skeletal muscle atrophy caused by disuse , unloading , weightlessness , poor nutrition and 
 disease leads to decreased muscle fiber area and muscle strength .
by contrast , skeletal 
 muscle hypertrophy caused by mechanical stimulation such as resistance training and loading 
 with terrestrial gravitation results in an increase in muscle fiber area and muscle 
 strength .
this enlargement in the morphological size of the muscle is mainly due to an 
 increase in the size of muscle fibers1 , 2 .
consequently , when skeletal muscle atrophies due to disuse 
 caused by local immobilization or unloading during treatment after an external injury , 
 physical therapy is applied to encourage early recovery .
resistance training is the standard method of inducing muscle hypertrophy in humans and 
 other animals3,4,5 , and it is important for 
 physical therapy to generate muscle hypertrophy and to intensify muscle power .
exercise with 
 relatively high external resistance equivalent to 7085% of the one - repetition maximum ( 1rm ) 
 is generally recommended for increasing strength via resistance training .
dons et al.6 reported that dynamic muscle strength 
 increases in response to lifting weight at 80% 1rm .
high - intensity resistance training is 
 effective in humans from the viewpoint of the rate of muscle protein synthesis7 , 8 . 

tamaki et al.9 showed that resistance 
 training with 6575% 1rm for 12 weeks induced muscle hypertrophy in rats .
suetta et al.10 also reported that retraining with a 
 6570% 1rm load for 4 weeks recovered the strength of the human quadriceps muscle with 
 disuse atrophy induced by immobilization for two weeks .
further , campbell et al.11 indicated that 3 weeks of resistance 
 training at 80% 1rm after unilateral lower limb suspension for 3 weeks can temporarily 
 recover a decrease in isometric torque , muscle volume , fascicle length , and physiological 
 cross - sectional area in the quadriceps muscle of young men .
however , the effect of 
 resistance training has not been investigated with respect to the repetition maximum concept 
 when targeting an atrophic muscle in an animal model of muscle atrophy caused by disuse .
the ubiquitin - proteasome system contributes to a specific proteolysis mechanism and is 
 involved in muscle atrophy , as is the autophagy - phagosome system . in this system ,
myostatin 
 ( involved in the control of muscle size ) , muscle atrophy f - box protein ( atrogin-1 ; plays a 
 role in the proteolysis of myogenic differentiation 1 ( myod ) ) , eukaryotic translation 
 inhibition factor 3 subunit f ( eif3f ) , desmin and vimentin , and muscle ring - finger protein-1 
 ( murf1 ; involved in the ubiquitination of myosin heavy chain , myosin light chain , actin , 
 troponin i , and myosin binding protein c ) are all expressed during muscle atrophy via the 
 ubiquitin - proteasome system12,13,14,15,16,17,18,19 .
resistance training decreases the mrna expression of 
 myostatin , atrogin-1 , and murf120 , 21 .
expression of insulin - like growth factor-1 ( igf-1 ) mrna activates the akt / mammalian target 
 of rapamycin ( mtor)/p70s6 kinase ( p70s6  k ) pathway via eccentric exercise and increases the 
 size of the rat gastrocnemius muscle22 . 

resistance training during the initial stages of unloading - induced muscle atrophy can 
 suppress this decrease in muscle size by increasing levels of igf-1 mrna and the activity of 
 anabolic signaling intermediates such as p70s6  k in rats23 . however , chronic resistance training increases igf-1 mrna 
 expression and suppresses the decrease in muscle size , although such training suppresses 
 akt / mtor phosphorylation in aged rats4 . 

thus , muscle atrophy and hypertrophy are controlled by highly specific and complex molecular 
 mechanisms .
consequently , disuse atrophy is treated by resistance training and/or physical stimulation 
 during physical therapy .
however , the one - repetition maximum is an important basis of 
 training intensity applied for resistance training in physical therapy , although many 
 contrasting studies suggest that it is not the optimal value for recovering the strength of 
 atrophic muscle . therefore , we examined the effect of resistance training using the 
 repetition maximum concept on recovery of disused atrophic muscle in physical therapy .
animal care and handling proceeded according to the guidelines for the care and use of 
 laboratory animals at kanazawa university , and the committee on animal experimentation at 
 kanazawa university approved all protocols .
thirty - six male wistar rats aged 9 weeks ( kiwa 
 laboratory animals co. , ltd .
, misato - cho , wakayama , japan ) were housed in a 
 temperature - controlled room ( 2024  c ) with a 12-h light/12-h dark cycle and access to 
 laboratory chow and water ad libitum .
we determined the histological effect of one regime of 
 squat exercise at 1rm in six rats after 5 weeks .
thirty rats were then randomly allocated 
 into the following groups ( n = 6 per group ) : the control , unload ( hindlimb unloaded as 
 described by morey - holton et al.24 for 2 
 weeks ) , reload ( unloading for 2 weeks followed by reloading for 3 weeks without training ) , 
 50% 1rm ( unloading for 2 weeks followed by reloading for 3 weeks with training at 50% 1rm ) 
 and 70% 1rm ( unloading for 2 weeks followed by reloading for 3 weeks with training at 70% 
 1rm groups ) .
both resistance training and 1rm measurements were achieved using the modified animal squat 
 training device developed by krisan et al.25 we used an electric stimulator ( nihon kohden , tokyo , japan ) to 
 stimulate the soles of both feet with 5 v of electricity , which caused the rats to react in 
 a manner resembling plyometric squats , thus generating substantial concentric and eccentric 
 contraction of the quadriceps muscle .
the 1rm in the reload , 50% 1rm and 70% 1rm groups was 
 measured after 1 day of reloading , and training at the 50% 1rm and 70% 1rm intensities was 
 started as follows : 14 repetitions / set , three sets / day , three times / week at 50% 1rm and 10 
 repetitions / set at 70% 1rm , with 1-minute intervals between sets .
the resistance weight 
 comprised 10  g or 25  g stainless steel plumbs .
the 1rms of the reload , 50% 1rm , and 70% 1rm groups were measured after reloading for 3 
 weeks and were also measured at 2 days before tissue extraction . after euthanasia , the 
 rectus femoris muscle was extracted from the right hindlimbs of all rats , placed in 
 tissue - tek o.c.t .
compound ( sakura finetek japan , tokyo , japan ) and frozen in isopentane 
 cooled in liquid nitrogen .
the frozen tissues were cut using a cm-41 cryostat ( sakura 
 finetek ) into 10-m - thick transverse sections for myosin atpase and hematoxylin and eosin 
 ( he ) staining and then dried for 2 hours at room temperature .
type i and ii fibers in 
 transverse sections were stained with myosin atpase at ph 10.4 .
sections were visualized 
 using a biozero bz-8000 microscope ( keyence , osaka , japan ) .
faint and intensely stained 
 muscle fibers were classified as type i and ii fibers in five regions per section , and the 
 ratios ( % ) of type ii fibers among the totals were calculated in the control group . 

cross - sectional areas of 200 unclassified myofibers per muscle were digitized and assessed 
 using the imagej image analysis software .
samples of muscle tissues from the reload , 50% 1rm , and 70% 1rm groups were soaked in 
 rnalater rna stabilization reagent ( qiagen , tokyo , japan ) overnight at 4  c and stored at 
 80  c .
total rna was then isolated from the stored muscle samples using an 
 rneasy fibrous tissue mini kit ( qiagen ) according to the manufacturer s 
 protocol .
the rnas were reverse transcribed using primescript 1st strand cdna 
 synthesis kits ( takara bio inc . , otsu , shiga , japan ) .
quantitative real - time polymerase 
 chain reaction ( qrt - pcr ) was performed using a lightcycler st300 ( roche 
 diagnostics , tokyo , japan ) and sybr premix ex taq ii ( takara bio inc . ) with the 
 following primer sets ( nihon gene research laboratories inc . ,
sendai , japan ) : 
 5-ccaaggggcttttacttcaac-3 and 5-tggaacgagctgactttgta-3 for igf-1 , 
 5-cattaccatgcctaccgagtc-3 and 5-ggcctttaccactttgttgtactg-3 for myostatin , 
 5-atgagaaaagcggcacct-3 and 5-tggttttgctgttctgtatgtc-3 for atrogin-1 , 
 5-ggaggaggaggatcaaga-3 and 5-ctgagcagagagaagacacac-3 for murf1 , 
 5-aacgggaaacccatcacca-3 and 5-cggagatgatgacccttttg-3 for glyceraldehyde-3-phosphate 
 dehydrogenase ( gapdh ) .
the qrt - pcr conditions were as follows : 10  s at 95  c and then 40 
 cycles of 5  s at 95  c and 20  s at 60  c , and this was followed by melting curve analysis . 

the 
 specificity of each primer set was judged based on the amplification of a single product 
 determined as a single peak at the appropriate melting temperature .
expression levels of 
 igf-1 , myostatin , atrogin-1 and murf1 mrna were normalized by subtracting the corresponding 
 gapdh mrna .
the relative contents of the respective mrnas in trained muscles were compared 
 with reloaded muscle .
differences in body weight and 
 cross - sectional areas between groups were assessed using a one - way analysis of variance 
 followed by tukey s host hoc test .
differences between trained groups and the reload group 
 were detected by dunnett s or steel s multiple comparison tests .
values of p < 0.05 were 
 considered to indicate statistical significance in all tests .
the 1rm in normal rats was 1,842.7  144.1  g , and the ratio of the 1rm to body weight was 
 4.3  0.2 .
muscle fiber injury in the rectus femoris muscle induced by measuring the 1rm was 
 sparse ( fig .
1.hematoxylin and eosin staining of rectus femoral muscles in 14-week - old rats after 
 measuring the 1rmarrowhead : sparse inflammatory cell infiltration ) .
the body weights of the rats in the control , unload , reload , 50% 1rm and 70% 1rm 
 groups were 332.0  6.6  g , 285.5  12.6  g , 380.5  25.8  g , 362.0  26.2  g and 395.0  
 17.9  g , respectively .
the control group weighed significantly less than the reload , 50% 1rm 
 and 70% 1rm groups , and the unload group weighed significantly less than all other groups 
 ( table 1table 1.body weight of rats and cross - sectional areas of the rat rectus femoris 
 musclecontrolunloadreload50%1rm70%1rmbody weight ( g)332.0  6.6285.5  12.6 * 380.5 
25.8362.0  26.2395.0  17.9cross - sectional areas ( m)3,326.3  431.0**2,083.6  375.52,665.5  333.32,816.3  327.72,916.3  549.4**p < 0.05 and
* * p < 0.01 vs. unload ; p < 0.01 vs. control ) . the cross - sectional areas of the rectus femoris muscle , which were initially 
 decreased by hindlimb unloading for two weeks , showed significant recovery in the 70% 1rm 
 training group ( table 1 ) .
hematoxylin and eosin staining of rectus femoral muscles in 14-week - old rats after 
 measuring the 1rm arrowhead : sparse inflammatory cell infiltration * p < 0.05 and * * p < 0.01 vs. unload ; p < 0.01 vs. control the 1rm of rats at the end of 3 weeks of training after reloading was significantly lower 
 than that of the reload group training at 50% 1rm ( table 2table 2.one-repetition maximum of rats followed by reloading and training for 3 
 weeksreload50%1rm70%1rmone - repetition maximum ( g)1,876  1501,628  169 * 1,756  136one - repetition maximum / body weight ( g / g)5.0  0.64.5  0.74.5  0.5*p < 0.05 vs. reload ) .
however , the difference in the ratio of the 1rm to body weight did not 
 significantly differ among the three groups ( table 
 2 ) .
the rectus femoris muscles in the control group consisted almost entirely ( 97.0 
  1.0% ) of type ii fibers with high levels of atpase activity , and type i fibers were mostly 
 located in the periphery of the muscle ( fig .
2.histological analysis of rat rectus femoris muscletype i and ii fibers are intensely and faintly stained , respectively , with myosin 
 atpase in the control group ( a ) . the rectus femoris muscle is largely comprised of 
 type ii fibers in the control group ( a-1 ) ; type i fibers are mostly located in 
 periphery of the rectus femoris muscle ( a-2 ) , and regions without type i fibers are 
 evident ( a-3 ) .
histogram showing the distribution of cross - sectional area in the 
 control , unload , reload , 50% 1rm , and 70% 1rm groups ( b)a13 ) .
the frequency distribution in cross - sectional areas showed global muscle fiber 
 atrophy in unloaded muscles , increased numbers of middle dimension muscle fibers in reloaded 
 muscles , and 50%1rm trained muscles and curves with double peaks in 70% 1rm trained muscles 
 ( fig .
histological analysis of rat rectus femoris muscle type i and ii fibers are intensely and faintly stained , respectively , with myosin 
 atpase in the control group ( a ) . the rectus femoris muscle is largely comprised of 
 type ii fibers in the control group ( a-1 ) ; type i fibers are mostly located in 
 periphery of the rectus femoris muscle ( a-2 ) , and regions without type i fibers are 
 evident ( a-3 ) .
histogram showing the distribution of cross - sectional area in the 
 control , unload , reload , 50% 1rm , and 70% 1rm groups ( b ) next , we assessed the effects of unloading , reloading , and training on the rectus femoris 
 muscle using he staining ( fig . 3fig .
3.hematoxylin and eosin staining of cross sections of the rectus femoris musclesthe left and right columns show overview and pathognomonic findings in unload , 
 reload , 50% 1rm , and 70% 1rm groups , respectively .
though muscle fiber dimension decreased temporarily by unloading , it was recovered 
 by hindlimb unloading for 2 weeks ( fig . 3 ; 

some small fibers in the 70% 1rm trained 
 muscles split and swelled ( fig . 3 ;
the 
 expression of igf-1 , myostatin and atrogin-1 mrna was essentially undetectable in untrained 
 and 50% 1rm and 70% 1rm trained muscles , whereas murf1 mrna expression was significantly 
 downregulated in 70% 1rm trained muscles to the level of untrained muscles ( table 3table 3.real-time pcr analysis of igf-1 , myostatin , atrogin-1 and murf1 mrna expression 
 in rat rectus femoris muscles 3 weeks after reloadingreload50%1rm70%1rmigf-11.0  0.31.2  0.41.3  0.3myostatin1.0  0.41.0  0.31.0  0.3atrogin-11.0  0.50.9  0.20.9  0.3murf11.0  0.21.3  0.70.6  0.2**p < 0.05 vs. reload ) .
hematoxylin and eosin staining of cross sections of the rectus femoris muscles the left and right columns show overview and pathognomonic findings in unload , 
 reload , 50% 1rm , and 70% 1rm groups , respectively .
the right line expanded a square of 
 the left line , respectively . * central nuclei fibers in the reload and 50% 1rm groups . 

in the present study , measurement of the 1rm resulted in injury to muscle fibers in the rat 
 rectus femoris muscle .
a previous study reported similar findings , that is , that squat 
 training leads to the appearance of fibers with central nuclei , split and new small - diameter 
 fibers in the rat plantaris muscle , and increased blood levels of creatine kinase 
 activity9 . despite using different 
 target muscles , these data suggest that brief high intensity exercise such as those 
 performed at the 1rm cause muscle fiber injury in rats . to evaluate the effect of resistance training during reloading for 3 weeks on atrophied 
 rectus femoris muscle that had atrophied as a result of unloading , we measured the 1rms of 
 the reload , 50% 1rm and 70% 1rm groups .
previous studies reported that squat training for 12 
 weeks increases the 1rm9 , 25 .
however , the effect of short - term training for 3 weeks 
 was not investigated .
there was no change in the 1rm in the present study , although 
 measurement of the 1rm , which was performed once , and reloading caused damage to the rectus 
 femoris muscle ( figs . 1 and 3 ) .
reloading followed by unloading reportedly caused muscle fiber 
 injury in the rodent soleus and plantaris muscles26 , 
 27 , and flann et al.28 reported that acute training induces pain 
 in the human quadriceps muscle .
consequently , improving muscle power over the short term 
 seems difficult , particularly in atrophic muscles .
the density of oxidative type i fibers is high in the soleus muscle but only accounts for 
 approximately 7% of the rectus femoris muscle29 . in the present study ,
however , the rats 
 were 1618 weeks old in the study by diaz - herrera29 , whereas those in the present study were 11 weeks old .
the ratio of 
 type i fibers generally increases with age , which may explain these discrepant findings .
unloading for 2 weeks causes atrophy in most rat hindlimb muscles , including the 
 quadriceps30 .
conversely , resistance 
 training in non - artificially manipulated rats using ladder and jumping into water for 8 and 
 12 weeks increased fiber cross - sectional areas in the flexor digitorum longus and plantaris 
 muscles31 , 32 .
many previous studies have reported that recovery of a rat soleus 
 muscle with atrophy induced by hindlimb suspension is accelerated by reloading27 , 33,34,35,36 and running37 , 38 . in the present study , 
 the decreases in body weight and muscle fiber cross - sectional areas induced by unloading for 
 2 weeks were not recovered by reloading for 3 weeks .
however , the cross - sectional area was 
 significantly increased in the 70% 1rm group compared with the unload group .
further , the 
 distribution of cross - sectional areas showed an increased number of small fibers in the 
 unload group and an increased number of bimodal large fibers in the 70% 1rm group .
these 
 findings indicate that 70% 1rm training increased the numbers of muscle fibers that had 
 previously been atrophic .
we measured the total cross - sectional area without discriminating 
 between type i and ii fibers due to deflection of the position of type i fibers , although 
 type i and ii fibers were separated by atpase staining .
previous studies have found that resistance training can injure muscle fibers and lead to 
 elevated levels of regenerative myogenic regulatory factor in skeletal muscle9 , 39 . 
 in the present study ,
pathological alterations such as central nuclei and necrosis fibers 
 were evident after measuring the 1rm and resistance training for 3 weeks . however , secondary 
 muscle damage after exercise that is required for recovery is accelerated40,41,42 .
muscle cell apoptosis and satellite cell 
 activation occur simultaneously during muscle unloading and reloading ; thus , an alternative 
 process involves complete fiber degradation and cell death , followed by de 
 novo synthesis of new fibers via satellite cell activation43 , 44 .
small split 
 myofibers that appear after high - intensity exercise arise through pinching off a small 
 segment of parent muscle fiber or invagination of the sarcolemma deep in muscle fibers in a 
 plane parallel to sarcomeres45 .
we also 
 found evidence of split fibers , indicating that 70% 1rm training provided sufficient 
 high - intensity stimulation to recover the atrophic rat rectus femoris muscle .
for evaluating the effect of resistance training during reloading for three weeks on rectus 
 femoris muscle that had atrophied as a result of unloading , we assessed mrna levels of 
 igf-1 , myostatin , atrogin-1 , and murf1 in the reload , 50% 1rm and 70% 1rm groups .
resistance 
 training did not alter the expression of igf-1 , myostatin , or atrogin-1 mrna , whereas murf1 
 mrna expression was decreased by 70% 1rm training compared with no training .
the 
 muscle - specific ubiquitin e3-ligase murf1 , which is part of the ubiquitin proteasome pathway 
 used for protein degradation during muscle atrophy46 , is upregulated during muscle atrophy47 .
kobayashi et al.48 reported that chronic isometric training decreases murf1 and 
 atrogin-1 protein levels in the gastrocnemius muscles of sprague - dawley but not wistar rats , 
 indicating that wistar rats are less sensitive to resistance training .
louis et al.49 demonstrated an acute increase in murf1 
 mrna expression after resistance training in humans .
by contrast , zanchi et al.21 reported that chronic resistance training 
 for 12 weeks decreased murf1 and atrogin-1 levels in the plantaris muscles of wistar rats . 

these findings suggest that murf1 is upregulated by muscle atrophy and acute resistance 
 training and downregulated by chronic resistance training .
similarly , murf1 upregulation may 
 participate in removing useless tissue during muscle atrophy and in recycling in conjunction 
 with tissue reconstruction during acute resistance training . in the present study ,
the 
 decrease in murf1 expression induced by 70% 1rm training inhibited protein breakdown after 
 muscle disuse . in summary , our data suggest that high - intensity resistance training is an effective 
 physical therapy method for treatment of atrophic muscles .
however , reloading and/or 
 overloading of atrophic muscle induced by muscle fiber injury may also cause inflammation 
 and pain .
additional multiphasic studies are required to determine the appropriate level and 
 duration of high - intensity training for physical therapy of atrophic muscle in humans .
 [ purpose ] the aim of this study was to determine the effect of resistance training on 
 atrophied skeletal muscle in rats based on evidence derived from physical therapy . 
 [ subjects and methods ] rats were forced to undergo squats as resistance training for 3 
 weeks after atrophying the rectus femoris muscle by hindlimb suspension for 2 weeks . 
 the 
 intensity of resistance training was adjusted to 50% and 70% of the maximum lifted weight , 
 i.e. , 50% of the one - repetition maximum and 70% of the one - repetition maximum , 
 respectively . 
 [ results ] three weeks of training did not alter the one - repetition maximum , 
 and muscle fibers were injured while measuring the one - repetition maximum and reloading . 
 
 the decrease in cross - sectional area in the rectus femoris muscle induced by unloading for 
 2 weeks was significantly recovered after training at 70% of the one - repetition maximum . 
 
 the levels of muscle ring - finger protein-1 mrna expression were significantly lower in 
 muscles trained at 70% of the one - repetition maximum than in untrained muscles . 
 
 [ conclusion ] these results suggest that high - intensity resistance training can promote 
 atrophic muscle recovery , which provides a scientific basis for therapeutic exercise 
 methods for treatment of atrophic muscle in physical therapy . 
INTRODUCTION
SUBJECTS AND METHODS
RESULTS
DISCUSSION