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Expression Signatures of Single Isolated Myofibers of Mouse Hindlimb.

ABSTRACT: 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.

ORGANISM(S): Mus musculus

PROVIDER: GSE98325 | GEO | 2019/12/20

REPOSITORIES: GEO

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Project description:Background: skeletal muscle is a complex, versatile tissue composed of a variety of functionally diverse fiber types. Although the biochemical, structural and functional properties of myofibers have been the subject of intense investigation for the last decades, understanding molecular processes regulating fiber type diversity is still complicated by the heterogeneity of cell types present in the whole muscle organ. Methodology/Principal Findings: we have produced a first catalogue of genes expressed in mouse slow-oxidative (type 1) and fast-glycolytic (type 2B) fibers through transcriptome analysis at the single fiber level (microgenomics). Individual fibers were obtained from murine soleus and EDL muscles and initially classified by myosin heavy chain isoform content. Gene expression profiling on high density DNA oligonucleotide microarrays showed that both qualitative and quantitative improvements were achieved, compared to results with standard muscle homogenate. First, myofiber profiles were virtually free from non-muscle transcriptional activity. Second, thousands of muscle-specific genes were identified, leading to a better definition of gene signatures in the two fiber types as well as the detection of metabolic and signaling pathways that are differentially activated in specific fiber types. Several regulatory proteins showed preferential expression in slow myofibers. Discriminant analysis revealed novel genes that could be useful for fiber type functional classification. Conclusions/Significance: as gene expression analyses at the single fiber level significantly increased the resolution power, this innovative approach would allow a better understanding of the adaptive transcriptomic transitions occurring in myofibers under physiological and pathological conditions. EDL and soleus muscles were incubated with type I collagenase to dissociate intact myofibres that were separated under stereo microscope from the bulk of hyper contracted fibres. Isolated myofibres were divided in two parts: one was immersed in Laemmli buffer for fibre typing; the other was placed in RNA extraction buffer for RNA amplification. We analyzed the transcription profiles of 10 biological replicas of type 2B single muscle fibres from EDL and 10 biological replicas of type 1 single muscle fibres from soleus. Microarray competitive hybridizations were carried out against an artificial control with a balanced composition of type 1 and type 2B fibres (20 hybridizations). Each oligonucleotide is spotted in two replicates on the glass slide, so for every data two intra-slide replicas are present. The control was created as follows: three couples of soleus and EDL muscles were removed from 3 different mice and treated with collagenase. Total RNA was extracted separately from EDL and soleus muscles. By mixing about 1/3 RNA from EDL and 2/3 RNA from soleus muscles the control had approximately the same contributions of type 1 and type 2B fibres. Purified RNA samples from single fibres and from control were amplified twice using the Amino Allyl MessageAmpM-bM-^DM-" II aRNA Amplification Kit (Ambion).

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Expression Signatures of Single Isolated Myofibers of Mouse Hindlimb.

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