Microgenomic analysis in skeletal muscle: expression signatures of individual fast and slow myofibers

Abstract

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.

Figure 1. Experimental set up of microgenomic technologies in skeletal muscles.

A) Transmitted light images at 2.5X magnification of isolated muscle fibers from soleus (top) and EDL (bottom). Intact, unblemished myofibers appears as translucent cylinders. The inset shows details of the characteristic striated pattern (magnification 40X). Black scale bars: 250 µm; white scale bars: 25 µm. B) MyHC electrophoretic characterization of single fibers fragments from soleus (top) and EDL (below) muscles. A whole muscle sample has been used as marker of molecular weight (*). As shown in the examples, type 1 and type 2A fibers are abundant in the slow soleus muscle; type 2B and hybrid 2B/2X fibers are most frequent in the fast EDL muscle. C) Electropherogram of total RNA extracted from a single soleus myofiber, analyzed in the Agilent 2100 Bioanalyzer using a RNA 6000 Pico LabChip. About 1/3 of the full amount recovered was loaded in this experiment. The high quality of total RNA is confirmed by the presence of ribosomal peaks with no shift to lower fragments (RNA degradation) and no additional signals (DNA contamination).

Figure 2. Statistical analysis of microarray data.

A) Dendogram obtained by hierarchal clustering of expression data generated by 10 pure fibers expressing MyHC-1 (soleus) and 10 pure fibers expressing MyHC-2b (EDL). Microarrays mRNA expression profiling permitted a clear distinction between type 1 and type 2B fibers. Furthermore, technical replicas grouped together within each experiment, confirming the good quality of microarray data. Analysis performed with MeV tool on the set of 11,964 probes that passed the normalization and filtering steps, using Pearson correlation distance. EDL samples came from mice number 1 (1–2), 2 (3–5), 3 (6–8) and 4 (9–10); soleus samples from mice 5 (1–2), 6 (3–5), 7 (6–7) and 8 (8–10). The letters a, b refer to spot replicates present in each microarray slide. B) Venn diagram formed by DE genes identified after SAM analyses. Ovals: one-class test; circles, two-class test. Overlapping areas represent genes positive to both tests. FDR values were 0.15% in the one-class test and 0.21% in the two-class test.

Figure 3. Single fiber analyses allowed removal of non-muscle cells and enrichment for muscle specific genes.

A) Heat map of selected DE genes identified by one-class SAM analysis. Expression data are Log2 signal ratios values (see Dataset S1) which were converted to colors according to the bar shown at the top: positive values correspond to genes over-expressed in isolated myofibers (red), whereas negative values refer to genes over-expressed in whole muscles (green), and therefore under-expressed in myofibers. Mean values were calculated for two spot replicates. B) Validation by qPCR of four DE genes identified by one-class SAM analysis. Signal ratios (natural log values) were calculated independently in pools of 50 type 1 and 50 type 2B myofibers compared to the whole muscle control. The bars in the histogram correspond to the arithmetic mean of the two values separately calculated for type 1 and type 2B fibers. Normalization is relative to two internal references Mfn1 and Txn1; the vertical bars symbolize the intra-assay SD. Positive values correspond to genes over-expressed in myofibers (red bars), and negative values in whole muscles (green bars), as in the heat map.

Figure 4. Molecular signatures of fast and slow myofibers revealed by two-class SAM analysis.

Expression data are Log2 signal ratios values (see Dataset S1). The different color code emphasizes distinction of fiber types: positive values are in yellow and negative values in blue. Genes with differential expression between type 1 (soleus) and type 2B (EDL) myofibers were grouped according to functional classification: i) sarcomeric proteins (GO: contractile fiber part); ii) calcium signaling (GO: sarcoplasmic reticulum or calcium binding); iii) nucleus (GO: regulation of transcription or nucleus).

Figure 5. The discriminant analysis emphasized the discovery power of single cell analyses.

A) Discriminant genes identified by PAM tool. Expression data are Log2 signal ratios values (see Dataset S1). Positive values are in yellow color and negative values in blue color (according to the bar shown at the top). Results sorted by ranking were split in two parts, in order to show genes with preferential expression in type 1 (soleus) or type 2B (EDL) myofibers. B) Validation by qPCR of DE genes identified by PAM analysis. Signal ratios (natural log values) were calculated independently in pools of type 1 (gray bars) and type 2B (white bars) myofibers compared to whole muscle control. Normalization is relative to two internal references Mfn1 and Txn1; the vertical bars symbolize the intra-assay SD. Note that the expression of myostatin was not detectable in type 1 myofibers (*).

Figure 6. Differential expression among individual fast fibers.

Expression levels among individual type 2B fibers of three selected genes (JunB, Fos, RRad). Expression data are Log2 signal ratios values which were converted to colors according to the bar shown at the top: positive values correspond to genes over-expressed in isolated myofibers (red), whereas negative values refer to genes over-expressed in whole muscles (green), and therefore under-expressed in myofibers.