Significant findings: In the first year of the funding, we have performed an initial longitudinal scRNA-seq analysis to map the RNA expression profile of entire population of mononuclear muscle cells in WT and BlaJ mouse muscles at pre-symptomatic (4 months old), early symptomatic (7 months old), and late symptomatic (11 months old) stages. This effort has resulted in sequencing of single cell samples from mildly affected (TA) and a severely affected (Quadriceps) muscle from BlaJ and matched WT mice. Subsequent analysis of these scRNA-seq data identified samples that passed stringent quality control tests for the resulting data, providing RNAseq data for nearly 200,000 cells including all the known muscle cell types. This provided a longitudinal and cross-sectional single cell RNAseq data for dysferlinopathic and healthy mouse muscle. In addition, bulk sequencing analysis was performed on quadriceps muscle from 4, 7, and 11-months old mice.
The data is now being analyzed bioinformatically to be harmonized with single cell sequencing data. The analysis is focused on two stromal cell populations pertinent to dysferlinopathy, namely FAPs and macrophages as subclusters of cells in both these populations have changed. These cells types show subpopulation-specific markers that match gene markers identified by integrated analysis of healthy and regenerating murine skeletal muscle single-cell datasets. The enriched FAPs and macrophage cell subclusters in BLAJ Quadriceps are apparent from the pre-symptomatic stage and expand with disease progression identifying the disease relevance of these cell sub-clusters, which will be focus of future studies.