Dying myofibers in elderly mouse skeletal muscles are characterized by the appearance of dystrophin-encircled vacuoles

Abstract

The age-related loss of skeletal muscle mass and strength (sarcopenia) is predominantly attributed to myofiber atrophy, however the role or existence of myofiber death is currently unclear. We recently discovered dysmorphic myofibers in normal elderly mice resembling those that characterize the Autophagic Vacuolar Myopathies, and speculated that they may be myofibers caught in the act of dying. Since these myofibers were identifiable by Dystrophin Encircled Vacuoles and invaginations with Intracellular Localization we coined the acronym DEVILs and aimed to determine their frequency, pathogenesis and correlation with myofiber loss. In whole transverse sections of young (1-6 month) and elderly (22-26 month) C57Bl/6j mouse muscles, DEVILated myofiber number correlated with myofiber loss, being increasingly prevalent in aged extensor digitorum longus (R = 0.7, p < 0.001) and soleus (R = 0.6, p = 0.004) muscles, whilst rare in myofiber loss resistant muscles (cleido- and sternomastoid). In a cell viability dye-exclusion test, 17 ± 14% of DEVILated myofibers stained positive and were accompanied by immunoglobulin infiltration compared to 1 ± 1% of normal myofibers (p = 0.029). Virtually all DEVILs were acid-phosphatase reactive but contained p62 immunoreactivity and periodic acid-Schiff stained plaques. Compared to normal myofibers, BNIP3 immunostaining in DEVILated myofibers was reduced, whilst MAP-LC3b was indifferent. Cleaved-caspase 3 immunoreactivity was marginally elevated in DEVILated myofibers, but unaccompanied by nuclear DNA fragmentation. DEVILated myofibers were also identified in elderly rat (24 month) and cadaveric human (78 years) muscles. We argue that DEVIL formation reflects a previously undescribed fibre death process via a mechanism involving autophagic dysfunction and that the process may represent our first direct insight into the mechanism by which myofibers are lost in old age.

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

“…Possible sources of calcium leak include the sarcoplasmic reticulum or the muscle fiber membrane, both of which have been implicated in muscle aging (Andersson et al 2011 ; Ramaswamy et al 2011 ). Compromised sarcolemma integrity has previously been observed near the myotendinous junctions (MTJs) of muscle fibers from older mice (Lal and Sheard 2015 ). Membrane tears localized to MTJs might be involved in the pathogenesis of MTJ and tendinous elongation (Lal and Sheard 2015 ; Nielsen et al 2018 ; Lal et al 2021 ) and they may also lead to the activation of calcium dependent proteases which degrade sarcomeres (Tidball et al 1995 ).…”

“…Compromised sarcolemma integrity has previously been observed near the myotendinous junctions (MTJs) of muscle fibers from older mice (Lal and Sheard 2015 ). Membrane tears localized to MTJs might be involved in the pathogenesis of MTJ and tendinous elongation (Lal and Sheard 2015 ; Nielsen et al 2018 ; Lal et al 2021 ) and they may also lead to the activation of calcium dependent proteases which degrade sarcomeres (Tidball et al 1995 ). Age-related proliferation of connective tissue including lengthening of both tendon and MTJ along with fiber shortening via loss of sarcomeres (Hooper 1981 ) are likely contributors to the observed functional changes including the increase in passive tension at L 0 .…”