Syncytial skeletal muscle cells contain hundreds of nuclei in a shared cytoplasm. We investigated nuclear heterogeneity and transcriptional dynamics in the uninjured and regenerating muscle using single-nucleus RNA-sequencing (snRNAseq) of isolated nuclei from muscle fibers. This revealed distinct nuclear subtypes unrelated to fiber type diversity, previously unknown subtypes as well as the expected ones at the neuromuscular and myotendinous junctions. In fibers of the Mdx dystrophy mouse model, distinct subtypes emerged, among them nuclei expressing a repair signature that were also abundant in the muscle of dystrophy patients, and a nuclear population associated with necrotic fibers. Finally, modifications of our approach revealed the compartmentalization in the rare and specialized muscle spindle. Our data identifies nuclear compartments of the myofiber and defines a molecular roadmap for their functional analyses; the data can be freely explored on the MyoExplorer server (https://shiny.mdc-berlin.de/MyoExplorer/).

Coadministration of a potent CYP3A4 inhibitor leads to a higher and prolonged exposure with voriconazole that might increase the risk of the development of adverse drug reactions on a short-term basis, particularly in CYP2C19 PM patients.

15All cells must compartmentalize their intracellular space in order to properly function 1 . Syncytial 16 cells face an additional challenge to this fundamental problem, which is to coordinate gene 17 expression programs among many nuclei. Using the skeletal muscle fiber as a model, we 18 systematically investigated the functional heterogeneity among nuclei inside a syncytium. We 19 performed single nucleus RNA sequencing (snRNAseq) of isolated myonuclei from uninjured 20 and regenerating muscle, which revealed a remarkable and dynamic heterogeneity. We identified 21 distinct nuclear subtypes unrelated to fiber type diversity, completely novel subtypes as well as 22 the expected neuromuscular 2-5 and myotendinous 6-8 junction subtypes. snRNAseq of fibers from 23 the Mdx dystrophy mouse model uncovered additional nuclear populations. Specifically, we 24 identified the molecular signature of degenerating fibers and found a nuclear population that 25 expressed genes implicated in myofiber repair. We confirmed the existence of this population in 26 patients with muscular dystrophy. Finally, modifications of our approach revealed an astonishing 27 compartmentalization inside the rare and specialized muscle spindle fibers. In summary, our 28 work shows how regional transcription shapes the architecture of multinucleated syncytial 29 muscle cells, and provides an unprecedented roadmap to molecularly dissect distinct 30 compartments of the muscle. 31 32 33 34 35 36 37 38 39 40 41 3 Multinucleated skeletal muscle cells possess functionally distinct nuclear compartments to meet 42 the needs for specialized gene expression in different parts of the large syncytium. The best 43 documented nuclear subtype locates at the neuromuscular junction (NMJ) where the nerve 44 instructs myonuclei to express genes involved in synaptic transmission 2-5 . Another specialized 45 compartment is found at the myotendinous junction (MTJ) where the myofibers attach to the 46 tendon. It has been shown that specialized cell adhesion and cytoskeletal proteins are enriched at 47 the MTJ to allow force transmission 6-8 . However, whether the corresponding transcripts are 48 specifically produced from MTJ myonuclei is often unclear, especially in mammals. More 49 generally, a systematic analysis of the heterogeneity among myonuclei is lacking, and we do not 50 know what kinds of nuclear subtypes exist. Such knowledge will provide important insights into 51 how myofibers can orchestrate their many functions. 52 Previous studies on gene expression in the muscle relied on the analysis of selected candidates 53 by in situ hybridization or on profiling the entire muscle tissue. The former is difficult to scale up, 54whereas the latter averages the transcriptomes of all nuclei. More recently, several studies have 55 used single cell approaches to reveal the cellular composition of the entire muscle tissue 9-11 . 56 However, these approaches did not sample nuclei in the myofiber and thus did not investigate 57 myonuclear transcriptomes. Single-nucleu...