Lineage tracing of newly accrued nuclei in skeletal myofibers uncovers distinct transcripts and interplay between nuclear populations

Multinucleated skeletal muscle cells have an obligatory need to acquire additional nuclei through fusion with activated skeletal muscle stem cells when responding to both developmental and adaptive growth stimuli. A fundamental question in skeletal muscle biology has been the reason underlying this need for new nuclei in syncytial cells that already harbor hundreds of nuclei. To begin to answer this long-standing question, we utilized nuclear RNA-sequencing approaches and developed a lineage tracing strategy capable of defining the transcriptional state of recently fused nuclei and distinguishing this state from that of pre-existing nuclei. Our findings reveal the presence of conserved markers of newly fused nuclei both during development and after a hypertrophic stimulus in the adult. However, newly fused nuclei also exhibit divergent gene expression that is determined by the syncytial environment they fuse into. Moreover, accrual of additional nuclei through fusion is required for nuclei already resident in adult myofibers to mount a normal transcriptional response to a load-induced stimulus. We propose a model of mutual regulation in the control of skeletal muscle development and adaptations, where newly fused and pre-existing myonuclei influence each other to maintain optimal functional growth. For bulk nuclear sequencing, to obtain sufficient material for sequencing, skeletal muscles were pooled together for this analysis. For the early development stage, 4 × Gastrocnemius and 4 × tibialis anterior muscles per replicate were used, while for adulthood muscle overload, 4 × Plantaris muscles per replicate were used. Nuclei were extracted using the protocol described in the methods section above. The washed nuclei pellets were resuspended in sorting buffer (2% BSA/RNase-free PBS) and labeled with anti-PCM1 (1:500, HPA023374, Sigma-Aldrich) on ice for 45 minutes. Nuclei were washed twice in sorting buffer and then labeled with an Alexa Fluor 647-conjugated secondary antibody (1:100, Invitrogen) at 4°C for 30 minutes, followed by labeling with Hoechst dye. The nuclei were washed again and resuspended in sorting buffer with 0.2 U/μl Protector Rnase inhibitor prior to cell sorting to separate the PCM1-labeled fractions using a FACS cell sorter (BD Aria). The sorted nuclei were then pelleted at 3000 × g for 10 minutes before mRNA isolation using the Direct-zol RNA Microprep kit (Zymo research R2060). For snRNA-Seq datasets, plantaris muscles from HSArtTA;TREH2B-GFP mice were used for snRNA-seq in this study. Four pieces of plantaris muscle, pooled from two mice that either received or did not receive synergistic ablation, were used for nuclei isolation using the protocol described above. After filtration via a 40 μm strainer, uuclei were labeled with Hoechst dye and 0.2 U/μl Protector RNase inhibitor (Roche). All GFP+ 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.