Abstract
The loss of lean muscle mass and strength in sarcopenia and dynapenia (respectively) has been largely attributed to myofibre atrophy, leaving the role of myofibre death unclear. The reported extent of myofibre loss between investigations, species and gender is discrepant, and inadvertently led to the trivialisation of its significance. The following dissertation documents our discovery of myofibres that resembled those that pathognomonically characterise the Autophagic Vacuolar Myopathies and possibly represent myofibre death morphology. In acknowledgement of their features, we have named these myofibres Dystrophin Encircled Vacuoles and invaginations with Intracellular Localisation (DEVILs). DEVILated myofibres were increasingly prevalent with age in muscles susceptible to myofibre loss (extensor digitorum longus and soleus) and displayed strong inverse correlations with total myofibre numbers (r=-0.5 & -0.6; soleus & extensor digitorum longus, respectively). The occurrence of DEVILs was restricted to peripheral myofibres of muscles where tensile stresses were thought to be high. DEVILs were 21 ±13% larger than normal myofibres, suggesting that they were not denervated prior to DEVILation.
A dye exclusion test revealed 17 ±14% of DEVILated myofibres (compared to 1 ±1 % of normal myofibres) admitted Trypan Blue. There was a graded uptake of Trypan Blue which correlated with the severity of morphological abnormality. However, this may have been due to the infiltration of dye-accumulating fibrous connective tissue in DEVILated myofibres, rather than compromised sarcolemmal integrity. The most severely DEVILated myofibres often appeared ghosted in immunofluorescent sections. Evidence of increased cleaved-caspase 3 immunoreactivity was not accompanied by DNA fragmentation (TUNEL) typically seen in apoptotic cell death. Instead, DEVILated myofibres appeared to exhibit autophagic cell death features (prominent sarcoplasmic vacuolasation, upregulated acid phosphatase activity, and elevated LAMP-2 immunostaining) without visible nuclear disruptions or mitochondria-specific elimination (mitophagy) or macroautophagy (lack of BNIP3 and LC3b immunoreactivity). Micro-autophagic dysfunction was evident by the presence of Periodic Acid-Schiff stained plaques of non-diastase digestible material in virtually all DEVILated myofibres.
Longitudinal cross-sections of elderly soleus muscles revealed that many DEVILated myofibres were innervated, and their neuromuscular-junctions reflected the same range of morphologies as normal elderly soleus myofibres. If DEVILated myofibres are the population of myofibres that die with age, then this investigation’s observations oppose the hypothesis that long term denervation of myofibres precedes their death, and instead, highlights our limited knowledge of the triggers of myofibre death. We believe that the denervation and atrophy of myofibres in some muscles predisposes particular myofibres to bear extraneous mechanical/metabolic stresses, which later develop DEVILs (reported here in mice, rats and humans). As of yet, DEVILated myofibres represent the only plausible myofibre death candidate with obvious morphological abnormalities and appears to predominantly affect the remaining innervated myofibres that underpin the functional capacity of elderly skeletal muscle. The death of these myofibres would explain the accelerated severity of weakness during the late stages of sarcopenia and dynapenia. Further investigations on the exact nature of DEVILation may provide valuable insights for future therapeutic investigations.