The multimodal transcriptional response of denervated skeletal muscle involves alterations in cholesterol homeostasis impacting muscle size

The development and maintenance of the neuromuscular junction (NMJ) requires reciprocal signals between the nerve terminals and the multinucleate skeletal muscle fiber (myofiber). This interaction leads to highly specialized transcription in the sub-synaptic or NMJ myonuclei within mature myofibers leading to clustering of acetylcholine receptors (AChRs). Here we utilized single-nucleus RNA sequencing (snRNA-seq) to delineate the transcriptional response of myonuclei to denervation. Through snRNA-seq on skeletal muscle from two independent mouse models of denervation, sciatic nerve transection and amyotrophic lateral sclerosis, we identify a multimodal transcriptional response of NMJ-enriched genes and an alteration in cholesterol homeostasis in both slow and fast myofibers. Gramd1, a family of genes involved in non-vesicular cholesterol transport, are enriched at the NMJ at baseline and upregulated in both models of denervation by the NMJ and extrasynaptic myonuclei In vivo gain and loss of function studies indicate that NMJ-enriched Gramd1 genes regulate myofiber sizes independent of an obvious impact on AChR clustering. We uncovered a dynamic transcriptional response of myonuclei to denervation and highlight a critical role for cholesterol transport to maintain myofiber sizes. Tibialis anterior (TA) muscles from SOD1G93A or C57BL/6 mice were harvested immediately following euthanasia, minced, and placed in homogenization buffer (0.25 M sucrose and 1% BSA in Mg2+-free, Ca2+-free, RNase-free 1x PBS). Nuclei isolation was done as previously described [20]. After filtration via a 40 μm strainer, nuclei were labeled with Hoechst dye and 0.2 U/μL Protector RNase inhibitor (Roche). All Hoechst stained myonuclei were purified using FACS (BD Aria, 70 μm nozzle) and gathered in sorting buffer containing Protector RNase inhibitor (0.2 U/μL). The nuclei were counted with a hemocytometer, and the concentration was fine-tuned as needed to reach the optimal range for the 10X Chromium chip. The 10X Chromium system was then used to load the nuclei using the Single Cell 3′ Reagent Kit v3.1, following the guidelines provided by the manufacturer. Around 12,000 nuclei were loaded for each operation. Sequencing was performed on an Illumina NovaSeq 6000 System.