Dynamic resolution of functionally related gene sets in response to acute heat stress
© Szustakowski et al; licensee BioMed Central Ltd. 2007
Received: 07 August 2006
Accepted: 05 June 2007
Published: 05 June 2007
Using a gene clustering strategy we determined intracellular pathway relationships within skeletal myotubes in response to an acute heat stress stimuli. Following heat shock, the transcriptome was analyzed by microarray in a temporal fashion to characterize the dynamic relationship of signaling pathways.
Bioinformatics analyses exposed coordination of functionally-related gene sets, depicting mechanism-based responses to heat shock. Protein turnover-related pathways were significantly affected including protein folding, pre-mRNA processing, mRNA splicing, proteolysis and proteasome-related pathways. Many responses were transient, tending to normalize within 24 hours.
In summary, we show that the transcriptional response to acute cell stress is largely transient and proteosome-centric.
Damaged skeletal muscle tissue retains the capacity to self-repair through activation, expansion and fusion of resident satellite, progenitor cells [1–3]. However, the signaling mechanisms required to trigger satellite cell activation remain unclear in this context. Emerging pathways involved in the injury-response profile of skeletal muscle include Notch , Akt/Foxo  and NFκB [6, 7], which appear to play distinct roles in regeneration, hypertrophy and atrophy. Although these signaling pathways are well established, there remains a dearth of molecular detail, including the transcriptional consequence of atrophic stimuli, and this aspect forms the focus of the current study.
We hypothesized that subtle variance in gene expression may underlie significant functional events within the cell. Consistently, previous studies have addressed the transcriptional profile of recovering muscle tissue following acute injury . However, such studies rely on the analysis of individual genes, which is limiting from both statistical and biological perspectives. Here, we performed a study with the tenet that mechanistic data may be more readily revealed in the context of pathway-related gene clustering [9, 10]. We employed Gene Set Enrichment Analysis (GSEA), a bioinformatics approach conceived to exploit functional regulation at the pathway level. The study focused on the dynamic expression profile of acutely stressed skeletal muscle cells, and the resultant data revealed major transcriptional adjustments in a wide range of functional categories. These findings may underscore a coordinated pathway-centric response, and reveal rational strategies for target discovery in atrophic muscle diseases.
Atrogin-1 is expressed specifically in skeletal and cardiac muscle, and has been shown to regulate muscle atrophy [19, 20]. In response to heat shock, Atrogin-1 (Fbxo32) was upregulated after 8 hours, and this level of expression was maintained for at least 15 hours (Figure 1D). This dynamic profile is consistent with previous reports of muscle injury response, whereby the expression of atrogin-1 was concomitant with atrophy and recovery . Atrogin-1 is regulated by Foxo transcription factors, via nuclear localization of Foxo in response to various stimuli that inhibit PI3K/Akt signaling and subsequently drive atrophy [5, 22].
Transient profiles of gene expression
Significantly upregulated genes at specific time points following heat shock
Hsp40 homolog – gi:12839599
Cystein rich protein 61
FBJ osteosarcoma oncogene
Bcl2-associated athanogene 3
Developmentally and sexually retarded with transient immune abnormalities
Procollagen, type VI, alpha 3
Placental growth factor
Procollagen, type V, alpha 1
Solute carrier family 19 (thiamine transproter), member 2
Procollagen, type I, alpha 1
AXIN1 up-regulated 1
Actin, beta, cytoplasmic
Moderate similarity to protein ref:NP_004410.2 (H.sapiens) phosphatase
Ribosomal protein S24
Heme oxygenase (decycling) 1
Low density lipoprotein receptor-related protein 1
Growth arrest and DNA-damage-inducible 45 gamma
Perlecan (heparan sulfate proteoglycan 2)
Myeloid differentiation primary response gene 116
Myosin heavy chain IX
v-maf musculoaponeurotic fibrosarcoma oncogene family, protein F (avian)
Early growth response 1
Dual specificity phosphatase 2
Expressed sequence – gi:2918507
Diphtheria toxin receptor
EST – gi:8772531
Transformed mouse 3T3 cell double minute 2
Interferon-related developmental regulator 1
Activity regulated cytoskeletal-associated protein
mRNA – gi:16877863
DnaJ (Hsp40) homolog, subfamily A, member 4
Acidic (leucine-rich) nuclear phosphoprotein 32 family, member A
Lysosomal acid lipase 1
Lysyl oxidase-like 2
ATPase, Na+/K+ transporting, alpha 1 polypeptide
Guanine nucleotide binding protein, alpha inhibiting 2
Monocyte to macrophage differentiation-associated
Protein tyrosine phosphatase 4a2
Purinergic receptor P2X, ligand-gated ion channel, 7
Histone 1, H4i
Similar to hypothetical protein MGC4368 – gi:15655075
Aldehyde dehydrogenase family 3, subfamily A1
Expressed sequence – gi:2519775
Pyridoxal (pyridoxine, vitamin B6) kinase
cDNA – gi:9515377
CDP-diacylglycerol synthase (phosphatidate cytidylyltransferase) 2
Protein kinase C-like 1
Related to uridine kinase – gi:12844097
mRNA – gi:4779190
Gene set enrichment analysis
Most significantly upregulated gene sets at 8 hours post heat shock
No. of probes in set
Protein metabolism and modification
mRNA transcription regulation
Protein complex assembly
Discussion and Conclusion
This study was conducted to explore changes in gene expression following acute injury of C2C12 myotubes, and to reveal regulation of pathways during initial recovery. 866 individual transcripts represented on the microarray varied more than 2 fold over the course of the study, and the data revealed significant transcriptional changes in a wide variety of functional classes following heat shock damage to the cells. We devised the current study with two distinct dimensions. Firstly, to trace the time-course of recovery over a 24 hour period, and secondly, to analyze in a pathway-centric manner in order to extract hypotheses of coordinated molecular mechanisms that occur during cellular stress and recovery. Two notable pathways significantly modulated during recovery were the BMP signaling pathway, and the extracellular matrix-protein mediated signaling pathway, both of which are known to function in myogenesis, either by directing lineage specification (BMP) or structural/regenerative signaling (ECM-M). Additionally, the muscle-specific E3 ligase gene, Atrogin-1 was upregulated significantly, consistent with its reported role in mediating muscle atrophy in response to catabolic signals , and this in turn was coincident with our observation of nuclear translocation of Foxo3a following heat shock [5, 22]. This reconstruction of a well documented atrophic pathway served to validate the heat shock treatment paradigm as a useful model of acute muscle stress.
The experimental approach carried out in this study demonstrated the value of characterizing cellular responses in a temporal fashion, as transcriptional regulation was commonly found to be non-static between contiguous time points within the first 24 hours of treatment, and often presented transient regulation that required a time-course dimension to clarify.
A notable trend that emerged from the data was an apparent increase in protein turnover apparatus-related gene sets. The most significantly upregulated gene sets included those for both transcriptional activity, translational activity and protein modification, as well as the proteolysis and proteosome pathways. This suggests that a primary response of skeletal myotubes to heat shock treatment is to renew the protein complement of the cell, presumably as a defense mechanism against proteomic damage, and potential aberrant consequences in the function or integrity of the cell. The data also show that much of this response is transient, and appears to normalize within 24 hours of the acute insult at the transcriptional level.
In summary, the data presented in this report demonstrate a dynamic resolution of gene expression in response to acute stress that implicates waves of pathway activity in the cell's metabolic recovery. Further validation of these data are required, however, in order to demonstrate clearly the practical nature of these pathways as part of the functional stress response profile.
Cell culture and treatment
C2C12 mouse skeletal myoblasts were cultured in DMEM high glucose with 10% FBS and 1% penicillin/streptomycin (Gibco), and maintained at 37°C and 5% CO2. Cells were grown to confluence and then differentiated for seven days in media containing 3% fetal bovine serum. Cells were heat shocked at 47°C for 30 minutes, and returned to the incubator at 37°C. After heat shock, cells were harvested at 1, 2, 4, 8, and 24 hours and processed to isolate the RNA using the RNeasy kit (Qiagen). Myotubes were isolated for expression profiling by serially exposing the culture to increasing concentrations of trypsin. This allowed for the multinucleated myotubes to become detached from the plate, while the mononucleate myoblasts remained adherent. This process ensured that we were profiling differentiated skeletal myotubes only, without contamination of myoblasts. 20 μg RNA per sample was microarrayed using the Mouse Affymetrix MOE430 Plus 2.0 chip. The heat-shock treatment conditions were determined not to be cytotoxic, as continued culture in normal growth conditions was sufficient for full recovery of the cells (data not shown).
Raw intensity files were imported into GeneSpring® v.6.2 (Silicon Genetics, US) and filtered such that genes with a raw expression level of less than 100 were excluded from any future analyses. The resulting gene list was filtered to identify genes with a fold change increase or decrease of 2 or greater. Statistically significant genes were found using one-way ANOVA with time as variable and no multiple testing correction.
where μi,jis the average expression value for gene i under condition j . Genes were then sorted according to their expression ratios. Next, the collection of available gene sets were projected onto the sorted gene list. In essence, this step applies a priori biological knowledge to the experimental data to identify functionally related genes that are expressed in a coordinated fashion. Gene sets were processed individually and used to partition the genes into two groups: those gene in a pathway, and all other genes measured on the Genechip. A two-tailed Wilcoxon rank-sum test was then calculated to determine if the genes in each gene set were enriched at either the top or bottom of the sorted list. The Wilcoxon p-values were then transformed into false discovery rate q-values [24, 25] as a means of multiple hypothesis testing correction.
We thank Qicheng Ma for help in the development of GSEA.
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