ABSTRACT Background Measuring in vivo protein synthesis is important for testing how interventions, disease and ageing remodel the proteome. A limitation of approaches using short‐term infusions of labelled amino acids was that long‐term changes had to be extrapolated from short‐term measurements. Deuterium oxide (D 2 O) is now widely used because it can measure protein synthesis over periods of days to weeks. We previously showed that the duration of labelling impacts measured protein synthesis, but many still employ repeated tissue sampling during D 2 O provision. We hypothesized that the timing of tissue sampling alters which proteins contribute to the overall protein fractional synthesis rate (FSR), thereby invalidating comparisons between serial samples collected during continuous labelling. Methods To test this hypothesis, we used a five‐timepoint (0d, 4d, 15d, 30d and 45d) D 2 O labelling time course followed by liquid chromatography–tandem mass spectrometry proteomics to calculate individual protein synthesis rates and plateau values in mouse gastrocnemius muscle. Individual proteins were stratified into slow and fast groups, such that each group was responsible for the slowest or fastest 20% of total synthesis over the first 4 days of labelling, respectively. Results In a 4‐day period representing a baseline period, the synthesis rate of summed proteins was 2.09%/day. When the period was extended to 15, 30 or 45 days, the synthesis rates of summed proteins declined to 1.64%, 1.31% and 1.10%/day, respectively. The contribution of slow and fast proteins to these rates changed as a function of time. The fast and slow proteins accounted for 4.3% and 34.0% of measured proteome abundance, respectively. From Day 0 to Day 4, fast and slow proteins contributed 19.8% and 20.2% of the calculated FSR, respectively. By Day 45, these contributions shifted to 6.5% and 28.8%. Further, we demonstrated that the assumption that the fraction new of proteins linearly increases to 100% new is not true for the majority of proteins. Instead, most proteins plateaued at some point less than 100%, which stops their individual contribution to a summed protein synthesis rate. Conclusions In continuous long‐term labelling studies, biopsy timing changes the measured bulk FSR because the relative contributions of individual proteins to that measurement over time. Fast proteins that plateau early decrease their contribution as labelling time increases. These findings indicate that studies with different labelling periods, or multiple biopsies over time during continuous labelling of days to weeks cannot be compared to biopsies taken at other times in the labelling period.
ABSTRACT Background Skeletal muscle atrophy is a pathological condition characterized by a progressive loss of muscle mass and function. Its core mechanisms involve an imbalance between protein synthesis and degradation, caused by oxidative stress and inflammation. Tectorigenin (TG) shows therapeutic potential but suffers from poor solubility and bioavailability. This study developed a zeolitic imidazolate framework‐8 (ZIF‐8) nanocarrier for TG (ZIF‐8@TG) to enhance delivery efficacy against neurogenic muscle atrophy. Methods ZIF‐8@TG nanoparticles were synthesized and characterized for size, drug loading, release profile and cytotoxicity. Their therapeutic effects were investigated in vitro using a nutrient deprivation‐induced C2C12 myotube atrophy model and in vivo using a sciatic nerve crush injury–induced mouse model of muscle atrophy. Immunofluorescence staining and western blotting were performed to assess the effects of ZIF‐8@TG on atrophic myotubes. Behavioural tests, immunofluorescence staining and western blotting were used to evaluate its impact on muscle atrophy in vivo. The underlying mechanisms were further explored through western blotting, electron microscopy, immunofluorescence staining and H&E staining. Results ZIF‐8@TG nanoparticles (approx. 50 nm) exhibited pH‐responsive release and efficient cellular uptake. In vitro, nutrient deprivation reduced myotube diameter by 53% ( p < 0.01), whereas ZIF‐8@TG treatment restored it to 92% of control levels, showing significantly better efficacy than free TG. In vivo, sciatic nerve injury decreased tibialis anterior muscle mass by 35% ( p < 0.001) and mean myofiber cross‐sectional area (CSA) by 41% ( p < 0.001). ZIF‐8@TG treatment significantly increased muscle mass to 74% and CSA to 93% of control values, whereas free TG resulted in non‐significant improvements. Additionally, ZIF‐8@TG improved motor function in mice following sciatic nerve crush. Mechanistically, the alleviation of skeletal muscle atrophy by ZIF‐8@TG may be associated with regulation of excessive activation of the ubiquitin‐proteasome system and autophagy‐associated changes, along with enhanced protein synthesis–related Akt/mTOR signalling pathway during sciatic nerve crush–induced muscle atrophy, as well as an association with reduced oxidative stress and inflammation. Conclusions ZIF‐8@TG effectively delivers TG, alleviating muscle atrophy in vitro and in vivo. ZIF‐8@TG treatment is associated with reduced oxidative stress and inflammation, preserved protein homeostasis and improved functional recovery, offering a promising nanotechnology‐based therapy strategy for neurogenic skeletal muscle atrophy.
ABSTRACT Background Sarcopenia is a major age‐related condition associated with functional decline, disability and mortality in older adults, yet reliable circulating biomarkers for early risk stratification remain limited. Tsukushi (TSK), a secreted hepatokine involved in systemic metabolic regulation, has recently been implicated in skeletal muscle homeostasis. However, prospective clinical evidence linking circulating TSK to sarcopenia development is lacking. We therefore investigated the association between serum TSK levels and incident sarcopenia in a community‐based cohort of older adults. Methods A total of 1370 community‐dwelling individuals aged ≥ 65 years were followed for 4 years. Baseline serum TSK levels were measured using enzyme‐linked immunosorbent assay. Multivariable Cox proportional hazards models were used to assess the association between TSK and incident sarcopenia after adjustment for confounding factors. Restricted cubic spline analyses were applied to explore dose–response relationships, and predictive performance was evaluated using time‐dependent receiver operating characteristic (ROC) curves. Results Lower baseline TSK levels were associated with a higher cumulative incidence of sarcopenia. In multivariable analyses, higher TSK levels were associated with a lower risk of incident sarcopenia (hazard ratio per SD increase = 0.64, 95% CI = 0.53–0.76, p < 0.001). Dose–response analyses demonstrated a non‐linear association with overall sarcopenia risk, whereas associations with low muscle strength and low physical performance were more nearly linear. The association appeared more evident in women than in men. TSK alone showed moderate discrimination for incident sarcopenia (AUC = 0.700), while a combined clinical model improved discrimination (AUC = 0.808 at 4 years). Conclusions Lower circulating TSK levels are associated with a higher risk of incident sarcopenia in older adults, particularly subsequent muscle‐related functional decline. TSK may be a candidate biomarker for early sarcopenia risk stratification and may reflect systemic metabolic processes relevant to muscle health.
ABSTRACT Background Desminopathy is a progressive myopathy and the most common intermediate filament disease in humans. Desmin is an essential part of the filamentous network that aligns myofibrils, anchors nuclei and mitochondria and connects the z discs to the sarcolemma. We challenged a rat model with a mutation in R349P Des , analogous to the most frequent R350P DES missense mutation in humans, through eccentric‐biassed exercise training. Methods R349P Des rats (Des) and their wild type (WT) and heterozygous (HET) littermates were subjected to 4 weeks of downhill running. At the end of 4 weeks, muscles were collected and assessed histologically and biochemically. Results Des rats demonstrated significantly lower running capacity compared to wild type and heterozygous littermates ( p < 0.0001), which exacerbated over the course of the study ( p < 0.001). Running performance in Des rats decreased by ~50% after 4 weeks of downhill running. Desmin‐positive skeletal muscle aggregates, a histopathological hallmark of desminopathy, were ~2‐fold higher after downhill running in the Des group ( p < 0.05). On a protein level, we found a differential exercise response of Des compared to WT affecting various cellular pathways concerning autophagy, proteasomal activity, energy metabolism and cytoskeletal proteins. Conclusions Our results suggest an impaired ability of Des muscle to adapt to chronic eccentric‐biassed exercise and a subsequent exacerbation of the functional and histopathological phenotype.
ABSTRACT Background Denervation‐induced muscle atrophy progresses during the interval between nerve injury and reinnervation, and depletion of muscle satellite cells (MuSCs) contributes to impaired functional recovery. Although mitochondrial dysfunction and impaired quality control are implicated in muscle wasting, whether excessive mitochondrial turnover drives MuSC failure after denervation remains unclear. This study aimed to identify the pathogenic mechanisms underlying MuSC depletion and determine whether mitochondrial transplantation therapy (MTT) preserves regenerative capacity. Methods A sciatic nerve transection model was established in mice to induce denervation‐associated muscle atrophy. Mitochondria isolated from differentiated MuSCs were transplanted into denervated gastrocnemius muscles beginning 6 h after injury and repeated every 3 days for 6 weeks. RNA sequencing, calcium imaging, mitochondrial ultrastructural analysis, mito‐mKeima‐based mitophagic flux assessment, flow cytometry and molecular analyses were performed to investigate disease mechanisms and therapeutic effects. In vitro calcium overload models combined with Mdivi‐1 treatment and Pink1 knockdown were used to define the regulatory pathway linking calcium stress, mitophagy and ferroptosis. Results Denervated muscle exhibited early calcium dyshomeostasis (Fluo‐4 AM fluorescence increased 6.7‐fold vs. Sham, p < 0.0001) and mitochondrial dysfunction within 3 days, followed by iron accumulation (nonheme iron increased 1.5‐fold, p = 0.0046) and ferroptotic changes by 2 weeks. Mechanistically, sustained calcium overload activated the Ampk‐Tsc2‐mTorc1 pathway in MuSCs, inducing Pink1‐dependent excessive mitophagic flux. Mito‐mKeima flow cytometry confirmed increased mitophagic flux after denervation, with approximately tenfold elevation at Day 3 and persistent activation at 2 weeks. Excessive mitophagy promoted Fth1 degradation (reduced by 47.5%, p = 0.0181) and increased Tfr1 expression (1.9‐fold, p = 0.0035), thereby enhancing ferroptotic susceptibility. Inhibition of mitochondrial fission or Pink1 knockdown reduced iron accumulation (Fe 2+ fluorescence reduced by 58.3% and 69.2%, respectively, both p < 0.01) and protected MuSCs from ferroptosis. MTT restored mitochondrial homeostasis by reducing calcium overload (73.7% decrease vs. A23187, p < 0.0001), increasing ATP production (36.9%, p < 0.01), suppressing excessive mitophagic flux, normalizing iron metabolism (39.9% reduction, p = 0.0015) and preserving MuSCs (30.0% increase vs. DN_PBS, p = 0.0196). Consequently, MTT attenuated denervation‐induced muscle fibre atrophy and improved functional recovery following delayed nerve repair. Conclusions This study identifies an early pathogenic cascade linking calcium overload, excessive mitophagic flux and ferroptotic susceptibility as a critical driver of MuSC dysfunction during denervation‐induced muscle wasting. MTT preserves regenerative capacity by interrupting this degenerative circuit and restoring mitochondrial quality control, providing a potential therapeutic strategy for denervation‐associated muscle atrophy and other muscle wasting disorders.
