Time course of responses of human skeletal muscle to oxidative stress induced by nondamaging exercise.

Previous studies in animals have demonstrated that a single period of aerobic exercise induces a rise in the skeletal muscle activity of the antioxidant enzymes superoxide dismutase and catalase and an increase in the muscle content of heat shock proteins (HSPs). The purpose of this study was to examine the time course of response of human skeletal muscle superoxide dismutase and catalase activities and the content of HSP60 and HSP70 after a period of exhaustive, nondamaging aerobic exercise. Seven volunteers undertook one-legged cycle ergometry at 70% maximal oxygen uptake for 45 min. Biopsies were obtained from the vastus lateralis muscle 7 days before and at 1, 2, 3, and 6 days after exercise. Muscle superoxide dismutase activity increased to a peak at 3 days postexercise, muscle catalase activities were unchanged, and muscle content of HSP60 and the inducible HSP70 increased by variable amounts to reach means of 190% and 3,100% of preexercise values, respectively, by 6 days postexercise. These data indicate that human skeletal muscle responds to a single bout of nondamaging exercise by increasing superoxide dismutase activity and provide the first evidence of an increase in HSP content of human skeletal muscle after a submaximal exercise bout.     

Time course of myogenic and metabolic gene expression in response to acute exercise in human skeletal muscle.

The aim of this study was to examine the time course activation of select myogenic (MRF4, Myf5, MyoD, myogenin) and metabolic (CD36, CPT1, HKII, and PDK4) genes after an acute bout of resistance (RE) or run (Run) exercise. Six RE subjects [25 +/- 4 yr (mean +/- SD), 74 +/- 14 kg, 1.71 +/- 0.11 m] and six Run subjects (25 +/- 4 yr, 72 +/- 5 kg, 1.81 +/- 0.07 m, 63 +/- 8 ml.kg(-1).min(-1)) were studied. Eight muscle biopsies were taken from the vastus lateralis (RE) and gastrocnemius (Run) before, immediately after, and 1, 2, 4, 8, 12 and 24 h after exercise. RE increased mRNA of MRF4 (3.7- to 4.5-fold 2-4 h post), MyoD (5.8-fold 8 h post), myogenin (2.6- and 3.5-fold 8-12 h post), HKII (3.6- to 10.5-fold 2-12 h post), and PDK4 (14- to 26-fold 2-8 h post). There were no differences in Myf5, CD36, and CPT1 mRNA levels 0-24 h post-RE. Run increased mRNA of MyoD (5.0- to 8.0-fold), HKII (12- to 16-fold), and PDK4 (32- to 52-fold) at 8-12 h postexercise. There were no differences in MRF4, Myf5, myogenin, CD36 and CPT1 mRNA levels 0-24 h post-Run. These data indicate a myogenic and metabolic gene induction with RE and Run exercise. The timing of the gene induction is variable and generally peaks 4-8 h postexercise with all gene expression not significantly different from the preexercise levels by 24 h postexercise. These data provide basic information for the timing of human muscle biopsy samples for gene-expression studies involving exercise.     

Time course and differential responses of the major heat shock protein families in human skeletal muscle following acute nondamaging treadmill exercise.

The exercise-induced expression of heat shock proteins (HSPs) in rodent models is relatively well defined. In contrast, comparable data from human studies are limited and the exercise-induced stress response of human skeletal muscle is far from understood. This study has characterized the time course and magnitude of the HSP response in the skeletal muscles of a healthy active, but untrained, young male population following a running exercise protocol. Eight subjects performed 45 min of treadmill running at a speed corresponding to their lactate threshold (11.7 +/- 0.5 km/h; 69.8 +/- 4.8% maximum O2 uptake). Muscle biopsies were obtained from the vastus lateralis muscle immediately before and at 24 h, 48 h, 72 h, and 7 days postexercise. Exercise induced a significant (P < 0.05) but variable increase in HSP70, heat shock cognate (HSC) 70, and HSP60 expression with peak increases (typically occurring at 48 h postexercise) to 210, 170, and 139% of preexercise levels, respectively. In contrast, exercise did not induce a significant increase in either HSP27, alphaB-crystallin, SOD 2 (MnSOD) protein content, or the activity of SOD and catalase. When examining baseline protein levels, HSC70, HSP27, and alphaB-crystallin appeared consistently expressed between subjects, whereas HSP70 and MnSOD displayed marked individual variation of up to 3- and 1.5-fold, respectively. These data are the first to define the time course and extent of HSP production in human skeletal muscle following a moderately demanding and nondamaging running exercise protocol. Data demonstrate a differential effect of aerobic exercise on specific HSPs.     

Acute molecular responses of skeletal muscle to resistance exercise in able-bodied and spinal cord-injured subjects.

Spinal cord injury (SCI) results in muscle atrophy, which contributes to a number of health problems, such as cardiovascular deconditioning, metabolic derangement, and osteoporosis. Electromyostimulation (EMS) holds the promise of ameliorating SCI-related muscle atrophy and, therefore, improving general health. To date, EMS training of long-term SCI subjects has resulted in some muscle hypertrophy but has fallen short of normalizing muscle mass. The aim of this study was to compare the molecular responses of vastus lateralis muscles from able-bodied (AB) and SCI subjects after acute bouts of EMS-induced resistance exercise to determine whether SCI muscles displayed some impairment in response. Analysis included mRNA markers known to be responsive to increased loading in rodent muscles. Muscles of AB and SCI subjects were subjected to EMS-stimulated exercise in two 30-min bouts, separated by a 48-h rest. Needle biopsy samples were obtained 24 h after the second exercise bout. In both the AB and SCI muscles, significant changes were seen in insulin-like growth factor binding proteins 4 and 5, cyclin-dependent kinase inhibitor p21, and myogenin mRNA levels. In AB subjects, the mRNA for mechano-growth factor was also increased. Before exercise, the total RNA concentration of the SCI muscles was less than that of the AB subjects but not different postexercise. The results of this study indicate that acute bouts of resistance exercise stimulate molecular responses in the skeletal muscles of both AB and SCI subjects. The responses seen in the SCI muscles indicate that the systems that regulate these molecular responses are intact, even after extended periods of muscle unloading.     

Time course of proteolytic, cytokine, and myostatin gene expression after acute exercise in human skeletal muscle.

The aim of this study was to examine the time course induction of select proteolytic [muscle ring finger-1 (MuRF-1), atrogin-1, forkhead box 3A (FOXO3A), calpain-1, calpain-2], myostatin, and cytokine (IL -6, -8, -15, and TNF-alpha) mRNA after an acute bout of resistance (RE) or run (RUN) exercise. Six experienced RE (25 +/- 4 yr, 74 +/- 14 kg, 1.71 +/- 0.11 m) and RUN (25 +/- 4 yr, 72 +/- 5 kg, 1.81 +/- 0.07 m) subjects had muscle biopsies from the vastus lateralis (RE) or gastrocnemius (RUN) before, immediately after, and 1, 2, 4, 8, 12, and 24 h postexercise. RE increased (P < 0.05) mRNA expression of MuRF-1 early (3.5-fold, 1-4 h), followed by a decrease in atrogin-1 (3.3-fold) and FOXO3A (1.7-fold) 8-12 h postexercise. Myostatin mRNA decreased (6.3-fold; P < 0.05) from 1 to 24 h postexercise, whereas IL-6, IL-8, and TNF-alpha mRNA were elevated 2-12 h. RUN increased (P < 0.05) MuRF-1 (3.6-fold), atrogin-1 (1.6-fold), and FOXO3A (1.9-fold) 1-4 h postexercise. Myostatin was suppressed (3.6-fold; P < 0.05) 8-12 h post-RUN. The cytokines exhibited a biphasic response, with immediate elevation (P < 0.05) of IL-6, IL-8, and TNF-alpha, followed by a second elevation (P < 0.05) 2-24 h postexercise. In general, the timing of the gene induction indicated early elevation of proteolytic genes, followed by prolonged elevation of cytokines and suppression of myostatin. These data provide basic information for the timing of human muscle biopsy samples for gene expression studies involving exercise. Furthermore, this information suggests a greater induction of proteolytic genes following RUN compared with RE.     

Proteolytic mRNA expression in response to acute resistance exercise in human single skeletal muscle fibers.

The purpose of this study was to characterize changes in mRNA expression of select proteolytic markers in human slow-twitch [myosin heavy chain (MHC) I] and fast-twitch (MHC IIa) single skeletal muscle fibers following a bout of resistance exercise (RE). Muscle biopsies were obtained from the vastus lateralis of eight young healthy sedentary men [23 +/- 2 yr (mean +/- SD), 93 +/- 17 kg, 183 +/- 6 cm] before and 4 and 24 h after 3 x 10 repetitions of bilateral knee extensions at 65% of one repetition maximum. The mRNA levels of TNF-alpha, calpains 1 and 2, muscle RING (really interesting novel gene) finger-1 (MuRF-1), atrogin-1, caspase-3, B-cell leukemia/lymphoma (Bcl)-2, and Bcl-2-associated X protein (Bax) were quantified using real-time RT-PCR. Generally, MHC I fibers had higher (1.6- to 5.0-fold, P < 0.05) mRNA expression pre- and post-RE. One exception was a higher (1.6- to 3.9-fold, P < 0.05) Bax-to-Bcl-2 mRNA ratio in MHC IIa fibers pre- and post-RE. RE increased (1.4- to 4.8-fold, P < 0.05) MuRF-1 and caspase-3 mRNA levels 4-24 h post-RE in both fiber types, whereas Bax-to-Bcl-2 mRNA ratio increased 2.2-fold (P < 0.05) at 4 h post-RE only in MHC I fibers. These results suggest that MHC I fibers have a greater proteolytic mRNA expression pre- and post-RE compared with MHC IIa fibers. The greatest mRNA induction following RE was in MuRF-1 and caspase-3 in both fiber types. This altered and specific proteolytic mRNA expression among slow- and fast-twitch muscle fibers indicates that the ubiquitin/proteasomal and caspase pathways may play an important role in muscle remodeling with RE.     

Selected contribution: acute cellular and molecular responses to resistance exercise.

Training protocols apply sequential bouts of resistance exercise (RE) to induce the cellular and molecular responses necessary to produce compensatory hypertrophy. This study was designed to 1) define the time course of selected cellular and molecular responses to a single bout of RE and 2) examine the effects of interbout rest intervals on the summation of these responses. Rat muscles were exposed to RE via stimulation of the sciatic nerve in vivo. Stimulated and control muscles were obtained at various time points post-RE and analyzed via Western blot and RT-PCR. A single bout of RE increased intracellular signaling (i.e., phosphorylations) and expression of mRNAs for insulin-like growth factor-I system components and myogenic markers (e.g., cyclin D1, myogenin). A rest interval of 48 h between RE bouts resulted in much greater summation of myogenic responses than 24- or 8-h rest intervals. This experimental approach should be useful for studying the regulatory mechanisms that control the hypertrophy response. These methods could also be used to compare and contrast different exercise parameters (e.g., concentric vs. eccentric, etc.).     

Time course of vasodilatory responses in skeletal muscle arterioles: role in hyperemia at onset of exercise

At the onset of dynamic exercise, muscle blood flow increases within 1-2 s. It has been postulated that local vasodilatory agents produced by the vascular endothelium or the muscle itself contribute to this response. We hypothesized that only vasodilators that act directly on the vascular smooth muscle could produce vasodilation of skeletal muscle arterioles in <2 s. To test this hypothesis, we determined the time course of the vasodilatory response of isolated skeletal muscle arterioles to direct application of potassium chloride, adenosine, acetylcholine, and sodium nitroprusside. Soleus and gastrocnemius muscles were dissected from the hindlimbs of male Sprague-Dawley rats. First-order arterioles (100-200 microm) were isolated, cannulated on micropipettes, and pressurized to 60 cmH(2)O in an organ bath. Vasodilatory agents were added directly to the bath, and diameter responses of the arterioles were recorded in real time on a videotape recorder. Frame-by-frame analysis of the diameter responses indicated that none of the vasodilator agents tested produced significant diameter increases in <4 s in either soleus or gastrocnemius muscle arterioles. These results indicate that, although these local vasodilators produce significant vasodilation of skeletal muscle resistance arterioles, these responses are not rapid enough (within 1-2 s) to contribute to the initiation of the exercise hyperemic response at the onset of dynamic exercise.     

Angiogenic growth factor response to acute systemic exercise in human skeletal muscle.

We investigated whether acute systemic exercise increases vascular endothelial growth factor (VEGF), VEGF receptor (KDR and Flt-1) mRNA, and VEGF protein in sedentary humans. Twelve sedentary subjects were recruited and performed 1 h of acute, cycle ergometer exercise at 50% of maximal oxygen consumption. Muscle biopsies were obtained from the vastus lateralis before exercise and at 0, 2, and 4 h postexercise. Acute exercise significantly increased VEGF mRNA at 2 and 4 h and increased KDR and Flt-1 mRNA at 4 h postexercise. The sustained increase in VEGF mRNA through 4 h and the increases in KDR and Flt-1 at 4 h are different from their respective time course responses in rats. In contrast to the increase in VEGF mRNA postexercise, VEGF protein levels were decreased at 0 h postexercise. These results provide evidence in humans that 1) VEGF, KDR, and Flt-1 mRNA are increased by acute systemic exercise; 2) the time course of the VEGF, KDR, and Flt-1 mRNA responses are different from those previously reported in rats (Gavin TP and Wagner PD. Acta Physiol Scand 175: 201-209, 2002); and 3) VEGF protein is decreased immediately after exercise.     

Proteomic responses of skeletal and cardiac muscle to exercise

Regular exercise is effective in the prevention of chronic diseases and confers a lower risk of death in individuals displaying risk factors such as hypertension and dyslipidemia. Thus, knowledge of the molecular responses to exercise provides a valuable contrast for interpreting investigations of disease and can highlight novel therapeutic targets. While exercise is an everyday experience and can be conceptualized in simple terms, it is also a complex physiological phenomenon and investigation of exercise responses requires sophisticated analytical techniques and careful standardization of the exercise stimulus. Proteomic investigation of exercise is in its infancy but the ability to link changes in function with comprehensive changes in protein expression and post-translational modification holds great promise for advancing physiology. This article highlights recent pioneering work investigating the effects of exercise in skeletal and cardiac muscle that has uncovered novel mechanisms underlying the benefits of physical activity.     

Alterations in peroxisome proliferator-activated receptor mRNA expression in skeletal muscle after acute and repeated bouts of exercise

Molecular and Cellular Biochemistry - Reticulon3 (RTN3), as a member of the reticulon family, is generally regarded as a novel human apoptosis-inducing protein. But the extensional role of RTN3...     

Temporal response of desmin and dystrophin proteins to progressive resistance exercise in human skeletal muscle.

We have investigated the adaptations of the cytoskeletal proteins desmin and dystrophin in relationship to known muscular adaptations of resistance exercise. We measured desmin, dystrophin, and actin protein contents, myosin heavy chain (MHC) isoform distribution, muscle strength, and muscle cross-sectional area (CSA) during 8 wk of progressive resistance training or after a single bout of unaccustomed resistance exercise. Muscle biopsies were taken from the vastus lateralis of 12 untrained men. For the single-bout group (n=6) biopsies were taken 1 wk before the single bout of exercise (week 0) and 1, 2, 4, and 8 wk after this single bout of exercise. For the training group (n=6), biopsies were taken 1 wk before the beginning of the program (week 0) and at weeks 1, 2, 4, and 8 of the progressive resistance training program. Desmin, dystrophin, and actin protein levels were determined with immunoblotting, and MHC isoform distribution was determined using SDS-PAGE at each time point for each group. In the training group, desmin was significantly increased compared with week 0 beginning at week 4 (182% of week 0; P<0.0001) and remained elevated through week 8 (172% of week 0; P<0.0001). Desmin did not change at any time point for the single-bout group. Actin and dystrophin protein contents were not changed in either group at any time point. The percentage of MHC type IIa increased and MHC type IIx decreased at week 8 in the training group with no changes occurring in the single-bout group. Strength was significantly increased by week 2 (knee extension) and week 4 (leg press), and it further increased at week 8 for both these exercises in the training group only. Muscle CSA was significantly increased at week 4 for type II fibers in the training group only (5,719+/-382 and 6,582+/-640 microm2, weeks 0 and 4, respectively; P<0.05). Finally, a significant negative correlation was observed between the desmin-to-actin ratio and the percentage of MHC IIx (R=-0.31; P<0.05, all time points from both groups). These data demonstrate a time course for muscular adaptation to resistance training in which desmin increases shortly after strength gains and in conjunction with hypertrophy, but before changes in MHC isoforms, whereas dystrophin remains unchanged.     

Effect of captopril on skeletal muscle angiogenic growth factor responses to exercise.

Acute exercise increases vascular endothelial growth factor (VEGF), transforming growth factor-beta(1) (TGF-beta(1)), and basic fibroblast growth factor (bFGF) mRNA levels in skeletal muscle, with the greatest increase in VEGF mRNA. VEGF functions via binding to the VEGF receptors Flk-1 and Flt-1. Captopril, an angiotensin-converting enzyme inhibitor, has been suggested to reduce the microvasculature in resting and exercising skeletal muscle. However, the molecular mechanisms responsible for this reduction have not been investigated. We hypothesized that this might occur via reduced VEGF, TGF-beta(1), bFGF, Flk-1, and Flt-1 gene expression at rest and after exercise. To investigate this, 10-wk-old female Wistar rats were placed into four groups (n = 6 each): 1) saline + rest; 2) saline + exercise; 3) 100 mg/kg ip captopril + rest; and 4) 100 mg/kg ip captopril + exercise. Exercise consisted of 1 h of running at 20 m/min on a 10 degrees incline. VEGF, TGF-beta(1), bFGF, Flk-1, and Flt-1 mRNA were analyzed from the left gastrocnemius by quantitative Northern blot. Exercise increased VEGF mRNA 4.8-fold, TGF-beta(1) mRNA 1.6-fold, and Flt-1 mRNA 1.7-fold but did not alter bFGF or Flk-1 mRNA measured 1 h after exercise. Captopril did not affect the rest or exercise levels of VEGF, TGF-beta(1), bFGF, and Flt-1 mRNA. Captopril did reduce Flk-1 mRNA 30-40%, independently of exercise. This is partially consistent with the suggestion that captopril may inhibit capillary growth.     

Human growth hormone responses to repeated bouts of sprint exercise with different recovery periods between bouts.

This study examined the growth hormone (GH) response to repeated bouts of sprint cycling. Eight healthy men completed three trials consisting of two 30-s sprints on a cycle ergometer separated by either 60 min (Trial A) or 240 min (Trial B) of recovery and a single 30-s sprint carried out the day after Trial B (Trial C). Trials A and B were separated by at least 7 days. Blood samples were obtained at rest and during recovery from each sprint. In Trial A, GH was elevated immediately before sprint 2, and there was no further increase in GH following the second sprint [area under the curve: 460 (SD 348) vs. 226 min.mug(-1).l(-1) (SD 182), P = 0.05]. Free insulin-like growth factor I tended to be lower immediately before sprint 2 than sprint 1 (P = 0.06). Serum free fatty acids were not different immediately before each of the sprints. In Trial B, there was a trend for a smaller GH response to the second sprint [GH area under the curve: 512 (SD 396) vs. 242 min.mug(-1).l(-1) (SD 190), P = 0.09]. Free insulin-like growth factor I tended to be lower (P = 0.06), and serum free fatty acids were higher (P = 0.01) immediately before sprint 2 than sprint 1. There was no difference in the GH response to sprinting on consecutive days (Trials B and C). In conclusion, repeated bouts of sprint cycling on the same day result in an attenuation or even ablation of the exercise-induced increase in GH, depending on the recovery interval between sprints.