Expression profiling reveals altered satellite cell numbers and glycolytic enzyme transcription in nemaline myopathy muscle

Abstract

The nemaline myopathies (NMs) are a clinically and genetically heterogeneous group of disorders characterized by nemaline rods and skeletal muscle weakness. Mutations in five sarcomeric thin filament genes have been identified. However, the molecular consequences of these mutations are unknown. Using Affymetrix oligonucleotide microarrays, we have analyzed the expression patterns of >21,000 genes and expressed sequence tags in skeletal muscles of 12 NM patients and 21 controls. Multiple complementary approaches were used for data analysis, including geometric fold analysis, two-tailed unequal variance t test, hierarchical clustering, relevance network, and nearest-neighbor analysis. We report the identification of high satellite cell populations in NM and the significant down-regulation of transcripts for key enzymes of glucose and glycogen metabolism as well as a possible regulator of fatty acid metabolism, UCP3. Interestingly, transcript level changes of multiple genes suggest possible changes in Ca(2+) homeostasis. The increased expression of multiple structural proteins was consistent with increased fibrosis. This comprehensive study of downstream molecular consequences of NM gene mutations provides insights in the cellular events leading to the NM phenotype.

Figures

Figure 1

NM and normal samples cluster independently by hierarchical clustering. Dendrogram generated by treeview (at top) reveals correlations between data sets for 34 specimens (middle) and identifies separate clusters for NM and normal muscles. The bottom part of the figure depicts signal strengths for a representative gene cluster, in which each row reflects the expression levels of one probe set across all samples. Color indicates relative signal levels, with red indicating the highest and green indicating the lowest expression.

Figure 2

Confirmatory studies of UCP3 and β-tubulin, at the transcript and protein levels. (A) UCP3 gene expression levels by quantitative reverse transcriptase PCR, for three representative normal (T145, T147, and T178) and NM (T68, T80, and T39) samples. UCP3 mRNA concentration is higher in normal than NM samples, and therefore peaks at an earlier cycle. (B) Immunoblotting reveals higher UCP3 protein levels in NM than in normal samples (Top). Specimens are indicated above. Ponceau S staining (PonS, Bottom) was used to ensure even loading of all protein samples. β-Tubulin also appears increased by immunoblotting in NM samples (β-TU, Middle). IF for β-tubulin in representative samples T146 (normal) (C) and T41 (NM) (D) reveals localization to connective tissue and not muscle fibers. (Bar = 50 μm.)

Figure 3

Identification of satellite cells in NM muscle. Indirect IF for NCAM1 (A–C) or PAX7 (D–F) is shown in green. 4′,6-Diamidino-2-phenylindole (DAPI)-counterstained nuclei (B, C, E, and F) are shown in blue. Putative satellite cells (arrows) are located peripheral to the myofibers and are surrounded by NCAM1-positive plasma membrane. (G–I) Lower-power views of satellite cells stained for PAX7 (appearing as small white dots) in normal biopsy, T137 (G), and two NM biopsies, T41 (H) and T43 (I). [Bars = 20 μm (A–F) and 50 μm (G–I).]

Figure 4

Highlights of key changes in cell cycle, differentiation, and metabolism-related transcripts. (A) Representative steps from pathways involved in cell cycle regulation and myogenesis. Significantly underexpressed (in red) and overexpressed (in green) genes were identified by using fold and t test analysis (Table 3). The CCND2/CDK4 pathway seems to be overall promoted, whereas the c-Fos and c-Jun pathways appear inhibited at the transcriptional level. Myogenesis-related genes are underexpressed. (B) Overall underexpression of key enzymes from the glucose and glycogen metabolic pathways (Table 3).