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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online April 10, 2002 as doi:10.1096/fj.01-0792fje. |
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* Institute of Anatomy,
Department of Mathematical Statistics and Actuarial Sciences, University of Bern, Bern, Switzerland;
UMR 5123 CNRS, Université Lyon I, Lyon, France; and
School of Biomedical Sciences, University of Leeds, England
2Correspondence: Institute of Anatomy, University of Bern, Bühlstrasse 26, Switzerland. E-mail: flueck{at}ana.unibe.ch
SPECIFIC AIM
Using a commercially available rat-specific microarray (AtlasTM Rat 1.2 Array), we analyzed modulation of the expression profile of a broad range of transcripts involved in cellular maintenance, metabolism, and regulation with atrophy of m. soleus caused by 35 days of hind limb suspension (HS). It was asked whether a statistical cDNA array approach has the discriminatory capability to detect expected and not previously recorded or suspected changes in the m. soleus transcript after prolonged exposure to simulated microgravity.
PRINCIPAL FINDINGS
1. Microarrays are sensitive tools in analysis of the skeletal muscle gene profile
HS caused a significant 61% loss of m. soleus mass (P<0.001, n=8). A total of 395 of the 1200 transcripts probed were reliably detected on the microarrays with our skeletal muscle cDNAs. Their expression levels varied over a wide range.
The signal of the most abundant gastric inhibitory polypeptide (GIP) mRNA was 200-fold more intense than the least abundant transcripts detected (serotonin 5HT2 receptor mRNA). Based on the known expression level of ID1, a low-level transcript, it was estimated that the technique used has the sensitivity to detect genes expressed as low as 0.005% of the total population. Based on these considerations, it is calculated that 1–5% of totally expressed genes in control rat m. soleus were monitored with the AtlasTM Rat 1.2 array.
Analyzing the non-normalized RNAs levels from eight control and eight suspended m. solei individually, combined with a statistical approach involving linear regression and the sign test based on the conservative Bonferroni correction, allowed us to identify significant changes in 105 of the 395 detected transcripts with atrophy of m. soleus.
The relative levels of seven transcripts yielding low (TPP II and NVP-3), medium (Cat L, TIMP-2, and LCAD), and high signals (RPL19, H-FABP) in the array experiments were determined in parallel using real- time quantitative RT-PCR. Regression analysis showed that the average signal ratio (35 days suspended vs. control m. soleus) detected with RT-PCR and Atlas arrays showed a linear correlation (R2=0.74) of slope 1.13 with the confidence interval including unity. Test-retest experiments revealed a concordance correlation coefficient (R2) for reverse transcription and array hybridization of 0.96.
Biological post hoc analysis with background corrected and normalized values was used to estimate the relative ratios between normal and atrophied m. solei. Judging from their signal ratios of mRNA concentrations, the changes observed were less than twofold, with four exceptions (GAPDH, IGFBP-5, NRPYR5, and H-FABP). To evaluate the system physiological implications of the observed changes of the expression level of mRNA species, these were arranged into ‘functional units’ consisting of transcripts with closely related cellular functions.
2. HS causes concerted transcriptional adaptations of metabolic genes and a distinct expressional induction of genes involved in protein turnover
Abundant mRNAs encoding glycolytic enzymes (PFKM, ALDOA, and GAPDH) and proteins associated with carbohydrate transport by glut-4 vesicles (IRAP, M6P/IGFR2, and VAMP3) were discretely induced whereas transcripts for proteins related to fatty acid import (FATP and H-FABP) were reduced. Multiple low abundant mRNAs coding for small GTPases involved in vesicle trafficking were down-regulated in HS m. solei.
Genes related to protein degradation underwent discrete expression changes (cathepsins C and L, proteasome subunit rPA28) and factors associated with proteasomal protein degradation (TPP II, UBE2B, and carboxypeptidase D) were induced with HS; other proteasome subunit mRNAs were unchanged. The extracellular proteases MMP-2 and u-PA as well as their natural inhibitors TIMP-2 and -3 were up-regulated. Abundant mRNAs for components of the ribosome and translation factors were generally induced with HS.
3. The transcript levels of genes implicated in nerve-muscle interactions and in excitation-contraction coupling are modulated by prolonged HS
Evidence for a down-regulation by HS of low abundant mRNAs for proteins involved in the nerve–muscle interaction was eminent. The transcript levels of sarcolemmal neuroreceptors involved in excitatory neurotransmission (postsynaptic nAChR
and 
, mAChRM2, 5HT2A) of inhibitory presynaptic neurotransmission (metabotropic glutamate receptors) of the nerve-specific calcium channel CACNA1A, the neurotransmitter SGII mRNA, as well as morphogenic receptors and ligands (UNC5H2, NPTXR, and NT-3) were all reduced significantly but to small extents.
Conversely, the relatively abundant transcripts encoding multiple voltage-dependent ion channels were increased to a ratio 1.2–1.8 suspended vs. control (SCN1B, SCN2A1, Na,K-ATPase2, and Kir2.2). The other sodium/potassium channels accessed did not show expressional adaptations whereas SERCA2, the most abundant ion channel mRNA on our array, was increased with HS in m. solei.
4. The expression of regulatory genes is differentially affected by HS
The low abundant expression levels of genes encoding intracellular and extracellular regulators of the cell cycle in rat m. soleus were variably affected after HS.
Moreover, some mRNAs that code for proteins involved in generation of second messengers and downstream signal transduction were affected in HS.
CONCLUSIONS AND SIGNIFICANCE
Our study demonstrates that commercially available microarray technology combined with linear statistical regression analysis of raw data that circumvents background correction is a precise, efficient tool with which to investigate adaptations of the rat skeletal muscle gene profile. Small differences of low abundant genes could be detected. Changes identified with the microarrays of mRNAs, such as ALDOA and GAPDH, were comparable with previous independent observations.
A by-product of the wide variety of transcripts represented on the array used was that some expected low-level transcripts showed strong signals. The most abundant transcript encoded the insulinotropic polypeptide GIP that potentiates glucose-stimulated insulin secretion, glucose uptake, glucose oxidation and glycogenesis in various extrapancreatic target tissues. GIP also effects skeletal muscle, but its expression has not been described before in this tissue. Its high expression indicates that skeletal muscle may have unsuspected paracrine and eventually endocrine functions in glucose metabolism.
An adaptive change was assumed to be biologically relevant when the signals of several mRNAs coding for proteins involved in closely related cellular functions changed significantly and in the same direction. A limitation in the interpretation of the data is that many of the detected mRNAs are expressed in different tissue types. However, the specific cellular localization of expression is known for many of these mRNAs. In some cases, the signal intensity gave clues as to the compartmental origin of transcripts.
Major evidence is provided to suggest that an increase in expression of genes involved in metabolization of carbohydrates (glut-4-mediated glucose uptake and glycolysis) and a down-regulation of genes involved in fatty acid import may contribute to the changes in the substrate profile such as a higher reliance on carbohydrate and a reduced uptake of fatty acids from the blood into the atrophied m. soleus fibers. Changes in small GTPases indicate for the first time a global modulation of nutrient trafficking. The expression profile of metabolic genes implies that slow-to-fast fiber transformation in atrophic m. soleus involves concerted transcriptional adaptations, but to a lesser extent than expected from the observed shift in fiber type areas (Fig. 1
).
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Up-regulated expression of distinct components of the intra- and extracellular proteolytic, as well as the translation machinery, indicates that protein turnover in m. soleus may not have reached a new steady state after 5 wk HS as generally assumed. A shift toward enhanced degradation of intracellular small nonmyofibrillar proteins and a degradation of extracellular structures are suggested. The augmentation of mRNAs for ribosomal proteins and translation factors (as detected in fast-twitch rat m. gastrocnemius after immobilization) does not seem to be restricted to atrophy of a particular fiber type. Increased polysome size, and/or an increased percentage of connective tissue with unloading of m. soleus are explanations for transcriptional changes in the ribosome machinery (Fig. 1)
.
The strong trend for down-regulation of low abundant neuroreceptors involved in excitatory and inhibitory neurotransmission, of a neurotransmitter and a nerve-specific calcium channel, as well as morphogenic receptors and ligands involved in outgrowth of nerves and formation of synapses, demonstrate that transcriptional changes occur in the pre- and postsynaptic region of the neuromuscular junction and vegetative synapses with prolonged suspension of m. solei (Fig. 1)
. This may be linked to the observation that the percentage of slow-type units decreases in favor of fast-type motor units and vascular innervation modifications in suspended m. soleus and indicates regression of motor synapses as described after 12.5 days of space flight in m. adductor longus.
Concurrent adaptations in several abundant mRNAs of voltage-dependent ion channels point to undocumented adaptations in excitation-contraction coupling with atrophy of m. soleus (Fig. 1)
. If the observed adaptations in expression of voltage-dependent ion channels are translated in corresponding changes in protein, fine-tuning of excitation-contraction coupling in the atrophied rat m. soleus is suggested. An increased amplitude of the sodium current into the sarcoplasmic compartment and accelerated inactivation after initial membrane depolarization following AChR release at the end plate would be expected from increases of the central pore (SCN2A1) and modulatory subunit (SCN1B) of the voltage-gated sodium channel. Changes in the setting of the resting membrane potential and an improved capacity to regenerate the resting sarcolemmal potential are possible consequences of increased inward rectifier Kir2.2 and Na, K-ATPase2, respectively. SERCA2 data support the speculation that remodeling of the sarcoplasmic reticulum occurs during HS. These changes in ion channels may be at the start of a series of events that contribute to the shortening of twitch time-to-peak in 14 day hindlimb suspended rat m. soleus and eventually to reduced relaxation time with muscle unloading.
Observations on enhanced expression of voltage-dependent ion channels and on reduced expression of excitatory and inhibitory neuroreceptors, neurotransmitters, and morphogenic receptors and ligands corroborate the idea that slow-type motor units are transformed toward fast types in HS and support the conclusion that slow-to-fast transformation of muscle fibers is accompanied by reprogramming of associated nerves.
Original observations on changes in a series of transcripts encoding intra- and extracellular regulators of the cell cycle document enhanced growth arrest and characterize the expression profile of myonuclei and fiber-associated cell structures as reprogrammed from a slow toward a fast-type muscle (Fig. 1)
. The changes in signal transduction protein mRNAs are interpreted as an indication of the adaptations in the cellular regulatory network involved in Ca2+ and glucose homeostasis, respectively, to the new atrophied muscle steady state.
Overall, the results indicate that distinct changes of individual gene groups contribute to late-stage atrophic process in rat skeletal muscle. Novel findings identify venues for future research, whereby microarrays are a highly efficient tool for directing complementary investigations and an important source of information.
FOOTNOTES
1 To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.01-0792fje; to cite this article, use FASEB J. (April 10, 2002) 10.1096/fj.01-0792fje. 
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