereas here, even though it was the muscle that displayed the smallest increase nevertheless was 30% heavier than that of mock‐treated Ercc1 ^Δ/− mice.59 These results suggest that activity of myostatin and/or activin in Ercc1 ^Δ/− mice are considerably higher than in aged wild‐type mice. Importantly, we show that treatment with sActRIIB did not induce changes in the circulating levels of either GH, glucose, insulin, or IGF‐1.

In this study, we show that muscles of Ercc1 ^Δ/− mice not only are smaller but contain numerous subcellular and biochemical abnormalities. It is well documented that contractile force normalized per mass is preserved when muscle undergoes regulated loss in weight.60 Here, we see that Ercc1 ^Δ/− mice generated only 50% of normalized grip strength compared with controls but that this value was significantly improved by sActRIIB treatment. Abnormal specific force and relaxation times in the muscle of Ercc1 ^Δ/− muscle, both features of structure alterations,61 were improved by sActRIIB treatment. We therefore suggest that sActRIIB not only prevents muscle from undergoing atrophy but also prevents the subcellular abnormalities. This is clearly evident in the TEM profiles of skeletal muscle, which show that every abnormal feature of Ercc1 ^Δ/− muscle (sarcomeres and mitochondria) was largely prevented by the action of sActRIIB. We suggest that at least one key mechanistic driver in this process is the activation of the autophagic programme mediated by members of the FoxO family. Thus, we propose that abnormally low autophagic activity displayed in Ercc1 ^Δ/− muscle leads to accumulation of p62 puncta and presumably abnormal mitochondria as well as through hyper‐activation of the UPR^MT,62 elevated levels of genes encoding prohibitins that function to restore organelle function,36 change in the histone mark profile (down‐regulation of H3K9Me3 and up‐regulation of H4K20me3) as well as ROS superoxide levels. The build‐up of ROS causes protein oxidation ultimately compromising the workings of the contractile apparatus and leading to a deficit in specific force.63 Our interpretation of these finding is that sActRIIB treatment of Ercc1 ^Δ/− mice leads to the activation of autophagy (LC3II/I ratio), which prevents the accumulation of p62 puncta and also abnormally functioning mitochondria, thus counteracting the need to activate either the UPR^MT or prohibitin programmes, maintains a normal profile of histone modification as well as avoiding the build‐up of high levels of ROS, which ultimately translates in the preservation of organ reserve capacity. We note that the expression of Mul1, a proposed mitochondrion‐specific U3 ubiquitin ligase, was not affected by the progeroid condition or following treatment with sActRIIB compared with controls. However, it is worth bearing in mind that numerous mitochondria targeting U3 ubiquitin ligases have been identified including PARKIN and that these non‐investigated molecules could be executing mitophagy in our experiments.40

However, the concomitant increase in protein synthesis and autophagy levels in Ercc1 ^Δ/− mice following sActRIIB treatment remains to be further investigated. In a normal setting, the two processes are antagonistically driven in large part by FoxO proteins (reviewed in Bonaldo & Sandri56). Here, we suggest that normal parameters do not operate evidenced by the hyper‐activation of Akt in Ercc1 ^Δ/− muscle. Indeed, there is a growing body of evidence for dual activation of Akt‐mediated pathways and autophagy when the normal landscape of regulation is altered.64 Hyper‐activation leading to initiation of novel signalling pathways and cellular outcomes is quite a common outcome and has been extensively studied especially in scenarios of uncontrolled cell division that underpin the development of many cancers.65, 66 Future studies, beyond the scope of the present investigation, combining gene expression and proteomic platforms are planned to identify the pathways susceptible to hyper‐activation of Akt. Nevertheless, we propose that the role of autophagy is in maintaining cellular homeostasis rather than anabolism.26

We thank Y. van Loon, J.J.M. Kouwenberg, K. Smit, Y.M.A. Rijksen, and the animal caretakers for general assistance with mouse experiments. The financial support from the Higher Committee for Education Development in Iraq, Biotechnology and Biological Sciences Research Council, is acknowledged (Grants BB/J016454/1 to H. C. H. and BB/I015787/1 to R. M.); the National Institute of Health (NIH)/National Institute of Ageing (NIA) (PO1 AG017242); European Research Council Advanced Grants DamAge and Dam2Age to J. H. J. H.; the KWO Dutch Cancer Society (5030); the Royal Academy of Arts and Sciences of the Netherlands (academia professorship to J. H. J. H.); the NWO Building Blocks of Life (to W. P. V. and J. H. J. H.); and the ADPS Longevity Research Award (to W. P. V.). T. B. H. was supported by the DFG (CRC1140, CRC 992, HU 1016/8‐1); the BMBF (01GM1518C); the European Research Council‐ERC (Grant 616891); and the H2020‐IMI2 consortium BEAt‐DKD. Finally, we thank the anonymous reviewers of our study for their constructive comments that have guided us into improving our manuscript.