A Novel Research Paradigm for Sarcopenia of Limb Muscles: Lessons From the Perpetually Working Diaphragm's Anti‐Aging Mechanisms

BACKGROUND: Skeletal muscle function and mass continuously decrease during aging. Most studies target limb muscles owing to their direct impact on mobility and falls risk. The diaphragm (DIA), also a type of skeletal muscle with different phenotype, has received less attention. Comparative research of the DIA and limb muscles can reveal their distinct aging characteristics. Critically, the potential endogenous anti-aging mechanisms of DIA that may provide new insights into the mechanisms of sarcopenia in limb muscles remain scarce. METHODS: Treadmill and grip tests assessed limb muscle function, while a lung function system evaluated respiratory function in both adult (6-month-old) and old (22-month-old) mice. Histological assessments evaluated muscle mass in both the DIA and tibialis anterior (TA). Transcriptome sequencing identified differentially expressed genes (DEGs) between the DIA and TA with aging. Adeno-associated virus (AAV)-encoding short hairpin (sh) RNA targeting gene was injected into adult mice's TA muscles to knockdown target gene level in TA, and AAV-gene was injected into old mice's TA to overexpress target gene level. RESULTS: Old mice displayed significantly reduced running distance (p = 0.0026), maximal speed (p = 0.0019), time to exhaustion (p = 0.0033) and grip strength (p = 0.0055) compared with adult mice, alongside TA's weight loss, decreased myofibre cross-sectional area (CSA) and autophagy(definition) deficiency. However, lung function indicators (respiratory rate, tidal volume, minute ventilation volume, forced vital capacity and ratio of forced expiratory volume in 100 or 200 ms to forced vital capacity), as well as DIA weight and morphology remained stable in old mice. Transcriptional analysis revealed 61 DEGs, with significant upregulation or downregulation observed in TA, but without changes in DIA during aging. Smox (spermine oxidase) is one of the DEGs, responsible for catalysing the conversion of spermine to spermidine. It was reported that in muscle atrophy models such as limb immobilisation, fasting and denervation, Smox's levels are positively correlated with muscle mass and function. Additionally, an increase in Smox also promotes mitochondrial biogenesis. In our study, AAV-shSmox adult mice decreased running distance, speed and time, myofibre CSA alongside mitochondrial function, compared with controls. In contrast, old mice with Smox overexpression showed enhanced mitochondrial function. CONCLUSIONS: In conclusion, this study reveals aging diversities of TA and DIA, explores the sarcopenia of limb muscles based on the anti-aging properties of DIA, which offers a novel perspective on limb sarcopenia. Our findings suggest Smox as a potential target for developing strategies to mitigate sarcopenia progression.