V. Vihko

Effect of Isometric Strength Training on Mechanical, Electrical, and Metabolic Aspects of Muscle Function*

SummaryMonozygous twin pairs (two female and four male) were used in a strength training study so that one member of each pair served as training subject (TS) and the other members as nonexercising controls (CS). TS trained four times a week for 12 weeks with maximal isometric knee extensions of the right leg. The parameters studied included muscle strength, endurance time, electromyographic activity, and activities of several key enzymes in nonoxidative and oxidative muscle metabolism. The results disclosed that in addition to a 20% increase in isometric knee extension strength in the trained leg of TS, an average increase of 11% was observed in strength of TS untrained leg. CS did not demonstrate any change in muscle strength. Training also included an improvement in the maintenance of a static load of 60% of the pretraining maximum. Increase in the maximum integrated electromyographic activity (IEMG) of the rectus femoris muscle occurred concomitantly with the knee extension strength. Training also caused reduction in the IEMG/tension ratio at submaximal loads indicating a more economical usage of the rectus femoris muscle. Muscle biopsies taken from the vastus lateralis muscle showed that the enzyme activities of MDH, SDH, and HK were higher, and LDH and CPK lower in the trained leg as compared to the nontrained control leg of TS or to the values of the untrained member of the twin pair. It is concluded that isometric strength training as used in the present study can cause increased recruitment of the available motor unit pool, improved efficiency at submaximal loads, and surprisingly also enchancement of the oxidative metabolism in the muscle.

Effects of experimental type 1 diabetes and exercise training on angiogenic gene expression and capillarization in skeletal muscle

Diabetes alters microvascular structure and function and is a major risk factor for cardiovascular diseases. In diabetic skeletal muscle, impaired angiogenesis and reduced VEGF‐A expression have been observed, whereas in healthy muscle exercise is known to have opposite effects. We studied the effects of type 1 diabetes and combined exercise training on angiogenic mRNA expression and capillarization in mouse skeletal muscle. Microarray and real‐time PCR analyses showed that diabetes altered the expression of several genes involved in angiogenesis. For example, levels of proangiogenic VEGF‐A, VEGF‐B, neuropilin‐1, VEGFR‐1, and VEGFR‐2 were reduced and the levels of antiangiogenic thrombospondin‐1 and retinoblastoma like‐2 were increased. Exercise training alleviated some of these changes, but could not completely restore them. VEGF‐A protein content was also reduced in diabetic muscles. In line with the reduced levels of VEGF‐A and other angiogenic factors, and increased levels of angiogenesis inhibitors, capillary‐to‐muscle fiber ratio was lower in diabetic mice compared to healthy controls. Exercise training could not restore capillarization in diabetic mice. In conclusion, these data illustrate that type 1 diabetes is associated with reduced skeletal muscle capillarization and the dysregulation of complex angiogenesis pathways.—Kivelä, R., Silvennoinen, M., Touvra, A.‐M., Lehti, T. M., Kainulainen, H., Vihko, V. Effects of experimental type 1 diabetes and exercise training on angiogenic gene expression and capillarization in skeletal muscle. FASEB J. 20, E921–E930 (2006)

Acid hydrolase activity in red and white skeletal muscle of mice during a two-week period following exhausting exercise

The activities of β-glucuronidase, β-N-acetylglucosaminidase, arylsulphatase, ribonuclease,p-nitrophenylphosphatase, and malate dehydrogenase together with protein content were assayed from representative mixed (m. rectus femoris), predominantly red (proximal heads ofm. vastus lateralis, m.v. medius andm. v. intermedius), and predominantly white (distal head ofm. vastus lateralis) muscle homogenates of mice during a two-week period following one single exposure to exhausting intermittent running on a treadmill. The activities of cathepsin D and β-glycerophosphatase were assayed from mixed muscle only. In all three muscle types, particularly in red muscle, the activities of β-glucuronidase, β-N-acetylglucosaminidase, arylsulphatase, and ribonuclease progressively increased between one to five days after the exercise; thereafter the activities began to decrease, being near the control values 15 days after the exercise. In mixed muscle, cathepsin D activity increased. No corresponding changes were observed in the activities of acid phosphatases.The time course of the activity changes closely resembled that earlier found to be caused by ischaemia in rabbit muscles. It is tentatively concluded that the two treatments, exhaustive exercise and temporary ischaemia, cause similar cell injuries, and that the lysosomal system involved seems to function similarly in the post-stress recovery of the fibres from these injuries.

Increased mRNAs for procollagens and key regulating enzymes in rat skeletal muscle following downhill running.

Abstract The purpose of the study was to investigate pre-translational regulation of collagen expression after a single bout of exercise. We analysed steady-state messenger ribonucleic acid (mRNA) levels for collagen types I, III and IV, α- and β-subunits of prolyl 4-hydroxylase and lysyl oxidase (enzymes modifying procollagen chains), and enzyme activity of prolyl 4-hydroxylase from rat soleus muscle (MS) and the red parts of quadriceps femoris muscle (MQF) after 12 h and after 1, 2, 4, 7 and 14 days of downhill (–13.5°) treadmill running at a speed of 17 m·min–1 for 130 min. Histological and biochemical assays revealed exercise-induced muscle damage in MQF but not MS. Steady-state mRNA levels for the α- and β-subunits of prolyl 4-hydroxylase in MQF, lysyl oxidase in MS and MQF were increased 12 h after running, whereas prolyl 4-hydroxylase activity did not increase until 2 days after exercise. The mRNA levels for the fibrillar collagens (I and III) and basement membrane type IV collagen significantly increased 1 day and 12 h after exertion, respectively. Peak mRNA levels were observed 2–4 days after running, the increases being more pronounced in MQF than in MS. No significant changes were observed in types I or III collagen at the protein level. Strenuous downhill running thus causes an increase in gene expression for collagen types I and III and their post-translational modifying enzymes in skeletal muscle in a co-ordinated manner. These changes, together with the increased gene expression of type IV collagen, may represent the regenerative response of muscle extracellular matrix to exercise-induced injury and an adaptive response to running exertion.

Exhaustive physical exercise and acid hydrolase activity in mouse skeletal muscle

SummaryAdult, untrained NMRI mice were exhausted on a motor-driven treadmill by an intermittent-type running programme. Serial cryostate sections for the staining of NADH-tetrazolium reductase, β-glucuronidase, β-N-acetylglucosaminidase, and β-glycerophosphatase activities and for making hematoxylin-eosin staining were cut from m. quadriceps femoris 1, 2, 3, 5, 7, and 15 days after physical exhaustion. A strong increase in the activities of β-glucuronidase and β-N-acetylglucosaminidase, was observed 7 days after exhaustion and the activity changes, which were similar for the both glycosidases, were more prominent in the highly oxidative red compared to less oxidative white fibres. Activity granules were more numerous in the perinuclear than the interfibrillar area of red fibres. Spots were arranged like longitudinal chains between myofibrils. Activity in connective tissue was usually observed only in animals exhausted 3–7 days earlier. Simultaneous activity in fibres exceeded that in connective tissue β-Glycerophosphatase activity was not, by the method used, seen in histologically “healthy” or normal-looking fibres. in samples taken 2–5 days after exhaustion some degenerating and necrotic fibres were observed. Inflammatory reaction was also observed being at its strongest five days after loading when mononuclear cells were seen inside necrotic fibres. The number of regenerating muscle cells was most abundant 7 days after exhaustion. It is suggested that temporary hypoxia, which accompanies exhaustive physical exercise in skeletal muscle, upsets the energy metabolism and homeostasis of fibres and causes the observed histological and histochemical alterations, which posses features typical of both lethal and sublethal acute cell injury.

Exercise-induced expression of angiogenic growth factors in skeletal muscle and in capillaries of healthy and diabetic mice

BackgroundDiabetes has negative, and exercise training positive, effects on the skeletal muscle vasculature, but the mechanisms are not yet fully understood. In the present experiment the effects of running exercise on the mRNA expression of pro- and antiangiogenic factors were studied in healthy and diabetic skeletal muscle. The responses in capillaries and muscle fibers, collected from the muscle with laser capture microdissection, were also studied separately.MethodsHealthy and streptozotocin-induced diabetic mice were divided into sedentary and exercise groups. Exercise was a single bout of 1 h running on a treadmill. Gastrocnemius muscles were harvested 3 h and 6 h post exercise, and angiogenesis-related gene expressions were analyzed with real-time PCR. In addition to muscle homogenates, capillaries and muscle fibers were collected from the muscle with laser capture microdissection method and analyzed for vascular endothelial growth factor-A (VEGF-A) and thrombospondin-1 (TSP-1) mRNA expression.ResultsOf the proangiogenic factors, VEGF-A and VEGF receptor-2 (VEGFR-2) mRNA expression increased significantly (P < 0.05) in healthy skeletal muscle 6 h post exercise. VEGF-B also showed a similar trend (P = 0.08). No significant change was observed post exercise in diabetic muscles in the expression of VEGF-A, VEGFR-2 or VEGF-B. The expression of angiogenesis inhibitor TSP-1 and angiogenic extracellular matrix protein Cyr61 were significantly increased in diabetic muscles (P < 0.05–0.01). Capillary mRNA expression resembled that in the muscle homogenates, however, the responses were greater in capillaries compared to muscle homogenates and pure muscle fibers.ConclusionThe present study is the first to report the effects of a single bout of exercise on the expression of pro- and antiangiogenic factors in diabetic skeletal muscle, and it provides novel data about the separate responses in capillaries and muscle fibers to exercise and diabetes. Diabetic mice seem to have lower angiogenic responses to exercise compared to healthy mice, and they show markedly increased expression of angiogenesis inhibitor TSP-1. Furthermore, exercise-induced VEGF-A expression was shown to be greater in capillaries than in muscle fibers.

Effects of training and anabolic steroids on collagen synthesis in dog heart

SummaryThe effects of endurance training and anabolic steroid (Methandienone 1.5 mg · kg−1 p. o. daily) and their combination on regional collagen biosynthesis and concentration in the hearts of male beagle dogs were studied by measuring prolyl 4-hydroxylase (PH) activity and hydroxyproline (HYP) concentration. The PH (P<0.05) and HYP (P<0.05) were both greater in the subendocardinal layer than in the subepicardium (EPI) of the left ventricular wall in controls, whereas opposite gradients (P<0.05) were observed in the right ventricle. Endurance exercise caused an increase of PH activity in EPI of the left ventricular wall (P<0.01). The HYP concentration increased in both layers of the right ventricle in the exercise plus steroid group (P<0.05). The results suggest that transmural differences exist in the rate of collagen synthesis and concentration in canine cardiac ventricles and that endurance exercise may accelerate collagen synthesis in EPI of the left ventricle and the combination of exercise and anabolic steroid causes an increase in collagen concentration in the right ventricular wall.

Localisation of lymphatic vessels and vascular endothelial growth factors-C and -D in human and mouse skeletal muscle with immunohistochemistry

The present study was aimed to localise lymphatic vessels and their growth factors in human and mouse skeletal muscle with immunohistochemistry and specific antibodies (VEGFR-3, LYVE-1, VEGF-C and VEGF-D). The largest lymphatic vessels were found in perimysial connective tissue next to the arteries and veins, as has been shown earlier with electron microscopy. As a new finding, we also found small LYVE-1 positive vessels in the capillary bed between muscle fibres. These vessels were located next to CD31 positive blood capillaries and were of the same size, but fewer in number. In addition, we described the localisation of the two main lymphangiogenic growth factor proteins, vascular endothelial growth factor-C and -D. Both proteins were expressed in skeletal muscle at mRNA and protein levels. VEGF-D was located under the sarcolemma in some of the muscle fibres, in the endothelia of larger blood vessels and in fibroblasts. VEGF-C protein was localised to the nerves and muscle spindles, to fibroblasts and surrounding connective tissue, but was not found in muscle fibres or endothelial cells. Our results are the first to suggest the presence of lymphatic capillaries throughout the skeletal muscle, and to present the localisation of VEGF-C and -D in the muscles.

Exercise-induced necrotic muscle damage and enzyme release in the four days following prolonged submaximal running in rats

Male Wistar rats were made to run uphill on a treadmill 5.5° incline at 17 m min−1 for 4 h, and killed for muscle and serum sampling 2, 4, 12, 24, 48 or 96 h after the exertion. To estimate the degree of muscle damage,β-glucuronidase activity, total protein concentration, water content and morphology were examined in the red parts of quadriceps femoris (MQF) and soleus (MS) muscles, the distal white part of the rectus femoris muscle (MRF) and the superficial part of triceps brachii muscle (MTB). Simultaneous serum samples were assayed for creatine kinase (CK) activity and carbonic anhydrase III (CA III) concentration. Fibre swelling and interstitial oedema were detected in MS at 4 h and in MQF at 12 h and typical histopathological changes, including inflammation and fibre necrosis, in both muscles 12–96 h post-exertion.β-Glucuronidase activity, a quantitative marker of muscle damage, was increased in MS at 4 h, in MQF at 24 h and in MRF 48 h after the running. No increase occurred in MTB. Water and protein content increased or decreased respectively, faster in MS (2 h post-exercise) than in MQF (12 h) or MRF (12 h). Water content thus contributed to muscle damage by preceding the increase inβ-glucuronidase activity. Serum CK activity was increased 2, 4, and 48 h after the running. Changes in serum CA III concentration were rather similar to those in CK but were not significant. The increase in serum CK was not in concert with the necrotic events in the muscle but occurred considerably earlier (2 h vs. 12–24 h post-exercise). The second peak in CK, 48 h post-exercise (during the necrotic phase), was smaller than the first one. Our results show that serum CK activity is an inaccurate estimate of exercise-induced muscle damage as regards interpretation of the degree and the time course of pathological events in the muscle.

Effect of chronic exercise on glucose uptake and activities of glycolytic enzymes measured regionally in rat heart.

SummaryRegional glucose uptake in perfused hearts, and the activities of several glycolytic enzymes contributing to the glucose metabolism in perfused and nonperfused hearts were studied in male and female rats after 8–9 weeks of swimming training. The left ventricular glucose uptake showed a transmural gradient in the sedentary animals, the subendocardial uptake being 30% and 12% higher than that of the subepicardial layer in the males and females, respectively. Swimming exercise abolished the left ventricular glucose uptake gradient in male rats, and in female rats an opposite gradient was found, the subepicardial uptake being 23% higher than the subendocardial uptake. The activities of phosphofructokinase and 3-phosphoglyceraldehyde dehydrogenase also showed transmural gradients in the left ventricles. Training did not abolish these gradients. Training-induced changes in the activities of phosphofructokinase, 3-phosphoglyceraldehyde dehydrogenase, pyruvate kinase, lactate dehydrogenase, glucose-6-phosphate dehydrogenase, citrate synthase, and malate dehydrogenase were found in certain sites of the myocardium. Perfusion of isolated hearts for 50 min with insulin-containing Krebs-Ringer buffer especially affected the activities of phosphofructokinase, lactate dehydrogenase, and citrate synthase, increasing these activities in the left ventricles and decreasing them in the atria. These results indicate that there are regional differences between male and female rats in the cardiac glucose uptake rate after swimming training.