Differentially expressed genes and morphological changes during lengthened immobilization in rat soleus muscle

r 2006, Copyright the Authors Differentiation (2007) 75:147–157 DOI: 10.1111/j.1432-0436.2006.00118.x Journal compilation r 2006, International Society of Differentiation

O RI G INA L AR T I C L E

Ji Won Kim . Oh Yun Kwon . Myoung Hee Kim

Differentially expressed genes and morphological changes during lengthened immobilization in rat soleus muscle

Received February 20, 2006; accepted in revised form May 18, 2006

Abstract To examine the effect of lengthened immobilization on the expression of genes and concomitant morphological changes in soleus muscle, rat hindlimbs were immobilized at the ankle in full dorsiflexion by plaster cast. After removing the muscle (after 1 hr, 1, 4, and 7 days of immobilization), morphology and differential gene expression were analyzed through electron microscopy and differential display reverse transcription-polymerase chain reaction (DDRT-PCR), respectively. At the myotendinous junction (MTJ), a large cytoplasmic space appeared after 1 hr of immobilization and became enlarged over time, together with damaged Z lines. Interfibrillar space was detected after 1 day of immobilization, but diminished after 7 days. At the muscle belly, Z-line streaming and widening were observed following 1 hr of immobilization. Disorganization of myofilaments (misalignment of adjacent sarcomeres, distortion, or absence of Z lines) was Ji Won Kim Department of Physical Therapy College of Health Science Baekseok University Cheonan, Korea

Key words skeletal muscle
lengthened immobilization
differentially expressed genes
morphological changes

Oh Yun Kwon Department of Physical Therapy College of Health Science Yonsei University Wonju, Korea

Introduction

. )1 Myoung Hee Kim (* Department of Anatomy, Embryology Lab., Brain Korea 21 Project for Medical Science, Yonsei University College of Medicine, Seoul 120-752, Korea Tel: 182 2 2228 1647 Fax: 82 2 365 0700 E-mail: [email protected] 1 Present address: Department of Anatomy, Embryology Lab, Yonsei University College of Medicine, 134 Schinchondong, Sodaemoongu, Seoul, Korea.

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detected after 4 days. Furthermore, mitochondrial swelling and cristae disruption were observed after 1 day of stretching. A set of 15 differentially expressed candidate genes was identified through DDRT-PCR. Of 11 known genes, seven (Atp5g3, TOM22, INrf2, Slc25a4, Hdac6, Tpm1, and Sv2b) were up and three (Podxl, Myh1, and Surf1) were down-regulated following immobilization. In the case of Acyp2, 1-day stretching-specific expression was observed. Atp5g3, Slc25a4, TOM22, and Surf1 are mitochondrial proteins related to energy metabolism, except TOM22, which has a chaperone-like activity located in the mitochondrial outer membrane. Together with these, INrf2, Hdac6, Podxl, and Acyp2 are related more or less to stress-induced apoptosis, indicating the responses to apoptotic changes in mitochondria caused by stretching. The expression of both Tpm1 and Myh1, fast twitch isoforms, suggests adaption to the immobilization. These results altogether indicate that lengthened immobilization regulates the expression of several stress/apoptosis-related and muscle-specific genes responsible for the slow-to-fast transition in soleus muscle despite profound muscle atrophy.

Muscle stretch is a general therapeutic maneuver used to increase range of motion (ROM) by elongating structures that have adaptively shortened and become hypomobile for a long time. Several studies have examined the effects of muscle stretch in a lengthened position (Tabary et al., 1972; Williams and Goldspink, 1973; Dix and Eisenberg, 1990; Williams, 1990), and suggested that the stretched muscle fibers increase the number of sarcomeres to maintain a normal passive

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length–tension relationship (Caiozzo et al., 2002). For this reason, muscle stretch is often applied in clinical rehabilitation therapy. However, little is known about the molecular mechanisms occurring during this process. In general, muscle stretch is sensed at the sarcolemma, most likely by a complex of integral membrane proteins, and signal transduction leads cytoplasmic responses such as the phosphorylation of transcription factor(s), translocation of these factors into the nucleus, specific gene (DNA) transcription, and translation into specific proteins (De Deyne, 2001). Using methods like Northern blotting, immunoblot, and RNA protection analysis, a number of genes, such as insulin-like growth factor-1 (IGF-1; McKoy et al., 1999), myosin heavy chain (MHC; Galler et al., 1997), dihydropyridine receptor (DHPR; Radzyukevich and Heiny, 2004), sarcoplasmic/endoplasmic reticulum Ca (21)-ATPases (SERCA1; Zador et al., 1999), mammalian target of rapamycin (mTOR; Hornberger et al., 2004), myogenin (Ikeda et al., 2004), c-fos (Ikeda et al., 2003), and ankyrin repeat domain 2 (Ankrd2; Kojic et al., 2004), have been identified to be expressed during and/or after muscle stretch. Through differential screening, Cros et al. (2001) analyzed 34 differentially expressed genes in rat soleus muscle atrophied by hindlimb suspension. The serial analysis of gene expression (SAGE) method was also used to elucidate the molecular basis of muscle atrophy induced by a cast in a shortened position (StAmand et al., 2001). They reported 47 genes, categorized into three groups according to their functions: contraction, energy metabolism, and housekeeping. Until now, however, systematic gene expression analysis has not been performed in the muscle following different periods of stretch time. In the case of the immobilization of muscle in a shortened position, reduction of muscle fiber length has been reported due to a loss of sarcomeres. However, intermittent stretches of 1/2, 1, or 2 hr daily for a period of 2 weeks have been reported to increase the number of sarcomeres following immobilization in a shortened position (Williams, 1990). Electron microscopy (EM) studies have revealed a large cytoplasmic space containing polysomes, sarcoplasmic reticulum and T membranes, mitochondria, Golgi complexes, and nascent filament assemblies at the myotendinous junction (MTJ) of 4-day stretched muscle fiber. In situ hybridization analysis found a high level of MCH mRNA expression at the MTJ of stretched muscle fibers (Dix and Eisenberg, 1990). A novel stretch-responsive skeletal muscle gene (Smpx) was identified through expression on the muscle fiber after 7 days of passive stretching, and it may play a role during muscle hypertrophy (Kemp et al., 2001). Other studies have also reported an increase in sarcomere number after casting of hindlimbs in a lengthened position for 3, 4, or 1–7 weeks (Tabary et al., 1972; Williams and Goldspink, 1978).

Because previous studies have not examined the systematic gene expression pattern following different periods of stretch time, we applied a differential display reverse transcription-polymerase chain reaction (DDRT-PCR) in an attempt to analyze the differentially (up or down-regulated) expressed genes systematically according to different periods of muscle stretching (1 hr, 1, 4, and 7 days) as well as the concomitant morphological changes.

Material and methods Animals Thirty-five Sprague–Dawley male rats weighting 300–320 g were used. Both hindlimbs were immobilized at the ankle in full dorsiflexion (about 551) by plaster casts for 1 hr, 1, 4, and 7 days except in the control group. Hindlimbs of 25 rats were used for EM studies and their left hindlimbs were taken out for DDRT-PCR. Ten rats were used for in situ hybridization. For DDRT-PCR and in situ hybridization, soleus muscles were rapidly excised, immediately frozen in liquid nitrogen, and stored at  801C. For EM, the soleus muscles from each limb were isolated, their length were measured, and weighted. EM Small blocks of tissue (1 mm3) from the mid-portion and MTJ portion of the muscle were immersed overnight at 41C in 0.1 M phosphate buffer of pH 7.4 containing 2.5% glutaraldehyde, and then post-fixed with 1% osmium tetroxide for 1 hr. Tissue samples were longitudinally oriented and embedded in epoxy resin (Spurr’s). Four sample blocks were obtained from each muscle sample. The sample blocks from each biopsy were trimmed of excess plastic resin and sectioned using a glass knife for thick sections and a diamond knife (DIATOME, Hatfield, PA) for thin sections. Thick sections (1 mm) were cut from the blocks and stained with toluidine blue (0.2% in 2.0% sodium borate) at 601C for 1 min. Thin sections (90 nm) were then cut, stained with saturated uranyl acetate for 5 min and lead citrate for 3 min, and examined using a transmission electron microscope (JEM-1200 EX; JEOL, Tokyo, Japan). RNA isolation Total muscle RNA was isolated by the acid guanidium thiocyanate phenol-chloroform method (Chomczynski and Sacchi, 1987). Briefly, soleus muscles were homogenized in 4 M guanidium isothiocyanate solution. The following solutions were added in succession with vortexing 2–5 min each time: 2 M sodium acetate pH 4, followed by water-saturated phenol, and then by chloroform-isoamyl alcohol. After centrifugation, the upper aqueous phase was taken and 2 vol of ethanol were added and kept overnight at  201C. After centrifugation, the RNA pellet was washed with 75% ethanol and dried in air. The RNAs obtained were quantitated using a spectrophotometer Ultraspec 2000 (Pharmacia Biotech, Piscataway, NJ). The quality of the RNA was assessed by visual inspection of ethidium bromide-stained 18S and 28S rRNA under ultraviolet light. DDRT-PCR A GenHunter RNAimages kit 1 (GenHunter Co., Nashville, TN) was used for DDRT-PCR analysis. After synthesizing the cDNA

149 by reverse transcription using three different types of oligo-dT primers (HT11A-5 0 -AAGCTTTTTTTTTTTA-3 0 , HT11G-5 0 -AAGCTTTTTTTTTTTG-3 0 , HT11C-5 0 -AAGCTTTTTTTTTTTC-3 0 ), these three oligo-dT primers and eight different types of arbitrary primers (H-AP1-5 0 -AAGCTTGATTGCC-3 0 , AP2-5 0 -AAGCTTCGACTGT-3 0 , AP3-5 0 -AAGCTTTGGTCAG-3 0 , AP4-5 0 -AAGCTTCTCAACG-3 0 , AP5-5 0 -AAGCTTAGTAGGC-3 0 , AP6-5 0 -AAGCTTGCACCAT-3 0 , AP7-5 0 -AAGCTTAACGAGG-3 0 , AP8-5 0 AAGCTTTTACCGC-3 0 ) were combined for each PCR reaction harboring a-32P-dCTP: 40 cycles of 941C for 30 sec, 401C for 2 min, and 721C for 5 min. The amplified cDNAs were electrophoresed on a 6% denaturing polyacrylamide gel. After drying, the gel was exposed to an X-ray film for 24 hr. The gel bands of interest were cut out, placed in 100 ml of dH2O, boiled for 15 min, and then used as a template for the second amplification. After addition of 4 ml of extracted cDNA, 20 mM dNTP, 0.2 mM arbitrary primer, 0.2 mM oligo-dT primer, and 0.4 ml Taq DNA polymerase (5 U/ml, Takara Bio Inc., Shiga, Japan) were added in a final volume of 40 ml. The second amplification was performed under the same PCR conditions. cDNA synthesis and semiquantitative RT-PCR Reverse transcription of 2 mg of RNA was performed with 1 mg oligo-dT primer, followed by the addition of a reaction mixture containing 40 U of RNase inhibitor, 20 U of MMLV reverse transcriptase, and 1 mM dNTP mix in a final volume of 25 ml. The mixture was incubated at 371C for 1 hr. The PCR primers for specific genes were as follows: Podxl (F, 5 0 -ATG TCC TCT GGC CTT CTG CT; R, 5 0 -CAC AAC CAC CCA AGT TCT CG-3 0 ), Tpm1 (F, 5 0 -TGA CAA GCT GAA GGA GGC TG-3 0 ; R, 5 0 -TGC AGA GCT CAG AGA GGT G-3 0 ), and Myh1 (F, 5 0 -CCT CCG GAA GTC TGA AAA GG-3 0 ; R, 5 0 -CCA TGT CCT CGA TCT TGT CG-3 0 ). After 25 cycles of PCR reaction, the products were resolved by ethidium bromide-containing gel electrophoresis and photographed under an ultraviolet illuminator. The negatives were scanned (HP ScanJet 6300C, Hewlett-Packard, Palo Alto, CA) and densitometric analyses were performed (Image J program, National Institutes of Health). As a control, we also performed RTPCR for the b-actin gene with a gene-specific primer-pair (F, 5 0 CAT GTT TGA GAC CTT CAA CAC-3 0 ; R, 5 0 -GCC ATC TCC TGC TCG AAG TCT-3 0 ). Cloning, nucleotide sequencing, and sequence analysis DDRT-PCR amplification products were cloned into pGEM-T vector (Promega, Madison, WI) and transformed into Escherichia coli strain DH5a, using the CaCl2 method. After selecting the transformants showing antibiotic resistances on the plate containing 50 mg/ml of ampicillin, the insert was confirmed by the digestion pattern of EcoRI. The nucleotide sequence of each clone was determined using an Auto-sequencer (LONG READIR 4200, LICOR, Lincoln, NE), and then the sequence was analyzed using the BLAST Network Service (http://www.ncbi.nlm.nih.gov) provided by the National Center for Biotechnology Information (NCBI, Bethesda, MD). Preparation of riboprobes To prepare riboprobes for in situ hybridization, the a-tropomyosin cDNA fragment (241 bp) was cloned into the pGEM-T easy vector (Promega). Antisense digoxigenin (DIG)-labeled RNA riboprobes were synthesized using the DIG RNA labeling kit (Boehringer Mannheim, Germany). The transcription reaction mixture containing 6 ml of RNase-free water, 2 ml of 10  nucleotide mixture (10 mM ATP, 10 mM CTP, 10 mM GTP, 10 mM UTP), 4 ml of dithiothreitol (50 mM), 2 ml of 10  transcription buffer (400 mM Tris-HCl [pH 8.0], 60 mM MgCl2, 20 mM spermidine), 2 ml of tem-

plate DNA (100 ng/ml) digested with SalI, 3 ml of T7 polymerase (50 U/ml; Epicentre, Madison, WI), and 1 ml RNase inhibitor was incubated at 371C for 2 hr. Sense digoxigenin (DIG)-labeled RNA riboprobes were synthesized under the same conditions, except that the template DNA was digested by EcoRI and SP6 polymerase. To remove the template DNA, 1 ml of RNase-free DNase (1 U/ml, Epicentre) was added, incubated at 371C for another 15 min, and then subsequently purified by ethanol precipitation. After determining the concentration of probes photometrically, they were stored in TE buffer (10 mM Tris-HCl, 1 mM ethylenediamine tetraaceticacid, pH 7.4) at  701C until use.

In situ hybridization of frozen section Longitudinal serial sections were made with a Microm microtome using disposable blades at  201C. Sections were cut at 10 mm placed on Fisher Superfrost Plus (Pittsburgh, PA), dried for 1 hr, fixed for 20 min with 4% paraformaldehyde (PFA) in 1  phosphate buffered saline (PBS), pH 7.4, washed in 1  PBS, dehydrated, and stored at  801C. The slides were equilibrated to room temperature (RT) before removing them from the container. The sections were rehydrated in 1  PBS and post-fixed in cold 4% PFA/PBS for 10 min, and then rinsed three times with 1  PBS for 10 min each. Acetylaton (295 ml H2O, 4 ml triethanolamine, 0.525 ml HCl, and 0.75 ml acetic anhydride) was followed by washing three times with 1  PBS. After applying 500 ml of pre-hybridization buffer, slides were incubated in a humidified chamber containing 5  SSC for 2 hr. Then, the solution was changed with hybridization solution containing 200–400 ng/ml RNA probe at 801C for 5 min and slides were further incubated in a humidified chamber containing 5  SSC and 50% formamide at 721C overnight. After removing cover slips by submerging in 5  SSC at 721C, samples were incubated in 0.2  SSC for 3 hr, 0.2  SSC for 5 min, and Buffer B1 (0.1 M Tris pH 7.5, 0.15 M NaCl) including levamisole for 5 min. Then, anti-Digoxygenine antibody (Boehringer Mannheim, Mannheim, Germany) was added to the slides (1:5,000 dilution in B1 buffer), and incubated in the humidified chamber overnight at 41C. Washing with B1 buffer three times for 5 min each was followed by equilibration with B3 buffer (0.1 M Tris pH 9.5, 0.1 M NaCl, 50 mM MgCl2). After applying 60–70 ml of B4 solution (4.5 ml/ml NBT, 3.5 ml/ml BCIP, Boehringer Mannheim), slides were covered with a parafilm. Then, slides were incubated for 1–2 days in a humidified chamber in the dark and the reaction was stopped by washing with 1  PBS. After air-drying, slides were mounted with Universal Mount (Research Genetics, Huntsville, AL).

Results Morphological changes following different periods of stretching In order to analyze the differentially expressed genes following muscle stretch, rat hindlimbs were immobilized at the ankle in full dorsiflexion by plaster casts for 1 hr, 1, 4, or 7 days, and morphological changes were examined first. As shown in Figure 1, cytoplasmic space started to appear at the MTJ following 1 hr of stretching. This space was enlarged by the end of 1 day and inter-fibrillar space was also observed. By 4 days, a large cytoplasmic space devoid of myofibrils was observed at the MTJ. As casting time increased, larger cytoplasmic space and more damaged Z lines appeared. Inter-fibril-

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Fig. 1 Changes in the myotendinous junction (MTJ) following different periods of muscle stretching. Control (Co) muscle fiber shows regularly organized sarcomeres at the MTJ. Cytoplasmic spaces in 1 hr (1 hr), 1-, 4-, and 7-day (d) stretched muscles at the MTJ. Original magnification,  5,000. Scale bar, 2 mm.

lar spaces were diminished at the MTJ by 7 days, but not after 4 days. At the muscle belly, Z-line streaming and widening were observed following 1 hr of stretching while the control muscle showed normal striation (Fig. 2). By day 1, more Z-line streaming and widening were observed. Ultra-structural disarrangement, such as disorganization of myofilaments, misalignment of adjacent sarcomeres, and distortion or absence of Z lines, was detected in 4 and 7-day stretched muscles. Interestingly, drastic changes in mitochondria, such as swelling and cristae disruption, were observed after 1 day of stretching (Fig. 3).

Isolation and analysis of differentially expressed genes following different periods of stretching After confirming morphological changes in the muscle following different periods of stretching, total RNA was isolated from each group and DDRT-PCR was performed. After comparing the expression pattern along

stretching time (Fig. 4), 30 differentially expressed bands (No. 1–30) were selected for re-amplification, subcloning, and sequencing. Fifteen bands did not reamplify or re-amplified as several bands or very short bands, and so only 15 bands successfully subcloned and sequenced were analyzed. When the nucleotide sequences were analyzed using BLAST, four genes (No. 14, –24, –27, and –28) were novel and 11 were known genes (Table 1). Of these known genes, seven genes, such as cytosolic inhibitor of Nrf2 (No. 1, INrf2), solute carrier family 25 (mitochondrial adenine nucleotide translocator) member 4 (No. 4, Slc25a4), adenosine triphosphate (ATP) synthase (No. 13, Atp5g3), synaptic vesicle glycoprotein 2B (No. 18, Sv2b), a-tropomyosin (No. 22, Tpm1), histone deacetylase 6 (No. 26, Hdac6), and mitochondrial translocase of outer membrane 22 (No. 29, TOM22), were up-regulated following stretching time (Fig. 4 and Table 1). In the case of Atp5g3 and TOM22, expression was up-regulated following 1 hr of stretching, whereas INrf2, Slc25a4, and Hdac6 were highly expressed in the muscles after 1 day of stretching. The expression of Tpm1 and Sv2b was up-regulated

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Fig. 2 Changes at the muscle belly following different periods of muscle stretching. Control (Co) muscle belly fibers show regularly organized sarcomeres. 1 hr (h) and 1-day (d) stretched muscle belly fibers show Z-line streaming and widening. Four and 7-day stretched muscle belly fibers exhibit the distortion and absence of the Z line, as well as the disorganization of myofilaments. Original magnification,  5,000. Scale bar, 2 mm.

following 4 and 7 days of stretching, respectively. On the other hand, podocalyxin (No. 5, Podxl) and Surfeit 1 (No. 9, Surf1) were down-regulated following stretching, while the genes Acylphosphatase 2 (No. 17, acyp2) and skeletal muscle MCH (No. 23, Myh1) were stagespecifically up-regulated and then down-regulated as stretching time increased.

Expression of Podx1, Tpm1, and Myh1 genes during muscle stretching In order to confirm the expression pattern of the genes isolated through DDRT-PCR, RT-PCR was carried out using gene-specific primer sets. As shown in Figure 5, the expression patterns of Tpm1 and Myh1, contractile

Fig. 3 Representative micrographs of mitochondria in the soleus muscle after 1 day of stretching (A,  5,000; B,  10,000). Note the focal subsarcolemmal mitochondrial swelling. Scale bar in A, 2 mm; Scale bar in B, 1 mm.

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23 5 1

17 18

9

28

4

27

22

29

24

13 26

14

Fig. 4 Representatives of differentially displayed reverse transcription-polymerase chain reaction. RNA was isolated from rat soleus muscles after different periods of muscle stretching. Differentially expressed genes are indicated as white arrows. Lane 1, control; lane 2, 1 hr stretched muscle; lanes 3–5, 1-, 4-, and 7-day stretched muscle, respectively. The primers were HT11A and AP2 for No. 1 (INrf2) and No. 4 (Slc25a4), HT11A and AP1 for No. 5 (Podx1),

HT11A and AP5 for No. 9 (Surf1), HT11C and AP1 for No. 13 (Atp5g3), HT11C and AP8 for No. 14, HT11G and AP1 for No. 17 (Acyp2), HT11G and AP2 for No. 18 (Sv2b), HT11G and AP4 for No. 22 (Tpm1), HT11G and AP5 for No. 23 (Myh1), No. 24, and No. 26 (Hdac6), HT11C and AP2 for No. 27, and HT11C and AP3 for No. 28 and No. 29 (TOM22).

elements of muscle, and the arbitrarily selected Podx1 turned out to be similar to those detected in the DDRTPCR analysis (Fig. 4). The expression of Tpm1 was upregulated in the 4 and 7-day stretched muscles, whereas Podx1 and Myh1 were up-regulated following a short period of stretching. However, the expression of Myh1 and Podxl was drastically decreased after long periods of stretching, as at 4 and 7 days.

Spatial expression pattern of a-tropomyosin gene in stretching muscle In order to see where Tpm1 was expressed in the stretched muscle, in situ hybridization was performed using a Dig-labeled antisense riboprobe. As DDRTPCR (Fig. 4) and RT-PCR (Fig. 5) revealed that Tpm1 was expressed strongly in 4- and 7-day stretched soleus

Table 1 List of differentially expressed genes identified by DDRT-PCR following lengthened immobilization in rat soleus muscle Band no. 1 4 5 9 13 14 17 18 22 23 24 26 27 28 29

Differentially expressed genes

Chromosomal locus

Accession no.

Expression pattern

Inhibitor of Nrf2 (INrf2) Solute carrier family 25 (mitochondrial adenine nucleotide translocator) member 4 (Slc25a4) Podocalyxin (Podxl) Surfeit 1 (Surf1) ATP synthase (Atp5g3) Novel gene Acylphosphatase 2 (Acyp2) Synaptic vesicle glycoprotein 2B (Sv2b) a-tropomyosin (Tpm1) Skeletal muscle myosin heavy chain (Myh1) Novel gene Histone deacetylase (Hdac6) Novel gene Novel gene Mitochondrial translocase of outer membrane 22 (TOM22)

8q13 16q11

DT527753 DT527754

1 d, 4 d, 7 d 1 d, 4 d, 7 d

4q22 3p12 3q23 8q13 14q22 1q31 8q24 10q24 14q11 Xq13 6q16 17q12 7q34

DT605226 DT605227 DT605228 DT605229 DT605230 DT605231 DT605232 DT605233 DT605234 DT605235 DT605236 DT605237 DT605238

Co, 1 hr Co, 1 hr, 1 d 1 hr, 1 d, 4 d, 7 d 1d 1d 7d 4 d, 7 d 1 hr 1d 1 d, 4 d, 7 d 7d 1d 1 hr, 1 d, 4 d, 7 d

d, day; hr, hour; DDRT-PCR, differential display reverse transcription-polymerase chain reaction.

153 Co Podxl Tpm1

1h

1d

4d

7d

M 300bp 200bp 300bp 200bp

Myh1

300bp 200bp

β-actin

400bp

Fig. 5 Reverse transcription-polymerase chain reaction (RT-PCR) of Podxl, Tpm1, and Myh1 genes. RT-PCR was carried out with the total RNA from muscle stretched 1 hr, 1, 4, 7 days, and the control.

muscles, these muscles were isolated and fixed before sectioning. As shown in Figure 6, Tpm1 transcripts were localized in the MTJ of both 4- and 7-day stretched muscles, and a stronger expression was detected in the 7-day stretched muscle (Figs. 6C,6D), as in the RT-PCR analysis (Fig. 5). As expected, Tpm1 expression was not detected in control, 1 hr, or 1-day stretched muscles (data not shown). Interestingly, the expression of Tpm1 was detected at the distal rather than proximal MTJ. Furthermore, Tpm1 expression was not detected at the muscle belly of both stretched and non-stretched muscles.

Discussion This research was focused on identifying genes that are differentially expressed in muscles during different

periods of stretching. We identified a set of candidate genes derived from excised DDRT-PCR bands. DNA sequencing revealed 11 known and four novel genes. Of these known genes, seven genes (Atp5g3, TOM22, INrf2, Slc25a4, Hdac6, Tpm1, and Sv2b) were up-regulated after stretching, whereas genes such as Podxl, Myh1, and Surf1 were down-regulated as stretching time increased. In the case of Acyp2, stretching timespecific (1 day) expression was detected (Fig. 7). Among these, four (Slc25a4, Atp5g3, TOM22, and Surf1) are mitochondrial proteins: three (Slc25a4, Atp5g3, and TOM22) were up-regulated and one (Surf1) was down-regulated following the stretching. Atp5g3 and Slc25a4 are commonly known to be involved in energy metabolism. Up-regulation of Atp5g3, a mitochondrial ATP synthase subunit 9 (isoform 3), has been reported at the transcriptional level after chronic alcohol ingestion (Li et al., 2001), in parallel with mitochondrial damage. Slc25a4, adenine nucleotide translocase (ADP/ ATP translocator), has also been reported to play an important role during mitochondria-mediated apoptosis, in addition to its translocase activity (Marzo et al., 1998). Therefore, the up-regulation of Atp5g3 and Slc25a4 could be a part of an adaptive process for mitochondrial repair in skeletal muscle following passive stretching and the concomitant mitochondrial damage (Fig. 3). Along with morphological changes, large cytoplasmic space started to appear at the MTJ after 1 hr of stretching. Muscle fibers have been reported to be decreased in diameter by as much as 90% as they are blended with tendon tissue (Loeb et al., 1987). The cytoplasmic space at the MTJ might have appeared because tension produced per cross-sectional area was at

Fig. 6 Tpm1 mRNA expression in 4-day (A and B) and 7-day (C and D) stretched muscles. In situ hybridization using antisense riboprobes against Tpm1 (A and C,  40; B and D,  100). The digoxigeninlabeled in situ hybridization reaction with BCIP/NBT appears blue-purple. In the case of sense riboprobes, hybridization signals were not detected (data not shown).

154 0

1h

1d

2d

3d

4d

5d

6d

7d

MTJ

Belly DT605228

Atp5g3

DT605238

TOM22

DT527753

INrf2

DT527754

Slc25a4

DT605235

Hdac6 Tpm1

DT605232 DT605231

Sv2b

DT605236

Novel Gene

DT605226 DT605227 DT605233

Podxl Surf1 Myh1

DT605230

Acyp2

DT605229

Novel Gene

DT605234

Novel Gene

DT605237

Novel Gene

the peak. TOM22, still another mitochondrial protein up-regulated after stretching, has been known to have chaperone-like activity (Yano et al., 2004). As stretching is a kind of a stress, it is not surprising that TOM22 is overexpressed in the stretched muscle, considering that chaperone proteins usually up-regulate during stress. Another interesting protein is the mitochondrial inner membrane protein, Surf1, which was down-regulated after 4 days of stretching. Deficiency/down-regulation of Surf1 protein has been reported to cause a deficiency of cytochrome c oxidase, which is associated with the serious mitochondrial disorder (Tiranti et al., 1998; Zhu et al., 1998) of Leigh disease, a subacute necrotizing encephalopathy (Poyau et al., 1999; Pecina et al., 2004). The down-regulation of Surf1 could have caused problems in energy metabolism, leading to severe cell damage when muscles were stretched longer than 2–4 days or so. Hdac6, a member of the class IIB Hdacs having cytosolic a-tubulin and HSP90 deacetylase activity (Bali et al., 2005) was up-regulated in the muscles after 1 day of stretching. Depletion of Hdac6 using siRNA led to HSP90 acetylation and concomitant disruption of its chaperone function (Bali et al., 2005), suggesting that

Fig. 7 An overview showing muscle morphology during lengthened immobilization and differentially expressed genes. Orange, up-regulated genes; blue, down-regulated genes; green, stage-specifically expressed genes.

the observed up-regulation of Hdac6 seems to be similar to the up-regulation of TOM22 in the stress condition such as muscle stretching here. Furthermore, class II Hdacs were reported to inhibit myogenesis by interacting with myocyte enhancer factor-2 (MEF-2) and thus repressing skeletal muscle-specific genes during muscle development (McKinsey et al., 2001). INrf2 (also named as Keap1, Kelch-like ECH-associated protein 1), an inhibitor of nuclear-factor-E2-related factor-2 (Nrf2), was also up-regulated in the stretched muscles. Under normal conditions, INrf2 associates with Nfr2 in the cytoplasm (Jaiswal, 2004) and leads to the proteosomal degradation of Nrf2 (Stewart et al., 2003). When cells are exposed to oxidative stress, Nrf2 dissociates from INrf2, enters into the nucleus, and induces antioxidant/detoxifying enzymes (Kang et al., 2005), which are important in maintaining cellular redox homeostasis (Nguyen et al., 2005). Accordingly, the up-regulation of INrf2 in the stretched muscles could have interrupted redox homeostasis, leading to cell damage. Another two genes up-regulated after stretching were Tpm1 and Sv2b, whose expression started after 4 and 7 consecutive days of stretching, respectively (Figs. 4 and 5). Tpm1 up-regulation has already been reported in

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12-day immobilized gastrocnemius muscle (St-Amand et al., 2001). This result is interesting because adaptation to immobilization induces a shift to fast-twitch isoforms in skeletal muscle (Baldwin, 1996) and Tpm1 expression has been reported in the fast muscle as a thin filament component, which, in association with the troponin complex, regulates contraction (Perry, 2003). The specific expression and accumulation of Tpm1 at the MTJ (Fig. 7) might have some role in inducing the shift to fast-twitch fibers at the MTJ, although it is not clear why Tpm1 was expressed only at the distal MTJ. In the case of Sv2b, which is involved in the regulation of synaptic vesicle exocytosis, there are few reports on its expression, except in neuronal and endocrine tissues including the brain and eye (Walmsley and Gaines, 2004). Although this is the first report of Sv2b expression in the skeletal muscle, its function is still obscure. Contrary to the up-regulated genes, Podxl, usually highly expressed by podocytes, was down-regulated along with Surf1 in the stretched muscle (Doyonnas et al., 2001). Stretched skeletal muscle has been reported to overexpress p53 (Siu and Always, 2005), which is known to repress the expression of Podxl at the transcriptional level (Stanhope-Baker et al., 2004), and so the down-regulation of Podxl in the stretched muscle (Fig. 5) is not surprising. These data altogether indicate that muscle stretch induces apoptosis, particularly mitochondria-dependent apoptosis, which has been suggested by others (Liao et al., 2004). The genes Myh1 and Acyp2 were expressed stagespecifically during muscle stretching. As a predominant fast MCH isoform associated with the highest speed of shortening and energy cost (Bottinelli et al., 1991, 1994), Myh1 was induced dramatically in the soleus when the muscle was immobilized in a shortened position, whereas passive stretch caused a significant reduction of expression (Loughna et al., 1990; Jankala et al., 1997). Here, the expression of Myh1 was prevented when the slow postural soleus muscle was passively stretched more than 4 days (Fig. 5) in agreement with a previous report (Loughna et al., 1990). However, DDRT-PCR analysis showed that the Myh1 gene was slightly upregulated in 1 hr stretched muscle (Fig. 4), suggesting that the expression of Myh1 in the soleus might have been involved in changing its muscle type after 1 hr of stretching. In the case of Acyp2, up-regulation was observed only in the 1-day stretched muscles. As a cytosolic enzyme present in skeletal and heart muscle, Acyp2 catalyzes the hydrolysis of sarcoplasmic reticulum (SR) Ca21-ATPase (SERCA; Nediani et al., 2003). Although there have been no reports on Acyp2 expression during muscle stretch, hindlimb suspension upregulates the activity of skeletal SERCA and fast Ca21 pump protein (Schulte et al., 1993; Cros et al., 2001). The overexpression of Acyp2 has also been reported to induce differentiation and apoptosis (Giannoni et al.,

2000; Cecchi et al., 2004), indicating that short-period muscle stretch induces apoptosis. In conclusion, many genes are expressed differentially at different periods of muscle stretching. Of these genes, Atp5g3 and Slc25a4 are related to energy metabolism, and up-regulation of these genes seems to suggest that mitochondria are coping with the apoptotic changes after 1 hr or 1 day of stretching. Down-regulation of Surf1 also causes a severe deficiency of cytochrome c oxidase, resulting in problems in energy metabolism. Up-regulation of Hdac6 seems to prevent sarcomerogenesis by repressing muscle-specific genes. Putting these results together, immobilization in a lengthened position for more than 1 hr to 1 day may bring about apoptotic changes as well as chromatin remodeling to inhibit muscle-specific gene expression in stretched muscle. This is quite contrary to previous studies showing that the stretched muscle increases the number of sarcomeres in series to maintain a normal passive-tension curve. The discrepancy could be explained by the lengthened position and applied stretch force: here, full dorsiflexion (about 551) to the end of its ROM was applied at the ankle, whereas other groups might have applied casts in lengthened position between the resting length and the end of its ROM, although the immobilization joint angle or stretch force was not clearly stated. In manual stretching, a physical therapist or a trained practitioner usually applies an external force to move the involved body segment slightly beyond the point of tissue resistance and available ROM. Therefore, it would be interesting in the future to investigate the apoptosis/sarcomerogenesis according to the angle of dorsiflexion and stretch force. In EM studies, swelling and cristae disruption of mitochondria and disorganization of myofilaments support apoptotic changes. The Tpm1 gene, related to the shift to fasttwitch fibers, accumulated at the MTJ after 4 and 7 days of stretching. However, it is not clear at this moment whether up-regulation of Tpm1 correlates with a shift of muscle fiber or apoptotic changes. Down-regulation of Myh1 after up-regulation in 1-hr stretched muscle is reported for the first time in this study. This result is interesting, because this stretch technique could be applied to promote higher speed and more power in soleus exercise. Mode of stretch can be defined by who or how the stretch force is applied or whether or not the patient is actively participating in the stretching maneuver. Categories include but are not limited to manual, mechanical, or self-stretching as well as passive, assisted, or active stretching. Among these, passive prolonged stretch, known to be more effective among patients having chronic, fibrotic contractures (Light et al., 1984; McHugh et al., 1992; Bonutti et al., 1994; Jansen et al., 1996), was applied here and the results provide further insight into the morphological changes and molecular mechanisms occurring in muscle after rather short consecutive periods (from 1 hr to 7 days) of stretching.

156 Acknowledgments This work was supported by the Korea Research Foundation Grant KRF-2004-E000012 and -2005-204C00074, and partly by the YBSTI Program-2005 and the Research Project on the Production of Bio-Organs (No. 200508030901), Ministry of Agriculture and Forestry, Republic of Korea.

References Baldwin, K.M. (1996) Effect of spaceflight on the functional, biochemical, and metabolic properties of skeletal muscle. Med Sci Sports Exerc 28:983–987. Bali, P., Pranpat, M., Bradner, J., Balasis, M., Fiskus, W., Guo, F., Rocha, K., Kumaraswamy, S., Boyapalle, S., Atadja, P., Seto, E. and Bhalla, K. (2005) Inhibition of histone deacetylase 6 acetylates and disrupts the chaperone function of heat shock protein 90: a novel basis for antileukemia activity of histone deacetylase inhibitors. J Biol Chem 280:26729–26734. Bonutti, P.M., Windau, J.E., Ables, B.A. and Miller, B.G. (1994) Static progressive stretch to reestablish elbow range of motion. Clin Orthop Relat Res 303:128–134. Bottinelli, R., Betto, R., Schiaffino, S. and Reggiani, C. (1994) Maximum shortening velocity and coexistence of myosin heavy chain isoforms in single skinned fast fibres of rat skeletal muscle. J Muscle Res Cell Motil 15:413–419. Bottinelli, R., Schiaffino, S. and Reggiani, C. (1991) Force–velocity relations and myosin heavy chain isoform compositions of skinned fibres from rat skeletal muscle. Physiol 437:655–672. Caiozzo, V.J., Utkan, A., Chou, R., Khalafi, A., Chandra, H., Baker, M., Rourke, B., Adams, G., Baldwin, K. and Green, S. (2002) Effects of distraction on muscle length: mechanisms involved in sarcomerogenesis. Clin Orthop Relat Res 403: S133–S145. Cecchi, C., Liguri, G., Fiorillo, C., Bogani, F., Gambassi, M., Giannoni, E., Cirri, P., Baglioni, S. and Ramponi, G. (2004) Acylphosphatase overexpression triggers SH-SY5Y differentiation towards neuronal phenotype. Cell Mol Life Sci 61:1775–1784. Chomczynski, P. and Sacchi, N. (1987) Single-step method of RNA isolation by acid guanidinium thiocyanate—phenol–chloroform extraction. Anal Biochem 162:156–159. Cros, N., Tkatchenko, A.V., Pisani, D.F., Leclerc, L., Leger, J.J., Marini, J.F. and Dechesne, C.A. (2001) Analysis of altered gene expression in rat soleus muscle atrophied by disuse. J Cell Biochem 83:508–519. De Deyne, P.G. (2001) Application of passive stretch and its implications for muscle fibers. Phys Ther 81:819–827. Dix, D.J. and Eisenberg, B.R. (1990) Myosin mRNA accumulation and myofibrillogenesis at the myotendinous junction of stretched muscle fibers. J Cell Biol 111:1885–1894. Doyonnas, R., Kershaw, D.B., Duhme, C., Merkens, H., Chelliah, S., Graf, T. and McNagny, K.M. (2001) Anuria, omphalocele, and perinatal lethality in mice lacking the CD34-related protein podocalyxin. J Exp Med 194:13–27. Galler, S., Schmitt, T.L., Hilber, K. and Pette, D. (1997) Stretch activation and isoforms of myosin heavy chain and troponin-T of rat skeletal muscle fibres. J Muscle Res Cell Motil 18:555–561. Giannoni, E., Cirri, P., Paoli, P., Fiaschi, T., Camici, G., Manao, G., Raugei, G. and Ramponi, G. (2000) Acylphosphatase is a strong apoptosis inducer in HeLa cell line. Mol Cell Biol Res Commun 3:264–270. Hornberger, T.A., Stuppard, R., Conley, K.E., Fedele, M.J., Fiorotto, M.L., Chin, E.R. and Esser, K.A. (2004) Mechanical stimuli regulate rapamycin-sensitive signalling by a phosphoinositide 3-kinase-, protein kinase B- and growth factor-independent mechanism. Biochem J 380:795–804. Ikeda, S., Yoshida, A., Matayoshi, S., Horinouchi, K. and Tanaka, N. (2004) Induction of myogenin messenger ribonucleic acid in rat skeletal muscle after 1 hour of passive repetitive stretching. Arch Phys Med Rehabil 85:166–167.

Ikeda, S., Yoshida, A., Matayoshi, S. and Tanaka, N. (2003) Repetitive stretch induces c-fos and myogenin mRNA within several hours in skeletal muscle removed from rats. Arch Phys Med Rehabil 84:419–423. Jaiswal, A.K. (2004) Nrf2 signaling in coordinated activation of antioxidant gene expression. Free Radic Biol Med 36:1199–1207. Jankala, H., Harjola, V.P., Petersen, N.E. and Harkonen, M. (1997) Myosin heavy chain mRNA transform to faster isoforms in immobilized skeletal muscle: a quantitative PCR study. J Appl Physiol 82:977–982. Jansen, C.M., Windau, J.E., Bonutti, P.M. and Brillhart, M.V. (1996) Treatment of a knee contracture using a knee orthosis incorporating stress-relaxation techniques. Phys Ther 76:182–186. Kang, K.W., Lee, S.J. and Kim, S.G. (2005) Molecular mechanism of Nrf2 activation by oxidative stress. Antioxid Redox Signal 7:1664–1673. Kemp, T.J., Sadusky, T.J., Simon, M., Brown, R., Eastwood, M., Sassoon, D.A. and Coulton, G.R. (2001) Identification of a novel stretch-responsive skeletal muscle gene (Smpx). Genomics 74:251. Kojic, S., Medeot, E., Guccione, E., Krmac, H., Zara, I., Martinelli, V., Valle, G. and Faulkner, G. (2004) The Ankrd2 protein, a link between the sarcomere and the nucleus in skeletal muscle. J Mol Biol 339:313–325. Li, H.S., Zhang, J.Y., Thompson, B.S., Deng, X.Y., Ford, M.E., Wood, P.G., Stolz, D.B., Eagon, P.K. and Whitcomb, D.C. (2001) Rat mitochondrial ATP synthase ATP5G3: cloning and upregulation in pancreas after chronic ethanol feeding. Physiol Genomics 6:91–98. Liao, X.D., Wang, X.H., Jin, H.J., Chen, L.Y. and Chen, Q. (2004) Mechanical stretch induces mitochondria-dependent apoptosis in neonatal rat cardiomyocytes and G2/M accumulation in cardiac fibroblasts. Cell Res 14:16–26. Light, K.E., Nuzik, S., Personius, W. and Barstrom, A. (1984) Low-load prolonged stretch vs. high-load brief stretch in treating knee contractures. Phys Ther 64:330–333. Loeb, G., Pratt, C., Chanaud, C. and Richmond, F. (1987) Distribution and innervation of short, interdigitated muscle fibers in parallel-fiber muscles of the cat hindlimb. J Morphol 191:1–15. Loughna, P.T., Izumo, S., Goldspink, G. and Nadal-Ginard, B. (1990) Disuse and passive stretch cause rapid alterations in expression of developmental and adult contractile protein genes in skeletal muscle. Development 109:217–223. Marzo, I., Brenner, C., Zamzami, N., Jurgensmeier, J.M., Susin, S.A., Vieira, H.L., Prevost, M.C., Xie, Z., Matsuyama, S., Reed, J.C. and Kroemer, G. (1998) Bax and adenine nucleotide translocator cooperate in the mitochondrial control of apoptosis. Science 281:2027–2031. McHugh, M.P., Magnusson, S.P., Gleim, G.W., and Nicholas, J.A. (1992) Viscoelastic stress relaxation in human skeletal muscle. Med Sci Sports Exerc 24:1375–1382. McKinsey, T.A., Zhang, C.L. and Olson, E.N. (2001) Control of muscle development by dueling HATs and HDACs. Curr Opin Genet Dev 11:497–504. McKoy, G., Ashley, W., Mander, J., Yang, S.Y., Williams, N., Russell, B. and Goldspink, G. (1999) Expression of insulin growth factor-1 splice variants and structural genes in rabbit skeletal muscle induced by stretch and stimulation. Physiol 516:583–592. Nediani, C., Celli, A., Fiorillo, C., Ponziani, V., Giannini, L. and Nassi, P. (2003) Acylphosphatase interferes with SERCA2a-PLN association. Biochem Biophys Res Commun 301:948–951. Nguyen, T., Sherratt, P.J., Yang, C.S. and Pickett, C.B. (2005) Nrf2 controls constitutive and inducible expression of ARE-driven genes through a dynamic pathway involving nucleocytoplasmic shuttling by Keap1. J Biol Chem 280:32485–32492. Pecina, P., Gnaiger, E., Zeman, J., Pronicka, E. and Houstek, J. (2004) Decreased affinity for oxygen of cytochrome-c oxidase in Leigh syndrome caused by SURF1 mutations. Am J Physiol Cell Physiol 287:C1384–C1388.

157 Perry, S.V. (2003) What is the role of tropomyosin in the regulation of muscle contraction? J Muscle Res Cell Motil 24:593–596. Poyau, A., Buchet, K. and Godinot, C. (1999) Sequence conservation from human to prokaryotes of Surf1, a protein involved in cytochrome c oxidase assembly, deficient in Leigh syndrome. FEBS Lett 462:416–420. Radzyukevich, T.L. and Heiny, J.A. (2004) Regulation of dihydropyridine receptor gene expression in mouse skeletal muscles by stretch and disuse. Am J Physiol Cell Physiol 287:C1445– C1452. Schulte, L.M., Navarro, J. and Kandarian, S.C. (1993) Regulation of sarcoplasmic reticulum calcium pump gene expression by hindlimb unweighting. Am J Physiol 264:C1308–C1315. Siu, P.M. and Always, S.E. (2005) Subcellular responses of p53 and Id2 in fast and slow skeletal muscle in response to stretchinduced overload. J Appl Physiol 99:1897–1904. St-Amand, J., Okamura, K., Matsumoto, K., Shimizu, S. and Sogawa, Y. (2001) Characterization of control and immobilized skeletal muscle: an overview from genetic engineering. FASEB J 15:684–692. Stanhope-Baker, P., Kessler, P.M., Li, W., Agarwal, M.L. and Williams, B.R. (2004) The Wilms tumor suppressor-1 target gene podocalyxin is transcriptionally repressed by p53. J Biol Chem 279:33575–33385. Stewart, D., Killeen, E., Naquin, R., Alam, S. and Alam, J. (2003) Degradation of transcription factor Nrf2 via the ubiquitin-proteasome pathway and stabilization by cadmium. J Biol Chem 278:2396–2402. Tabary, J.C., Tabary, C., Tardieu, C., Tardieu, G. and Goldspink, G. (1972) Physiological and structural changes in the cat’s soleus muscle due to immobilization at different lengths by plaster casts. J Physiol 224:231–244.

Tiranti, V., Hoertnagel, K., Carrozzo, R., Galimberti, C., Munaro, M., Granatiero, M., Zelante, L., Gasparini, P., Marzella, R., Rocchi, M., Bayona-Bafaluy, M.P., Enriquez, J.A., Uziel, G., Bertini, E., Dionisi-Vici, C., Franco, B., Meitinger, T. and Zeviani, M. (1998) Mutations of SURF-1 in Leigh disease associated with cytochrome c oxidase deficiency. Am J Hum Genet 63:1609– 1621. Walmsley, S.J. and Gaines, P.J. (2004) Identification of two cDNAs encoding synaptic vesicle protein 2 (SV2)-like proteins from epithelial tissues in the cat flea, Ctenocephalides felis. Insect Mol Biol 13:225–230. Williams, P.E. (1990) Use of intermittent stretch in the prevention of serial sarcomere loss in immobilised muscle. Ann Rheum Dis 49:316–317. Williams, P.E. and Goldspink, G. (1973) The effect of immobilization on the longitudinal growth of striated muscle fibres. J Anat 116:45–55. Williams, P.E. and Goldspink, G. (1978) Changes in sarcomere length and physiological properties in immobilized muscle. J Anat 127:459–468. Yano, M., Terada, K. and Mori, M. (2004) Mitochondrial import receptors Tom20 and Tom22 have chaperone-like activity. J Biol Chem 279:10808–10813. Zador, E., Dux, L. and Wuytack, F. (1999) Prolonged passive stretch of rat soleus muscle provokes an increase in the mRNA levels of the muscle regulatory factors distributed along the entire length of the fibers. J Muscle Res Cell Motil 20:395–402. Zhu, Z., Yao, J., Johns, T., Fu, K., De Bie, I., Macmillan, C., Cuthbert, A.P., Newbold, R.F., Wang, J., Chevrette, M., Brown, G.K., Brown, R.M. and Shoubridge, E.A. (1998) SURF1, encoding a factor involved in the biogenesis of cytochrome c oxidase, is mutated in Leigh syndrome. Nat Genet 20:337–343.