Abstract
Declines in muscle mass and strength are hallmark features of sarcopenia, an age-related condition that contributes to morbidity and mortality in older people. Muscle mass is contingent on muscle fiber size and/or number; however, exactly which of these decline with age is unclear. The degree of weakness exceeds the amount of muscle atrophy in sarcopenia, indicating that mass loss is not the only factor driving weakness. Here, I sought to identify those factors contributing to the loss of muscle mass and strength in sarcopenia.
In a murine model (C57Bl/6j), I characterized age-related differences in muscle structure, function and proteomes, and also identified which features of muscle aging were amenable to voluntary long-term endurance exercise (running wheel-based; 20-24 months-of-age). Structural features (muscle length and girth, tendon lengths, and pennation angles) were measured from whole biceps brachii (BB), extensor digitorum longus (EDL), soleus (SOL), sternomastoid (SM) and tibialis anterior (TA) muscles. Fiber size and number were quantified using fluorescent immunohistochemistry to visualize dystrophin. Age-related differences in muscle structure (decreased girth, increased pennation angle and longer tendons) were consistent with fiber shortening and evident in lower- (EDL, SOL and TA), but not upper-body (BB and SM) muscles. Structural changes also caused an under counting of fibers in the muscles of older mice when conventional histology (transverse sections) was used (SOL: 1024 ±59 vs. 872 ±164, p=0.034; n=12; younger and older respectively), whereas a modified oblique sectioning technique showed no age-related differences in fiber number (SOL: 1075 ±53 vs. 1006 ±114, p=0.157) but decreased fiber diameter (SOL: 38.6 ±3.8μm vs. 34.2 ±3.9μm, p=0.041). Isometric tension from SOL muscles was measured in-situ, within mice given an intravenous injection of the fluorescent deoxyglucose analogue, 2-NBDG (2-[N-[7-Nitrobenz-2-oxa-1,3-diazol-4-yl]Amino]-2-Deoxyglucose). Post-hoc identification of denervated fibers was possible because 2-NBDG accumulates in active (innervated) but not inactive fibers (denervated) following nerve stimulation. I found age-related declines in absolute (-17 ±5%, p<0.001; n=23) and specific tension (force/anatomical cross-sectional area; -17 ±5%, p=0.040), and increased denervated fiber prevalence (+785 ±7%, p=0.030). Neither muscle atrophy (specific tension) nor denervation (specific tension adjusted for denervation) completely accounted for muscle weakness, indicating significant contributions from other factors (muscle structure or force transmission efficacy, but not single fiber weakness). Using SWATH-MS (sequential window acquisition of all theoretical fragment ion spectra mass spectrometry), I found age-related de-enrichment of syncoilin (involved in force transmission) and enrichment of dysferlin (membrane repair protein) within older SOL proteomes, implicating altered force transmission and membrane permeating events in muscle aging. Lastly, exercise reversed age-related changes in muscle structure, reduced denervated fiber prevalence (-50 ±0.1%, p=0.030), and improved absolute (+9 ±2%, p=0.006) but not specific tension. Syncoilin and dysferlin abundance were not different between the muscles of sedentary and exercise-exposed older mice.
My findings indicate that age-related muscular deterioration is due to fiber atrophy (reduced length and diameter), increased denervation, changes to muscle structure and reduced force transmission efficacy. Death of whole fibres does not contribute to muscle loss in the aged mouse. Endurance exercise attenuates some, but not all, features of muscle aging.