Characterizing Subsynaptic Myonuclei in Aged Muscle

Abstract

IntroductionDuring aging, the neuromuscular junction (NMJ) undergoes structural fragmentation and functional deficits that alter neuromuscular communication, impair muscle contraction, and contribute to muscle atrophy. At the NMJ, myonuclei accumulate and localize in a fashion that juxtaposes three‐to‐six myonuclei in the postsynaptic region. Although the entire role of these so‐called subsynaptic myonuclei remains enigmatic, previous literature suggests they contribute to gene regulation of synapse‐specific proteins required for maintaining the structural integrity of the synaptic architecture. Interestingly, a recent report find that NMJs in muscles of aged mice contain fewer subsynaptic myonuclei compared with muscles of adult mice in parallel with degenerating or degenerated endplates. Furthermore, disturbance of the nuclear envelope by in‐vivo knock‐out of Lmna appears to accelerate NMJ degeneration and reduces the number of myonuclei confined to the endplate. Therefore, with the observed reductions in subsynaptic myonuclei in degenerated NMJs, our study aims to further examine and characterize subsynaptic myonuclei and their role in NMJ degeneration.MethodsGastrocnemius muscles were harvested from Young (6 months) and Aged (28 months) wild type mice and 6‐ and 12‐month‐old Sod1KO mice (N=3/group) and briefly fixed in 4% PFA for 10 minutes. Myofiber bundles were mechanically teased under a dissecting microscope and subjected to immunohistochemistry for visualization of motor neurons (neurofilament‐NF and synaptic vesicle glycoprotein 2‐SV2), motor end plates (alpha‐bungarotoxin), and myonuclei (nesprin 1 and 4’,6‐diamidino‐2‐phenylindole‐DAPI). Myofiber bundles were then mounted onto a slide and imaged via High‐sensitivity confocal microscopy. Captured images were then 3D projected in FIJI for assessment and quantification of subsynaptic myonuclei, innervation status, and post synaptic area assessed via aNMJ morph.ResultsIn young mice with primarily fully innervated endplates, the number of myonuclei associated with the endplate was lower than the number of synaptic myonuclei in muscles of aged and Sod1KO mice, both of which contain greater numbers of partially and non‐innervated endplates.ConclusionThere may be a greater number of subsynaptic myonuclei localized to degenerating or degenerated endplates, although whether the change in nuclear number is a consequence of NMJ degeneration or contributes to the denervation cannot be established from this study.