Methylome-proteome integration after late-life voluntary exercise training reveals regulation and target information for improved skeletal muscle health.

Exercise is a potent stimulus for combatting skeletal muscle ageing. To study the effects of exercise on muscle in a preclinical setting, we developed a combined endurance-resistance training stimulus for mice called progressive weighted wheel running (PoWeR). PoWeR improves molecular, biochemical, cellular and functional characteristics of skeletal muscle and promotes aspects of partial epigenetic reprogramming when performed late in life (22-24 months of age). In this investigation, we leveraged pan-mammalian DNA methylome arrays and tandem mass-spectrometry proteomics in skeletal muscle to provide detailed information on late-life PoWeR adaptations in female mice relative to age-matched sedentary controls (n = 7-10 per group). Differential CpG methylation at conserved promoter sites was related to transcriptional regulation genes as well as Nr4a3, Hes1 and Hox genes after PoWeR. Using a holistic method of -omics integration called binding and expression target analysis (BETA), methylome changes were associated with upregulated proteins related to global and mitochondrial translation after PoWeR (P = 0.03). Specifically, BETA implicated methylation control of ribosomal, mitoribosomal, and mitochondrial complex I protein abundance after training. DNA methylation may also influence LACTB, MIB1 and UBR4 protein induction with exercise - all are mechanistically linked to muscle health. Computational cistrome analysis predicted several transcription factors including MYC as regulators of the exercise trained methylome-proteome landscape, corroborating prior late-life PoWeR transcriptome data. Correlating the proteome to muscle mass and fatigue resistance revealed positive relationships with VPS13A and NPL levels, respectively. Our findings expose differential epigenetic and proteomic adaptations associated with translational regulation after PoWeR that could influence skeletal muscle mass and function in aged mice. KEY POINTS: Late-life combined endurance-resistance exercise training from 22-24 months of age in mice is shown to improve molecular, biochemical, cellular and in vivo functional characteristics of skeletal muscle and promote aspects of partial epigenetic reprogramming and epigenetic age mitigation. Integration of DNA CpG 36k methylation arrays using conserved sites (which also contain methylation ageing clock sites) with exploratory proteomics in skeletal muscle extends our prior work and reveals coordinated and widespread regulation of ribosomal, translation initiation, mitochondrial ribosomal (mitoribosomal) and complex I proteins after combined voluntary exercise training in a sizeable cohort of female mice (n = 7-10 per group and analysis). Multi-omics integration predicted epigenetic regulation of serine ß-lactamase-like protein (LACTB - linked to tumour resistance in muscle), mind bomb 1 (MIB1 - linked to satellite cell and type 2 fibre maintenance) and ubiquitin protein ligase E3 component N-recognin 4 (UBR4 - linked to muscle protein quality control) after training. Computational cistrome analysis identified MYC as a regulator of the late-life training proteome, in agreement with prior transcriptional analyses. Vacuolar protein sorting 13 homolog A (VPS13A) was positively correlated to muscle mass, and the glycoprotein/glycolipid associated sialylation enzyme N-acetylneuraminate pyruvate lyase (NPL) was associated to in vivo muscle fatigue resistance.

Muscle Fiber Cross-Sectional Area Is Associated With Quadriceps Strength and Rate of Torque Development After ACL Injury.

ABSTRACT: Graham, MC, Thompson, KL, Hawk, GS, Fry, CS, and Noehren, B. Muscle fiber cross-sectional area is associated with quadriceps strength and rate of torque development after ACL injury. J Strength Cond Res 38(6): e273-e279, 2024-The purpose of this study was to investigate the relationship between muscle fiber type-specific properties of the vastus lateralis and quadriceps muscle performance in individuals after an anterior cruciate ligament (ACL) tear. 26 subjects (22.0 ± 5.4 years) were included in this cross-sectional study, and all data were collected before ACL reconstruction. Quadriceps peak torque (QPT) and early (0-100 ms) and late (100-200 ms) rate of torque development (RTD) were obtained from maximal voluntary isometric quadriceps strength testing. Muscle fiber cross-sectional area (fCSA) and percent fiber type distribution (FT%) were evaluated through immunohistochemical analysis of a muscle biopsy. Between-limb differences in fiber characteristics were assessed using paired t-tests (with α-level 0.05). Relationships between fiber-specific properties and quadriceps muscle performance were determined using separate multiple linear regression analyses for ACL-injured and noninjured limbs. There were significant differences in fCSA between ACL-injured and noninjured limbs across all fiber types, but no differences in FT%. Type 1 fCSA, type 2a fCSA, and their interaction effect were the explanatory variables with the strongest relationship to all performance outcomes for the ACL-injured limb. The explanatory variables in the ACL-injured limb had a significant relationship to QPT and late RTD, but not early RTD. These findings suggest that QPT and late RTD are more heavily influenced by fCSA than FT% in ACL-injured limbs. This work serves as a foundation for the development of more specific rehabilitation strategies aimed at improving quadriceps muscle function before ACL reconstruction or for individuals electing nonsurgical management.

Decoding the decline: unveiling drivers of sarcopenia.

There remains a critical need to define molecular pathways underlying sarcopenia to identify putative therapeutic targets. Research in the mechanisms of aging and sarcopenia relies heavily on preclinical rodent models. In this issue of the JCI, Kerr et al. implemented a clinically-relevant sarcopenia classification system of aged C57BL/6J mice, capturing sarcopenia prevalence across both sexes. The authors performed detailed physiological, molecular, and energetic analyses and demonstrated that mitochondrial biogenesis, oxidative capacity, and AMPK-autophagy signaling decreased as sarcopenia progressed in male mice. Sarcopenia was less prevalent in female mice with fewer alterations compared with the male-affected processes. The findings highlight factors beyond age as necessary for classifying the sarcopenic phenotype in rodent models, reveal sexual dimorphism across the trajectory of age-related declines in muscle mass and function in a commonly used rodent model, and provide insight into sex-dependent molecular alterations associated with sarcopenia progression.

Sex Differences in Quadriceps Atrophy After Anterior Cruciate Ligament Tear.

BACKGROUND: Female athletes lag behind their male counterparts in recovery from anterior cruciate ligament (ACL) injury. Quadriceps muscle size and strength are crucial factors for regaining function after ACL injury, but little is known about how these metrics vary due to biological sex. HYPOTHESIS: Female patients have reduced vastus lateralis fiber cross-sectional area (CSA) and lower quadriceps strength after ACL injury than male patients. STUDY DESIGN: Cross-sectional study. LEVEL OF EVIDENCE: Level 4. METHODS: A total of 60 participants with recent ACL tear were evaluated for vastus lateralis muscle fiber CSA, isometric quadriceps peak torque, and quadriceps rate of torque development. Linear mixed models were fit to determine differences across sex and limb for each variable of interest. RESULTS: The female group averaged almost 20% atrophy between limbs (P < 0.01), while the male group averaged just under 4% (P = 0.05). Strength deficits between limbs were comparable between female and male groups. CONCLUSION: Immediately after ACL injury, female patients have greater between-limb differences in muscle fiber CSA but between-limb strength deficits comparable with those of male patients. CLINICAL RELEVANCE: These results indicate that the underpinnings of strength loss differ based on biological sex, and thus individual patients could benefit from a sex-specific treatment approach to ACL injury.

Overexpression of manganese superoxide dismutase mitigates ACL injury-induced muscle atrophy, weakness and oxidative damage.

Oxidative stress has been implicated in the etiology of skeletal muscle weakness following joint injury. We investigated longitudinal patient muscle samples following knee injury (anterior cruciate ligament tear). Following injury, transcriptomic analysis revealed downregulation of mitochondrial metabolism-related gene networks, which were supported by reduced mitochondrial respiratory flux rates. Additionally, enrichment of reactive oxygen species (ROS)-related pathways were upregulated in muscle following knee injury, and further investigation unveiled marked oxidative damage in a progressive manner following injury and surgical reconstruction. We then investigated whether antioxidant protection is effective in preventing muscle atrophy and weakness after knee injury in mice that overexpress Mn-superoxide dismutase (MnSOD+/-). MnSOD+/- mice showed attenuated oxidative damage, atrophy, and muscle weakness compared to wild type littermate controls following ACL transection surgery. Taken together, our results indicate that ROS-related damage is a causative mechanism of muscle dysfunction after knee injury, and that mitochondrial antioxidant protection may hold promise as a therapeutic target to prevent weakness and development of disability.

Nicotinamide N-methyltransferase inhibition mimics and boosts exercise-mediated improvements in muscle function in aged mice.

Human hallmarks of sarcopenia include muscle weakness and a blunted response to exercise. Nicotinamide N-methyltransferase inhibitors (NNMTis) increase strength and promote the regenerative capacity of aged muscle, thus offering a promising treatment for sarcopenia. Since human hallmarks of sarcopenia are recapitulated in aged (24-month-old) mice, we treated mice from 22 to 24 months of age with NNMTi, intensive exercise, or a combination of both, and compared skeletal muscle adaptations, including grip strength, longitudinal running capacity, plantarflexor peak torque, fatigue, and muscle mass, fiber type, cross-sectional area, and intramyocellular lipid (IMCL) content. Exhaustive proteome and metabolome analyses were completed to identify the molecular mechanisms underlying the measured changes in skeletal muscle pathophysiology. Remarkably, NNMTi-treated aged sedentary mice showed ~ 40% greater grip strength than sedentary controls, while aged exercised mice only showed a 20% increase relative to controls. Importantly, the grip strength improvements resulting from NNMTi treatment and exercise were additive, with NNMTi-treated exercised mice developing a 60% increase in grip strength relative to sedentary controls. NNMTi treatment also promoted quantifiable improvements in IMCL content and, in combination with exercise, significantly increased gastrocnemius fiber CSA. Detailed skeletal muscle proteome and metabolome analyses revealed unique molecular mechanisms associated with NNMTi treatment and distinct molecular mechanisms and cellular processes arising from a combination of NNMTi and exercise relative to those given a single intervention. These studies suggest that NNMTi-based drugs, either alone or combined with exercise, will be beneficial in treating sarcopenia and a wide range of age-related myopathies.

The 24-Hour Time Course of Integrated Molecular Responses to Resistance Exercise in Human Skeletal Muscle Implicates MYC as a Hypertrophic Regulator That is Sufficient for Growth.

Molecular control of recovery after exercise in muscle is temporally dynamic. A time course of biopsies around resistance exercise (RE) combined with -omics is necessary to better comprehend the molecular contributions of skeletal muscle adaptation in humans. Vastus lateralis biopsies before and 30 minutes, 3-, 8-, and 24-hours after acute RE were collected. A time-point matched biopsy-only group was also included. RNA-sequencing defined the transcriptome while DNA methylomics and computational approaches complemented these data. The post-RE time course revealed: 1) DNA methylome responses at 30 minutes corresponded to upregulated genes at 3 hours, 2) a burst of translation- and transcription-initiation factor-coding transcripts occurred between 3 and 8 hours, 3) global gene expression peaked at 8 hours, 4) ribosome-related genes dominated the mRNA landscape between 8 and 24 hours, 5) methylation-regulated MYC was a highly influential transcription factor throughout the 24-hour recovery and played a primary role in ribosome-related mRNA levels between 8 and 24 hours. The influence of MYC in human muscle adaptation was strengthened by transcriptome information from acute MYC overexpression in mouse muscle. To test whether MYC was sufficient for hypertrophy, we generated a muscle fiber-specific doxycycline inducible model of pulsatile MYC induction. Periodic 48-hour pulses of MYC over 4 weeks resulted in higher muscle mass and fiber size in the soleus of adult female mice. Collectively, we present a temporally resolved resource for understanding molecular adaptations to RE in muscle and reveal MYC as a regulator of RE-induced mRNA levels and hypertrophy.