Integrated expression analysis of muscle hypertrophy identifies Asb2 as a negative regulator of muscle mass

Abstract

The transforming growth factor-β (TGF-β) signaling network is a critical regulator of skeletal muscle mass and function and, thus, is an attractive therapeutic target for combating muscle disease, but the underlying mechanisms of action remain undetermined. We report that follistatin-based interventions (which modulate TGF-β network activity) can promote muscle hypertrophy that ameliorates aging-associated muscle wasting. However, the muscles of old sarcopenic mice demonstrate reduced response to follistatin compared with healthy young-adult musculature. Quantitative proteomic and transcriptomic analyses of young-adult muscles identified a transcription/translation signature elicited by follistatin exposure, which included repression of ankyrin repeat and SOCS box protein 2 (Asb2). Increasing expression of ASB2 reduced muscle mass, thereby demonstrating that Asb2 is a TGF-β network-responsive negative regulator of muscle mass. In contrast to young-adult muscles, sarcopenic muscles do not exhibit reduced ASB2 abundance with follistatin exposure. Moreover, preventing repression of ASB2 in young-adult muscles diminished follistatin-induced muscle hypertrophy. These findings provide insight into the program of transcription and translation events governing follistatin-mediated adaptation of skeletal muscle attributes and identify Asb2 as a regulator of muscle mass implicated in the potential mechanistic dysfunction between follistatin-mediated muscle growth in young and old muscles.

Figure 1. Follistatin gene delivery promotes muscle hypertrophy, which is diminished in old mice.

C57Bl/6 mice of indicated age were injected with rAAV:MCS in the left TA and rAAV:FST in the right TA. (A) The mass of TA muscles from rAAV:FST-injected mice were compared with the contralateral rAAV:MCS control (ratio-paired t test, P < 0.05, n = 10, ± SEM). The change in muscle mass between control and follistatin treatment is presented as a percentage of maximal response in 6-month-old mice (unpaired t test, P < 0.05, n = 10, ± SEM) (B) The muscle weights and response to follistatin treatment as presented in A for 7- and 32-month-old mice (ratio-paired t test; P < 0.05; 7 month, n = 5; 32 month, n = 4; ± SEM). Maximal response is defined by the follistatin response in 7-month-old mice (unpaired t test; P < 0.05; 7 month, n = 5; 32 month, n = 4; ± SEM). (C) The diameter of TA muscle fibers from mice reported in B (Holm-Sidak multiple t test; P < 0.05; 7 month, n = 5; 32 month, n = 4; ± SEM). (D) Immunoblot analysis of mice from A for detection of follistatin expression and SMAD signaling. Quantification of immunoblot analysis from D for (E) follistatin expression (ratio-paired t test, P < 0.05, n = 6, ± SEM). (F) Ratio of phosphorylated to total SMAD3 (ratio-paired t test, P < 0.05, n = 6, ± SEM), and (G) ratio of phosphorylated to total SMAD1/5 (ratio-paired t test, P < 0.05, n = 6, ± SEM). (H) Time course analysis of follistatin expression in TA muscles injected with rAAV:indFST. DOX was administered in food ad libitum for the indicated number of days. n = 2. (I) The mass of TA muscles from rAAV:indFST-injected mice were compared with the contralateral rAAV:MCS control (ratio-paired t test; P < 0.05; no DOX, n = 9; 2 days DOX, n = 7; 28 days DOX, n = 12; ± SEM). Recombinant adeno-associated virus, rAAV; control construct, MCS; follistatin, FST; tibialis anterior muscle, TA; doxycycline, DOX; tetracycline responsive follistatin, indFST.

Figure 2. Quantitative proteomic analysis reveals changes in protein signature associated with acute and chronic follistatin expression in skeletal muscle.

(A) Schematic displaying the experimental workflow for iTRAQ sample labeling and tandem mass spectrometry (MS/MS) analysis. (B) Distribution of the quantified proteins relative to no-DOX samples with a ± 1.5–fold expression change cut-off (moderated t test, adjusted P < 0.05, n = 3). (C) Pearson correlation between replicates from 2- and 28-day follistatin treatments. (D) Gene set enrichment analysis predicted pathways regulated across the indicated time points in the proteomics data set. The upregulated pathways are shown in red, downregulated are shown in blue, and pathways showing a mixed response are highlighted in green (P < 0.05). (E) Z-score scatterplot of the proteins concordantly changed at 2 and 28 days from the 2-dimensional directional pathway analysis. Significantly upregulated proteins are highlighted in red in quadrant I, and downregulated proteins are cultured in blue in quadrant III (integrated P < 0.05); proteins that remained unaltered under the tested hypotheses are shown in gray. Isobaric labeling for relative and absolute quantitation, iTRAQ; hydrophilic interaction chromatography, HILIC; nano-ultra high performance liquid chromatography, nanoUHPLC; differentially expressed, DE; recombinant adeno-associated virus, rAAV; tibialis anterior muscle,TA; doxycycline, DOX; tetracycline responsive follistatin, indFST.

Figure 3. Differential expression of sequenced transcripts and proteins in response to acute and chronic follistatin expression in skeletal muscle.

Overlap between significant differentially expressed (DE) proteins and genes from the proteomic and RNA-Seq data sets were categorized into 8 groups. Shapes are used to annotate changes specific to each data set. Proteins and genes significantly altered in both the proteome and transcriptome analysis are represented in circles, transcript-only changes are represented by triangles, and protein-only changes are represented by squares. Colors are used to code for the temporal factor; changes across all time points are shown in red, acute transcript changes are shown in green, and chronic transcript changes are shown in magenta. Significantly changed proteins during acute and chronic treatment without any transcript level change observed are shown in blue. Unchanged genes/proteins are shown in gray unfilled circles. Significance of each protein/gene was determined from the bioinformatics analysis in their respective data sets.

Figure 4. Asb2 is an atrogene that is downregulated during follistatin treatment.

(A) C57Bl/6 mice were injected with rAAV:MCS in the left TA and rAAV:indFST in the right TA. Immunoblot analysis of ASB2 expression in response to indicated follistatin treatments. (B) Quantification of ASB2 expression presented in A (ratio-paired t -test; P < 0.05; no DOX, n = 6; 2 days DOX, n = 7; 28 days DOX, n = 8; ± SEM). (C) Quantitative PCR analysis of Asb2 mRNA in response to indicated treatments (ratio-paired t-test; P < 0.05; no DOX, n = 6; 2 days DOX, n = 5; 28 days DOX, n = 4; ± SEM). (D) The mass of TA muscles of C57Bl/6 mice after injection of rAAV:MCS in the left TA and rAAV:SMAD7 in the right TA. Mass values were compared between contralateral TA muscles from individual mice (ratio-paired t-test, P < 0.05, n = 5, ± SEM). (E) Immunoblot analysis of mice from D for ASB2 expression and SMAD signaling. Quantification of immunoblots from E for (F) ASB2 expression (ratio-paired t test, P < 0.05, n = 5, ± SEM) and (G) ratio of phosphorylated to total SMAD3 and ratio of phosphorylated to total SMAD2 (ratio-paired t test, P < 0.05, n = 5, ± SEM). (H) C57Bl/6 mice were injected with rAAV:MCS in the left TA and rAAV:Asb2 in the right TA. The mass of TA muscles from rAAV:Asb2-injected muscles were compared with contralateral control muscles (ratio-paired t test; P < 0.05; 14 days, n = 5; 28 days, n = 4; ± SEM). (I) Change in muscle mass between rAAV:Asb2- and control-treated TA muscles from H at indicated time points (unpaired t test; P < 0.05; 14 days, n = 5; 28 days, n = 4; ± SEM). Recombinant adeno-associated virus, rAAV; ankyrin repeat-containing protein with a suppressor of cytokine signaling box 2, Asb2; control construct, MCS; follistatin, FST; tibialis anterior muscle, TA; doxycycline, DOX; tetracycline responsive follistatin, indFST.

Figure 5. ASB2 is not downregulated in aged muscle exposed to follistatin, but suppression is required for maximal follistatin response.

C57Bl/6 mice of indicated age were injected with rAAV:MCS in the left TA and rAAV:FST in the right TA. (A) Immunoblot analysis of ASB2 expression in response to indicated follistatin treatment. (B) Quantification of ASB2 expression presented in A (ratio-paired t test, P < 0.05, n = 6, ± SEM). (C) Quantitative PCR analysis of Asb2 mRNA from mice in A (ratio-paired t test; P < 0.05; 6 month, n = 4; 24 month, n = 5; ± SEM). (D) Immunoblot analysis of TBC1D1 expression in response to indicated follistatin treatment. (E) Quantification of TBC1D1 expression presented in D (ratio-paired t test, P < 0.05, n = 6, ± SEM). (F) C57Bl/6 mice were injected with rAAV:FST plus rAAV:MCS in the left TA and rAAV:FST plus rAAV:Asb2 in the right TA. The rAAV:FST plus rAAV:Asb2–coinjected muscles were compared against the rAAV:FST plus rAAV:MCS control muscles (ratio paired t test; P < 0.05; 14 days, n = 4; 28 days, n = 8; ± SEM). (G) Muscle fiber diameter in TA muscles examined 28 days after treatment with rAAV:FST plus rAAV:MCS or rAAV:FST plus rAAV:Asb2 (Holm-Sidak multiple t test, P < 0.05, n = 4, ± SEM). (H) Comparison in relative changes in muscle mass due to Asb2 expression in static (Figure 4E) versus follistatin (F) treatment (unpaired t test; P < 0.05; rAAV:Asb2 14 days, n = 5; rAAV:FST + rAAV:Asb2 14 days, n = 4; rAAV:Asb2 28 days, n = 4; rAAV:FST + rAAV:Asb2 28 days, n = 8; ± SEM). (I) C57Bl/6 mice underwent surgical denervation or sham operation on contralateral TA muscles. Immunoblot analysis of indicated time course after surgery for ASB2 expression. (J) Quantification of immunoblots from I for ASB2 expression (ratio-paired t test, P < 0.05, n = 4, ± SEM). Recombinant adeno-associated virus, rAAV; ankyrin repeat-containing protein with a suppressor of cytokine signaling box 2, Asb2; control construct, MCS; follistatin, FST; tibialis anterior muscle, TA; doxycycline, DOX; tetracycline responsive follistatin, indFST; TBC1 domain family member 1, TBC1D1.