Mapping the non-coding RNA landscape in skeletal muscle health and disease

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Project Summary Skeletal muscle tissues are developed and maintained through the coordinated action of myogenic and non- myogenic cells. Dysregulation of myogenic cell identities and functions are commonly observed in skeletal muscle disease. Facioscapulohumeral muscular dystrophy (FSHD) is the second most common inherited muscular dystrophy and results in progressive muscle weakness without any effective therapies. Numerous cellular etiologies are observed in FSHD, such as loss of myogenic cells, including muscle stem cells and myofibers, and increased fibrogenic, adipogenic, and immune cells. The most common form of FSHD arises from aberrant expression of the DUX4 gene caused by epigenetic de-repression of the D4Z4 locus. DUX4 expression in FSHD individuals is regionally varied and highly sporadic within skeletal muscle tissue. Notably, DUX4 expression is both induced by and has pathogenic mechanisms related to noncoding RNAs (ncRNAs). Noncoding RNAs (including miRNAs, lncRNAs, snoRNAs, and eRNAs) are critical regulators of skeletal muscle cell identities and functions in health and diseases and act through modulation of transcriptional networks. Comprehensive understanding of ncRNA networks and mechanisms is lacking due to a paucity of ncRNA profiling technologies. Conventional single-cell and spatial RNA-sequencing technologies preferentially detect polyadenylated, protein-coding mRNAs, and do not efficiently capture most ncRNAs due to their lack of polyadenylation. In this proposal, we will apply a new RNA mapping technology called STRS-HD that is uniquely capable of efficiently and comprehensively detecting the total transcriptome, including both polyadenylated and non-adenylated transcripts, with single-cell spatial resolution to reveal global ncRNA expression heterogeneity in diverse cell types within skeletal muscles. We will leverage this new spatial total RNA-sequencing method to broadly interrogate noncoding RNAs in healthy skeletal myogenesis and in FSHD pathogenesis. In Aim 1, we will implement this total transcriptomic method to investigate how noncoding RNAs impact cell fate regulation adult skeletal muscle regeneration in mice. We will explore cell type-specific ncRNA expression variation and use spatial transcriptomics to map ncRNA features onto spatially resolved cell-cell communication interactions to provide insights into ncRNA regulation of myogenic cell fates. In Aim 2, we apply these methods to resolve how ncRNAs vary in FSHD pathologies using two mouse models subject to DUX4 anti-sense oligonucleotide therapy. We will integrate spatial total transcriptome maps with histopathology to reveal ncRNA determinants of altered myogenic cell specification and myofiber damage in FSHD. In Aim 3, we will extend the STRS-HD approach to human FSHD biopsies and compare ncRNA features to unaffected familial controls and contrast spatial ncRNA maps to cell-free RNA-sequencing in donor plasma to identify new total RNA biomarkers of FSHD. These new total transcriptomic technologies will be broadly applicable to the study of ncRNAs in developmental and disease biology of skeletal muscle and other tissues.