Muscle cell growth in protein deficient rats following administration of sheep red blood cells.

In order to observe the effects of sheep red blood cells (SRBC) administration on the muscle cell growth in malnourished states, adult male Wistar rats (135 +/- 10 g 10 animals per group) subjected during 30 days to 1% and 10% protein diets, were injected (i.v.) either 15.5 x 10(8) sheep red blood cells or 0.5 ml saline/100 g b.w. after 20 days of experiment. On the 10th day after injection the animals were sacrificed and the gastrocnemius muscle was removed, weighed and homogenized. The supernatant fluids were used to evaluate muscle protein, DNA and RNA rates and acid DNase activity. All parameters were depleted in malnourished rats, indicating a muscle cellular atrophy as well as a decrease in muscle protein synthesis per DNA-unit. Muscle hyperplasia and hypertrophy were found in antigenically stimulated rats fed 10% protein against non-stimulated control. In contrast, muscle growth in protein-deficient rats SRBC-treated was unmodified when compared to non-stimulated malnourished muscle, although RNA functionality seems to be enhanced (RNA/DNA). These data suggest that a redistribution of essential nutrients occurred for muscle growth adaptation rather than for defensive mechanism.     

Plasma membrane biogenesis in the avian salt gland: a biochemical and quantitative electron microscopic autoradiographic study

The avian salt gland provides an ideal system for the study of plasma membrane (PM) biogenesis. Feeding ducklings 1% sodium chloride (salt stress) induces the secretory cells of the gland to synthesize large amounts of PM, which forms an extensive basolateral PM domain after 7-9 days of treatment. In the present study, the initial biosynthetic events following salt stress were investigated. In vivo studies using 3H-uridine indicated that increased rates of RNA synthesis could be detected by 2 hr after the beginning of salt stress and continued through at least 12 hr. Under in vitro conditions, increased rates of protein and glycoprotein synthesis (as monitored by 3H-leucine and 3H-fucose incorporation, respectively) were also detected after 2 hr and continued through 7-9 days. Increased levels of Na,K-ATPase, a specific secretory cell PM marker, were detected after 8 hr of treatment as monitored by specific activity and 3H-ouabain binding. Sodium dodecyl sulfate-polyacrylamide slab gel electrophoresis coupled with fluorography indicated that both 3H-leucine and 3H-fucose were incorporated into partially purified preparations of Na,K-ATPase isolated after 12 hr. Light microscopic autoradiographic analysis of pulse-chase experiments indicated that in secretory cells of 12-hr salt-stressed glands, 3H-leucine- and 3H-fucose-labelled products reached the cell periphery by 1-2 hr after the initial pulse. The incorporation of both tritiated precursors was predominantly associated with the secretory cells. Quantitative electron microscopic autoradiography indicated that 3H-leucine is initially taken up by elements of the rough endoplasmic reticulum (RER) and cytoplasm (5 min postpulse), subsequently transported to and concentrated within components of the Golgi apparatus (10 min of chase), and ultimately incorporated into all domains of the plasma membrane of secretory cells by 1-2 hr of chase. The data is consistent with a flow of newly synthesized membrane components from RER to Golgi to plasma membrane and is analogous to the pattern previously found for the synthesis and processing of PM proteins in a wide variety of cell types.     

Nuclear translocation of EndoG at the initiation of disuse muscle atrophy and apoptosis is specific to myonuclei

Skeletal muscle atrophy is associated with an increase in apoptosis, and we showed previously that endonuclease G (EndoG) is localized to nuclei following unloading. The goal of this study was to determine whether the onset of apoptosis in response to disuse was consistent with the hypothesis that EndoG is involved in myofiber nuclear loss. Atrophy was induced by hindlimb suspension for 12 h or 1, 2, 4 and 7 days in 6-mo-old rats. Soleus myofiber cross-sectional area decreased significantly by 2 days, whereas muscle mass and muscle-to-body mass ratio decreased by 4 and 7 days, respectively. By contrast, a significant increase in apoptosis, evidenced by terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL)-positive nuclei, occurred as early as 12 h after suspension, preceding the elevation in muscle atrophy F-box gene expression. The early increase in apoptosis appeared to be specific to myofiber nuclei, whereas TUNEL-positive interstitial cells did not become significantly elevated until 2 days after suspension. Furthermore, TUNEL-positive myofiber nuclei colocalized with EndoG as early as 12 h after suspension, and no such localization was observed in interstitial cells. Although no significant change in total activated caspase-3, -7, or -12 protein abundance was apparent, activated caspase-3 was expressed in interstitial cells undergoing apoptosis, some of which were endothelial cells. These data indicate that apoptosis is an early, and therefore possibly causative, event in the process of muscle atrophy, and that EndoG nuclear translocation is specific for myofiber nuclear apoptosis, whereas interstitial cells may undergo apoptosis via a more classical, caspase-dependent pathway.     

放线菌素D和环已亚胺对伊贝母胚性愈伤组织中胚性细胞团和球形胚发生及大分子代谢动态的影响

伊贝母(F.pallidiflora Schrenk)胚性愈伤组织接种于NAA 1.0mg/L+6-BA2.0 mg/L的MS培养基上,在培养10天前可产生大量单细胞到多细胞胚性细胞团,培养10至15天,逐渐形成大量球形胚。利用这样一个实验体系,在培养0、1、2、3和4天后加入放线菌素D(AMD,20μg/ml)和环己亚胺(CHM,20μg/ml),继续培养至第6天,分析大分子代谢动态和观察胚性细胞团的形成情况;培养6和10天后加入同样浓度的AMD和CHM。继续培养至第15天,分析大分子代谢动态及观察球形胚形成情况。结果表明:(1)培养0、1、2、3和4天加入AMD的分别抑制胚性细胞团的100%、63%和45%,加入CHM的抑制100%、85%和75%,培养6和10天后加入CHM抑制球形胚的100%和75%;(2)DNA、RNA和蛋白质在胚性细胞团和球形胚形成时出现两个峰值,其中RNA变化剧烈,最早出现峰值。AMD和CHM分别抑制RNA和蛋白质的合成;(3)过氧化物酶同工酶带在胚性细胞团和球形胚形成过程中顺序表达,AMD和CHM分别在转录和转译水平上对其进行规律性抑制。根据以上结果,本文对伊贝母体细胞胚胎发生的机制进行了初步讨论。     

RNA synthesis in the ultrastructural and biochemical components of the nucleolus of Chinese hamster ovary cells

A correlated autoradiographic and biochemical study of RNA synthesis in the nucleoli of chinese hamster ovary cells has been made. Quantitative analysis of the labeling indicates that the fibrillar ribonucleoprotein (RNP) component is labeled faster than 80S RNP and 45S RNA molecules, but approaches simultaneously a steady-state 3H to 14C ratio or grains/mum2 after 30 min of [3H]uridine incorporation. On the other hand, the 55S RNP, the 36S + 32S RNA, and the granular RNP components have the same kinetic of labeling with [3H]uridine. These results suggest that the fibrillar and granular RNP components of the nucleolus are the ultrastructural substratum of, respectively, the 80S RNP (45S RNA) and 55S RNP (36S + 32S RNA). The possibility that precursors to 80S RNP exist also in the fibrillar region of the nucleolus is strongly suggested by the rapid labeling of the fibrils on the autoradiographs.     

Cell accumulation in the junctional region of denervated muscle

If skeletal muscles are denervated, the number of mononucleated cells in the connective tissue between muscle fibers increases. Since interstitial cells might remodel extracellular matrix, and since extracellular matrix in nerve and muscle plays a direct role in reinnervation of the sites of the original neuromuscular junctions, we sought to determine whether interstitial cell accumulation differs between junctional and extrajunctional regions of denervated muscle. We found in muscles from frog and rat that the increase in interstitial cell number was severalfold (14-fold for frog, sevenfold for rat) greater in the vicinity of junctional sites than in extrajunctional regions. Characteristics of the response at the junctional sites of frog muscles are as follows. During chronic denervation, the accumulation of interstitial cells begins within 1 wk and it is maximal by 3 wk. Reinnervation 1-2 wk after nerve damage prevents the maximal accumulation. Processes of the cells form a multilayered veil around muscle fibers but make little, if any, contact with the muscle cell or its basal lamina sheath. The results of additional experiments indicate that the accumulated cells do not originate from terminal Schwann cells or from muscle satellite cells. Most likely the cells are derived from fibroblasts that normally occupy the space between muscle fibers and are known to make and degrade extracellular matrix components.     

Effect of methotrexate on cells in a keratinized epithelium with an active thymidine salvage pathway assessed by autoradiography

In the partially synchronized cell system of the hamster cheek pouch epithelium, the inhibitory effect of a bolus injection of methotrexate (Mtx) (2 g/m2, injected at 1200 hr) was analysed by means of both autoradiography and flow cytometry (FCM) in a 21-hr experiment. For autoradiography [3H]TdR and [3H]UdR were used as tracers for salvage and de novo pathways of thymidylate (TMP) synthesis, respectively. For FCM no tracers were injected. The autoradiographic studies demonstrated an active TdR salvage pathway for DNA synthesis, not affected by the impaired de novo TMP synthesis. The blocked de novo TMP synthesis was partially released 7 hr after Mtx injection, but it had not totally recovered at the end of the experiment. The decrease in the fraction of S-phase cells detected about 10 hr after Mtx injection by autoradiographic labelling with [3H]TdR and by FCM was found to be caused by a decrease in the number of cells entering S phase. However, Mtx did not influence the salvage TMP synthesis rate of cells entering S phase.     

NUCLEIC ACID AND PROTEIN METABOLISM DURING THE MITOTIC CYCLE IN VICIA FABA

In order to investigate some of the cytochemical processes involved in interphase growth and culminating in cell division, a combined autoradiographic and microphotometric study of nucleic acids and proteins was undertaken on statistically seriated cells of Vicia faba root meristems. Adenine-8-C¹⁴ and uridine-H³ were used as ribonucleic acid (RNA) precursors, thymidine-H³ as a deoxyribonucleic acid (DNA) precursor, and phenylalanine-3-C¹⁴ as a protein precursor. Stains used in microphotometry were Feulgen (DNA), azure B (RNA), pH 2.0 fast green (total protein), and pH 8.1 fast green (histone). The autoradiographic data (representing rate of incorporation per organelle) and the microphotometric data (representing changes in amounts of the various components) indicate that the mitotic cycle may be divided into several metabolic phases, three predominantly anabolic (net increase), and a fourth phase predominantly catabolic (net decrease). The anabolic periods are: 1. Telophase to post-telophase during which there are high rates of accumulation of cytoplasmic and nucleolar RNA and nucleolar and chromosomal total protein. 2. Post-telophase to preprophase characterized by histone synthesis and a diphasic synthesis of DNA with the peak of synthesis at mid-interphase and a minor peak just preceding prophase. The minor peak is coincident with a relatively localized DNA synthesis in several chromosomal regions. This period is also characterized by minimal accumulations of cytoplasmic RNA and chromosomal and nucleolar total protein and RNA. 3. Preprophase to prophase in which there are again high rates of accumulation of cytoplasmic RNA, and nucleolar and chromosomal total protein and RNA. The catabolic phase is: 4. The mitotic division during which there are marked losses of cytoplasmic RNA and chromosomal and nucleolar total protein and RNA.     

Cardiac hypertrophy in rats after supravalvular aortic constriction. I. Size and number of cardiomyocytes, endothelial and interstitial cells

The ascending aorta of 22 adult male Sprague-Dawley rats was constricted with a silver ring, and 25 animals were subjected to a sham-operation. The hearts, including the main arteries, were fixed by retrograde perfusion 3, 7, 14, 21 and 35 days after the operation. The cross-sectional area of the aorta was reduced by the constriction to an average of 20% of the values found after sham-operation. Twenty-one days after the constriction the weight of the left ventricular myocardium including the septum was increased 1.7-fold compared with controls. No further increase in weight was observed 35 days after the operation. The relative volumes of the tissue components remained largely constant in the subepicardial myocardium. In the subendocardial myocardium, however, the volume fraction of interstitial and, to a lesser extent, of endothelial tissue was significantly increased. Twenty-one days after constriction the estimated total volumes of the different myocardial components per left ventricle were increased 1.7-fold for heart muscle parenchyma, 1.8-fold for endothelial tissue, 2.9-fold for interstitial tissue, and 1.3-fold for capillary lumina compared with controls. At 35 days, only the interstitial tissue showed a further increase to 4.8-fold of control values. The mean cardiomyocyte volume was increased after aortic constriction in proportion to the increase in left ventricular weight, i.e. 1.7-fold over controls at 21 days. After 35 days its value was 29,500 +/- 790 micron 3 in rats subjected to aortic constriction compared with 16,800 +/- 640 micron 3 in controls. At this time the estimated number of cardiomyocytes per left ventricle showed no significant differences between experimental animals (2.9 X 10(7)) and controls (3.1 X 10(7)). Endothelial and interstitial cells were not only increased in average single cell volume (1.3-fold and 2.0-fold, respectively), but also in number per left ventricle (1.4-fold and 2.7-fold, respectively). Two-dimensional parameters indicated that during hypertrophy the capillary supply lagged behind the overall mass increase but achieved control levels on termination of hypertrophic growth at 35 days. These results show that even in pronounced hypertrophy the increase in mass of the myocardial parenchyma in the rat is due exclusively to an enlargement of cardiomyocytes (hypertrophy), whereas in endothelial and interstitial tissues enlargement of cells as well as increase in cell number (hyperplasia) also plays a role.     

Stimulation of epidermal protein synthesis in vivo by topical triamcinolone acetonide.

The rate of epidermal protein synthesis in vivo was determined in the hairless mouse by a method in which a large dose of [3H]phenylalanine (150 mumol/100 g body wt.) is administered via the tail vein. The epidermal free phenylalanine specific radioactivity rapidly rose to a plateau value which by 10 min approached that of plasma, after which it declined. This dose of phenylalanine did not of itself alter protein synthesis rates, since incorporation of co-injected tracer doses of [3H]lysine and [14C]threonine was unaffected. The fractional rate of protein synthesis obtained for epidermis was 61.6%/day, whereas values for liver and gastrocnemius muscle in the same group of mice were 44%/day and 4.8%/day respectively. When expressed on the basis of RNA content, the value for epidermis (18.6 mg of protein/day per mg of RNA) was approx. 3-fold higher than those for liver and gastrocnemius muscle. Topical administration of 0.1% triamcinolone acetonide increased the epidermal fractional protein synthesis rate by 33% after 1 day and by 69% after 7 days, compared with vehicle-treated controls. These effects were entirely accounted for by the increase in protein synthesis rates per mg of RNA. RNA/protein ratios were unaffected by this treatment.     

Reducing plasma HIV RNA improves muscle amino acid metabolism

We reported (Yarasheski KE, Zachwieja JJ, Gischler J, Crowley J, Horgan MM, and Powderly WG. Am J Physiol Endocrinol Metab 275: E577-E583, 1998) that AIDS muscle wasting was associated with an inappropriately low rate of muscle protein synthesis and an elevated glutamine rate of appearance (Ra Gln). We hypothesized that high plasma HIV RNA caused dysregulation of muscle amino acid metabolism. We determined whether a reduction in HIV RNA (> or =1 log) increased muscle protein synthesis rate and reduced R(a) Gln and muscle proteasome activity in 10 men and 1 woman (22-57 yr, 60-108 kg, 17-33 kg muscle) with advanced HIV (CD4 = 0-311 cells/microl; HIV RNA = 10-375 x 10(3) copies/ml). We utilized stable isotope tracer methodologies ([13C]Leu and [15N]Gln) to measure the fractional rate of mixed muscle protein synthesis and plasma Ra Gln in these subjects before and 4 mo after initiating their first or a salvage antiretroviral therapy regimen. After treatment, median CD4 increased (98 vs. 139 cells/microl, P = 0.009) and median HIV RNA was reduced (155,828 vs. 100 copies/ml, P = 0.003). Mixed muscle protein synthesis rate increased (0.062 +/- 0.005 vs. 0.078 +/- 0.006%/h, P = 0.01), Ra Gln decreased (387 +/- 33 vs. 323 +/- 15 micromol.kg fat-free mass(-1).h(-1), P = 0.04), and muscle proteasome chymotrypsin-like catalytic activity was reduced 14% (P = 0.03). Muscle mass was only modestly increased (1 kg, P = not significant). We estimated that, for each 10,000 copies/ml reduction in HIV RNA, approximately 3 g of additional muscle protein are synthesized per day. These findings suggest that reducing HIV RNA increases muscle protein synthesis and reduces muscle proteolysis, but muscle protein synthesis relative to whole body protein synthesis rate is not restored to normal, so muscle mass is not substantially increased.     

Myostatin short interfering hairpin RNA gene transfer increases skeletal muscle mass

BACKGROUND: Myostatin negatively regulates skeletal muscle growth. Myostatin knockout mice exhibit muscle hypertrophy and decreased interstitial fibrosis. We investigated whether a plasmid expressing a short hairpin interfering RNA (shRNA) against myostatin and transduced using electroporation would increase local skeletal muscle mass. METHODS: Short interfering RNAs (siRNAs) targeting myostatin were co-transfected with a myostatin-expressing plasmid into HEK293 cells and identified for myostatin silencing by Western blot. Corresponding shRNAs were cloned into plasmid shRNA expression vectors. Myostatin or a randomer negative control shRNA plasmid was injected and electroporated into the tibialis anterior or its contralateral muscle, respectively, of nine rats that were sacrificed after 2 weeks. Six other rats received a beta-galactosidase reporter plasmid and were sacrificed at 1, 2, and 4 weeks. Uptake of plasmid was examined by beta-galactosidase expression, whereas myostatin expression was determined by real-time polymerase chain reaction (PCR) and Western blotting. Muscle fiber size was determined by histochemistry. Satellite cell proliferation was determined by PAX7 immunohistochemistry. Myosin heavy chain type II (MHCII) expression was determined by Western blot. RESULTS: beta-Galactosidase reporter plasmid was expressed at 1 and 2 weeks but diminished by 4 weeks in tibialis anterior skeletal muscle. Myostatin shRNA reduced myostatin mRNA and protein expression by 27 and 48%, respectively. Tibialis anterior weight, fiber size, and MHCII increased by 10, 34, and 38%, respectively. Satellite cell number was increased by over 2-fold. CONCLUSIONS: This is the first demonstration that myostatin shRNA gene transfer is a potential strategy to increase muscle mass.     

MACROMOLECULAR EVENTS LEADING TO CELL DIVISION IN TETRAHYMENA PYRIFORMIS AFTER REMOVAL AND REPLACEMENT OF REQUIRED PYRIMIDINES

Tetrahymena pyriformis were brought to a non-growing state by removal of pyrimidines from their growth medium. During pyrimidine deprivation cell number increased 3- to 4 fold, and this increase was accompanied by one or more complete cycles of macronuclear DNA replication. Autoradiographic studies show that endogenous protein and RNA were turning over throughout starvation and that RNA breakdown products were used to support the DNA synthesis that occurred during the early period of starvation. However, after 72 hours of starvation all DNA synthesis and cell division had ceased. Feulgen microspectrophotometry shows the macronuclei of these cells to have been stopped at a point prior to DNA replication (G1 stage). After pyrimidine replacement the incorporation of H³-uridine, H³-adenosine, and H³-leucine was measured by the autoradiographic grain counting method. The results indicate that RNA synthesis began to increase almost immediately, but that there was a lag of almost an hour before an increase in protein synthesis. In agreement with the autoradiographic data, chemical data also show that cellular content of RNA began to increase shortly after pyrimidine replacement but that cellular protein content did not increase until about one hour later. Pulse labeling of the cells with H³-thymidine at intervals after pyrimidine replacement shows that labeled macronuclei first began to appear at 150 minutes; that 98 per cent of the macronuclei were in DNA synthesis at 240 to 270 minutes; and that the percentage then began to decrease from 300 to 390 minutes, at which time only 25 per cent of the macronuclei were labeled. Cellular content of DNA did not increase for at least 135 minutes after pyrimidine replacement; however, just before the first cells divided (360 minutes) the DNA content had doubled. After pyrimidine replacement the cells first began to divide at 360 minutes, and 50 per cent had divided at 420 minutes; however, all cells had not divided until 573 minutes. This technique of chemical synchronization of cells in mass cultures makes feasible detailed biochemical analysis of events leading to nuclear DNA replication and cell division.     

Interstitial cell proliferation in the testis of the ethylene dimethane sulfonate-treated rat.

This study was conducted to examine interstitial cell proliferation in the testis of the ethylene dimethane sulfonate (EDS)-treated rat. Initial autoradiographic studies demonstrated a peak of [3H]thymidine incorporation by interstitial cells at 2 and 4 days post-EDS treatment. Subsequent studies were designed using in vivo pulse labeling regimens in an attempt to identify interstitial cell proliferation associated with Leydig cell regeneration. Rats were injected with [3H]thymidine at days 2 and 4 post-EDS and were killed 6 hours later or at 30 days post-EDS. Although cells labeled at 2 and 4 days post-EDS appeared to undergo subsequent division, the Leydig cells visible at 30 days post-EDS were not labeled. In a second study, rats were injected with [3H]thymidine at days 10 and 20 post-EDS and were killed either 6 hours later or at 24 days post-EDS. In the 10-day post-EDS group, interstitial cells were labeled at both the 6-hour and 24-day time points; however, Leydig cells present at 24 days were not labeled. In contrast, the testes of rats that were killed at 20 days post-EDS (6 hours labeling period) contained Leydig cells that displayed grains over the nucleus, thus suggesting that Leydig cell proliferation had occurred. In addition, a high number of the Leydig cells observed at 24 days post-EDS were labeled, suggesting that they arose from divisions occurring during the 20- to 24-day post-EDS period. These studies demonstrate that interstitial cell proliferation occurs in several stages following EDS treatment.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of Methotrexate On Cells In A Keratinized Epithelium With an Active Thymidine Salvage Pathway Assessed By Autoradiography*

In the partially synchronized cell system of the hamster cheek pouch epithelium, the inhibitory effect of a bolus injection of methotrexate (Mtx) (2 g/m², injected at 1200 hr) was analysed by means of both autoradiography and flow cytometry (FCM) in a 21-hr experiment. For autoradiography [³H]TdR and [³H]UdR were used as tracers for salvage and de nouo pathways of thymidylate (TMP) synthesis, respectively. For FCM no tracers were injected. the autoradiographic studies demonstrated an active TdR salvage pathway for DNA synthesis, not affected by the impaired de novo TMP synthesis. the blocked de novo TMP synthesis was partially released 7 hr after Mtx injection, but it had not totally recovered at the end of the experiment. the decrease in the fraction of S-phase cells detected about 10 hr after Mtx injection by autoradiographic labelling with [³H]TdR and by FCM was found to be caused by a decrease in the number of cells entering S phase. However, Mtx did not influence the salvage TMP synthesis rate of cells entering S phase.)     

Distinct roles for Pax7 and Pax3 in adult regenerative myogenesis

We assessed viable Pax7(-/-) mice in 129Sv/J background and observed reduced growth and marked muscle wasting together with a complete absence of functional satellite cells. Acute injury resulted in an extreme deficit in muscle regeneration. However, a small number of regenerated myofibers were detected, suggesting the presence of residual myogenic cells in Pax7-deficient muscle. Rare Pax3(+)/MyoD+ myoblasts were recovered from Pax7(-/-) muscle homogenates and cultures of myofiber bundles but not from single myofibers free of interstitial tissues. Finally, we identified Pax3+ cells in the muscle interstitial environment and demonstrated that they coexpressed MyoD during regeneration. Sublaminar satellite cells in hind limb muscle did not express detectable levels of Pax3 protein or messenger RNA. Therefore, we conclude that interstitial Pax3+ cells represent a novel myogenic population that is distinct from the sublaminar satellite cell lineage and that Pax7 is essential for the formation of functional myogenic progenitors from sublaminar satellite cells.     

Effect of luteinizing hormone on RNA synthesis in Leydig cells of immature rats

Luteotrophic hormone acts on testicular interstitial cells, promoting the activation of several cellular events that culminate in steroids synthesis. Since the interstitial tissue include several cell types, purified Leydig cells were used in this work. Isolated interstitial cells from immature rats were purified through a 0-40% metrizamide gradient. Either LH, HCG or Bt2-cAMP significantly stimulated the incorporation of [3H]uridine into RNA, when compared to control. The effect of HCG on RNA synthesis was developed within 30 min after the addition of the hormone and was dose-dependent. The maximum effect was attained with 10 mIU/ml of HCG. These results indicate that HCG/LH or Bt2-cAMP but not FSH, promote an acute stimulation of RNA synthesis by Leydig cells from immature rats.     

Serum response factor plays an important role in the mechanically overloaded plantaris muscle of rats

Molecular signaling pathways linking the hypertrophy after mechanical overloading in vivo have not been identified. Using western blot analysis, immunoprecipitation, and immunohistochemistry, we investigated the effect of the mechanical overloading state on RhoA, serum response factor (SRF), and MyoD in the rat plantaris muscle. Adult male rats (10 weeks of age) were used in this experiment. Compensatory enlargement of the plantaris muscle was induced in one leg of each rat by surgical removal of the ipsilateral soleus and gastrocnemius muscles. In the normal plantaris muscle of rats, slight expression of RhoA and SRF was observed in the quiescent satellite cells possessing CD34 and c-Met. Western blotting using the homogenate of whole muscle clearly showed that mechanical overloading of the plantaris muscle significantly increased the amount of RhoA during 3-6 days postsurgery. Threonine phosphorylation of SRF occurred at 2-4 h after mechanical overloading. The most marked increase in SRF protein was observed in the hypertrophied muscle at 6 days postsurgery. At 2 days postoperation, SRF immunoreactivity was not detected in the proliferating satellite cells possessing bromodeoxyuridine and in the infiltrating macrophages expressing ED1 in the overloaded muscle by surgical removal. The SRF protein was colocalized with RhoA, FAK, and myogenin but not Myf-5 in many mononuclear cells at 6 days of functional overload. At this time, MyoD immunoreactivity was detected in the cytoplasm of mononuclear cells (possibly satellite cell-derived myoblasts) possessing SRF protein at the nucleus. These results suggest that the signaling pathway through RhoA-FAK-SRF is important to the differentiation of satellite cells by interacting MyoD and myogenin in the hypertrophied muscle of rats.