Gene expression and muscle fiber function in a ... - Semantic Scholar

Physiol Genomics 39: 141–159, 2009. First published August 25, 2009; doi:10.1152/physiolgenomics.00026.2009.

TRANSLATIONAL PHYSIOLOGY

Gene expression and muscle fiber function in a porcine ICU model Varuna C. Banduseela,1 Julien Ochala,1 Yi-Wen Chen,2,3 Hanna Go¨ransson,4 Holly Norman,1,5 Peter Radell,6 Lars I. Eriksson,6 Eric P. Hoffman,2,3 and Lars Larsson1,7 1

Department of Clinical Neurophysiology, Uppsala University Hospital, Uppsala, Sweden; 2Research Center for Genetic Medicine, Children National Medical Center; 3Department of Pediatrics, The George Washington University Medical Center, Washington, District of Columbia; 4Department of Medical Sciences, Uppsala University Hospital, Uppsala, Sweden; 5 Department of Physiology, University of Wisconsin, Madison, Wisconsin; and 6Department of Anesthesiology, Karolinska Institute, Stockholm, Sweden; 7Department of Biobehavioral Health, Pennsylvania State University, University Park, Pennsylvania Submitted 12 February 2009; accepted in final form 17 August 2009

Banduseela VC, Ochala J, Chen Y-W, Go¨ransson H, Norman H, Radell P, Eriksson LI, Hoffman EP, Larsson L. Gene expression and muscle fiber function in a porcine ICU model. Physiol Genomics 39: 141–159, 2009. First published August 25, 2009; doi:10.1152/physiolgenomics.00026.2009.—Skeletal muscle wasting and impaired muscle function in response to mechanical ventilation and immobilization in intensive care unit (ICU) patients are clinically challenging partly due to 1) the poorly understood intricate cellular and molecular networks and 2) the unavailability of an animal model mimicking this condition. By employing a unique porcine model mimicking the conditions in the ICU with long-term mechanical ventilation and immobilization, we have analyzed the expression profile of skeletal muscle biopsies taken at three time points during a 5-day period. Among the differentially regulated transcripts, extracellular matrix, energy metabolism, sarcomeric and LIM protein mRNA levels were downregulated, while ubiquitin proteasome system, cathepsins, oxidative stress responsive genes and heat shock proteins (HSP) mRNAs were upregulated. Despite 5 days of immobilization and mechanical ventilation single muscle fiber cross-sectional areas as well as the maximum force generating capacity at the single muscle fiber level were preserved. It is proposed that HSP induction in skeletal muscle is an inherent, primary, but temporary protective mechanism against protein degradation. To our knowledge, this is the first study that isolates the effect of immobilization and mechanical ventilation in an ICU condition from various other cofactors. mechanical ventilation; immobilization; muscle function; gene expression; ubiquitin proteasome system; heat shock proteins; Lim proteins; intensive care unit SEVERE MUSCLE WASTING and impaired muscle function accompany critical illness in intensive care unit (ICU) patients with negative consequences for recovery from primary disease and weaning from the respirator. While ICU outcome has traditionally focused simply on survival, modern critical care also addresses post-ICU complications and quality of life. Several recent studies show unambiguously that neuromuscular dysfunction, resulting in muscle wasting and weakness, is the most persistent and debilitating of problems for survivors from the ICU for as long as 2 yr after hospital discharge, which is the longest observation period reported to date (18, 28). There is,

Address for reprint requests and other correspondence: L. Larsson, Dept. of Neuroscience, Clinical Neurophysiology, Univ. Hospital, Entrance 85, 3rd Fl., SE-751 85 Uppsala, Sweden (e-mail: [email protected]).

accordingly, a significant need for more research focusing on mechanisms underlying the muscle wasting and weakness in ICU patients (36). Primary disease, sepsis, and multiorgan failure undoubtedly contribute to the impaired muscle function. Nevertheless, it is highly likely that muscle unloading, lack of voluntary muscle activation, and mechanical ventilation are directly involved in the progressive impairment of muscle function during long-term ICU treatment. Critically ill ICU patients are frequently exposed to immobilization and mechanical ventilation, but the contribution of these procedures to the muscle weakness observed in ICU is masked by other prominent cofactors described above. The effects of muscle unloading in combination with lack of voluntary activation and mechanical ventilation are therefore often neglected in the studies of muscle weakness in ICU patients, despite the fact that they may play a key role in the pathophysiology of the muscle wasting. Furthermore, the lack of an animal model mimicking the ICU conditions has hindered the deciphering of the multifaceted and complex underlying mechanisms. In this context it is critical to have an experimental ICU model mimicking the key elements such as absence of voluntary activation of skeletal muscle, muscle unloading, an intact motoneuron, sedation, and mechanical ventilation. This model differs from established muscle wasting models, such as peripheral denervation, cast immobilization, bed rest, or hindlimb suspension. Furthermore, the precise onset of the muscle weakness and/or protein degradation in ICU patients is unclear due to prior sedation and typically noticed when the weaning from mechanical ventilation is attempted; muscle weakness may accordingly go undetected several weeks after admission to the ICU. However, studies aimed at investigating the molecular events and functional and structural characteristics in the early stages of the ICU stay are very limited. To address these issues, we have used a unique porcine ICU model (63) and evaluated muscle fiber size, force-generating capacity of individual muscle fibers, and gene expression in muscles from piglets immobilized, sedated, and mechanically ventilated for 5 days. By integrating physiological parameters with gene expression data and using a unique animal model, we aim to decipher the functional characteristics and molecular networks of skeletal muscle in early stages of the ICU condition.

1094-8341/09 $8.00 Copyright © 2009 the American Physiological Society

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PROTECTIVE ROLE OF HEAT SHOCK PROTEINS IN ICU

MATERIALS AND METHODS

Animals and Tissue Collection Sixteen female domestic piglets (Sus scrofa, average body wt 26.5 kg) were used in this study. All piglets originated from the same farm (Vallrums Lantbruk, Ransta, Sweden) and were kept in 12-squaremeter pens with hay, straw, and wood shavings as bedding material. They were housed at 18 –19°C and relative humidity of 45–55% under natural day-night rhythm with liberal access to feed (Sma˚grisfoder Solo 331; Lantma¨nnen, Stockholm, Sweden), water, and environmental enrichment. Food, but not water, was withheld for 12 h before induction of anesthesia. The pigs were sedated (Zoletil, Domitor, Orion Pharma Animal Health) before an intravenous line was prepared, and 100 mg of ketamine (Ketaminol, Intervet) was administered. After tracheostomy anesthesia was maintained through the anesthetic conserving device, which permits administration of isoflurane to the breathing circuit via a syringe pump (72). All piglets were immobilized by anesthesia and mechanically ventilated (Servo 900C; Siemens-Elema, Solna, Sweden), and the first biopsies (day 1) from the m. biceps femoris were obtained from all animals after administration of anesthetics. During the 5-day study period, four animals were sedated with isoflurane inhalation (0.8 – 1.3% end-tidal concentration; Abbott Laboratories, Chicago, IL) supplemented by intravenous bolus doses of morphine and ketamine as needed. Biopsies from the m. biceps femoris were obtained on two further separate occasions (days 3 and 5) in these animals. Core body temperature (blood) was maintained in the range of 38.5– 40°C by a servo-controlled heating pad. The animals received intravenous crystalloid fluid (Ringer’s acetate) to maintain stable blood pressure and urinary output and a glucose infusion (25 mg glucose/ml Rehydrex; Fresenius Kabi, Stockholm, Sweden) in the range of 0.5–1.5 mg 䡠 kg⫺1 䡠 min⫺1 to decrease the effects of catabolism. Each animal received prophylactic streptomycin 750 mg/day and benzylpenicillin 600 mg/day (Streptocillin Vet; Boeringer-Ingelheim, Hellerup, Denmark). Arterial blood gas analysis as well as electrolytes and blood glucose levels were monitored regularly and kept in the normal range throughout the study period. The biopsies were split in two portions. One part was frozen in liquid propane cooled by liquid nitrogen and stored at ⫺80°C for extraction of RNA and protein. The other part was immediately placed in an ice-cold relaxing solution (in mmol/l: 100 KCl, 20 Imidazole, 7 MgCl2, 2 EGTA, 4 ATP, pH 7.0; 4°C). Small bundles of ⬃25–50 fibers were dissected free from the muscle and tied to a glass micro capillary tube at ⬃110% resting length. The bundles were then placed in a skinning solution (relaxing solution containing glycerol; 50:50 vol/vol) at 4°C for 24 h and subsequently stored at ⫺20°C for use within 3 wk or treated with a cryoprotectant (sucrose solution) for long-term storage at ⫺80°C as described earlier (18). The Ethical Committee at the Karolinska Institute approved all aspects of this study (Dnr N71/98, N54/02, and N75/04). Single Muscle Fiber Experimental Procedure On the day of an experiment, a fiber segment length of 1–2 mm was left exposed to the solution between connectors leading to a force transducer (model 400A, Aurora Scientific) and a lever arm system (model 308B, Aurora Scientific). The total compliance of the attachment system was carefully controlled and remained similar for all the single muscle fibers tested (6 ⫾ 0.5% of fiber length). The apparatus was mounted on the stage of an inverted microscope (model IX70, Olympus). While the fiber segments were in relaxing solution, we set sarcomere length to 2.65–2.75 ␮m by adjusting the overall segment length. The sarcomere length was controlled during the experiments with a high-speed video analysis system (model 901A HVSL, Aurora Scientific). The fiber segment width, depth, and length between the connectors were measured (45). Fiber cross-sectional area (CSA) was calculated from the diameter and depth, assuming an elliptical cirPhysiol Genomics • VOL

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cumference, and was corrected for the 20% swelling that is known to occur during skinning (62). The maximum force normalized to fiber CSA was measured in each muscle fiber segment (P0/CSA) (45). After the mechanical measurements, each fiber was placed in urea buffer (120 g urea, 38 g thiourea, 70 ml H2O, 25 g mixed bed resin, 2.89 g dithiothreitol, 1.51 g Trizma base, 7.5 g SDS, 0.004% bromphenol blue) in a plastic micro centrifuge tube and stored at ⫺80°C. Myosin heavy chain isoform (MyHC) expression of single fibers was determined by 6% SDS-PAGE. The acrylamide concentration was 4% (wt/vol) in the stacking gel and 6% in the running gel, and the gel matrix included 30% glycerol. Sample loads were kept small (equivalent to ⬃0.05 mm of fiber segment) to improve the resolution of the MyHC bands (types I, IIa, and IIx). Electrophoresis was performed at 120 V for 24 h with a Tris-glycine electrode buffer (pH 8.3) at 15°C (SE 600 vertical slab gel unit, Hoefer Scientific Instruments). The gels were subsequently scanned in a soft laser densitometer (Molecular Dynamics) with a high spatial resolution (50 ␮m pixel spacing) and 4,096 optical density levels. Expression Profiling Three micrograms of total RNA from the muscle samples were extracted and processed to generate biotin-labeled cRNA as previously described (16). Each sample was then hybridized to Affymetrix Porcine Genome Array, which contains 23,937 probes representing 20,201 genes. Standard operating procedure and quality control were done as previously described (16). Muscle samples from three groups (16 animals day 1, 4 animals day 3 and 5) were profiled. All profiles have been made publicly accessible via National Center for Biotechnology Information Gene Expression Omnibus (no GSE16348; http:// www.ncbi.nlm.nih.gov/geo/) and the Children’s National Medical Center Public Expression Profiling Resource (http://pepr.cnmcresearch. org). Microarray Data Normalization and Analysis Subsequent analysis of the gene expression data was carried out in the freely available statistical computing language R (http://www. r-project.org) using packages available from the Bioconductor project (www.bioconductor.org). The raw data were normalized using the robust multiarray average (37) background-adjusted, normalized, and log-transformed summarized values first suggested by Li and Wong in 2001 (50). To search for the differentially expressed genes between the samples from the different days, an empirical Bayes moderated t-test was applied (80), using the “limma” package. A linear model was fitted to the data, and the day 3 vs. day 1 effect, day 5 vs. day 1 effect, and day 5 vs. day 3 were estimated. To address the problem with multiple testing, the P values were adjusted according to Benjamini and Hochberg. Significant probe sets with an adjusted P value ⬍0.05 were selected for further investigation, and those showing more than twofold change were included in further analyses. Since the porcine array is minimally annotated and is not identified by webbased analysis software, we used published putative human homologs (87). Up- and downregulated transcripts were further analyzed and categorized using DAVID web-based functional annotation tool (http://david.abcc.ncifcrf.gov/) (35). Some of the functional categories were combined and some categorization was done manually, to improve the interpretative value of the data. Clustering images were developed using Genesis software (83). Quantitative Real-time RT-PCR Reverse transcription and quantitative PCR analysis was performed as previously described (63). Briefly, total RNA (100 ng) was reverse transcribed to cDNA using Ready-To-Go You-Prime First Strand Beads (Amersham Biosciences, Uppsala, Sweden), random hexamers (Amersham Biosciences) and oligo-dT primers (Amersham Biosciences), or Qscript cDNA supermix (Quanta Biosciences). cDNA www.physiolgenomics.org


was amplified in triplicate using MyiQ single-color real-time PCR detection system (Bio-Rad Laboratories, Hercules, CA) and used to quantify the mRNA levels for porcine MyHC type I, IIb, and IIx, actin, atrogin-1, heat shock protein 110/105, and 18S. The thermal cycling conditions include 95°C for 9 min, followed by 50 cycles of amplification at 95°C for 15 min, followed by 60°C for 1 min. Each reaction was performed in a 25 ␮l volume with 0.4 ␮M of each primer and 0.2 ␮M probe or SYBR green (1988123; Roche Diagnostics, Ulm, Germany). Taq man primers and probes were designed using Primer Express program (Applied Bio System, Foster City, CA). Porcine 18S gene was used as the internal control. Primer sequences for porcine MyHC isoforms, actin, and atrogin-1 are published elsewhere (63, 66). The primer sequences for heat shock protein (HSP) 105/110 are forward-TCA AGA GGG CTG TGA TTA AGC A, reverse-TGG AGA GCA AAA TGC AAG AAA A, amplicon size 63 bp (GenBank accession NM_001097504). Unpaired t-test was used to test statistical significance. Mean ct values for 18s, actin, MyHC 7 (type 1), MyHC 1 (type IIx), atrogin-1, and HSP 110 were 14.8, 26.1, 29.5, 28.8, 26.6, and 31.4, respectively. Immunoblotting Approximately equal amounts of muscle tissue were dissolved in urea buffer. Equal volumes of lysate were separated by SDS-PAGE and transferred to PVDF membranes (GE Healthcare). Membranes were incubated with HSP 70 (SMC 100A/B, Stress Marq Biosciences, Victoria, BC, Canada), HSP 110 (SPA-1101, Assay Designs-Stressgen, Ann Arbor, MI), HSP 90 (SPA-830, Assay Designs-Stressgen), ␣B-crystallin (SMC 159 A, Stress Marq Biosciences), myosin (MF20; Developmental Studies Hybridoma bank, University of Iowa, Department of Biology, Iowa City, IA 52242), and actin (sc-1616; Santa Cruz Biotechnology, Santa Cruz, CA) primary antibodies. Protein detection was performed incubating the membranes with secondary antibodies NXA931, NA931, or NA934 (GE Healthcare) and using ECL Advance western blotting detection kit (RPN 2135, Amersham Biosciences) according to manufacturer’s instructions. All signal intensities were normalized to actin signal intensity. Autoradiograms were scanned (Molecular Dynamics), and intensity volumes of each band corresponding to specific antibodies were measured (ImageQuant TLv 2003, Amersham Biosciences). Determination of Myosin and Actin Composition Myofibrillar myosin and actin ratios were determined by 12% SDS-PAGE (45). The separating gels were stained with Coomassie blue and subsequently scanned in a soft laser densitometer (Molecular Dynamics). Intensity volumes of myosin and actin were measured for each sample (ImageQuant TLv 2003, Amersham Biosciences). In addition, separate immunoblots were stained with specific antibodies targeting sarcomeric myosin and actin as described above. Statistical Analysis For the single muscle fiber size and function, Sigma Stat software (Jandel Scientific) was used to generate descriptive statistics. Given the small number of hybrid type I/IIa fibers observed in the single muscle fiber experiments, comparisons were restricted to type I, IIa, IIa/IIx, and IIx. A two-way ANOVA (day ⫻ fiber type) was performed followed by the Tukey’s test. Values are means and standard error of means (SE). RESULTS

Single Muscle Fiber Size and Function (Day 5 vs. Day 1) After permeabilization, biceps femoris muscle fibers were isolated from the bundles and mounted for analysis of CSA. Maximal force production at optimal sarcomere length for force production was measured at the single muscle fiber level Physiol Genomics • VOL

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and normalized to CSA, i.e., P0/CSA. The MyHC isoform expression in each muscle fiber was determined by sensitive SDS-PAGE, and a total of 28 type I, 20 type IIa, 9 type IIa/IIx, and 11 type IIx fibers were included in the analyses. No significant difference was observed in MyHC isoform expression between day 5 and day 1 (Fig. 1A). The CSA was maintained in fibers expressing type I, IIa, and IIa/IIx MyHC isoforms from piglets exposed to mechanical ventilation for 5 days (Fig. 1, B and C). On day 1, the mean CSAs were 950 ⫾ 90 (type I, n ⫽ 15), 1,250 ⫾ 100 (type IIa, n ⫽ 10), 1,480 ⫾ 170 (type IIa/IIx, n ⫽ 5), and 1,320 ⫾ 180 ␮m2 (type IIx, n ⫽ 4). On day 5, the corresponding mean CSAs were 1,160 ⫾ 90 (type I, n ⫽ 13), 990 ⫾ 100 (type IIa, n ⫽ 10), 840 ⫾ 190 (type IIa/IIx, n ⫽ 4), and 1,100 ⫾ 150 ␮m2 (type IIx, n ⫽ 7). P0/CSA was also preserved in fibers expressing type I, IIa, and IIa/IIx MyHC isoforms from piglets exposed to mechanical ventilation for 5 days (Fig. 1D). On day 1, the mean P0/CSA was 14.00 ⫾ 1.70 (type I, n ⫽ 15), 13.80 ⫾ 2.10 (type IIa, n ⫽ 10), 12.70 ⫾ 2.80 (type IIa/IIx, n ⫽ 5), and 12.30 ⫾ 2.50 Ncm⫺2 (type IIx, n ⫽ 4). On day 5, the mean P0/CSA was 10.50 ⫾ 1.80 (type I, n ⫽ 13), 12.90 ⫾ 2.10 (type IIa, n ⫽ 10), 12.80 ⫾ 2.70 (type IIa/IIx, n ⫽ 4), and 12.70 ⫾ 1.50 Ncm⫺2 (type IIx, n ⫽ 7). Gene Expression (Day 5 vs. Day 3 vs. Day 1) Primary visualization of gene expression data distribution in each biopsy sample was acquired by an unsupervised hierarchical clustering method. Profiles of the biopsies taken at similar time points clustered together and showed a similar expression pattern. Differential expressions of each transcript (gene) in different profiles (each sample) and in different clusters (group/cluster of samples) are shown by the heat map (Fig. 2). In the following, transcription and expression refer to mRNA expression and does not indicate protein localization unless stated otherwise. Day 3 vs. Day 1 A total of 378 differentially expressed probe sets showed a twofold or greater change on day 3. Of these probes, 133 were upregulated and 245 were downregulated. The summary of major functional groups for upregulated genes is presented in Table 1, and the summary of downregulated genes at day 3 is presented in Table 2. Upregulated transcripts day 3. A summary of genes listed and fold change is given in Table 1. PROTEIN CATABOLISM/PROTEIN METABOLISM. Upregulated transcripts of atrogin-1, a 26S proteasome subunit, ubiquitinbinding protein, and ubiquitylation factor E4a, a E4 u-box type ubiquitin ligase suggest that ubiquitin proteasome system (UPS) is active at the mRNA level, in the early phase of muscle unloading (12). Upregulation of cathepsin D mRNA, a protease associated with the lysosome autophagy system, implies that more than one protein degradation mechanism is likely to be active at the transcriptional level (10). SERINE/THREONINE-PROTEIN KINASE. This functional group includes glycogen synthase kinase 3␤ (GSK-3␤), a negative regulator of muscle growth, protein kinase-C␣ (PKC-␣), and calcium-calmodulin-dependent protein kinase II beta. PKC-␣ www.physiolgenomics.org

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Fig. 1. Fiber type distribution, muscle fiber cross-sectional area (CSA) and function. A: fiber type distribution of day 1 (gray) and day 5 (black) based on single fiber experiments. Values are means ⫹ SE. B: pictures of 2 fibers, one taken on day 1, the other on day 5. The scale bar represents 50 ␮m. C: CSA of muscle fibers expressing different MyHC isoforms on day 1 (black bars) and day 5 (gray bars). D: specific tension (P0/CSA) of type I, IIa, IIa/IIx, and IIx muscle fibers on day 1 (black bars) and day 5 (gray bars). Values are means ⫹ SE.

has been recently implicated in initiating UPS-driven protein degradation (90). REGULATION OF TRANSCRIPTION. Signal transducer and activator of transcription 5B, histone deacetylase 4 (HDAC4); a class II HDAC involved in repressing myogenic enhancing factor 2 (MEF 2), and inhibitor of DNA binding 1 (ID1); a negative regulator of MyoD, were among this set of genes (9, 43). CELL CYCLE/CELL SURVIVAL. This group includes p21, Gadd 45␣, and Bcl-2, all of which are involved in cell cycle arrest and cell survival. Downregulated transcripts day 3. A summary of genes listed and fold change are given in Table 2. COLLAGEN/SIGNAL PEPTIDE GLYCOPROTEINS/EXTRACELLULAR MATRIX. Downregulated expression of extracellular matrix (ECM)

proteins, mainly collagen isoforms, was a prominent feature in the early phase. mRNA of leucine-rich repeat-proteoglycans fibromodulin, asporin, keratocan, osteoglycin, lumican, all involved in assembly of ECM, and expression of ECM-glycoproteins were also downregulated. Microfibrillar associated protein 4 and 5 mRNA were downregulated more than fivefold. Transcripts of fibronectin and tenascin, two other proteins that modulate cell and ECM interactions, were also downregulated. CALCIUM BINDING EF-HAND. Expression of troponin C, a key regulatory protein in the initiation and relaxation of muscle contraction, was downregulated. This is the only sarcomeric protein that showed differential expression at this early phase. CHAPERONE DNAJ, COOH-TERMINAL/CHAPERONE/PROTEIN METABOLISM. Expressions of several HSP 40 family members with

unknown functions were downregulated in the early phase of Physiol Genomics • VOL

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immobilization and mechanical ventilation. Among these HSP 40 family members, DNAJB4, which is reported to be extensively localized in skeletal muscle involved in protein folding, was downregulated. MLF1, a highly expressed protein in skeletal muscle involved in suppressing intracellular protein aggregate formation in conjunction with chaperone MRJ (HSP 40), was also downregulated at the mRNA level (51). GLUCOSE METABOLISM. Three days of muscle unloading and mechanical ventilation resulted in decreased transcription of several genes involved in glucose metabolism, such as pyruvate kinase, enolase ␣, fructose 1– 6 biphosphatase, 2.3 biphosphoglycerate mutase, and glycogen synthase. TRANSCRIPTION AND TRANSCRIPTION REGULATION. mRNAs of peroxisome proliferative-activated receptor gamma, coactivator beta (PGC-1␤), a gene involved in mitochondrial biogenesis, was markedly downregulated (7.6-fold) (3), while actinbinding Rho-activating protein (STARS), implicated in stimulating SRF, was also downregulated (2). Downregulated transcription of insulin-like growth factor I (IGF-I) is consistent with the well-documented role it plays in muscle remodeling and fits with our observation of increased expression of atrogin-1, i.e., the decreased suppression of atrogin-1 via IGF/PI3K/Akt pathway will lead to increased expression of UPS (13, 74). Day 5 vs. Day 1 A total of 1,479 differentially expressed probe sets showed twofold or larger change on day 5. Of these probes, 790 transcripts were upregulated and 689 transcripts were downregulated. Sevwww.physiolgenomics.org

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5

5

5

3

3

3

3


Fig. 2. Hierarchical clustering of the gene expression data. Out of all the transcripts, the expression values were filtered to include the values within one standard deviation, as the complete expression image is too large. Note the clustering of the expression data of each probe into day 1, day 3, and day 5 (experiments) and visually verifiable similar expression pattern in each cluster.

enty-six transcripts were upregulated on both day 3 and 5. Of the 245 downregulated transcripts on day 3, 160 were downregulated on day 5. The approximate doubling of significantly altered transcripts on day 5 indicates a growing number of molecular networks involved in these physiological processes as the duration of the mechanical ventilation and muscle unloading increased. Upregulated transcripts day 5. A summary of genes listed and fold change is given in Tables 3 and 4. PROTEIN METABOLISM/CHAPERONE/HSPS. The muscle-specific Skp1-cul1-fbox (SCF) E3 ligase MAFbx (atrogin-1) and Fbxo 33, another SCF E3 ligase, which specifically target Y box protein 1 (YB-1), showed increased transcription (54). Other E3 ligases, CHFR, SUGT1, and Ubox protein RN 37 (UBOX5), showed a similar heightened expression. The upregulation of E4 enzyme associated with multiubiquitylation, UBE4A (UFD2), and E2 ubiquitin-conjugating enzymes UBE2L3 and UBE2J1, together with the majority of proteasome subunits, indicate that UPS is active at the mRNA level. Increased expression of cathepsins D, B, K, and Z suggests that autophagic lysosomal pathways are active at the mRNA level in parallel with UPS at this stage. FoxO3 regulates autophagic lysosomal pathways in vitro via induction of autophagy-related genes in skeletal muscle (55, 92). HSPs showed increased expression, suggesting a key role for molecular chaperones and a severe cellular stress situation. Expression of several HSP 70 family members was upregulated, namely HSP 70-2 (8-fold), HSP 70-6, and HSP 70-4. Increased HSP 70 expression has recently been associated with antiapoptotic signaling in long-term cultured myotubes (25). Members of the HSP 40 family, which enhance the chaperone activity of HSP 70, DNAJC16, and DNAJA1, were upregulated (58). mRNA of Physiol Genomics • VOL

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HSP 60, which assists in folding and assembly of proteins and protecting proteins under stress, was also upregulated (17, 56). Within the HSP 90 family, HSP90AA1, HSP90AB1, and HSP90B1 (GRP94) show increased transcription around threefold or higher. HSP90AB1 is a cochaperone associated with myosin assembly (8). Transcription of HSP 110/105, a primary HSP in mammalian cells that selectively recognize and protect denatured proteins, was upregulated more than eightfold (65). Furthermore, HSP 70 cochaperone Bcl2-associated athanogene 3 (BAG3), which has recently been implicated in maintaining Z-disk integrity under stress, showed a twofold increased expression (32). In addition, small heat shock proteins (sHSP) ␣B-crystalline (CRYAB) and HSPB8 (HSP22) were also upregulated. CRYAB and HSP 22 are molecular chaperones and show enhanced synthesis in response to stress (4, 79). RNA METABOLISM AND PROCESSING. EIF2C2 (argonaute2), exportin 5, and mir21 mRNA were upregulated. These molecules act in concert in an mRNA-silencing mechanism (68). It was recently shown that miR21 could silence troponin mRNA (93). 1EIF3S3, subunit of eukaryotic initiation factor 3, was upregulated 20-fold. Upregulation of many nucleolar genes involved in ribosome biogenesis and pre-mRNA processing indicates an active transcription machinery at this stage. TRANSCRIPTION/REGULATION OF TRANSCRIPTION. PPAR-␤/␦, the predominant isoform present in muscle, which plays a major role in fatty acid catabolism and tissue remodeling in skeletal muscle, showed increased expression (76). Surprisingly, mRNA of MyoD and MEF 2a was also upregulated twofold. Increased expression of ID1 and ID2 suggests that they may repress MyoD-mediated transcription by binding to E www.physiolgenomics.org

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Table 1. Upregulated transcripts day 3 Human Homolog

Gene Name

Functional Category

Fold Change

Protein Catabolism/Protein Metabolism 200766_at 202038_at 208777_s_at 224878_at 209943_at 225801_at

CTSD UBE4A PSMD11 NP_061989 FBXL4 FBXO32

209945_s_at 203935_at 213093_at 210404_x_at

GSK3B ACVR1 PRKCA CAMK2B

cathepsin D (lysosomal aspartyl peptidase) ubiquitination factor E4A (UFD2 homolog, yeast) proteasome 26S subunit, nonATPase, 11 ubiquitin-binding protein homolog F-box and leucine-rich repeat protein 4 F-box protein 32

2.03 2.05 2.07 2.67 2.63 3.38

Serine/Threonine-Protein Kinase glycogen synthase kinase 3␤ activin A receptor, type I protein kinase C␣ calcium/calmodulin-dependent protein kinase II␤

2.31 2.21 2.17 2.02

Regulation of Transcription 212550_at 1554322_a_at 208937_s_at 210426_x_at

STAT5B HDAC4 ID1 RORA

signal transducer and activator of transcription 5B histone deacetylase 4 inhibitor of DNA binding 1 rar-related orphan receptor A

2.49 2.58 3.37 2.12

Cell Cycle/Cell Survival 202284_s_at 203725_at 203685_at

CDKN1A GADD45A BCL2

cyclin-dependent kinase inhibitor 1A (P21, CIP1) growth arrest and DNA damage-inducible-␣ B-cell cell/lymphoma 2

242458_at 221523_s_at 226576_at 212651_at

RALGPS2 RRAGD ARHGAP26 RHOBTB1

3.24 4.30 2.15

G Protein-related ral GEF with pH domain and Sh3 binding motif 2 ras-related GTP binding D rho GTPase-activating protein 26 rho-related BTB domain containing 1

2.30 2.03 2.10 2.29

P ⬍ 0.05.

proteins, thereby delaying downstream expression (38). Moreover, increased C-MYC expression may explain the enhanced HSP 70 expression, as C-MYC has been implicated in binding to the promoter region of HSP 70 and regulating its expression (85). Heightened expression of Runx1 (5-fold), a protein directly associated with preventing atrophy and myofibrillar disorganization, suggests that compensatory mechanisms are likely to play a decisive role in this phase of the muscle immobilization (88). CELL CYCLE REGULATION/PROTEIN KINASE/ATP BINDING. A massive upregulation was observed in Gadd45␣ and p21 mRNA, two proteins associated with survival signaling and cell cycle arrest. Increased expression of GSK-3␤ could lead to decreased protein synthesis by inhibiting translation, if protein localization follows a similar pattern (71). OXIDATIVE STRESS RESPONSE. Heightened expression of oxidative stress response genes, namely sulfiredoxin 1 (⬎30-fold), thioredoxin system, and SOD2, suggests that oxidative stress may be a major effect in skeletal muscle immobilization. SMALL G PROTEIN/GTPASE RELATED. Members of the small GTP binding protein superfamily are known to regulate a spectrum of cellular responses, ranging from reorganizing actin cytoskeleton to gene expression. In this study, nearly 20 transcripts related to small G proteins were upregulated. These include GEFs (guanine nucleotide exchange factors), which are responsible for activating small G proteins, as well as GAPs (GTPase-activating proteins), which are involved in inactivating G protein activity. Downregulated transcripts day 5. Summary of gene list and fold change are given in Tables 5 and 6. Physiol Genomics • VOL

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HYDROGEN ION TRANSPORTER/OXIDATIVE PHOSPHORYLATION.

Components of the mitochondrial electron transport chain and several genes belonging to complexes of the electron transport chain showed decreased mRNA expression. Namely, ubiquinol-cytochrome c reductase core protein 1 (complex III), Succinate dehydrogenase complex, subunit C, integral membrane protein (complex II), cytochrome C-1 (complex II), cytochrome c oxidase subunit VA (complex IV), ubiquinol cytochrome c reductase, subunit VII (complex III), 16 subunits of NADH dehydrogenase (complex I), and ATPase synthase subunits (complex V) were downregulated. Provided that the protein localization shows a similar trend as transcription, the cellular energy pathways would be severely affected in this phase of the immobilization. The decreased transcription of adenylate kinase and pyruvate kinase further suggest that 5 days of immobilization and mechanical ventilation may lead to an energy crisis in skeletal muscle. CARBOHYDRATE METABOLISM. Continued downregulation of gene transcripts involved in glycolysis and gluconeogenesis, namely phosphofrucktokinase, biphosphoglycerate mutase, fructose 1– 6 biphosphatase, and triosephosphate isomerase, suggests that the energy pathways may be further affected. Decreased mRNA expression of PGC-1␤ (11-fold) and PGC-1␣, the two gene products involved in mitochondrial biogenesis and mitochondrial energy metabolism, strengthens the notion of energy crisis in skeletal muscle at 5 days of immobilization (3, 81, 89). HEME BINDING PROTEINS. Hemoglobin-␣, hemoglobin-␤, and myoglobin mRNA were downregulated, which could result in a possible hypoxic condition at 5 days of immobilization, at the protein level. www.physiolgenomics.org

147


Table 2. Downregulated transcripts day 3 Human Homolog

Gene Name

Functional Category

Fold Change

Collagen/Signal Peptide/Glycoprotein/ECM collagen, type VI, ␣1 C1QTNF3 collagen, type XXI, ␣1 collagen, type XIV, ␣1 (undulin) collagen, type I, ␣2 collagen, type V, ␣1 collagen, type XII, ␣1 collagen, type I, ␣1 frizzled-related protein microfibrillar-associated protein 4 microfibrillar-associated protein 5 fibromodulin asporin (lrr class 1) keratocan osteoglycin (osteoinductive factor, mimecan) lumican fibronectin 1 tenascin XB

212091_s_at 220988_s_at 208096_s_at 216866_s_at 202404_s_at 203325_s_at 225664_at 202312_s_at 244419_at 212713_at 209758_s_at 202709_at 224396_s_at 220504_at 222722_at 229554_at 211719_x_at 213451_x_at

COL6A1 C1QTNF3 COL21A1 COL14A1 COL1A2 COL5A1 COL12A1 COL1A1 FRZB MFAP4 MFAP5 FMOD ASPN KERA OGN LUM FN1 TNXB

201063_at 209904_at 202855_s_at 207050_at

RCN1 TNNC1 SLC16A3 CACNA2D1

⫺2.30 ⫺5.79 ⫺2.60 ⫺2.35 ⫺2.76 ⫺3.00 ⫺3.26 ⫺3.39 ⫺2.79 ⫺5.21 ⫺5.65 ⫺3.76 ⫺4.95 ⫺4.84 ⫺2.78 ⫺2.82 ⫺3.07 ⫺3.42

Calcium-binding EF-hand/Protein Metabolism/Ion Transport reticulocalbin 1 troponin C type 1 (slow) solute carrier family 16 member 3 CACNA2D1

⫺2.51 ⫺5.69 ⫺6.10

Chaperone DnaJ, COOH-terminal/Chaperone/Protein Metabolism 225061_at 207453_s_at 203810_at 204784_s_at

DNAJA4 DNAJB5 DNAJB4 MLF1

DnaJ (HSP 40) homolog, subfamily A, member 4 DnaJ (HSP 40) homolog, subfamily B, member 5 DnaJ (HSP 40) homolog, subfamily B, member 4 myeloid leukemia factor 1

⫺4.18 ⫺4.15 ⫺2.79 ⫺4.26

Glucose Metabolism 206844_at 201251_at 203502_at 217294_s_at 201673_s_at

FBP2 PKM2 BPGM ENO1 GYS1

fructose-1,6-bisphosphatase 2 pyruvate kinase, muscle 2,3-bisphosphoglycerate mutase enolase 1␣ glycogen synthase 1 (muscle)

⫺2.43 ⫺2.64 ⫺4.81 ⫺2.14 ⫺3.05

Transcription/Transcription Regulation/Growth Factor 1553639_a_at 204203_at 1552732_at 202241_at 209542_x_at 206254_at 203851_at

PPARGC1B CEBPG NP_631905 TRIB1 IGF1 EGF IGFBP6

207574_s_at 224836_at 216952_s_at 219829_at 221051_s_at 202362_at 225166_at

GADD45B TP53INP2 LMNB2 ITGB1BP2 ITGB1BP3 RAP1A ARHGAP18

PPARGC1B CCAAT/enhancer binding protein (C/EBP) ␥ actin-binding rho-activating protein tribbles homolog 1 (Drosophila) insulin-like growth factor 1 (somatomedin C) epidermal growth factor (␤-urogastrone) insulin-like growth factor binding protein 6

⫺7.64 ⫺2.32 ⫺3.38 ⫺3.30 ⫺2.45 ⫺7.62 ⫺2.90

Miscellaneous growth arrest and DNA damage-inducible-␤ tumor protein P53 inducible nuclear protein 2 lamin B2 integrin-␤1 binding protein (melusin) 2 integrin-␤1 binding protein 3 RAP1A, member of ras oncogene family rho GTPase-activating protein 18

⫺2.33 ⫺8.58 ⫺2.26 ⫺3.37 ⫺11.44 ⫺2.34 ⫺2.21

P ⬍ 0.05. COLLAGEN. The number of downregulated collagen transcripts continued to increase after 5 days of immobilization, suggesting that ECM is further affected by decreased synthesis. SARCOMERIC PROTEINS. The reduced expression of the regulatory thin filament proteins, troponin T1, troponin I3, troponin I1, troponin C1; the subunits of troponin complex, together with tropomyosin-␤ and tropomyosin-␣ suggests that the synthesis of Ca2⫹-dependent contractile machinery is decreased in

Physiol Genomics • VOL

39 •

this phase of immobilization. Expression of ␣-actinin, a constituent of z-line was downregulated fivefold. Significant downregulation was also observed in the expression of thick filament proteins such as in the M-band associated myomesin 1 and myomesin 2, MyHC 8 (skeletal, perinatal), MyHC 7 (beta cardiac or type I), MyHC 1 (type IIx/IId), and the myosin-associated myosin binding protein C (MyBP-C, slow type). The giant sarcomeric proteins titin and nebulin were also www.physiolgenomics.org

148



Gene Name

Functional Category

Fold Change

Protein Metabolism/Chaperone/Heat Shock Proteins 225801_at 227521_at 218803_at 224309_s_at 215544_s_at 202038_at 200682_s_at 217826_s_at 200766_at 227961_at 202450_s_at 210042_s_at 200830_at 201388_at 201274_at 208777_s_at 212296_at 200039_s_at 200820_at 204219_s_at 235573_at 221667_s_at 211015_s_at 200599_s_at 200807_s_at 211538_s_at 213418_at 211969_at 214359_s_at 200881_s_at 212911_at 221667_s_at 212009_s_at 230031_at 217911_s_at 200825_s_at 209283_at

FBXO32 FBXO33 CHFR SUGT1 RN37_HUMAN UBE4A UBE2L3 UBE2J1 CTSD CTSB CTSK CTSZ PSMD2 PSMD3 PSMA5 PSMD11 PSMD14 PSMB2 PSMD8 PSMC1 HSPH1 HSPB8 HSPA4 TRA1 HSPD1 HSPA2 HSPA6 HSPCA HSPCB DNAJA1 Q9Y2G8 HSPB8 STIP1 HSPA5 BAG3 HYOU1 CRYAB

F-box protein 32 F-box protein 33 CHFR SGT1, suppressor of G2 allele of SKP1 U-box domain containing 5 ubiquitination factor E4A ubiquitin-conjugating enzyme E2L 3 ubiquitin-conjugating enzyme E2, J1 cathepsin D cathepsin B cathepsin K (pycnodysostosis) cathepsin Z proteasome 26S subunit, nonATPase, 2 proteasome 26S subunit, nonATPase, 3 proteasome subunit, ␣-type, 5 proteasome 26S subunit, nonATPase, 11 proteasome 26S subunit, nonATPase, 14 proteasome subunit, ␤-type, 2 proteasome 26S subunit, nonATPase, 8 proteasome 26S subunit, ATPase, 1 heat shock 105 kDa/110 kDa protein 1 heat shock 22 kDa protein 8 heat shock 70 kDa protein 4 heat shock 90 kDa ␤ (GRP94), member 1 heat shock 60 kDa protein 1 (chaperonin) heat shock 70 kDa protein 2 heat shock 70 kDa protein 6 (HSP70B’) heat shock protein 90 kDa ␣, A1 heat shock protein 90 kDa ␣, B1 DnaJ (HSP40) homolog, subfamily A, member 1 DnaJ (HSP40) homolog, subfamily C, member 16 heat shock 22 kDa protein 8 stress-induced-phosphoprotein 1 heat shock 70 kDa protein 5 (GRP 78) BCL2-associated athanogene 3 hypoxia upregulated 1 crystallin ␣B

3.00 2.08 2.06 2.54 2.17 2.04 2.12 2.40 4.52 2.31 2.88 2.47 3.62 3.50 3.50 3.20 3.18 3.14 3.05 3.02 8.01 2.77 2.93 2.91 2.00 8.18 3.66 3.50 2.85 5.61 2.33 2.77 3.63 2.10 2.46 2.08 3.14

RNA Metabolism and Processing 224917_at 223055_s_at 225827_at 201592_at

Q96GC9 XPO5 EIF2C2 EIF3S3

microRNA 21 exportin 5 eukaryotic translation initiation factor 2C, 2 EIF3S3

2.57 2.93 2.41 20.74

P ⬍ 0.05.

downregulated at the mRNA level. Muscle-specific ring finger protein, MuRF 2, also showed decreased transcription. TRANSCRIPTION, DIFFERENTIATION, INTRACELLULAR SIGNALING, AND GROWTH FACTORS. Expression of MEF 2c, a protein that

regulates sarcomere integrity and myofibril assembly, was downregulated (30, 69). The two growth factors responsible for muscle hypertrophy, IGF-I and IGF-II mRNA, were also downregulated. Epidermal growth factor (EGF) expression was drastically downregulated 18-fold. Two circadian genes, PER 2 and PER 3, showed decreased expression above twofold. Recently, 215 genes were identified as circadian transcripts in skeletal muscle, most of which were associated with transcription, protein synthesis, and degradation (59). Caveolin-1, ATF2, CREB5, Triadin, Calsequestrin 2, and STARS also showed decreased expression. TRANSITION METAL ION BINDING/LIM PROTEINS. A number of genes that belongs to LIM (Lin11, Isl-1, and Mec) proteins were downregulated at the transcriptional level. Elfin, an abundantly Physiol Genomics • VOL

39 •

expressed protein in skeletal muscle and shown to be colocalized with ␣-actinin in rat myocardium, showed a decreased expression. FHL3 mRNA was downregulated at a similar scale. LIM and cysteine rich domain 1 (LIMCD1/dyxin) showed a more than eightfold decreased transcription. Expression of cysteine and glycine-rich protein 3 (CSRP3), also known as MLP, was downregulated 16-fold, while expression of LIM domain binding 3 (LDB3/ Cypher), a protein associated with maintenance of Z-line integrity during contraction, was also downregulated drastically. Nebulin related anchoring protein (NRAP), a protein implicated in the assembly of myofibrils, also showed decreased expression (20). BIOPOLYMER MODIFICATION/KINASE ACTIVITY. Integrin beta binding protein 3 showed an almost sevenfold decreased transcription. Calcium/calmodulin-dependent kinase-␣ (CamKII-␣) mRNA was downregulated. SRF and CREB are reported to be substrates of CamKII in skeletal muscle. Mitogen-activated protein kinase kinase 6 (MAP2K6), an upstream activator of www.physiolgenomics.org


149


Gene Name

Functional Category

Fold Change

Transcription/Regulation of Transcription 206657_s_at 208328_s_at 201471_s_at 208937_s_at 201565_s_at 202431_s_at 211181_x_at 212695_at

MYOD1 MEF2A OSIL ID1 ID2 MYC RUNX1 CRY2

myogenic differentiation 1 myocyte enhancer factor 2A sequestosome 1 inhibitor of DNA binding 1, inhibitor of DNA binding 2, myc runt-related transcription factor 1 cryptochrome 2 (photolyase-like)

2.09 2.03 3.46 2.45 2.15 2.13 5.72 2.20

Cell Cycle Regulation/Protein Kinase/ATP Binding 203725_at 202284_s_at 209945_s_at 218181_s_at 218205_s_at

GADD45A CDKN1A GSK3B Q9NX89 MKNK2

growth arrest and DNA damage-inducible-␣ cyclin-dependent kinase inhibitor 1A (P21, CIP1) glycogen synthase kinase 3␤ mitogen-activated protein kinase kinase kinase kinase 4 MAP kinase-interacting serine/threonine kinase 2

9.78 4.53 3.44 2.31 2.41

Oxdative Stress Response 225252_at 215223_s_at 225302_at 208680_at 208864_s_at 201588_at 201266_at 225609_at

SCRT2 SOD2 TXNDC10 PRDX1 TXN TXNL1 TXNRD1 GSR

204141_at 242458_at 204803_s_at 223691_at 204819_at 219026_s_at 225347_at 227633_at 202810_at

TUBB RALGPS2 RRAD Q8NE09 FGD1 NP_060895 ARL10B RHEB DRG1

sulfiredoxin 1 homolog (S. cerevisiae) superoxide dismutase 2, mitochondrial thioredoxin domain containing 10 peroxiredoxin 1 thioredoxin thioredoxin-like 1 thioredoxin reductase 1 glutathione reductase

30.44 2.76 2.05 2.87 3.58 3.10 3.33 4.36

GTP Binding/Small GTPase Regulator Activity tubulin-␤2a RALGPS2 ras-related associated with diabetes regulator of G protein signaling 22 fyve, rhoGEF and pH domain containing 1 ras protein activator like 2 ADP-ribosylation factor-like 8a ras homolog enriched in brain DRG1

4.12 3.65 3.10 2.80 2.79 2.78 2.56 2.53 2.51

P ⬍ 0.05.

p38MAPK and MAPK12 (p38 ␥), showed decreased expression. Activin receptor II b (ActR-IIb), which is associated with myostatin binding, also showed decreased transcription (70). SARCOLEMMAL AND SARCOPLASMIC PROTEINS. Many members of the dystrophin glycoprotein complex (DGC) were downregulated at the mRNA level, namely dystrophin, syntrophin-␤, dystrobrevin-␣, dystroglycan-␣, sarcoglycan-␥, sarcoglycan-␦, and sarcospan. Expression of sarcoplasmic/endoplasmic reticulum Ca2⫹ ATPase 1 (SERCA 1) and SERCA 2 was downregulated more than threefold. SMALL G PROTEINS/G PROTEIN RELATED. Several small G protein-related transcripts belong to RAP and RAB subfamilies, G protein-activating GEFs were downregulated together with decreased expression of RGS3, a G protein-inhibiting (GTPase activating protein; GAP) protein, and G protein-coupled receptor GPR133. Upregulated transcripts day 5 vs. day 3. A summary of the gene list and fold change is given in Table 7. HSP transcripts and related chaperone expression were significantly upregulated on day 5 when compared with day 3. The oxidative stress response transcripts also showed the same trend. Furthermore, Cathepsin mRNA and transcripts of the proteasome subunits showed increased expression compared with day 3. Physiol Genomics • VOL

39 •

Downregulated transcripts day 5 vs. day 3. A summary of the gene list and fold change is given in Table 8. Sarcomeric protein mRNA were downregulated including two myosin isoforms, titin, MyBP-C, and MuRF 2. LIM protein transcription showed a similar downregulation. VALIDATION OF MICROARRAY DATA. According to real-time RT-PCR analyses, type I (⫺6-fold, P ⬍ 0.038) and IIx (⫺8fold, P ⬍ 0.001) MyHCs were significantly downregulated, while atrogin-1 (3.2-fold, P ⬍ 0.047) and HSP 110/105 (6fold, P ⬍ 0.029) were significantly upregulated after 5 days of muscle unloading and mechanical ventilation (Fig. 3). Actin expression did not change significantly. Immunoblotting for HSP 70, ␣B-crystallin, and HSP 90 showed a trend toward increased protein levels on day 5, but HSP 110 protein was unchanged on day 5 (Fig. 4). The myosin-actin ratio calculated from 12% SDS-PAGE showed similar ratios on day 1 (2.12 ⫾ 0.04, mean ⫾ SE) vs. day 5 (2.04 ⫾ 0.04). Similar results were obtained by targeting myosin and actin by specific antibodies (Fig. 4) for day 1 (3.97 ⫾ 0.371, mean ⫾ SE) and day 5 (2.95 ⫾ 0.357). The lack of an altered myosin-actin protein ratio despite a significant downregulation of myosin at the transcriptional level is not surprising due to the slow turnover www.physiolgenomics.org

150



Gene Name

Functional Category

Fold Change

Hydrogen Ion Transporter/Oxidative Phosphorylation 201903_at 216591_s_at 201066_at 203663_s_at 201568_at 203621_at 203371_s_at 218320_s_at 202839_s_at 211752_s_at 201740_at 208714_at 203039_s_at 201966_at 203613_s_at 203606_at 218201_at 217773_s_at 207335_x_at 207573_x_at 208972_s_at 201322_at 228168_at 202587_s_at 201251_at

UQCRC1 SDHC CYC1 NDUFB3 UCRQ_HUMAN NDUFB5 NDUFB3 NDUFB11 NDUFB7 NDUFS7 NDUFS3 NDUFV1 NDUFS1 NDUFS2 NDUFB6 NDUFS6 NDUFB2 NDUFA4 ATP5I ATP5L ATP5G1 ATP5B ATP5G3 AK1 PKM2

ubiquinol-cytochrome C reductase core protein I succinate dehydrogenase complex, subunit C, 15 kDa cytochrome c-1 cytochrome c oxidase subunit VA ucrq_human NADH dehydrogenase 1 ␤-subcomplex, 5, 16 kDa NADH dehydrogenase 1 ␤-subcomplex, 3, 12 kDa NADH dehydrogenase 1 ␤-subcomplex, 11, 17.3 kDa NADH dehydrogenase 1 ␤-subcomplex, 7, 18 kDa NADH dehydrogenase Fe-S protein 7, 20 kDa NADH dehydrogenase Fe-S protein 3, 30 kDa NADH dehydrogenase flavoprotein 1, 51 kDa NADH dehydrogenase Fe-S protein 1, 75 kDa NADH dehydrogenase Fe-S protein 2, 49 kDa NADH dehydrogenase 1 ␤-subcomplex, 6, 17 kDa NADH dehydrogenase Fe-S protein 6, 13 kDa NADH dehydrogenase 1 ␤-subcomplex, 2, 8 kDa NADH dehydrogenase 1 ␣-subcomplex, 4, 9 kDa ATP5I ATP5L ATP5G1 ATP5B ATP5G3 adenylate kinase 1 pyruvate kinase, muscle

⫺2.09 ⫺2.88 ⫺2.32 ⫺2.05 ⫺2.36 ⫺2.36 ⫺2.08 ⫺2.16 ⫺2.06 ⫺3.53 ⫺3.17 ⫺3.03 ⫺3.55 ⫺2.23 ⫺2.39 ⫺2.21 ⫺2.75 ⫺2.13 ⫺2.22 ⫺3.04 ⫺2.66 ⫺2.11 ⫺2.24 ⫺6.34 ⫺2.78

Carbohydrate Metabolism 210976_s_at 203502_at 206844_at 200822_x_at 1553639_a_at 219195_at

PFKM BPGM FBP2 TPI1 PPARGC1B PPARGC1A

209116_x_at 214414_x_at 204179_at

HBB HBA1 MB

phosphofructokinase, muscle 2,3-bisphosphoglycerate mutase fructose-1,6-bisphosphatase 2 triosephosphate isomerase 1 PGC-1␤ PGC-1␣

⫺5.07 ⫺4.63 ⫺4.44 ⫺4.09 ⫺11.80 ⫺2.97

Heme Binding Proteins hemoglobin ␤ hemoglobin ␣1 myoglobin

⫺4.91 ⫺5.82 ⫺4.61

Sarcomeric Proteins 213201_s_at 205742_at 205177_at 222976_s_at 204083_s_at 205589_at 34471_at 216265_x_at 205951_at 214087_s_at 208195_at 205766_at 203862_s_at 205610_at 205826_at 205054_at 236175_at

TNNT1 TNNI3 TNNI1 TPM3 TPM2 MYL3 MYH8 MYH7 MYH1 MYBPC1 TTN TCAP ACTN3 MYOM1 MYOM2 Q6P516 MuRF2

troponin T type 1 (skeletal, slow) troponin I type 3 (cardiac) troponin I type 1 (skeletal, slow) tropomyosin 3 tropomyosin 2 (␤) myosin, light polypeptide 3, myosin, heavy polypeptide 8, perinatal myosin, heavy polypeptide 7, cardiac ␤ myosin, heavy polypeptide 1 myosin binding protein C, slow type titin titin-cap (telethonin) actinin ␣2 myomesin 1 (skelemin) 185 kDa myomesin (m-protein) 2, 165 kDa nebulin tripartite motif-containing 55

⫺4.58 ⫺2.95 ⫺4.13 ⫺2.20 ⫺3.25 ⫺6.37 ⫺2.01 ⫺2.85 ⫺2.12 ⫺2.79 ⫺2.44 ⫺4.00 ⫺5.16 ⫺4.50 ⫺3.57 ⫺2.78 ⫺2.04

P ⬍ 0.05.

rate of myosin, i.e., half-life of myosin has been reported to vary between 14 and 30 days (64, 75). DISCUSSION

The aim of this study was to analyze muscle fiber size and function together with the gene expression pattern of skeletal Physiol Genomics • VOL

39 •

muscle using a porcine ICU model, i.e., 5 days exposure to muscle unloading and mechanical ventilation. In a pilot study, we have previously shown that muscle fiber CSA is not affected by 5 days of mechanical ventilation and muscle unloading according to morphological analyses of enzymehistochemically stained cryo-sections (63). Due to the uncerwww.physiolgenomics.org

151



Gene Name

Functional Category

Fold Change

Collagen/Collagen Triple Helix Repeat collagen, type VI ␣1 collagen, type IV ␣5 C1QTNF3 collagen, type XXI ␣1 collagen, type XIV ␣1 (undulin) collagen, type XI ␣1 collagen, type I ␣2 scavenger receptor class A, member 5 collagen, type V ␣1 collagen, type I ␣1 collagen, type III ␣1

212091_s_at 213110_s_at 220988_s_at 208096_s_at 216866_s_at 37892_at 202404_s_at 235849_at 203325_s_at 202312_s_at 215076_s_at

COL6A1 COL4A5 C1QTNF3 COL21A1 COL14A1 COL11A1 COL1A2 Q6ZMJ2 COL5A1 COL1A1 COL3A1

207968_s_at 212984_at 205931_s_at 209542_x_at 202410_x_at 206254_at 211527_x_at 1552791_a_at 207317_s_at 1555131_a_at 208518_s_at 1552732_at

MEF2C ATF2 CREB5 IGF-I IGF-II EGF VEGF TRDN CASQ2 PER3 PER2 STARS

208690_s_at 218818_at 218574_s_at 205553_s_at 213717_at 235312_s_at

PDLIM1 FHL3 LMCD1 CSRP3 LDB3 NRAP

221051_s_at 213108_at 205698_s_at 206106_at 220028_at

ITGB1BP3 CAMK2A MAP2K6 MAPK12 ACVR2B

205444_at 212361_s_at 214708_at 211493_x_at 208086_s_at 212128_s_at 207302_at 210329_s_at 204963_at

ATP2A1 ATP2A2 SNTB1 DTNA DMD DAG1 SGCG SGCD SSPN

209539_at 202362_at 204974_at 219167_at 204681_s_at 225930_at 232267_at 220027_s_at 203823_at

ARHGEF6 RAP1A RAB3A RASL12 RAPGEF5 NKIRAS1 GPR133 RASIP1 RGS3

221232_s_at 208353_x_at

ANKRD2 ANK1/sANK1

⫺2.02 ⫺2.32 ⫺6.68 ⫺4.13 ⫺2.09 ⫺3.21 ⫺2.40 ⫺2.21 ⫺2.36 ⫺3.10 ⫺3.08

Transcription/Differentiation/Intracellular Signaling/Growth Factors myocyte enhancer factor 2c activating transcription factor 2 cAMP-responsive element binding protein 5 insulin-like growth factor 1 insulin-like growth factor 2 epidermal growth factor vascular endothelial growth factor triadin calsequestrin 2 (cardiac muscle) period homolog 3 (Drosophila) period homolog 2 (Drosophila) actin-binding rho-activating protein

⫺2.10 ⫺2.22 ⫺5.12 ⫺2.59 ⫺2.19 ⫺18.28 ⫺2.04 ⫺2.41 ⫺2.39 ⫺2.34 ⫺2.36 ⫺5.82

Metal Ion Binding/Transition Metal Ion Binding PDZ and LIM domain 1 (elfin) four and a half LIM domains 3 lim and cysteine-rich domains 1 cysteine and glycine-rich protein 3 LIM domain binding 3 nebulin-related anchoring protein

⫺2.58 ⫺2.42 ⫺8.13 ⫺16.23 ⫺5.08 ⫺4.58

Biopolymer Modification/Kinase Activity Integrin-␤1 binding protein 3 CAMK2A mitogen-activated protein kinase kinase 6 mitogen-activated protein kinase 12 activin A receptor, type IIB

⫺6.93 ⫺3.47 ⫺3.82 ⫺2.21 ⫺2.59

Sarcolemmal and Sarcoplasmic Proteins ATP2A1 ATP2A2 syntrophin-␤1 dystrobrevin-␣ dystrophin dystroglycan 1 sarcoglycan-␥ sarcoglycan-␦ sarcospan

⫺3.94 ⫺3.33 ⫺2.19 ⫺2.75 ⫺2.71 ⫺2.19 ⫺2.01 ⫺2.14 ⫺2.46

Small G Protein Related RAC/CDC42 (GEF) 6 rap1A, member of ras oncogene family rab3A, member ras oncogene family ras-like, family 12 rap (GEF) 5 NF-␬B inhibitor interacting ras-like 1 g protein-coupled receptor 133 Ras-interacting protein 1 regulator of G protein signaling 3

⫺2.29 ⫺2.40 ⫺3.00 ⫺2.22 ⫺2.54 ⫺2.02 ⫺2.21 ⫺2.86 ⫺2.86

Ankyrin Repeat ankyrin repeat domain 2 ankyrin 1, erythrocytic

⫺6.76 ⫺4.60 Continued


39 •

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152


Table 6.—Continued Human Homolog

207950_s_at 202921_s_at 1554577_a_at 230972_at 226425_at 229052_at 233292_s_at

Gene Name

ANK3 ANK2 PSMD10 ANKRD9 RSNL2 ANKRD23 ANKHD1

Functional Category

Fold Change

ankyrin 3, node of ranvier (ankyrin G) ankyrin 2, neuronal proteasome 26S subunit, nonATPase, 10 ankyrin repeat domain 9 restin-like 2 ankyrin repeat domain 23 ankyrin repeat and KH domain containing 1

⫺2.94 ⫺2.01 ⫺2.55 ⫺3.35 ⫺2.40 ⫺3.32 ⫺2.33

Heat Shock Related 204784_s_at 207453_s_at 203810_at 214767_s_at

MLF1 DNAJB5 DNAJB4 HSPB6

myeloid leukemia factor 1 DnaJB5 DnaJB4 Heat shock protein ␤6 (HspB6)

⫺23.67 ⫺3.93 ⫺2.13 ⫺6.19

P ⬍ 0.05.

tainty in muscle fiber CSA measurements in frozen muscle biopsies and the variations in sarcomere lengths between muscle samples (46, 47), measurements were done in single muscle fibers at a fixed sarcomere length in a larger group of animals on day 1 and day 5. The previous observation of a maintained muscle fiber size was confirmed and extended with regard to the maintained force-generation capacity at the single muscle fiber level on day 5 compared with control samples on day 1. These observations suggest that the size of the immobilized

muscle is not affected by the observed changes in gene expression; alternatively a primary protein protection mechanism is activated in the early phase, counteracting the adverse effects of immobilization. At the early phase of immobilization on day 3, two different proteolytic mechanisms were initiated, namely the ubiquitinproteasome pathway and the autophagic lysosomal system, indicated by the upregulation of atrogin-1 mRNA and cathepsin D mRNA, respectively. Furthermore, the increased expres-

Table 7. Upregulated transcripts day 5 vs. day 3 Human Homolog

Gene Name

Functional Category

235573_at 211969_at 214359_s_at 200807_s_at 230031_at 211538_s_at 221891_x_at 211015_s_at 213418_at 200881_s_at 200825_s_at 202558_s_at 212009_s_at 209283_at 221667_s_at 206375_s_at 202295_s_at 200766_at 200786_at 201274_at 200039_s_at 212296_at

HSPH1 HSPCA HSPCB HSPD1 HSPA5 HSPA2 HSPA1L HSPA4 HSPA6 DNAJA1 HYOU1 STCH STIP1 CRYAB HSPB8 HSPB3 CTSH CTSD PSMB7 PSMA5 PSMB2 PSMD14

heat shock 105 kDa/110 kDa protein 1 heat shock protein 90 kDa ␣, class A member 1 heat shock protein 90 kDa ␣, class B member 1 heat shock 60 kDa protein 1 (chaperonin) heat shock 70 kDa protein 5 heat shock 70 kDa protein 2 heat shock 70 kDa protein 8 heat shock 70 kDa protein 4 heat shock 70 kDa protein 6 (HSP70B’) DnaJ (HSP 40) homolog, subfamily A, member 1 hypoxia upregulated 1 stress 70 protein chaperone, microsome-associated, 60 kDa stress-induced-phosphoprotein 1 (HSP 70/HSP 90-organizing protein) crystallin, ␣B heat shock 22 kDa protein 8 heat shock 27 kDa protein 3 cathepsin H cathepsin D (lysosomal aspartyl peptidase) proteasome subunit, ␤-type, 7 proteasome subunit, ␣-type, 5 proteasome subunit, ␤-type, 2 proteasome 26S subunit, nonATPase, 14

208680_at 225252_at 225609_at 201266_at 208864_s_at 215223_s_at

PRDX1 SCRT2 GSR TXNRD1 TXN SOD2

peroxiredoxin 1 sulfiredoxin 1 homolog (S. cerevisiae) glutathione reductase thioredoxin reductase 1 thioredoxin superoxide dismutase 2, mitochondrial

Fold Change

Heat Shock and Protein Metabolism 15.18 4.80 2.31 2.62 2.81 4.19 2.54 4.30 6.47 10.05 2.79 4.33 2.62 3.21 4.28 2.05 2.61 2.22 2.00 2.63 2.10 2.39

Oxidative Stress Responsive Genes 2.17 23.99 2.54 4.38 3.07 4.69

Transcription Factors 211181_x_at 202723_s_at

RUNX1 FOXO1A

runt-related transcription factor 1 forkhead box O1A (rhabdomyosarcoma)

P ⬍ 0.05. Physiol Genomics • VOL

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5.93 2.04


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Table 8. Downregulated transcripts day 5 vs. day 3 Human Homolog

Gene Name

Functional Category

Fold Change

Sarcomeric Proteins and Related 214087_s_at 205826_at 205951_at 216265_x_at 205610_at 34471_at 222976_s_at 223762_at 205177_at 204083_s_at 213201_s_at 205766_at 203862_s_at 236175_at 208195_at

MYBPC1 MYOM2 MYH1 MYH7 MYOM1 MYH8 TPM3 TMOD4 TNNI1 TPM2 TNNT1 TCAP ACTN3 RNF29 TTN

myosin binding protein C, slow type myomesin (m-protein) 2, 165 kDa myosin, heavy polypeptide 1, skeletal muscle, adult myosin, heavy polypeptide 7, cardiac muscle-␤ myomesin 1 (skelemin) 185 kDa myosin, heavy polypeptide 8, skeletal muscle, perinatal tropomyosin 3 tropomodulin 4 (muscle) troponin i type 1 (skeletal, slow) tropomyosin 2 (␤) troponin t type 1 (skeletal, slow) titin-cap (telethonin) actinin, ␣2 MuRF-2 titin

208690_s_at 218574_s_at 205553_s_at 235312_s_at 218818_at 213717_at

PDLIM1 LMCD1 CSRP3 NRAP FHL3 LDB3

PDZ and LIM domain 1 (elfin) LIM and cysteine-rich domains 1 cysteine and glycine-rich protein 3 nebulin-related anchoring protein four and a half LIM domains 3 LIM domain binding 3

⫺3.22 ⫺4.28 ⫺2.33 ⫺3.27 ⫺3.60 ⫺2.23 ⫺2.42 ⫺2.09 ⫺3.94 ⫺4.71 ⫺4.24 ⫺3.26 ⫺3.43 ⫺2.11 ⫺2.42

LIM Proteins ⫺2.45 ⫺3.38 ⫺14.55 ⫺3.04 ⫺3.32 ⫺4.14

Miscellaneous 204784_s_at 213093_at 210329_s_at 209112_at 214767_s_at

MLF1 PRKCA SGCD CDKN1B HSPB6

myeloid leukemia factor 1 protein kinase C, ␣ sarcoglycan-␦ cyclin-dependent kinase inhibitor 1B (P27, KIP1) heat shock protein, ␣-crystallin-related, B6

⫺8.17 ⫺3.90 ⫺2.39 ⫺2.55 ⫺3.15

P ⬍ 0.05.

sion of GSK-3␤ suggests that basal protein turnover may be affected not only by an enhanced degradation but also by a reduction in protein synthesis (13). Upregulation of Gadd45-␣, p21, and Bcl 2 expression suggests that cell cycle arrest is a likely possibility preventing apoptosis and maintaining cell survival signals at this early stage. Gadd45-␤ and p21 expression has been shown to increase in acute quadriplegic myopathy (AQM) patients with severe atrophy, muscle weakness, and apoptotic features, representing later stages of the disease (21). HDAC4 is a transcriptional repressor of muscle-specific MEF 2 (61). Increased expression of HDAC4 indicates that muscle specific gene repression is initiated at this stage. This hypothesis is further strengthened by the increased transcription of ID1 protein, since ID1, by binding to E proteins inhibit MyoD-mediated transcription (38). Downregulation of IGF-I mRNA is consistent with the increased atrogin-1 expression, which is associated with myotube atrophy when the IGF-I/ PI3K/Akt protein synthesis pathway was inhibited (13). Downregulation of EGF mRNA may also play a role in the decreased protein synthesis, since EGF has been shown to mimic IGF-I acting via PI3K/Akt. Moreover EGF induces a stronger MAP kinase phosphorylation than IGF-I (26). PGC1-␤ is a major regulator of mitochondrial biogenesis and energy metabolism in skeletal muscle (3, 81), and the decreased expression of PGC1-␤ might be the initiating signal for the disrupted energy metabolism we observed on day 5. A wide range of collagen isoforms and other constituents of ECM were downregulated at the mRNA level, while only one myofibrillar protein was downregulated on day 3, i.e., troponin C (slow) mRNA, sugPhysiol Genomics • VOL

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gesting that decreased biosynthesis of collagen and ECM constituents is a primary characteristic of skeletal muscle immobilization. Intramuscular connective tissue accounts for 10% of skeletal muscle, and it is involved in passive elastic support and force transmission and provides mechanical support for vessels (40). In addition, ECM and integrins also play an important role as mechanosensors, influencing protein synthesis and degradation in skeletal muscle (40). Regulation of Sarcomeric Protein Synthesis and Degradation On day 5, it is evident that not only the expression of ECM proteins but also the majority of the components of sarcomere and DGC gene expression are downregulated. The subsarcolemmal DGC proteins are not only a structural component in skeletal muscle but are also included in a sarcolemmal signaling network and are disrupted in several myopathies (23). The mRNA of thick filament proteins such as type I and IIx/IId MyHC isoforms, as well as thin filament proteins, seems to be coordinately downregulated, which suggests that reduction in total muscle protein turnover is partly achieved by decreased expression of the majority of sarcomeric and many ECM proteins. ECM proteins play a vital role in providing mechanical support for nerves and vessels and, most importantly, lateral transmission of force between fibers and fascicles (40). The preferential loss of thick filament proteins, e.g., myosin, in critically ill ICU patients with AQM is well documented (44). Downregulation of myosin gene expression is also reported in other muscle remodeling conditions, which may indicate that www.physiolgenomics.org

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Fig. 3. Microarray results validated by real time RT-PCR. Atrogin-1 (A) and heat shock protein (HSP) 110/105 (B) are upregulated and type I (C), and type IIx (D) thick filament proteins are downregulated.

myosin is preferably decreased at the mRNA and protein levels (1, 5). However, results from our unique immobilization model, which is different from traditional bed rest, hindlimb unloading, and disuse models, suggest that not only myosin but also the majority of other sarcomeric proteins are coordinately downregulated before fiber atrophy and loss in force generation occur. Codownregulation of MEF 2c mRNA, a muscle transcription factor that is associated with myogenic differentiation, may explain the selective transcriptional downregulation

of thick filament proteins observed on day 5. Morpholino knockdowns show downregulation of thick filament mRNA and proteins as well as disrupted sarcomere assembly (30). MEF 2c mutant mice show sarcomere disorganization, mainly in M-band regions. Furthermore, myomesin 1 and myomesin 2 appear to be direct targets of MEF 2c (69). The results suggest that 5 days of sedation, muscle unloading, and mechanical ventilation initiate a program at the gene level for myofibrillar disassembly by reduced synthesis of

Fig. 4. Immunoblotting for HSP 70, HSP 90, and HSP 110 proteins. A: total protein samples (C1–C4) (individual and pooled) from day 1 compared with day 5, shows a trend in increasing signal intensities at day 5 with HSP 70, ␣B-crystallin (CRYAB) and HSP 90. B: HSP 70, ␣B-crystallin, HSP 90, and HSP 110 fold-changes were determined by signal volumes normalized to actin signal volumes. Physiol Genomics • VOL

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sarcomeric, DGC, and ECM proteins in a highly regulated manner. The constituent members of the two transverse centers of sarcomere organization, Z-line and M-band, seem to be targeted by this disassembly process. Titin and Murf2 are two proteins located at the M-band, involved in mechano-transduction-dependent transcriptional regulation in skeletal muscle (42). It has been proposed that the LIM domain-containing proteins, which localize in close proximity to the Z-disk, function as mechano/stress/stretch sensors (11, 33). MARP family ANKRD2 and DARP are also implicated in mechanostretch sensing (7, 41, 60). ANKRD2 is specifically shown to interact with Telethonin, a Z-disk protein, and also shown to enhance p53-mediated p21 expression (41). Thus, immobilization may result in loss of tensile/mechano sensors in the porcine model as an adaptive or feedback mechanism that in turn may play a critical role in initiating myofibril disassembly (Tables 5, 6). However, the maintained muscle fiber size and force generation capacity observed at this stage and, preceding the concomitant severe atrophy, loss of force and force-generating capacity (loss of force normalized to CSA) suggest that the dramatic changes at the gene level had no or only minor effects at the protein level. This is in part due to the fact that several of the affected proteins have half-lives that are significantly longer than 5 days (64, 75), but also to other mechanisms discussed below that may protect proteins from degradation. Increased transcription of atrogin-1, along with proteasome subunits and E2 ligases, demonstrates that UPS is activated early in the muscle unloading. Furthermore, parallel increased transcription of cathepsins B, D, K, and Z suggests that the autophagic lysosomal pathway is also activated in response to immobilization. It was recently shown that, in culture, the autophagic lysosomal pathway is the main contributor of proteolysis of myotubes (92). However, despite the increased transcription of the two major proteolytic systems in skeletal muscle, we did not observe any change in fiber CSA or force generation. HSPs Surprisingly, a large number of heat shock and related protein-mRNAs were upregulated, demonstrating a high molecular chaperone activity. In contrast, HSP downregulation has been described in gene expression studies of disuse atrophy (82), atrophy due to denervation, and atrophying nonmammalian muscle (73). Specifically, HSP 70 and other HSPs are mainly stress induced and involved in protein folding, refolding, and assembly (52, 53). Titin M-band-deficient mice show ␣B-crystallin and HSP 27 as well as proteasome upregulation (67). These two proteins were reported to be accumulated as insoluble complexes in disused rat soleus muscle, suggesting its chaperone activity in maintaining quality control of proteins (39). MEF 2 knockout zebrafish embryos with defects in sarcomere assembly also show upregulation of HSP 90, a cochaperone involved in myosin folding (8, 30). Recent publications suggest that HSP 90 act as a cochaperone with myosin chaperone UNC-45, regulating assembly of thick filaments (24). The protective role of HSPs against immobilization-induced muscle atrophy has been reported in numerous studies, and heating reverses immobilization-induced atrophy by induction Physiol Genomics • VOL

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of HSP 27 and HSP 70 protein (77). The downregulation of HSP 70 and HSP 25 proteins in response to prolonged hindlimb unloading (28 days) results in a 55% reduction in rat soleus muscle weight (48), although HSP 25 protein was reported to increase in response to short-term unloading (39). Furthermore, HSP 70 protein overexpression protects against immobilization-induced muscle fiber atrophy (78). In aged rats, endogenous HSP 70 protein is decreased compared with young rats, and a further reduction in HSP 70 protein level along with muscle atrophy is observed in immobilized old rats. However, overexpression of the HSP 70 protein in immobilized old-aged rats protected muscle cells from under going atrophy (22). The massive induction of a battery of HSP mRNAs in this study may accordingly be secondary to the young age of the piglets in this study. This is consistent with the lower incidence of muscle weakness in critically ill children in the ICU than in adult patients (6). Preliminary unpublished observations from our group in piglets exposed to corticosteroids, neuromuscular blocking agents, sepsis, immobilization, and mechanical ventilation showed a maintained muscle fiber size and dramatic decrease in force generation capacity at the end of the 5-day period. These piglets did not, on the other hand, show an upregulation of HSPs or related chaperones. Based on previous reports and our unpublished observations taken together, we speculate that the induction of HSPs in the immobilized porcine model acts as a protective mechanism for preventing myofibrillar protein degradation during the first 5 days of muscle unloading. Small G Proteins and Actin Cytoskeleton More than 20 small G proteins and related proteins (GEFs and GAPs) were significantly up- and downregulated at the mRNA level in this study. Small G proteins regulate multiple cellular functions by exerting multiple responses and mutual cross talk with other G protein cascades (57). A subfamily of small G proteins, Rho GTPases, plays a critical role in myofibrillogenesis (15, 34). It is speculated that Obscurin, a giant protein in the sarcomere with a Rho-GEF domain, may play a role in the remodeling of actin cytoskeleton during myofibrillogenesis (91) and is demonstrated to regulate titin assembly at the Z-disk (14). Furthermore, it has been shown that member proteins of DGC are codownregulated with H-Ras, Rac1, and Cdc42 expression and activity (19), and Ras-Raf-MAPK pathway has been reported to be involved in muscle protein degradation in a nonvertebrate model (84). How small G proteins act in concert in response to and/or initiating myofibrillar-protective mechanism is yet to be elucidated. However, the majority of small G proteins are implicated in actin cytoskeleton remodeling. It may be that small G proteins in their collective action are responsible for maintaining and preserving actin thin filament cytoskeleton, while the thick filaments are sacrificed first when proteolysis occur in immobilized muscle. Single Muscle Fiber Structure and Function Analyses The skinned muscle fiber preparation allows investigation of the function of myofilament proteins in a cell with an intact filament lattice, but without the confounding effects related to intercellular connective tissue or protein heterogeneity between cells of multicellular preparations (31). Other parameters of www.physiolgenomics.org

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importance for in vivo muscle function, which may be affected by long-term ICU treatment, are not assessable for study using the skinned fiber preparation, such as excitation-contraction coupling, muscle metabolism, energy stores, and motor recruitment pattern. The lack of muscle atrophy during the first 5 days in this study is in accordance with a previous pilot study (63). In the pilot study, a maintained muscle fiber size, according to morphometrical measurements on enzyme-histochemically stained muscle cross-sections, was observed in a piglet exposed to an endotoxin-induced sepsis, systemic corticosteroid hormone treatment, postsynaptic block of neuromuscular transmission, and mechanical ventilation for 5 days. Preliminary results from time-resolved analyses of muscle size and regulation of muscle contraction in 48 mechanically ventilated and pharmacologically paralyzed rats at durations varying from 6 h to 3 wk show a maintained muscle weight in five distal hindlimb muscles during the first 4 days and followed by an almost linear decrease resulting in a 50% loss in muscle mass and body

weight after 3 wk paralysis and mechanical ventilation (J. Ochala, A.-M. Gustafson, M. Llano Diez, G. Renaud, M. Li, S. Aare, R. Qaisar, V. Banduseela, Y. Hedstro¨m, X. Tang, B. Dworkin, and L. Larsson; unpublished observations). During this period, specific force declined by almost 90%, indicating a dramatic and preferential loss of contractile proteins. This is in accordance with our previous observations in critically ill ICU patients (44). Conclusion In this study we have analyzed the expression profile in response to immobilization and mechanical ventilation over a 5-day period in a unique porcine ICU model (63). We have observed a general downregulation in the transcripts of sarcomeric proteins, LIM proteins, ECM constituents, and transcripts of molecules involved in energy metabolism, while expression of genes involved in the UPS system, including

Fig. 5. Schematic representation. Proposed major molecular events and pathways of interest after 5 days of immobilization and mechanical ventilation. This diagram illustrates the hypothesis that despite increased ubiquitin-proteasome system and lysosome autophagy pathway, upregulation of HSPs protects myofibrils from undergoing protein degradation. Main molecular events depicted here derived from the expression profiling and single fiber force measurement are as follows. A: sarcomeric and sarcolemmal protein synthesis is downregulated. B: mitochondrial dysfunction by decreased expression of mitochondria and related genes. C: oxidative stress is indicated by the oxidative stress responsive genes, which may be a direct effect of mitochondrial dysfunction. D: upregulation of E2, E3 ligases, proteasome components, and cathepsins. E: upregulation of HSPs and molecular chaperones leading to protection from protein degradation and normal force generation.


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muscle-specific E3 ligase atrogin, transcripts of cathepsins, and transcripts of HSPs, was upregulated. However, we have observed no decrease in fiber CSA or force generation, suggesting that the expected atrophic changes have been countered by a protective mechanism. Hibernating wild black bears are able to conserve muscle strength and show no loss of fiber size or number (27, 86). Data from captive brown bears have also confirmed the sustained muscle strength in hibernation, but no specific molecular pathway was implicated in either study (29). Recently it has been shown that despite prolonged dormancy, hibernating bat muscles exhibit an elevation of HSPs without any atrophic changes. Induction of HSPs in response to periodic arousals, which is common to all hibernators, may in turn result in protection of myofibrillar proteins (49). Furthermore, HSP 70 overexpression attenuates immobilization-induced muscle atrophy in rats (78). Moreover, unpublished preliminary results from our group have shown a maintained muscle fiber size and a 40% reduction in force generation capacity in the absence of HSP upregulation in response to 5 days exposure to postsynaptic block of neuromuscular transmission, systemic treatment with corticosteroids, and sepsis, in addition to immobilization (anesthesia) and mechanical ventilation in young piglets. Taking these results together, we speculate that induction of HSPs may play an inherent temporary protective mechanism in skeletal muscle in the early stages of immobilization and mechanical ventilation. Further studies on the effects of various triggering factors common in the ICU condition, such as systemic corticosteroid hormone treatment and sepsis, together with immobilization and mechanical ventilation, on muscle morphology, regulation of muscle contraction, and chaperone mRNA and protein expression, may elucidate the exact role of HSPs in skeletal muscle at early stages of ICU conditions. The proposed molecular events leading to our final hypothesis are illustrated in Fig. 5. ACKNOWLEDGMENTS We are grateful to Yvette Hedstro¨m, Ann-Marie Gustafson, and Rebeca Corpen˜o Rodriguez for excellent technical assistance. We thank Dr. Rongye Shi for generating array data. GRANTS This study was supported by Swedish Research Council Grant 8651, Association Francaise contre les Myopathies, and the National Center for Medical Rehabilitation Research. REFERENCES 1. Acharyya S, Butchbach ME, Sahenk Z, Wang H, Saji M, Carathers M, Ringel MD, Skipworth RJ, Fearon KC, Hollingsworth MA, Muscarella P, Burghes AH, Rafael-Fortney JA, Guttridge DC. Dystrophin glycoprotein complex dysfunction: a regulatory link between muscular dystrophy and cancer cachexia. Cancer Cell 8: 421– 432, 2005. 2. Arai A, Spencer JA, Olson EN. STARS, a striated muscle activator of Rho signaling and serum response factor-dependent transcription. J Biol Chem 277: 24453–24459, 2002. 3. Arany Z, Lebrasseur N, Morris C, Smith E, Yang W, Ma Y, Chin S, Spiegelman BM. The transcriptional coactivator PGC-1beta drives the formation of oxidative type IIX fibers in skeletal muscle. Cell Metab 5: 35– 46, 2007. 4. Arrigo AP, Simon S, Gibert B, Kretz-Remy C, Nivon M, Czekalla A, Guillet D, Moulin M, Diaz-Latoud C, Vicart P. Hsp27 (HspB1) and alphaB-crystallin (HspB5) as therapeutic targets. FEBS Lett 581: 3665– 3674, 2007. 5. Banduseela V, Ochala J, Lamberg K, Kalimo H, Larsson L. Muscle paralysis and myosin loss in a patient with cancer cachexia. Acta Myol 26: 136 –144, 2007. Physiol Genomics • VOL

39 •

157

6. Banwell BL, Mildner RJ, Hassall AC, Becker LE, Vajsar J, Shemie SD. Muscle weakness in critically ill children. Neurology 61: 1779 –1782, 2003. 7. Barash IA, Bang ML, Mathew L, Greaser ML, Chen J, Lieber RL. Structural and regulatory roles of muscle ankyrin repeat protein family in skeletal muscle. Am J Physiol Cell Physiol 293: C218 –C227, 2007. 8. Barral JM, Hutagalung AH, Brinker A, Hartl FU, Epstein HF. Role of the myosin assembly protein UNC-45 as a molecular chaperone for myosin. Science 295: 669 – 671, 2002. 9. Bassel-Duby R, Olson EN. Signaling pathways in skeletal muscle remodeling. Annu Rev Biochem 75: 19 –37, 2006. 10. Bechet D, Tassa A, Taillandier D, Combaret L, Attaix D. Lysosomal proteolysis in skeletal muscle. Int J Biochem Cell Biol 37: 2098 –2114, 2005. 11. Boateng SY, Goldspink PH. Assembly and maintenance of the sarcomere night and day. Cardiovasc Res 77: 667– 675, 2008. 12. Bodine SC, Latres E, Baumhueter S, Lai VK, Nunez L, Clarke BA, Poueymirou WT, Panaro FJ, Na E, Dharmarajan K, Pan ZQ, Valenzuela DM, DeChiara TM, Stitt TN, Yancopoulos GD, Glass DJ. Identification of ubiquitin ligases required for skeletal muscle atrophy. Science 294: 1704 –1708, 2001. 13. Bodine SC, Stitt TN, Gonzalez M, Kline WO, Stover GL, Bauerlein R, Zlotchenko E, Scrimgeour A, Lawrence JC, Glass DJ, Yancopoulos GD. Akt/mTOR pathway is a crucial regulator of skeletal muscle hypertrophy and can prevent muscle atrophy in vivo. Nat Cell Biol 3: 1014 – 1019, 2001. 14. Bowman AL, Catino DH, Strong JC, Randall WR, KontrogianniKonstantopoulos A, Bloch RJ. The rho-guanine nucleotide exchange factor domain of obscurin regulates assembly of titin at the Z-disk through interactions with Ran binding protein 9. Mol Biol Cell 19: 3782–3792, 2008. 15. Bryan BA, Li D, Wu X, Liu M. The Rho family of small GTPases: crucial regulators of skeletal myogenesis. Cell Mol Life Sci 62: 1547– 1555, 2005. 16. Chen YW, Zhao P, Borup R, Hoffman EP. Expression profiling in the muscular dystrophies: identification of novel aspects of molecular pathophysiology. J Cell Biol 151: 1321–1336, 2000. 17. Cheng MY, Hartl FU, Martin J, Pollock RA, Kalousek F, Neupert W, Hallberg EM, Hallberg RL, Horwich AL. Mitochondrial heat-shock protein hsp60 is essential for assembly of proteins imported into yeast mitochondria. Nature 337: 620 – 625, 1989. 18. Cheung AM, Tansey CM, Tomlinson G, Diaz-Granados N, Matte A, Barr A, Mehta S, Mazer CD, Guest CB, Stewart TE, Al-Saidi F, Cooper AB, Cook D, Slutsky AS, Herridge MS. Two-year outcomes, health care use, and costs of survivors of acute respiratory distress syndrome. Am J Respir Crit Care Med 174: 538 –544, 2006. 19. Chockalingam PS, Cholera R, Oak SA, Zheng Y, Jarrett HW, Thomason DB. Dystrophin-glycoprotein complex and Ras and Rho GTPase signaling are altered in muscle atrophy. Am J Physiol Cell Physiol 283: C500 –C511, 2002. 20. Dhume A, Lu S, Horowits R. Targeted disruption of N-RAP gene function by RNA interference: a role for N-RAP in myofibril organization. Cell Motil Cytoskeleton 63: 493–511, 2006. 21. Di Giovanni S, Molon A, Broccolini A, Melcon G, Mirabella M, Hoffman EP, Servidei S. Constitutive activation of MAPK cascade in acute quadriplegic myopathy. Ann Neurol 55: 195–206, 2004. 22. Dodd S, Hain B, Judge A. Hsp70 prevents disuse muscle atrophy in senescent rats. Biogerontology 10: 605– 611, 2009. 23. Durbeej M, Campbell KP. Muscular dystrophies involving the dystrophin-glycoprotein complex: an overview of current mouse models. Curr Opin Genet Dev 12: 349 –361, 2002. 24. Etard C, Behra M, Fischer N, Hutcheson D, Geisler R, Strahle U. The UCS factor Steif/Unc-45b interacts with the heat shock protein Hsp90a during myofibrillogenesis. Dev Biol 308: 133–143, 2007. 25. Ferrer-Martinez A, Montell E, Montori-Grau M, Garcia-Martinez C, Gomez-Foix AM, Roberts MA, Mansourian R, Mace K. Long-term cultured human myotubes decrease contractile gene expression and regulate apoptosis-related genes. Gene 384: 145–153, 2006. 26. Gogg S, Smith U. Epidermal growth factor and transforming growth factor alpha mimic the effects of insulin in human fat cells and augment downstream signaling in insulin resistance. J Biol Chem 277: 36045– 36051, 2002. 27. Harlow HJ, Lohuis T, Beck TD, Iaizzo PA. Muscle strength in overwintering bears. Nature 409: 997, 2001. www.physiolgenomics.org

158


28. Herridge MS, Cheung AM, Tansey CM, Matte-Martyn A, DiazGranados N, Al-Saidi F, Cooper AB, Guest CB, Mazer CD, Mehta S, Stewart TE, Barr A, Cook D, Slutsky AS. One-year outcomes in survivors of the acute respiratory distress syndrome. N Engl J Med 348: 683– 693, 2003. 29. Hershey JD, Robbins CT, Nelson OL, Lin DC. Minimal seasonal alterations in the skeletal muscle of captive brown bears. Physiol Biochem Zool 81: 138 –147, 2008. 30. Hinits Y, Hughes SM. Mef2s are required for thick filament formation in nascent muscle fibres. Development 134: 2511–2519, 2007. 31. Hofmann PA, Greaser ML, Moss RL. C-protein limits shortening velocity of rabbit skeletal muscle fibres at low levels of Ca2⫹ activation. J Physiol 439: 701–715, 1991. 32. Homma S, Iwasaki M, Shelton GD, Engvall E, Reed JC, Takayama S. BAG3 deficiency results in fulminant myopathy and early lethality. Am J Pathol 169: 761–773, 2006. 33. Hoshijima M. Mechanical stress-strain sensors embedded in cardiac cytoskeleton: Z disk, titin, and associated structures. Am J Physiol Heart Circ Physiol 290: H1313–H1325, 2006. 34. Hoshijima M, Sah VP, Wang Y, Chien KR, Brown JH. The low molecular weight GTPase Rho regulates myofibril formation and organization in neonatal rat ventricular myocytes. Involvement of Rho kinase. J Biol Chem 273: 7725–7730, 1998. 35. Huang da W, Sherman BT, Tan Q, Collins JR, Alvord WG, Roayaei J, Stephens R, Baseler MW, Lane HC, Lempicki RA. The DAVID Gene Functional Classification Tool: a novel biological module-centric algorithm to functionally analyze large gene lists. Genome Biol 8: R183, 2007. 36. Hudson LD, Lee CM. Neuromuscular sequelae of critical illness. N Engl J Med 348: 745–747, 2003. 37. Irizarry RA, Hobbs B, Collin F, Beazer-Barclay YD, Antonellis KJ, Scherf U, Speed TP. Exploration, normalization, and summaries of high density oligonucleotide array probe level data. Biostatistics 4: 249 –264, 2003. 38. Jen Y, Weintraub H, Benezra R. Overexpression of Id protein inhibits the muscle differentiation program: in vivo association of Id with E2A proteins. Genes Dev 6: 1466 –1479, 1992. 39. Kato K, Ito H, Kamei K, Iwamoto I, Inaguma Y. Innervation-dependent phosphorylation and accumulation of alphaB-crystallin and Hsp27 as insoluble complexes in disused muscle. FASEB J 16: 1432–1434, 2002. 40. Kjaer M. Role of extracellular matrix in adaptation of tendon and skeletal muscle to mechanical loading. Physiol Rev 84: 649 – 698, 2004. 41. Kojic S, Medeot E, Guccione E, Krmac H, Zara I, Martinelli V, Valle G, Faulkner G. The Ankrd2 protein, a link between the sarcomere and the nucleus in skeletal muscle. J Mol Biol 339: 313–325, 2004. 42. Lange S, Xiang F, Yakovenko A, Vihola A, Hackman P, Rostkova E, Kristensen J, Brandmeier B, Franzen G, Hedberg B, Gunnarsson LG, Hughes SM, Marchand S, Sejersen T, Richard I, Edstrom L, Ehler E, Udd B, Gautel M. The kinase domain of titin controls muscle gene expression and protein turnover. Science 308: 1599 –1603, 2005. 43. Langlands K, Yin X, Anand G, Prochownik EV. Differential interactions of Id proteins with basic-helix-loop-helix transcription factors. J Biol Chem 272: 19785–19793, 1997. 44. Larsson L, Li X, Edstrom L, Eriksson LI, Zackrisson H, Argentini C, Schiaffino S. Acute quadriplegia and loss of muscle myosin in patients treated with nondepolarizing neuromuscular blocking agents and corticosteroids: mechanisms at the cellular and molecular levels. Crit Care Med 28: 34 – 45, 2000. 45. Larsson L, Moss RL. Maximum velocity of shortening in relation to myosin isoform composition in single fibres from human skeletal muscles. J Physiol 472: 595– 614, 1993. 46. Larsson L, Skogsberg C. Effects of the interval between removal and freezing of muscle biopsies on muscle fibre size. J Neurol Sci 85: 27–38, 1988. 47. Larsson L, Skogsberg C, Gur H. Influence of the interval between removal and freezing of muscle samples on muscle fibre size with special reference to sample size and fibre type. Acta Physiol Scand 139: 451– 458, 1990. 48. Lawler JM, Song W, Kwak HB. Differential response of heat shock proteins to hindlimb unloading and reloading in the soleus. Muscle Nerve 33: 200 –207, 2006. 49. Lee K, Park JY, Yoo W, Gwag T, Lee JW, Byun MW, Choi I. Overcoming muscle atrophy in a hibernating mammal despite prolonged Physiol Genomics • VOL

39 •

50. 51. 52. 53. 54.

55.

56. 57. 58. 59.

60.

61. 62. 63.

64. 65. 66. 67. 68. 69. 70. 71.

72. 73.

disuse in dormancy: proteomic and molecular assessment. J Cell Biochem 104: 642– 656, 2008. Li C, Wong WH. Model-based analysis of oligonucleotide arrays: expression index computation and outlier detection. Proc Natl Acad Sci USA 98: 31–36, 2001. Li ZF, Wu X, Jiang Y, Liu J, Wu C, Inagaki M, Izawa I, Mizisin AP, Engvall E, Shelton GD. Non-pathogenic protein aggregates in skeletal muscle in MLF1 transgenic mice. J Neurol Sci 264: 77– 86, 2008. Liu Y, Gampert L, Nething K, Steinacker JM. Response and function of skeletal muscle heat shock protein 70. Front Biosci 11: 2802–2827, 2006. Liu Y, Steinacker JM. Changes in skeletal muscle heat shock proteins: pathological significance. Front Biosci 6: D12–25, 2001. Lutz M, Wempe F, Bahr I, Zopf D, von Melchner H. Proteasomal degradation of the multifunctional regulator YB-1 is mediated by an F-Box protein induced during programmed cell death. FEBS Lett 580: 3921–3930, 2006. Mammucari C, Milan G, Romanello V, Masiero E, Rudolf R, Del Piccolo P, Burden SJ, Di Lisi R, Sandri C, Zhao J, Goldberg AL, Schiaffino S, Sandri M. FoxO3 controls autophagy in skeletal muscle in vivo. Cell Metab 6: 458 – 471, 2007. Martin J, Horwich AL, Hartl FU. Prevention of protein denaturation under heat stress by the chaperonin Hsp60. Science 258: 995–998, 1992. Matozaki T, Nakanishi H, Takai Y. Small G-protein networks: their crosstalk and signal cascades. Cell Signal 12: 515–524, 2000. Mayer MP, Bukau B. Hsp70 chaperones: cellular functions and molecular mechanism. Cell Mol Life Sci 62: 670 – 684, 2005. McCarthy JJ, Andrews JL, McDearmon EL, Campbell KS, Barber BK, Miller BH, Walker JR, Hogenesch JB, Takahashi JS, Esser KA. Identification of the circadian transcriptome in adult mouse skeletal muscle. Physiol Genomics 31: 86 –95, 2007. McKoy G, Hou Y, Yang SY, Vega Avelaira D, Degens H, Goldspink G, Coulton GR. Expression of Ankrd2 in fast and slow muscles and its response to stretch are consistent with a role in slow muscle function. J Appl Physiol 98: 2337–2343, 2005. Miska EA, Karlsson C, Langley E, Nielsen SJ, Pines J, Kouzarides T. HDAC4 deacetylase associates with and represses the MEF2 transcription factor. EMBO J 18: 5099 –5107, 1999. Moss RL. Sarcomere length-tension relations of frog skinned muscle fibres during calcium activation at short lengths. J Physiol 292: 177–192, 1979. Norman H, Kandala K, Kolluri R, Zackrisson H, Nordquist J, Walther S, Eriksson LI, Larsson L. A porcine model of acute quadriplegic myopathy: a feasibility study. Acta Anaesthesiol Scand 50: 1058 – 1067, 2006. Obinata T, Maruyama K, Sugita H, Kohama K, Ebashi S. Dynamic aspects of structural proteins in vertebrate skeletal muscle. Muscle Nerve 4: 456 – 488, 1981. Oh HJ, Chen X, Subjeck JR. Hsp110 protects heat-denatured proteins and confers cellular thermoresistance. J Biol Chem 272: 31636 –31640, 1997. Ooi PT, da Costa N, Edgar J, Chang KC. Porcine congenital splayleg is characterised by muscle fibre atrophy associated with relative rise in MAFbx and fall in P311 expression. BMC Vet Res 2: 23, 2006. Peng J, Raddatz K, Labeit S, Granzier H, Gotthardt M. Muscle atrophy in titin M-line deficient mice. J Muscle Res Cell Motil 26: 381–388, 2005. Pillai RS. MicroRNA function: multiple mechanisms for a tiny RNA? RNA 11: 1753–1761, 2005. Potthoff MJ, Arnold MA, McAnally J, Richardson JA, Bassel-Duby R, Olson EN. Regulation of skeletal muscle sarcomere integrity and postnatal muscle function by Mef2c. Mol Cell Biol 27: 8143– 8151, 2007. Rebbapragada A, Benchabane H, Wrana JL, Celeste AJ, Attisano L. Myostatin signals through a transforming growth factor beta-like signaling pathway to block adipogenesis. Mol Cell Biol 23: 7230 –7242, 2003. Rommel C, Bodine SC, Clarke BA, Rossman R, Nunez L, Stitt TN, Yancopoulos GD, Glass DJ. Mediation of IGF-1-induced skeletal myotube hypertrophy by PI(3)K/Akt/mTOR and PI(3)K/Akt/GSK3 pathways. Nat Cell Biol 3: 1009 –1013, 2001. Sackey PV, Martling CR, Granath F, Radell PJ. Prolonged isoflurane sedation of intensive care unit patients with the Anesthetic Conserving Device. Crit Care Med 32: 2241–2246, 2004. Salem M, Kenney PB, Rexroad CE 3rd, Yao J. Microarray gene expression analysis in atrophying rainbow trout muscle: a unique non-



74.

75. 76.

77. 78. 79. 80. 81.

82.

mammalian muscle degradation model. Physiol Genomics 28: 33– 45, 2006. Sandri M, Sandri C, Gilbert A, Skurk C, Calabria E, Picard A, Walsh K, Schiaffino S, Lecker SH, Goldberg AL. Foxo transcription factors induce the atrophy-related ubiquitin ligase atrogin-1 and cause skeletal muscle atrophy. Cell 117: 399 – 412, 2004. Schiaffino S, Reggiani C. Molecular diversity of myofibrillar proteins: gene regulation and functional significance. Physiol Rev 76: 371– 423, 1996. Schuler M, Ali F, Chambon C, Duteil D, Bornert JM, Tardivel A, Desvergne B, Wahli W, Chambon P, Metzger D. PGC1alpha expression is controlled in skeletal muscles by PPARbeta, whose ablation results in fiber-type switching, obesity, and type 2 diabetes. Cell Metab 4: 407– 414, 2006. Selsby JT, Dodd SL. Heat treatment reduces oxidative stress and protects muscle mass during immobilization. Am J Physiol Regul Integr Comp Physiol 289: R134 –R139, 2005. Senf SM, Dodd SL, McClung JM, Judge AR. Hsp70 overexpression inhibits NF-kappaB and Foxo3a transcriptional activities and prevents skeletal muscle atrophy. FASEB J 22: 3836 –3845, 2008. Shemetov AA, Seit-Nebi AS, Gusev NB. Structure, properties, and functions of the human small heat-shock protein HSP22 (HspB8, H11, E2IG1): a critical review. J Neurosci Res 86: 264 –269, 2008. Smyth GK. Linear models and empirical bayes methods for assessing differential expression in microarray experiments. Stat Appl Genet Mol Biol 3: Article 3, 2004. Sonoda J, Mehl IR, Chong LW, Nofsinger RR, Evans RM. PGC-1beta controls mitochondrial metabolism to modulate circadian activity, adaptive thermogenesis, and hepatic steatosis. Proc Natl Acad Sci USA 104: 5223–5228, 2007. Stevenson EJ, Giresi PG, Koncarevic A, Kandarian SC. Global analysis of gene expression patterns during disuse atrophy in rat skeletal muscle. J Physiol 551: 33– 48, 2003.


39 •

159

83. Sturn A, Quackenbush J, Trajanoski Z. Genesis: cluster analysis of microarray data. Bioinformatics 18: 207–208, 2002. 84. Szewczyk NJ, Jacobson LA. Activated EGL-15 FGF receptor promotes protein degradation in muscles of Caenorhabditis elegans. EMBO J 22: 5058 –5067, 2003. 85. Taira T, Negishi Y, Kihara F, Iguchi-Ariga SM, Ariga H. c-myc protein complex binds to two sites in human hsp70 promoter region. Biochim Biophys Acta 1130: 166 –174, 1992. 86. Tinker DB, Harlow HJ, Beck TD. Protein use and muscle-fiber changes in free-ranging, hibernating black bears. Physiol Zool 71: 414 – 424, 1998. 87. Tsai S, Cassady JP, Freking BA, Nonneman DJ, Rohrer GA, Piedrahita JA. Annotation of the Affymetrix porcine genome microarray. Anim Genet 37: 423– 424, 2006. 88. Wang X, Blagden C, Fan J, Nowak SJ, Taniuchi I, Littman DR, Burden SJ. Runx1 prevents wasting, myofibrillar disorganization, and autophagy of skeletal muscle. Genes Dev 19: 1715–1722, 2005. 89. Wu Z, Puigserver P, Andersson U, Zhang C, Adelmant G, Mootha V, Troy A, Cinti S, Lowell B, Scarpulla RC, Spiegelman BM. Mechanisms controlling mitochondrial biogenesis and respiration through the thermogenic coactivator PGC-1. Cell 98: 115–124, 1999. 90. Wyke SM, Tisdale MJ. Induction of protein degradation in skeletal muscle by a phorbol ester involves upregulation of the ubiquitin-proteasome proteolytic pathway. Life Sci 78: 2898 –2910, 2006. 91. Young P, Ehler E, Gautel M. Obscurin, a giant sarcomeric Rho guanine nucleotide exchange factor protein involved in sarcomere assembly. J Cell Biol 154: 123–136, 2001. 92. Zhao J, Brault JJ, Schild A, Cao P, Sandri M, Schiaffino S, Lecker SH, Goldberg AL. FoxO3 coordinately activates protein degradation by the autophagic/lysosomal and proteasomal pathways in atrophying muscle cells. Cell Metab 6: 472– 483, 2007. 93. Zhu S, Si ML, Wu H, Mo YY. MicroRNA-21 targets the tumor suppressor gene tropomyosin 1 (TPM1). J Biol Chem 282: 14328 –14336, 2007.
