From Denervation to Reinnervation: The Role of Subsynaptic Myonuclei and Lmna in Skeletal Muscle Physiology

Abstract

This dissertation investigates the roles of subsynaptic myonuclei (SSM) and the nuclear envelope protein Lamin A/C in maintaining neuromuscular junction (NMJ) health, particularly during aging and after nerve injury. SSM, a specialized subset of myonuclei located beneath the motor endplate, are thought to support NMJ development and maintenance, yet their relationship to NMJ integrity and response to denervation remains unclear. To begin to address this knowledge gap, SSM counts and NMJ innervation status were analyzed in three mouse models of acute and chronic NMJ disruption: surgical denervation, aging, and chronic oxidative stress (Sod1-/- mice). SSM were identified via Nesprin 1 immunolabeling and anatomical localization. SSM numbers remained stable during aging, chronic oxidative stress, and after 7 days of denervation following a nerve transection injury. Despite significant NMJ degeneration and denervation in aged and Sod1-/- mice, SSM counts showed no relationship to NMJ innervation status, challenging the hypothesis that SSM loss drives NMJ decline. These findings highlight the need to explore other SSM characteristics, such as transcriptional profiles and structural roles, to better understand their contribution to NMJ health. We then shifted focus to explore the critical role of Lamin A/C, a protein encoded by the Lmna gene, in maintaining myonuclear integrity and reinnervation. Lamin A/C, a key intermediate filament protein in the nuclear lamina, is expressed in skeletal muscle and decreases with age, and with muscle-specific deletion reported to accelerate NMJ degradation, resembling age-associated NMJ decline. The role of Lamin A/C was examined in the context of NMJ regeneration following sciatic nerve injury in wildtype (WT) and muscle-specific heterozygous knockout mice (AC-Lmna+/−). Lamin A/C expression, significantly upregulated post-injury at both transcript and protein levels, returned to baseline by 14–28 days. NMJ morphology analysis revealed no structural differences across several features of the NMJ between genotypes. Despite intact NMJ morphology, AC-Lmna+/− mice exhibited delayed recovery of nerve-stimulated force generation and impaired NMJ transmission fidelity post-injury, implicating Lamin A/C in functional recovery rather than or perhaps in addition to structural maintenance. Notably, this response was specific to Lamin A/C and not part of a general nuclear envelope response. These results suggest that while Lamin A/C is non-essential for NMJ reinnervation, its partial loss delays functional recovery, raising questions about nuclear integrity disruptions in age-associated NMJ decline. Together, this work provides new insights into NMJ maintenance and regeneration by decoupling SSM count from NMJ integrity and highlighting Lamin A/C’s role in functional recovery. These findings underscore the complexity of NMJ regulation and reinnervation, suggesting Lamin A/C has a marginal role and additional targets influence muscle decline and NMJ deterioration.