Skeletal Muscle-Specific Deletion of E3 Ligase Asb2 Enhances Muscle Mass and Strength

Abstract

Background: Maintaining skeletal muscle mass and strength is crucial to prevent sarcopenia during healthy ageing. Ankyrin repeat and suppressor of cytokine signalling box protein 2 (Asb2), an E3 ligase, has been implicated in regulating muscle mass; however, its roles on muscle strength remain unclear, with mixed findings from previous studies. Overexpression of Asb2 decreases muscle mass, whereas its knockdown delays myoblast differentiation and reduces contractile proteins. Given these contradictory findings, we aimed to clarify the role of Asb2 in muscle mass and strength using a skeletal muscle-specific Asb2 knockout (Asb2 MKO) mouse model. Additionally, we investigate the long-term effects of Asb2 on aged muscle, underlying mechanisms on muscle regulation and metabolic effects of Asb2 MKO mice to better understand its role in muscle function and age-related metabolic diseases.

Methods: Asb2 MKO mice were generated using Acta1-Cre recombinase. Body composition was quantified in male and female mice up to 18 months of age. Muscle strength, energy expenditure and glucose metabolism were evaluated using the grip strength test, mitochondrial oxygen consumption measurement, indirect calorimetry and glucose/insulin tolerance tests. Transcriptomic analyses and siRNA studies were performed to elucidate the mechanisms underlying the Asb2 deletion.

Results: The MKO mice were born healthy and exhibited selective Asb2 deletion in the skeletal muscle, leaving the cardiac muscle unaffected. This deletion led to an increase in the mass of various skeletal muscles (9%-23%, p < 0.05) and improved grip strength (~10%, p < 0.05), both of which were sustained throughout the ageing process. The MKO mice also revealed enhanced mitochondrial function, energy expenditure and whole-body insulin sensitivity. Transcriptomic data supported the muscle phenotype observed in the MKO mice. Notably, desmin, a protein critical for structural integrity and mitochondrial function, was identified as a target protein of the ASB2 E3 ligase.

Conclusions: Skeletal muscle-specific deletion of Asb2 led to increased muscle mass and strength, potentially through preservation of desmin levels. These findings suggest that targeting Asb2 may enhance muscle growth and prevent age-related muscle decline, with potential benefits for metabolic health, particularly by improving mitochondrial function and insulin sensitivity.

Keywords: Asb2; ageing; desmin; mitochondrial function; skeletal muscle mass.

FIGURE 1

Skeletal muscle‐specific Asb2 knockout mice revealed increased lean body mass and skeletal muscle tissue weight. Both WT and Asb2 MKO male mice fasted overnight prior to sacrifice. (A) Body composition of both young 6‐month‐old WT and Asb2 MKO male mice was measured before fasting (n = 6–7 each). (B) Skeletal muscle and heart tissue weights of young 6‐month‐old WT and Asb2 MKO male mice (n = 6–7 each). (C) Body composition of aged 18‐month‐old WT and Asb2 MKO male mice was measured before fasting (n = 6 each). (D) Skeletal muscle and heart tissue weights of aged 18‐month‐old WT and Asb2 MKO male mice (n = 5–6 each). (E) Representative haematoxylin and eosin (H&E)‐stained images and cross‐sectional areas (CSA) of tibialis anterior (TA) muscle from young 6‐month‐old (left) and old 18‐month‐old (right) of WT and Asb2 MKO mice (n = 3–5 each). Asterisks (*) indicate muscle fibres with a CSA greater than 3000 μm². Scale bars represent 40 μm. (F) Grip strength of aged 18‐month‐old WT and Asb2 MKO male mice. Data are presented as means ± standard error of the mean (SEM). Statistical significance was determined by Student's t test. CSA, cross‐sectional area; EDL, extensor digitorum longus; GAS, gastrocnemius; H&E, haematoxylin and eosin; ns, not significant; QD, quadriceps; TA, tibialis anterior.

FIGURE 2

Skeletal muscle‐specific Asb2 knockout mice revealed increased energy expenditure. Whole‐body energy balance in young 6‐month‐old WT and Asb2 MKO male mice fed a regular chow diet. (A) Oxygen consumption (V_O2), (B) carbon dioxide production (V_CO2), (C) energy expenditure, (D) respiratory exchange ratio (RER), (E) food intake and (F) activity were measured in mice housed in individual metabolic cages for 48 h (n = 10 each). Data are presented as means ± standard error of the mean (SEM). ^$ p < 0.05 and ^$$ p < 0.01 by two‐way ANOVA. ^# p < 0.05, ^## p < 0.01 and ^### p < 0.001 by Student's t test. RER, respiratory exchange ratio; V_CO2, carbon dioxide production; V_O2, oxygen consumption.

FIGURE 3

Skeletal muscle‐specific Asb2 knockout mice displayed modest metabolic advantages when subjected to a high‐fat diet. Six‐month‐old WT and Asb2 MKO male mice fed a high‐fat diet (HFD) for a period of 4 weeks. (A) Body weight change curve for 4 weeks of HFD feeding (n = 10–14 each). (B) Final body weight and body composition measured before fasting (left) and changes in lean body and fat mass before and after 4 weeks of HFD feeding (right) (n = 10–14 each). (C, D) Analyses were performed on HFD‐fed WT and Asb2 MKO male mice that were fasted overnight prior to sacrifice. (C) Skeletal muscle and heart tissue weights (n = 10–14 each). (D) Glucose tolerance test (GTT) (left) and glucose‐stimulated insulin secretion (GSIS) (right) were performed on HFD‐fed WT and Asb2 MKO male mice after overnight fasting (n = 4–5 each). (E, F) Insulin tolerance test (ITT) and insulin signalling analysis were performed on HFD‐fed WT and Asb2 MKO male mice after 6 h of fasting. (E) ITT (left) and area under the curve (AUC) (for 60 min) (right) (n = 3–6 each). (F) Representative immunoblots of phospho‐Akt (Ser473), Akt and GAPDH in gastrocnemius (GAS) muscle of WT and Asb2 MKO mice at 60 min after insulin injection following 4 weeks on HFD (n = 3–5 each). Data are presented as means ± standard error of the mean (SEM). Statistical significance was determined by Student's t test. AUC, area under curve; EDL, extensor digitorum longus; GAS, gastrocnemius; GAPDH, glyceraldehyde‐3‐phosphate dehydrogenase; GSIS, glucose‐stimulated insulin secretion; GTT, glucose tolerance test; HFD, high‐fat diet; ITT, insulin tolerance test; ns, not significant; QD, quadriceps; TA, tibialis anterior.

FIGURE 4

RNA‐Seq analysis revealed enriched gene sets related to muscle mass in skeletal muscle‐specific Asb2 knockout mice. RNA‐Seq analyses using gastrocnemius (GAS) muscles of young 6‐month‐old WT and Asb2 MKO male mice fasted overnight prior to sacrifice (n = 3 each). (A) Hierarchically clustered differentially expressed genes (DEGs) presented as heat maps according to the Z score (red indicates upregulated genes; green indicates downregulated genes). (B) Number of DEGs. (C) Gene Ontology Biological Processes (GOBP) analyses of the transcripts derived from RNA‐seq revealed statistically significant differences using the Molecular Signatures Database (MSigDB). (D) Gene set enrichment analysis (GSEA) on the hallmark gene sets for RNA‐seq analyses between Asb2 MKO versus WT (red indicates gene sets for upregulated genes; green indicates gene sets for downregulated genes). DEGs, Differentially expressed genes; GAS, gastrocnemius; GOBP, gene ontology biological processes; GSEA, gene set enrichment analysis; MSigDB, molecular signatures database.

FIGURE 5

Skeletal muscle‐specific Asb2 knockout mice revealed increased protein expression of desmin in skeletal muscle. (A) Representative immunoblots of ankyrin repeat and suppressor of cytokine signalling box protein 2 (ASB2) and its target proteins' expression in the tibialis anterior (TA) muscle of 6‐month‐old WT and Asb2 MKO male mice fasted overnight prior to sacrifice (left). Bar charts display the quantification of optical densities of blot bands for filamin B, transcription factor 3 (TCF3), torsin 1A interacting protein 1 (TOR1AIP1), desmin and ASB2 (right) (n = 8 each). (B) Immunoblots of desmin protein expression in myofibrillar fraction isolated from gastrocnemius (GAS) muscle of young 6‐month‐old WT and Asb2 MKO mice fasted overnight prior to sacrifice (left). Bar charts show the quantification of optical densities of blot bands for desmin (right) (n = 6 each). (C) Representative immunoblots of ASB2 and desmin protein expression in Asb2 knockdown or MG132 treated myotube cells (left). Bar charts show the quantification of optical densities of blot bands for ASB2 and desmin (right) (n = 3 each). (D) Respiration of mitochondria isolated from GAS muscle of young 6‐month‐old WT and Asb2 MKO mice fasted overnight prior to sacrifice (n = 4 each). (E) mRNA expression of mitochondrial‐related genes in GAS muscle of young 6‐month‐old WT and Asb2 MKO mice fasted overnight prior to sacrifice (n = 4–8 each). Data are represented as means ± standard error of the mean (SEM). ^$ p < 0.05 and ^$$ p < 0.01 by two‐way ANOVA (D). *p < 0.05, **p < 0.01 and ***p < 0.001 by one‐way ANOVA (C). Statistical significance was determined by Student's t test (A, B, E). ADP, adenosine diphosphate; ASB2, ankyrin repeat and suppressor of cytokine signalling box protein 2; Atp5b, ATP synthase F1 subunit beta; Cox, cytochrome C oxidase subunit; FCCP, carbonyl cyanide‐4‐(trifluoromethoxy) phenylhydrazone; GAPDH, glyceraldehyde‐3‐phosphate dehydrogenase; GAS, gastrocnemius; Ndufv2, NADH–ubiquinone oxidoreductase core subunit V2; Nrf1, nuclear respiratory factor 1; ns, not significant; OCR, oxygen consumption rate; Pgc1‐α, peroxisome proliferator‐activated receptor 1 alpha; TA, tibialis anterior; TCF3, transcription factor 3; Tfam, mitochondrial transcription factor A; TOR1AIP1, torsin 1A interacting protein 1.