Project description:MicroRNA-expression profile of dystrophic single fibres vs wild type single fibers isolated from different muscle of mdx and c57bl mice. Myofibers were isolated from different muscle type (tibialis, diaphragm and quadriceps of gender- (male) and age- (3 month old and half) matched wt and dystrophic mice). 9 total samples per animal model, 3 replicates per muscle type sample
Project description:MicroRNA-expression profile of dystrophic single fibres vs wild type single fibers isolated from different muscle of mdx and c57bl mice. Myofibers were isolated from different muscle type (tibialis, diaphragm and quadriceps of gender- (male) and age- (3 month old and half) matched wt and dystrophic mice).
Project description:MicroRNA-expression profile of dystrophic single fibers compared to wild type single fibers isolated from different muscles of mdx and C57BL mice. Myofibers were isolated from different muscle types (tibialis, diaphragm and quadriceps) of gender- (male) and age- (3 month old and half) matched wt and dystrophic mice. 9 total samples per animal model (C57BL, mdx), 3 replicates per muscle type.
Project description:MicroRNA-expression profile of dystrophic single fibers compared to wild type single fibers isolated from different muscles of mdx and C57BL mice.
Project description:Skeletal muscles are composed by different myofiber types with a wide range of metabolic and functional properties. In the past years, biophysical analyses on isolated muscle fibers were extensively used to study the different contractile features of each myofiber type. To better clarify the complex mechanisms determining and regulating this heterogeneity, we applied genomic technologies at the level of single isolated myofibers. Fibers were prepared from soleus and EDL mouse muscles in order to obtain a comprehensive collection of fiber types classified according to myosin heavy chain (MyHC) isoforms. Transcriptomic analysis performed using microarray technology produced expression profiles of myofibers free from the background of non-contractile cell of the muscles. This permitted to identify a complete catalogue of genes differentially expressed among fiber types, mainly coding for metabolic enzymes, isoforms of the contractile proteins, and proteins involved in calcium homeostasis. Interestingly, myofibers transcriptionally grouped in 3 different transcriptional clusters, named transcriptional slow (tS), intermediate (tI), and fast(tF), mainly according to their metabolism and speed of contraction. This transcriptionally based myofibers grouping only partially fit with myofibers classification based on MyHC isoforms. In addition, using microarray data, we identified a limited set of transcriptional markers that can be used to unequivocally classify myofibers in one of the three clusters (tS, tI, or tF). This approach based on single cell analysis would allow a better description and comprehension of the adaptive transitions at transcriptional level occurring in myofibers under physiological and pathological conditions.
Project description:Skeletal muscle is a heterogeneous tissue. Individual myofibers that make up the contractile muscle tissue exhibit variation in their metabolic and contractile properties. Although there are biochemical and histological assays to study myofiber heterogeneity, methods to analyze the transcriptomes of individual myofibers are lacking. We have developed single myofiber RNA-Seq (smfRNA-Seq) to analyze the whole transcriptome of an individual myofiber by combining single fiber isolation with Switching Mechanisms at 5’ end of RNA Template (SMART) technology. Our method provides high-resolution genome wide expression profiles of single myofibers. This method will be useful to study developmental and age-related dynamics in the composition of skeletal muscle.
Project description:We report the application of single myofiber ATAC-Seq (smfATAC-Seq) to investigate the chromatin accessibility of a single myofiber without the presence of confounding muscle resident cell types. This method demonstrates that open chromatin regions of myonuclei can be tagmentated and high-quality sequencing ready libraries can be generated from these fragments. To perform comparative analysis as well as to demonstrate the applicability of the smfATAC-Sew to study changes in chromatin of myonuclei within different contexts, smfATAC-Seq was performed both on uninjured myofibers as well as injured myofibers seven days after induced injury. Furthermore, ATAC-Seq on 5000 muscle stem cells (MuSCs) was also performed according to the previously described OMNI ATAC-Seq protocol (Corces, M.R. et al. Nature Methods, 2017) in order to compare the sequencing quality of the smfATAC-Seq as well as to demonstrate the changes in open chromatin that occur from the stem cell state to the fully differentiated myofibers. smfATAC-seq resulted in comparable coverage and sequencing depth to the ATAC-seq performed on MuSCs and allowed for peak calling and differential peak analysis. These analysis revealed that the open chromatin state of uninjured and injured myofibers after seven days are mostly similar although some regions invovled in immune response remain in an open state in the injured myofibers compared to the uninjured myofibers and the regions involved in structural formation of the muscle are more accessible in the case of regeneration. Even though certain differences in the open chromatin are observed, smfATAC-Seq analysis suggest that overall, the open chromatin state of the myonuclei returns back to homeostasis after seven days of regeneration. Furthermore, smfATAC-Seq comparison with the ATAC-Seq from MuSCs show the differences in open chromatin regions between these conditions. Increased accessibility of genes involved in myogenesis and structural components can be observed in the myofibers compared to MuSCs while MuSCs show increased accessibility in regions involved in membrane permeability and signalling pathways. In addition, the regions that are accessible for both conditions include genes involved in mitochondrial transport, regulation of transcription and regulation of metabolites and energy. Overall, this study introduces smfATAC-Seq that succesfully assesses the genome-wide chromatin accessibility of a single myofiber with relatively high sequencing depth. The smfATAC-Seq can be used to perform comparative analysis between different conditions such as injury. However, this method can be readily applied to study differences between young and old myofibers or in the context of muscular dystrophy, cachexia, and exercise.