Use of a novel technique to assess impact of age-related denervation on mouse soleus muscle function.

Denervation contributes to loss of force-generating capacity in aged skeletal muscles, but problems with quantification of denervated fibers mean the precise impact of denervation on muscle function remains unclear. This study therefore looked to develop a reliable assay for identifying denervated muscle fibers, and used this to explore the impact of denervation on age-related force-generation in mouse skeletal muscle. Thirteen young (6-month-old) and 10 old (24-months-old) C57Bl/6 J female mice were utilized. Anaesthetized mice were infused with the fluorescent deoxyglucose analog 2[N-(7-nitrobenz-2-oxa-1,2-diaxol-4-yl)amino]-2-deoxyglucose (2-NBDG) and the tibial nerve was repeatedly stimulated to label active skeletal muscle fibers by activity-dependent uptake of 2-NBDG. Data on muscle force generation were acquired as part of the stimulation routine. Labeled muscles were removed, snap frozen, sectioned, and slide mounted. Sections were imaged to show accumulation of 2-NBDG in activated fibers and lack of 2-NBDG accumulation in quiescent (denervated) fibers, then processed using immunohistochemistry to allow collection of data on fiber number and morphology. Soleus muscles from older mice had nine times as many denervated fibers as those from young mice (average n = 36 vs 4, old vs young). Older muscles developed significantly more passive force and less specific force, but denervation only partly accounted for age-related deficits in specific force. Further investigations are required to definitively identify contributors to the decrease in force generation that remain unaccounted for.

Age-related structural changes show that loss of fibers is not a significant contributor to muscle atrophy in old mice.

Age-related loss of skeletal muscle mass is widely considered a consequence of both fiber atrophy and fiber death. Evidence for fiber death derives largely from an age-related reduction in fiber numbers in muscle cross-sections, however it is unclear how age-related alterations in muscle morphology affect accuracy of such counts. To explore this we performed an examination of muscle and tendon length, muscle mass and girth, and pennation angle, in addition to histological section fiber counts of parallel-fibered (sternomastoid), fusiform (biceps brachii), and pennate (tibialis anterior, extensor digitorum longus, soleus) muscles from 31 mice aged 6-32 months. Age-related decline in mass and girth occurred in soleus (p = 0.026; p = 0.040), tibialis anterior (p = 0.004; p = 0.039), and extensor digitorum longus (p = 0.040; p = 0.022) muscles, for which location of maximal girth also changed. Tendon length and pennation angle remained consistent across the lifespan in all except soleus which showed elongation of both proximal and distal tendons coupled with alterations in pennation angle. Age-related decreases in fiber number were observed in transversely sectioned soleus and extensor digitorum longus muscles however when age-related changes in morphology were accounted for via oblique sectioning the age-related decrease in fiber number was eliminated. Findings show loss of fibers is not a significant contributor to age-related muscle wasting in mice, and that age-related changes in connective tissue selectively impact muscle structure. Fiber shortening is a likely contributor to loss of mass and change in function in muscles of old mice.

Age-related remodelling of the myotendinous junction in the mouse soleus muscle.

The age-related loss of muscle mass and function predominantly affect muscles of the lower limbs and have largely been associated with decline in muscle fibre size and number, although the exact mechanisms underlying these losses are poorly understood. In addition, consistent reports that the loss of muscle strength exceeds that which can be explained by declines in muscle mass has widened the search for causes of sarcopenia to include supporting tissues such as the extracellular matrix and tendons. Although the changes to both muscle and tendon with age are well characterised, little work has focused on the interface between these two tissues, the myotendinous junction (MTJ). Given the crucial role for this structure in force transfer between muscle and tendon, we asked whether the myotendinous junction underwent structural changes with age in lower limb muscle. We used whole muscle to assess gross muscle and tendon morphology, and immunohistochemistry to determine fibre and MTJ profile number in young (6 months), middle aged (18 months) and elderly (24 months) C57BL/6 female mice. MTJ length was quantified using serial cross sections of the soleus muscle. We found an apparent 3.5-fold increase in MTJ profiles per cross section with no increase in fibre number in old mice, and found this to be a result of a doubling in length of the MTJ region with age. This coincided with an increase in proximal tendon length (31%), as well as an increase in collagen deposition between 6 and 24-months of age consistent with an expansion of the fibre termination area. These findings uncover a previously undescribed effect of ageing on the MTJ and open up new lines of investigation into the role of this structure in the age-related loss of muscle function.