Soleus, plantaris and gastrocnemius VEGF mRNA responses to hypoxia and exercise are preserved in aged compared with young female C57BL/6 mice

Aims: In humans, skeletal muscle capillarization and the vascular endothelial growth factor (VEGF) mRNA response to acute exercise are lower in aged compared with young. The exercise‐induced increase in VEGF mRNA has been proposed to involve hypoxic regulation of VEGF and is believed to be fibre type‐dependent. We hypothesized that attenuated VEGF mRNA responses to hypoxia and exercise with advanced age would be greatest in oxidative vs. glycolytic muscles. Methods: 3‐ and 24‐month‐old female C57BL/6 mice were exposed to acute hypoxia (F_IO₂ = 0.06) or performed a single exercise (65% of maximum treadmill running speed) bout. Capillarization and VEGF mRNA were analysed in the soleus, plantaris and gastrocnemius muscles. Results: In each muscle, VEGF mRNA was greater in aged compared with young, while the VEGF mRNA response to acute hypoxia or acute exercise was similar between young and aged. Morphological analysis revealed that type IIA fibre percentage and type IIB capillarization in the plantaris were greater and type IIB fibre cross‐sectional area (FCSA) in the gastrocnemius was smaller in aged compared with young. Conclusions: These findings suggest that ageing does not impair the potential for non‐pathological angiogenesis in mice and that acute exercise increases VEGF mRNA in the soleus, plantaris and gastrocnemius muscles, which differ considerably in fibre type percentage.     

Acute resistance exercise increases skeletal muscle angiogenic growth factor expression

AIMS: Both aerobic and resistance exercise training promote skeletal muscle angiogenesis. Acute aerobic exercise increases several pro-angiogenic pathways, the best characterized being increases in vascular endothelial growth factor (VEGF). We hypothesized that acute resistance exercise also increases skeletal muscle angiogenic growth factor [VEGF and angiopoietin (Ang)] expression. METHODS: Seven young, sedentary individuals had vastus lateralis muscle biopsies and blood drawn prior to and at 0, 2 and 4 h post-resistance exercise for the measurement of VEGF; VEGF receptor [KDR, Flt-1 and neuropilin 1 (Nrp1)]; Ang1 and Ang2; and the angiopoietin receptor--Tie2 expression. Resistance exercise consisted of progressive knee extensor (KE) exercise to determine one repetition maximum (1-RM) followed by three sets of 10 repetitions (3 x 10) of KE exercise at 60-80% of 1-RM. RESULTS: Resistance exercise significantly increased skeletal muscle VEGF mRNA and protein and plasma VEGF protein at 2 and 4 h. Resistance exercise increased KDR mRNA and Tie2 mRNA at 4 h and Nrp1 mRNA at 2 and 4 h. Skeletal muscle Flt-1, Ang1, Ang2 and Ang2/Ang1 ratio mRNA were not altered by resistance exercise. CONCLUSIONS: These findings suggest that acute resistance exercise increases skeletal muscle VEGF, VEGF receptor and angiopoietin receptor expression. The increases in muscle angiogenic growth factor expression in response to acute resistance exercise are similar in timing and magnitude with responses to acute aerobic exercise and are consistent with resistance exercise promoting muscle angiogenesis.     

Effect of high intensity training on capillarization and presence of angiogenic factors in human skeletal muscle

The effect of intense training on endothelial proliferation, capillary growth and distribution of vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) was examined in human skeletal muscle. Two intermittent knee extensor training protocols (at ∼150% (Study 1) versus ∼90% (Study 2) of leg ̇ _O2 max) were conducted. Muscle biopsies were obtained throughout the training periods for immunohistochemical assessment of capillarization, cell proliferation (Ki-67-positive cells), VEGF and bFGF. In Study 1, microdialysis samples were collected from the trained and untrained leg at rest and during exercise and added to endothelial cells to measure the proliferative effect. After 4 weeks of training there was a higher ( P < 0.05) capillary-to-fibre ratio (Study 1: 2.4 ± 0.1 versus 1.7 ± 0.1) and number of Ki-67-positive cells (Study 1: 0.18 ± 0.05 versus 0.00 ± 0.01) than before training. Neither the location of proliferating endothelial cells nor capillarization was related to muscle fibre type. The endothelial cell proliferative effect of the muscle microdialysate increased from rest to exercise in both the untrained leg (from 262 ± 60 to 573 ± 87% of control perfusate) and the trained leg (from 303 ± 75 to 415 ± 108% of perfusate). VEGF and bFGF were localized in endothelial and skeletal muscle cells and training induced no changes in distribution. The results demonstrate that intense intermittent endurance training induces capillary growth and a transient proliferation of endothelial cells within 4 weeks, with a similar growth occurring around type I versus type II muscle fibres.     

Effects of aerobic exercise training on antioxidant enzyme activities and mRNA levels in soleus muscle from young and aged rats

The aim of this study was to investigate the effect of aerobic exercise training on activities and mRNA levels of catalase (CAT), glutathione peroxidase (GPX), Cu,Zn- and Mn-superoxide dismutases (SOD), TBARS content, and xanthine oxidase (XO) activity, in soleus muscle from young and aged rats. The antioxidant enzyme activities and mRNA levels were markedly increased in soleus muscle with aging. TBARS content of soleus muscle from the aged group was 8.3-fold higher as compared with that of young rats. In young rats, exercise training induced an increase of all antioxidant enzyme activities, except for Cu,Zn-SOD. XO also did not change. The TBARS content was also increased (2.9-fold) due to exercise training in soleus muscle from young rats. In aged rats, the activities of CAT, GPX and Cu,Zn-SOD in the soleus muscle did not change with the exercise training, whereas the activities of Mn-SOD (40%) and XO (27%) were decreased. The mRNA levels of Mn-SOD and CAT were decreased by 42% and 24%, respectively, in the trained group. Exercise training induced a significant decrease of TBARS content (81%) in the soleus muscle from aged rats. These findings support the proposition that exercise training presents an antioxidant stress effect on skeletal muscle from both young and aged rats.     

Exercise induces interleukin-8 receptor (CXCR2) expression in human skeletal muscle

Exercise induces a marked increase in interleukin-8 (IL-8) mRNA and protein expression within skeletal muscle fibres. Interleukin-8 belongs to a subfamily of CXC chemokines containing a Glu-Leu-Arg (ELR) motif. CXC chemokines with ELR motifs are potent angiogenic factors in vivo, and IL-8 has been shown to act as an angiogenic factor in human microvascular endothelial cells by binding to the CXC receptor 2 (CXCR2). In the present study, we examined the expression of the interleukin-8 receptor CXCR2 in human skeletal muscle biopsies after concentric exercise. Healthy volunteers were randomized to either 3 h of cycle ergometer exercise at 60% of maximum oxygen uptake (n = 8) or rest (n = 7). Muscle biopsy samples were obtained from the vastus lateralis before exercise (0 h), immediately after exercise (3 h), and at 4.5, 6, 9 and 24 h. Skeletal muscle CXCR2 mRNA increased significantly in response to exercise (3 and 4.5 h) when compared with pre-exercise samples. Expression of the CXCR2 protein was low in skeletal muscle biopsies before exercise and at the end of the exercise period (3 h). However, at 4.5-9 h, an increase in CXCR2 protein was seen in the vascular endothelium, and also slightly within the muscle fibres, as determined by immunohistochemistry. The present study demonstrates that concentric exercise induces CXCR2 mRNA and protein expression in the vascular endothelial cells of the muscle fibres. These findings suggest that muscle-derived IL-8 may act locally to stimulate angiogenesis through CXCR2 receptor signalling.     

Muscle adaptation to extreme endurance training in man.

To evaluate the effect of extreme endurance training on muscle fibre composition and activities of oxidative enzymes in different fibre types biopsies were taken from vastus lateralis, gastrocnemius and deltoideus of elite orienteers. Comparisons were made between the (trained) leg muscles and the (relatively untrained) arm muscles, and with leg muscles of 16--18 years old boys. The orienteers had the same percentage type I fibres and vastus lateralis and gastrocnemius as in deltoideus, but higher percentage type I fibres in vastus lateralis compared with the controls. The similarity between trained and untrained muscle in the orienteers suggests that training had not caused the high percentage type I fibres which rather might be the result of selection of individuals with the best prerequisites for high oxidative capacity. However, the distribution of type II subgroups in the leg muscles of the orienteers differed from both their own deltoideus and leg muscles of the controls, the relationship IIA/IIB being altered in favour of the more oxidative IIA. The leg muscles of the orienteers also showed an increased occurrence of the normally IIC fibre. These latter findings point at the possibility of a training induced alteration in the subgroup pattern. Unlike in the controls there was no significant difference in succinate dehydrogenase activity, measured in single fibres, between type I and II fibres in gastrocnemius of the orienteers. Thus, type II fibres have the ability metabolically to adapt to high oxidative demands. This might to some extent be mediated by a conversion from IIB to IIA form.     

Muscle Adaptation to Extreme Endurance Training in Man

To evaluate the effect of extreme endurance training on muscle fibre composition and activities of oxidative enzymes in different fibre types biopsies were taken from vastus lateralis, gastrocnemius and deltoideus of elite orienteers. Comparisons were made between the (trained) leg muscles and the (relatively untrained) arm muscles, and with leg muscles of 16–18 years old boys. The orienteers had the same percentage type I fibres in vastus lateralis and gastrocnemius as in deltoideus, but higher percentage type I fibres in vastus lateralis compared with the controls. The similarity between trained and untrained muscle in the orienteers suggests that training had not caused the high percentage type I fibres which rather might be the result of selection of individuals with the best prerequisites for high oxidative capacity. However, the distribution of type II subgroups in the leg muscles of the orienteers differed from both their own deltoideus and leg muscles of the controls, the relationship IIA/IIB being altered in favour of the more oxidative IIA. The leg muscles of the orienteers also showed an increased occurrence of the normally rare IIC fibre. These latter findings point at the possibility of a training induced alteration in the subgroup pattern. Unlike in the controls there was no significant difference in succinate dehydrogenase activity, measured in single fibres, between type I and II fibres in gastrocnemius of the orienteers. Thus, type II fibres have the ability metabolically to adapt to high oxidative demands. This might to some extent be mediated by a conversion from IIB to IIA form.     

Preferential utilization of perilipin 2-associated intramuscular triglycerides during 1 h of moderate-intensity endurance-type exercise

The lipid droplet (LD)-associated protein perilipin 2 (PLIN2) appears to colocalize with LDs in human skeletal muscle fibres, although the function of PLIN2 in the regulation of intramuscular triglyceride (IMTG) metabolism is currently unknown. Here we investigated the hypothesis that the presence of PLIN2 in skeletal muscle LDs is related to IMTG utilisation during exercise. We therefore measured exercise-induced changes in IMTG and PLIN2 distribution and changes in their colocalization. Muscle biopsies from the vastus lateralis were obtained from seven lean, untrained men (22 ± 2 years old, body mass index 24.2 ± 0.9 kg m(-2) and peak oxygen uptake 3.35 ± 0.13 l min(-1)) before and after 1 h of moderate-intensity cycling at ~65% peak oxygen uptake. Cryosections were stained for perilipin 2, IMTG and myosin heavy chain type I and viewed using wide-field and confocal fluorescence microscopy. Exercise induced a 50 ± 7% decrease in IMTG content in type I fibres only (P < 0.05), but no change in PLIN2 content. Colocalization analysis showed that the fraction of PLIN2 associated with IMTG was 0.67 ± 0.03 before exercise, which was reduced to 0.51 ± 0.01 postexercise (P < 0.05). Further analysis revealed that the number of PLIN2-associated LDs was reduced by 31 ± 10% after exercise (P < 0.05), whereas the number of PLIN2-null LDs was unchanged. No such changes were seen in type II fibres. In conclusion, this study shows that PLIN2 content in skeletal muscle is unchanged in response to a single bout of endurance exercise. Furthermore, the PLIN2 and IMTG association is reduced postexercise, apparently due to preferential utilization of PLIN2-associated LDs. These results confirm the hypothesis that the PLIN2 association with IMTG is related to the utilization of IMTG as a fuel during exercise.     

Histochemical and biochemical changes in human skeletal muscle with age in sedentary males, age 22--65 years.

Biopsies for histochemical and biochemical analyses were taken from the vastus lateralis muscle of 55 untrained, healthy male subjects from 22 to 65 years of age. Fibre type distribution changed towards a decrease in the percentage of type II fibres, both in type IIA and type IIB fibres, whereas type IIB/IIA fibre ratio and type IIC percentage did not change with increasing age. It was found that the type IIB/IIA fibre ratio was inversely related to type I fibres, i.e. subjects rich in type I fibres had a relatively smaller proportion of type IIB fibres. Fibre area determinations revealed a selective decrease in type II fibre area. Consequently, the type II/I fibre area ratio and relative type II fibre area decreased. No changes in the specific activities of Mg2+ stimulated ATPase and myokinase were observed, while the activity of lactate dehydrogenase (LDH) was higher in the youngest groups than in the oldest. LDH isozyme pattern shifted towards a decrease in percentage distribution of the muscle specific isozymes and a corresponding decrease in muscle specific activity while the activity of the heart specific isozymes did not change.     

Exercise training induces transitions of myosin isoform subunits within histochemically typed human muscle fibres

Fibre type composition based on histochemical myosin ATPase reaction was studied in cross section of biopsies from the vastus lateralis muscle of men. In addition, protein composition as well as peptide patterns of isolated myosin heavy chains were examined in batches of individually classified fibres from the same biopsies. High intensity endurance training during 8 weeks induces significant decreases by 31–70% of the type IIB fibre population in 3 of 4 subjects (in one case no change was observed). These decreases were offset by corresponding increases in either type I or type IIA fibres with the type IIC fibres remaining always below 3%. A total of 13 professional cyclists with training periods over several years have a 20 times lower content of type IIB fibres than 4 sedentary controls and a concomitant high content of 80% of type I fibres. The content of type I and type IIB fibres of 8 sprinter athletes did almost not differ from that of controls. Thus the type IIB fibres respond most sensitively with a decrease to aerobic endurance training. Since both type IIA and IIB fibres were identical in protein composition containing the same fast variety of myosin light chains and heavy chains as well as troponin-I, their interconversion could not be seen at the molecular level. However, the slow variety of myosin light chains and of troponin-I started accumulating after 8 weeks of training in type IIA fibres. Furthermore, the myosin heavy chain isoform started shifting by producing new peptide patterns that resemble the digestion pattern of slow myosin heavy chains in fibres which still classified as type IIA. These changes on the molecular level in type IIA fibres mark the beginning of their transition over the intermediate and variable type IIC fibres, towards the slow type I fibre.Abbreviations VLM vastus lateralis muscle - maximal oxygen uptake capacity - MVD total mitochondrial volume density - I slow-twitch fibre type - IIA IIB, fast-twitch fibre types - IIC intermediate fibre type - HC myosin heavy chain - LC myosin light chain - TN-I troponin-I - ATPase adenosine 5-triphosphatase (EC 3.6.1.3) - SAV-8 Staphylococcus aureus V-8 proteinase (EC 3.4.21.19) - SDS sodium dodecyl sulfate - kD kilo Dalton     

Expression of angiogenic regulators and skeletal muscle capillarity in selectively bred high aerobic capacity mice

Selective breeding for high voluntary wheel running in untrained mice has resulted in a 'mini muscle' (MM) phenotype, which has increased skeletal muscle capillarity compared with muscles from non-selected control lines. Vascular endothelial growth factor (VEGF) and thrombospondin-1 (TSP-1) are essential mediators of skeletal muscle angiogenesis; thus, we hypothesized that untrained MM mice with elevated muscle capillarity would have higher basal VEGF expression and lower basal TSP-1 expression, and potentially an exaggerated VEGF response to acute exercise. We examined skeletal muscle morphology and skeletal muscle protein expression of VEGF and TSP-1 in male mice from two (untrained) mouse lines selectively bred for high exercise capacity (MM and Non-MM), as well as one non-selected control mouse line (normal aerobic capacity). In the MM mice, gastrocnemius (GA) and plantaris (PLT) muscle capillarity (i.e. capillary-to-fibre ratio and capillary density) were greater compared with control mice (P < 0.05). In Non-MM mice, only muscle capillarity in PLT was greater than in control mice (P < 0.001). The soleus (SOL) showed no statistical differences in muscle capillarity among groups. In the GA, MM mice had 58% greater basal VEGF (P < 0.05), with no statistical difference in basal TSP-1 when compared with control mice. In the PLT, MM mice had a 79% increase in basal VEGF (P < 0.05) and a 39% lower basal TSP-1 (P < 0.05) compared with the control animals. Non-MM mice showed no difference in basal VEGF in either the GA or the PLT compared with control mice. In contrast, basal TSP-1 was elevated in the PLT, but not in the GA, of Non-MM mice compared with control mice. Neither VEGF nor TSP-1 was significantly different in SOL muscle among the three mouse lines. In response to acute exercise, MM mice displayed a 41 and 28% increase (P < 0.05) in VEGF in the GA and PLT, respectively, whereas neither control nor Non-MM mice showed a significant VEGF response to acute exercise. In contrast, TSP-1 levels were decreased by 90% in GA (P < 0.05) but increased by 50% in PLT (P < 0.05) in response to acute exercise in MM mice. The SOL showed no response to exercise for either VEGF or TSP-1 for any of the mouse lines. These data, with the exception of the Non-MM plantaris muscle, suggest that elevated capillarity is associated with altered balance between positive and negative angiogenic regulators (i.e. VEGF versus TSP-1, respectively). Based on the greater capillarity and significant VEGF response to exercise in MM mice, these data suggest that VEGF expression may, at least in part, be genetically determined.     

Expression of the Na+/H+ exchanger isoform NHE1 in rat skeletal muscle and effect of training

The expression of the Na⁺/H⁺ exchanger isoform NHE1 was quantified in homogenates of various rat skeletal muscles by means of immunoblotting, and the effect of 3 weeks of treadmill training on NHE1 expression was determined in a red (oxidative) as well as a white (glycolytic)‐muscle preparation. The NHE1 antibodies recognized a glycosylated protein at 101–111 kDa. There was a positive correlation between the NHE1 expression in the muscle and percent type IIB fibres and percent type IID/X fibres, whereas the NHE1 expressions were negatively correlated to percent type I fibres and percent type I + IIA fibres. Thus the highest NHE1 expression was evident in the most glycolytic fibres. Treadmill training increased (P < 0.05) the NHE1 content by 29 and 36% in oxidative and glycolytic fibres, respectively, suggesting that training enhanced the NHE1 content of all muscle‐fibre types. Therefore training may improve the capacity for pH regulation in skeletal muscle.     

Enzyme levels in pools of microdissected human muscle fibres of identified type: Adaptive response to exercise

Enzyme activities were determined in pools of type I (slow twitch) and IIA and II B (fast twitch) fibres of the thigh muscle from individuals engaged to a high degree in physical training of an endurance character and from non-endurance-trained controls. The endurance-trained (ET) group had significantly higher activity levels of the mitochondrial enzymes citrate synthase, malate dehydrogenase, and 3-OH-acylCoA dehydrogenase both in type I (2.1×, 1.7×, 1.4×) and in type IIA (2.3×, 1.8×, 1.4×) and IIB fibres (2.0×, 1.5 ×, 1.5×) than the non-endurance-trained (NET) group. Of the glycolytic enzymes, phosphofructokinase (PFK) in type I fibres was significantly higher (I.8×) in the ET than in the NET group whereas glyceraldehydephosphate dehydrogenase (GAPDH) in type I fibres was similar in the two groups. In type II fibres both PFK and GAPDH levels tended to be higher in the ET group. Lactate dehydrogenase (LDH) of both fibre types were not different in the two groups. Type 1 fibres differed significantly from type II fibres for all the six enzymes measured in both groups. However, no significant difference between fibres of types IIA and IIB was found. The results indicate that fibres of types I, IIA and IIB in human skeletal muscle all possess great adaptability with regard to their oxidative capacity. Furthermore, the data suggest that extensive endurance training may enhance the glycolytic capacity in both type I and type II fibres although the glycolytic capacity of the muscle as a whole generally is low in endurance trained subjects owing to a predominance of type I fibres. It is concluded that further studies are needed to determine whether there is a metabolic distinction between fibres of types IIA and IIB.     

Expression of the Na(+)/H(+) exchanger isoform NHE1 in rat skeletal muscle and effect of training

The expression of the Na(+)/H(+) exchanger isoform NHE1 was quantified in homogenates of various rat skeletal muscles by means of immunoblotting, and the effect of 3 weeks of treadmill training on NHE1 expression was determined in a red (oxidative) as well as a white (glycolytic)-muscle preparation. The NHE1 antibodies recognized a glycosylated protein at 101-111 kDa. There was a positive correlation between the NHE1 expression in the muscle and percent type IIB fibres and percent type IID/X fibres, whereas the NHE1 expressions were negatively correlated to percent type I fibres and percent type I + IIA fibres. Thus the highest NHE1 expression was evident in the most glycolytic fibres. Treadmill training increased (P < 0.05) the NHE1 content by 29 and 36% in oxidative and glycolytic fibres, respectively, suggesting that training enhanced the NHE1 content of all muscle-fibre types. Therefore training may improve the capacity for pH regulation in skeletal muscle.     

Skeletal muscle adaptations in elastic resistance-trained young men and women

Skeletal muscle adaptations (fiber-type composition, cross-sectional area, myosin heavy chain (MHC) content, and capillarity) were assessed in the vastus lateralis muscle of young men and women after 8 weeks of training with the Sportcord, an elastic resistance device. Ten men [mean (SD) age 20 (1.1) years] and 13 women [20 (1.2) years] performed two sets each to failure of single leg squats and leg extensions at approximately 50 repetitions x min(-1). Biopsy samples were taken from the right vastus lateralis muscle before and after training. Six fiber types (I, IC, IIC, IIA, IIAB, and IIB) were classified using myofibrillar ATPase histochemistry. Training with the Sportcord caused a small, but significant, increase in one-repetition maximum using free weights and a large increase in repetitions to failure. In addition, elastic resistance training caused an increase in the percentage of fibers classified as type IIAB for both men and women, and a decrease in the percentage of type IIB fibers in the men. MHC analysis supported these findings (a significant increase in the percentage of MHCIIa for the men). The cross-sectional areas ofboth the type I and IIAB + IIB fibers increased after training for the men, whereas no area changes were found for the women. The capillary:fiber ratio and capillary contacts per fiber type increased significantly for the men, and similar trends were noted for the women. Capillary density did not change in either the men or the women. These data suggest minor changes in fiber type composition (IIB-->IIAB), fiber size, and capillarization following short-term training with elastic resistance. Although muscular changes did occur using the Sportcord, the extent of these changes was less than those reported previously for short-term resistance-training programs using free weights.     

Exercise-induced metallothionein expression in human skeletal muscle fibres

Exercise induces free oxygen radicals that cause oxidative stress, and metallothioneins (MTs) are increased in states of oxidative stress and possess anti-apoptotic effects. We therefore studied expression of the antioxidant factors metallothionein I and II (MT-I + II) in muscle biopsies obtained in response to 3 h of bicycle exercise performed by healthy men and in resting controls. Both MT-I + II proteins and MT-II mRNA expression increased significantly in both type I and II muscle fibres after exercise. Moreover, 24 h after exercise the levels of MT-II mRNA and MT-I + II proteins were still highly increased and the MT-II mRNA expression reached a 15-fold increase. As expected, immunohistochemical detection of malondialdehyde (MDA) and nitrotyrosine (NITT) showed that formation of free radicals and oxidative stress were clearly increased in exercising muscle peaking shortly after the end of exercise in both type I and II muscle fibres. This is the first report demonstrating that MT-I + II are significantly induced in human skeletal muscle fibres following exercise. As MT-I + II are antioxidant factors that protect various tissues during pathological conditions, the MT-I + II increases post exercise may represent a mechanism whereby contracting muscle fibres are protected against cellular stress and injury.