Real time RT-PCR analysis of housekeeping genes in human skeletal muscle following acute exercise

Studies examining gene expression with RT-PCR typically normalize their mRNA data to a constitutively expressed housekeeping gene. The validity of a particular housekeeping gene must be determined for each experimental intervention. We examined the expression of various housekeeping genes following an acute bout of endurance (END) or resistance (RES) exercise. Twenty-four healthy subjects performed either a interval-type cycle ergometry workout to exhaustion (~75 min; END) or 300 single-leg eccentric contractions (RES). Muscle biopsies were taken before exercise and 3 h and 48 h following exercise. Real-time RT-PCR was performed on ß-actin, cyclophilin (CYC), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and ß2-microglobulin (ß2M). In a second study, 10 healthy subjects performed 90 min of cycle ergometry at ~65% of O2 max, and we examined a fifth housekeeping gene, 28S rRNA, and reexamined ß2M, from muscle biopsy samples taken immediately postexercise. We showed that CYC increased 48 h following both END and RES exercise (3- and 5-fold, respectively; P < 0.01), and 28S rRNA increased immediately following END exercise (2-fold; P = 0.02). ß-Actin trended toward an increase following END exercise (1.85-fold collapsed across time; P = 0.13), and GAPDH trended toward a small yet robust increase at 3 h following RES exercise (1.4-fold; P = 0.067). In contrast, ß2M was not altered at any time point postexercise. We conclude that ß2M and ß-actin are the most stably expressed housekeeping genes in skeletal muscle following RES exercise, whereas ß2M and GAPDH are the most stably expressed following END exercise.

Age and walking speed effects on muscle recruitment in gait termination

During gait termination at normal walking speed, older adults more frequently employ two-step responses, increasing their stopping distance and stopping time more than younger controls. This study investigated ageing effects on lower limb muscle recruitment patterns during stopping at three walking speeds. Twelve young male (26±3.7 years, range 19–30) and 12 gender-matched older participants (72±4.3 years, range 65–82) terminated walking at normal, medium and maximum speed. A visual stopping stimulus was presented 10 ms following either left or right heel-contact with no stimulus (catch) on 30% of trials. Electromyographic (EMG) activity was recorded from the tibialis anterior (TA), soleus (SOL), biceps femoris (BF), vastus lateralis (VL) and gluteus medius (GM). Older males more frequently (46% of trials) took two-steps to stop than young males (20%). The stance leg muscles responded significantly faster than the swing leg, and with increased speed, fewer swing limb muscles contributed to stopping. Older males were slower to respond with the stance leg, at 215 ms following the stimulus compared with 176 ms for the younger group. They also recruited fewer swing leg muscles with less frequent activation of the soleus and gluteus medius. Failure to activate muscles would provide less extensor torque to maintain the centre of gravity anterior to the forward base of support. This would decrease the total force opposing horizontal velocity in order to bring the body to rest and, as a consequence, encourage an additional step prior to stopping.

Interaction of contractile activity and training history on mRNA abundance in skeletal muscle from trained athletes

Skeletal muscle displays enormous plasticity to respond to contractile activity with muscle from strength- (ST) and endurance-trained (ET) athletes representing diverse states of the adaptation continuum. Training adaptation can be viewed as the accumulation of specific proteins. Hence, the altered gene expression that allows for changes in protein concentration is of major importance for any training adaptation. Accordingly, the aim of the present study was to quantify acute subcellular responses in muscle to habitual and unfamiliar exercise. After 24-h diet/exercise control, 13 male subjects (7 ST and 6 ET) performed a random order of either resistance (8 x 5 maximal leg extensions) or endurance exercise (1 h of cycling at 70% peak O2 uptake). Muscle biopsies were taken from vastus lateralis at rest and 3 h after exercise. Gene expression was analyzed using real-time PCR with changes normalized relative to preexercise values. After cycling exercise, peroxisome proliferator-activated receptor- coactivator-1 (ET 8.5-fold, ST 10-fold, P < 0.001), pyruvate dehydrogenase kinase-4 (PDK-4; ET 26-fold, ST 39-fold), vascular endothelial growth factor (VEGF; ET 4.5-fold, ST 4-fold), and muscle atrophy F-box protein (MAFbx) (ET 2-fold, ST 0.4-fold) mRNA increased in both groups, whereas MyoD (3-fold), myogenin (0.9-fold), and myostatin (2-fold) mRNA increased in ET but not in ST (P < 0.05). After resistance exercise PDK-4 (7-fold, P < 0.01) and MyoD (0.7-fold) increased, whereas MAFbx (0.7-fold) and myostatin (0.6-fold) decreased in ET but not in ST. We conclude that prior training history can modify the acute gene responses in skeletal muscle to subsequent exercise.

Effects of Alcohol and Training on Exercise Performance and Muscle Recovery Biochemistry in the Brown Anole (Anolis sagrei)

Contrary to previous research, training may improve exercise performance in a lizard, the brown anole. A brief, two-week training period resulted in increased performance speed and distance before exhaustion in trained lizards. Trained lizards were also able to more effectively use leg glycogen stores, however each of these improvements were not found in lizards treated with alcohol. Liver glycogen concentrations were also lower in alcohol-treated lizards, and patterns of liver glycogen concentrations during recovery indicate some hepatic lactate gluconeogenesis.

Local nitric oxide synthase inhibition reduces skeletal muscle glucose uptake but not capillary blood flow during in situ muscle contraction in rats

OBJECTIVE: We have previously shown in humans that local infusion of a nitric oxide synthase (NOS) inhibitor into the femoral artery attenuates the increase in leg glucose uptake during exercise without influencing total leg blood flow. However, rodent studies examining the effect of NOS inhibition on contraction-stimulated skeletal muscle glucose uptake have yielded contradictory results. This study examined the effect of local infusion of an NOS inhibitor on skeletal muscle glucose uptake (2-deoxyglucose) and capillary blood flow (contrast-enhanced ultrasound) during in situ contractions in rats.

RESEARCH DESIGN AND METHODS: Male hooded Wistar rats were anesthetized and one hindleg electrically stimulated to contract (2 Hz, 0.1 ms) for 30 min while the other leg rested. After 10 min, the NOS inhibitor NG-nitro-L-arginine methyl ester (L-NAME) (arterial concentration of 5 µmol/l) or saline was infused into the epigastric artery of the contracting leg.

RESULTS: Local NOS inhibition had no effect on blood pressure, heart rate, or muscle contraction force. Contractions increased (P < 0.05) skeletal muscle NOS activity, and this was prevented by L-NAME infusion. NOS inhibition caused a modest significant (P < 0.05) attenuation of the increase in femoral blood flow during contractions, but importantly there was no effect on capillary recruitment. NOS inhibition attenuated (P < 0.05) the increase in contraction-stimulated skeletal muscle glucose uptake by ~35%, without affecting AMP-activated protein kinase (AMPK) activation.

CONCLUSIONS: NOS inhibition attenuated increases in skeletal muscle glucose uptake during contraction without influencing capillary recruitment, suggesting that NO is critical for part of the normal increase in skeletal muscle fiber glucose uptake during contraction.

Cellular adaption to repeated eccentric exercise-induced muscle damage

Eccentrically biased exercise results in skeletal muscle damage and stimulates adaptations in muscle, whereby indexes of damage are attenuated when the exercise is repeated. We hypothesized that changes in ultrastructural damage, inflammatory cell infiltration, and markers of proteolysis in skeletal muscle would come about as a result of repeated eccentric exercise and that gender may affect this adaptive response. Untrained male (n = 8) and female (n = 8) subjects performed two bouts (bout 1 and bout 2), separated by 5.5 wk, of 36 repetitions of unilateral, eccentric leg press and 100 repetitions of unilateral, eccentric knee extension exercises (at 120% of their concentric single repetition maximum), the subjects' contralateral nonexercised leg served as a control (rest). Biopsies were taken from the vastus lateralis from each leg 24 h postexercise. After bout 2, the postexercise force deficit and the rise in serum creatine kinase (CK) activity were attenuated. Women had lower serum CK activity compared with men at all times (P < 0.05), but there were no gender differences in the relative magnitude of the force deficit. Muscle Z-disk streaming, quantified by using light microscopy, was elevated vs. rest only after bout 1 (P < 0.05), with no gender difference. Muscle neutrophil counts were significantly greater in women 24 h after bout 2 vs. rest and bout 1 (P < 0.05) but were unchanged in men. Muscle macrophages were elevated in men and women after bout 1 andbout 2 (P < 0.05). Muscle protein content of the regulatory calpain subunit remained unchanged whereas ubiquitin-conjugated protein content was increased after both bouts (P < 0.05), with a greater increase after bout 2. We conclude that adaptations to eccentric exercise are associated with attenuated serum CK activity and, potentially, an increase in the activity of the ubiquitin proteosome proteolytic pathway.

Gender differences in muscle inflammation after eccentric exercise

Unaccustomed exercise is followed by delayed-onset muscle soreness and morphological changes in skeletal muscle. Animal studies have demonstrated that women have an attenuated response to muscle damage. We studied the effect of eccentric exercise in untrained male (n = 8) and female (n = 8) subjects using a unilateral exercise design [exercise (Ex) and control (Con) legs]. Plasma granulocyte counts [before (Pre) and 48 h after exercise (+48h)] and creatine kinase activity [Pre, 24 h after exercise (+24h), +48h, and 6 days after exercise (+6d)] were determined before (Pre) and after (+24h, +48h, +6d) exercise, with biopsies taken from the vastus lateralis of each leg at +48h for determination of muscle damage and/or inflammation. Plasma granulocyte counts increased for men and decreased for women at +48h (P < 0.05), and creatine kinase activity increased for both genders at +48h and +6d (P < 0.01). There were significantly greater areas of both focal (P < 0.001) and extensive (P < 0.01) damage in the Ex vs. Con leg for both genders, which was assessed by using toluidine blue staining. The number of leukocyte common antigen-positive cells/mm2 tissue increased with exercise (P < 0.05), and men tended to show more in their Ex vs. Con leg compared with women (P = 0.052). Men had a greater total (Ex and Con legs) number of bcl-2-positive cells/mm2 tissue vs. women (P < 0.05). Atrophic fibers with homogeneous bcl-2-positive staining were seen only in men (n = 3). We conclude that muscle damage is similar between genders, yet the inflammatory response is attenuated in women vs. men. Finally, exercise may stimulate the expression of proteins involved in apoptosis in skeletal muscle.

Muscle determinants of bone mass, geometry and strength in prepubertal girls

Purpose: The aim of this study was to compare the relative contribution of peak muscle force (isokinetic peak torque) with surrogate estimates of muscle force, including leg lean tissue mass (LTM) and vertical jump height (VJH), on bone mass, geometry and strength in healthy prepubertal girls (n = 103).

Methods: Total leg and FN BMC and leg LTM were measured by DXA; the hip strength analysis program was used to assess FN diameter, cross-sectional area (CSA) and section modulus (Z). Isokinetic peak torque of the knee extensors and flexors (60°·s-1) were used as direct measures of peak muscle force. VJH was measured as an estimate of neuromuscular function. Total leg length or femoral length was used as a surrogate measure of moment arm length.

Results: All estimates of muscle function, except VJH, were positively associated with leg BMC (r = 0.72 - 0.90) and FN BMC, geometry and strength (r = 0.35-0.65) (all, P < 0.001). Multiple linear regression analyses revealed that leg LTM and isokinetic peak torque were independently and equally predictive of leg BMC and FN BMC, bone geometry and strength, explaining 8 to 28% of the variance in each of the bone traits after accounting for moment arm length. When isokinetic peak torque was corrected for both leg LTM and moment arm length, it remained an independent predictor of BMC, CSA and Z, but only accounted for an additional 2 to 5% of the variance.

Conclusion: These data suggest that DXA-derived leg LTM can be used as a reasonable surrogate for isokinetic peak muscle forces when assessing bone strength in relation to muscular function in healthy pre-pubertal girls.

Altered movement patterns but not muscle recruitment in moderately trained triathletes during running after cycling

Previous studies have shown that cycling can directly influence neuromuscular control during subsequent running in some highly trained triathletes, despite these triathletes' years of practice of the cycle-run transition. The aim of this study was to determine whether cycling has the same direct influence on neuromuscular control during running in moderately trained triathletes. Fifteen moderately trained triathletes participated. Kinematics of the pelvis and lower limbs and recruitment of 11 leg and thigh muscles were compared between a control run (no prior exercise) and a 30 min run that was preceded by a 15 min cycle (transition run). Muscle recruitment was different between control and transition runs in only one of 15 triathletes (<7%). Changes in joint position (mean difference of 3°) were evident in five triathletes, which persisted beyond 5 min of running in one triathlete. Our findings suggest that some moderately trained triathletes have difficulty reproducing their pre-cycling movement patterns for running initially after cycling, but cycling appears to have little influence on running muscle recruitment in moderately trained triathletes.

Reduction in corticospinal inhibition in the trained and untrained limb following unilateral leg strength training

This study used transcranial magnetic stimulation to measure the corticospinal responses following 8 weeks of unilateral leg strength training. Eighteen healthy, non-strength trained participants (14 male, 4 female; 18–35 years of age) were matched for age, gender, and pre-training strength; and assigned to a training or control group. The trained group participated in unilateral horizontal leg press strength training, progressively overloaded and wave periodised, thrice per week for 8 weeks. Testing occurred prior to the intervention, at the end of 4 weeks and at the completion of training at 8 weeks. Participants were tested in both legs for one repetition maximum strength, muscle thickness, maximal electromyography (EMG) activity, and corticospinal excitability and inhibition. No changes were observed in muscle thickness in either leg. The trained leg showed an increase in strength of 21.2% (P = 0.001) and 29.0% (P = 0.007, compared to pre-testing) whilst the untrained contralateral leg showed 17.4% (P = 0.01) and 20.4% (P = 0.004, compared to pre-testing) increases in strength at 4 and 8 weeks, respectively. EMG and corticospinal excitability did not change; however, corticospinal inhibition was significantly reduced by 17.7 ms (P = 0.003) and 17.3 ms (P = 0.001) at 4 and 8 weeks, respectively, in the trained leg, and 25.1 ms (P = 0.001) and 20.8 ms (P = 0.001) at 4 and 8 weeks, respectively, in the contralateral untrained leg. This data support the theory of corticospinal adaptations underpinning cross-education gains in the lower limbs following unilateral strength training.

Muscle atrophy, pain, and damage in bed rest reduced by resistive (vibration) exercise

The purpose of this study was to investigate the effectiveness of a short-duration (5-6 min, 3 d·wk) resistive exercise program with (RVE) or without (RE) whole-body vibration in reducing muscle atrophy in the lower limb during prolonged inactivity when compared with that in an inactive control group. METHODS: As part of the second Berlin BedRest Study, 24 male subjects underwent 60 d of head-down tilt bed rest. Using magnetic resonance imaging, muscle volumes of the individual muscles of the lower limb were calculated before and at various intervals during and after bed rest. Pain levels and markers of muscle damage were also evaluated during and after bed rest. Adjustment of P values to guard against false positives was performed via the false discovery rate method. RESULTS: On the "intent-to-treat" analysis, RE reduced atrophy of the medial and lateral gastrocnemius, soleus, vasti, tibialis posterior, flexor hallucis longus, and flexor digitorum longus (P ≤ 0.045 vs control group) and RVE reduced atrophy of the medial and lateral gastrocnemius and tibialis posterior (P ≤ 0.044). Pain intensity reports after bed rest were lower in RE at the foot (P ≤ 0.033) and whole lower limb (P = 0.01) and in RVE at the thigh (P ≤ 0.041), lower leg (P ≤ 0.01), and whole lower limb (P ≤ 0.036). Increases in sarcomere-specific creatine kinase after bed rest were less in RE (P = 0.020) and RVE (P = 0.020). No differences between RE and RVE were observed. CONCLUSIONS: In conclusion, a short-duration RVE or RE can be effective in reducing the effect of prolonged bed rest on lower extremity muscle volume loss during bed rest and muscle damage and pain after bed rest. Copyright © 2014 by the American College of Sports Medicine.

Muscle strength and areal bone mineral density at the hip in women: a cross-sectional study

BACKGROUND: Muscle strengthening exercises are promoted for building and maintaining a healthy skeleton. We aimed to investigate the relationship between muscle strength and areal bone mineral density (BMD) at the hip in women aged 26-97 years.

METHODS: This cross-sectional study utilises data from 863 women assessed for the Geelong Osteoporosis Study. Measures of hip flexor and abductor strength were made using a hand-held dynamometer (Nicholas Manual Muscle Tester). The maximal measure from three trials on each leg was used for analyses. BMD was measured at the hip using dual energy x-ray absorptiometry (DXA; Lunar DPX-L). Total lean mass, body fat mass and appendicular lean mass were determined from whole body DXA scans. Linear regression techniques were used with muscle strength as the independent variable and BMD as the dependent variable. Models were adjusted for age and indices of body composition.

RESULTS: Measures of age-adjusted hip flexor strength and hip abductor strength were positively associated with total hip BMD. For each standard deviation (SD) increase in hip flexor strength, the increase in mean total hip BMD (SD) was 10.4 % (p = 0.009). A similar pattern was observed for hip abductor strength, with an increase in mean total hip BMD of 22.8 % (p = 0.025). All associations between hip muscle strength and total hip BMD were independent of height, but were nullified after adjusting for appendicular lean mass or total lean mass.

CONCLUSIONS: There was a positive association observed between muscle strength and BMD at the hip. However, this association was explained by measures of lean mass.

Persisting side-to-side differences in bone mineral content, but not in muscle strength and tendon stiffness after anterior cruciate ligament reconstruction

Tendon stiffness may be involved in limiting peak musculoskeletal forces and thus may constitute an upper limit for bone strength. The patellar tendon bone (PTB) graft, which is harvested from the patellar tendon during surgical reconstruction of the anterior cruciate ligament (ACL), is an ideal scenario to test this hypothesis. Eleven participants were recruited who had undergone surgical reconstruction of the ACL with a PTB graft 1-10 years prior to study inclusion. As previously reported, there was no side-to-side difference in thigh muscle cross-sectional area, in maximum voluntary knee extension torque, or in patellar tendon stiffness, suggesting full recovery of musculature and tendon. However, in the present study bone mineral content (BMC), assessed by peripheral quantitative computed tomography, was lower on the operated side than on the control side in four regions studied (P = 0·0019). Differences were less pronounced in the two sites directly affected by the operation (patella and tibia epiphysis) when compared to the more remote sites. Moreover, significant side-to-side differences were found in BMC in the trabecular compartment in the femoral and tibial epiphysis (P = 0·004 and P = 0·047, respectively) with reductions on the operated side, but increased in the patella (P = 0·00016). Cortical BMC, by contrast, was lower on the operated side at all sites except the tibia epiphysis (P = 0·09). These findings suggest that impaired recovery of BMC following ACL reconstruction is not because of lack of recovery of knee extensor strength or patellar tendon stiffness. The responsible mechanisms still remain to be determined.

Resistive vibration exercise attenuates bone and muscle atrophy in 56 days of bed rest: biochemical markers of bone metabolism

UNLABELLED: During and after prolonged bed rest, changes in bone metabolic markers occur within 3 days. Resistive vibration exercise during bed rest impedes bone loss and restricts increases in bone resorption markers whilst increasing bone formation. INTRODUCTION: To investigate the effectiveness of a resistive vibration exercise (RVE) countermeasure during prolonged bed rest using serum markers of bone metabolism and whole-body dual X-ray absorptiometry (DXA) as endpoints. METHODS: Twenty healthy male subjects underwent 8 weeks of bed rest with 12 months follow-up. Ten subjects performed RVE. Blood drawings and DXA measures were conducted regularly during and after bed rest. RESULTS: Bone resorption increased in the CTRL group with a less severe increase in the RVE group (p = 0.0004). Bone formation markers increased in the RVE group but decreased marginally in the CTRL group (p < 0.0001). At the end of bed rest, the CTRL group showed significant loss in leg bone mass (-1.8(0.9)%, p = 0.042) whereas the RVE group did not (-0.7(0.8)%, p = 0.405) although the difference between the groups was not significant (p = 0.12). CONCLUSIONS: The results suggest the countermeasure restricts increases in bone resorption, increased bone formation, and reduced bone loss during bed rest.

Relationship of upper-limb and thoracic muscle strength to 6-min walk distance in COPD patients

Study objectives: This study was developed to investigate the influence of thoracic and upperlimb muscle function on 6-min walk distance (6MWD) in patients with COPD.Design: A prospective, cross-sectional study.Setting: the pulmonary rehabilitation center of a university hospital.Patients: Thirty-eight patients with mild to very severe COPD were evaluated.Measurements and results: Pulmonary function and baseline dyspnea index (BDI) were assessed, handgrip strength, maximal inspiratory pressure (Pimax), and 6MWD were measured, and the one-repetition maximum (1RM) was determined for each of four exercises (bench press, lat pull down, leg extension, and leg press) performed on gymnasium equipment. Quality of life was assessed using the St. George Respiratory Questionnaire (SGRQ). We found statistically significant positive correlations between 6MWD and body weight (r = 0.32; p < 0.05), BDI (r = 0.50; p < 0.01), FEV, (r = 0.33; p < 0.05), PImax (r = 0.53; p < 0.01), and all values of 1RM. A statistically significant negative correlation was observed between 6MWD and dyspnea at the end of the 6-min walk test (r = -0.29; p < 0.05), as well as between 6MWD and the SGRQ activity domain (r = -0.45; p < 0.01) and impact domain (r = -0.34; p < 0.05) and total score (r = -0.40; p < 0.01). Multiple regression analysis selected body weight, BDI, Pimax, and lat pull down IRM as predictive factors for 6MWD (R-2 = 0.589).Conclusions: the results of this study showed the importance of the skeletal musculature of the thorax and upper limbs in submaximal exercise tolerance and could open new perspectives for training programs designed to improve functional activity in COPD patients.