Single Nuclear Analyses Reveal Compromised Integrity of Neuromuscular Junction Myonuclei Following Joint Injury

Abstract

The neuromuscular junction is an essential structure that links electrical signals from the nervous system to contraction of skeletal muscle. The post synaptic region of a muscle cell is maintained by a subset of specialized myonuclei responsible for clustering of post synaptic structural proteins and acetylcholine receptors. Disruptions in the transcriptional program of these myonuclei may result in ineffective myofiber recruitment, slowed force generation, and muscle weakness. We have shown that traumatic joint injury results in protracted deficits in muscle strength and power, but the peripheral mechanistic underpinnings remain unclear. Phenotypically, muscle pathology occurring after traumatic joint injury resembles that of neuromuscular destabilization, yet molecularly, this remains uninvestigated. Therefore, we hypothesize that disruption of neuromuscular regulatory gene programs and destabilization of the neuromuscular junction following joint injury will result in rapid muscle functional deficits. Human muscle biopsies were obtained from the vastus lateralis of anterior cruciate ligament (ACL) injured and healthy limbs of 26 participants. Injured biopsies were taken before and 7 days following reconstruction surgery. Bulk tissue RNA-seq analysis was performed on all participants and single-nucleus RNAseq (snRNAseq) was performed on a subset of participants. Briefly, for snRNAseq, muscle was dounce homogenized, filtered, and nuclei isolated via fluorescent activated cell sorting. Following sequencing, CellRanger output files containing count matrices were filtered, and analyzed using Partek Flow and Seurat. Biopsy sections were processed for histochemistry to analyze fiber size and motor end plate innervation. All participants underwent strength testing on a biodex to assess isometric quadriceps torque and rate of torque development. RNA sequencing displayed >3 fold upregulation of CHRNA1 (P< 0.001) after injury alone and >18 fold upregulation 7 days following reconstruction surgery (P< 0.001). This was accompanied by a host of other neuromuscular regulatory genes including HDAC4 (P< 0.001), GDNF (P< 0.001), and RUNX1 (P< 0.001). Single nucleus sequencing revealed a complete disappearance of neuromuscular junction regulatory myonuclei following reconstruction surgery coupled with delocalization of acetylcholine receptor expression. These findings were confirmed with fluorescent in situ hybridization of CHRNA1 RNA on biopsy sections. Functionally, CHRNA1 RNA expression strongly correlates with deficits in rate of torque development following injury (R^2 = 0.85, P< 0.001). Our results display rapid remodeling of the postsynaptic neuromuscular junction following joint injury that underscores sustained deficits in strength and power. Interventions targeting the preservation of peripheral innervation may be warranted to enhance outcomes following joint injury. R01AR072061. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.