Acid proteolytic capacity in mouse cardiac and skeletal muscles after prolonged submaximal exercise

Acid proteolytic capacity in mouse cardiac muscle and in predominantly white (distal head of m. vastus lateralis) or predominantly red (proximal red heads of m. vastus lateralis, m. v. medialis, and m. v. intermedius) skeletal muscle was estimated 5 days after 3 h, 6 h or 9 h prolonged running at a speed of 13.5 m/min. The activities of acid protease and β-glucuronidase together with the rate of acid autolysis considerably increased in both skeletal muscle types, especially in red muscle, but did not increase in cardiac muscle. Acid proteolytic capacity and β-glucuronidase activity increased in relation to the duration of running. Protein content and oxidative capacity (the activities of citrate synthase and malate dehydrogenase) decreased in red skeletal muscle after 6 h and 9 h running. In white muscle only protein content slightly decreased after 9 h running. No corresponding changes were observed in cardiac muscle. Histopathological changes were traced in mixed skeletal muscle (m. rectus femoris). Necrotic lesions were observed in the red superficial area of m. rectus femoris after 6 h and, in particular, after 9 h running. The results show that prolonged submaximal running also produces lethal and sublethal skeletal muscle fibre injuries, as well as exhaustive exercise or temporary ischaemia as reported earlier. It is suggested that sublethal injuries precede lethal ones and that acid proteolytic capacity increases especially in the sublethally injured muscle fibres.     

Acid proteolytic capacity in mouse cardiac and skeletal muscles after prolonged submaximal exercise

Acid proteolytic capacity in mouse cardiac muscle and in predominantly white (distal head of m. vastus lateralis) or predominantly red (proximal red heads of m. vastus lateralis, m. v. medialis, and m. v. intermedius) skeletal muscle was estimated 5 days after 3 h, 6 h or 9 h prolonged running at a speed of 13.5 m/min. The activities of acid protease and -glucuronidase together with the rate of acid autolysis considerably increased in both skeletal muscle types, especially in red muscle, but did not increase in cardiac muscle. Acid proteolytic capacity and -glucuronidase activity increased in relation to the duration of running. Protein content and oxidative capacity (the activities of citrate synthase and malate dehydrogenase) decreased in red skeletal muscle after 6 h and 9 h running. In white muscle only protein content slightly decreased after 9 h running. No corresponding changes were observed in cardiac muscle. Histopathological changes were traced in mixed skeletal muscle (m. rectus femoris). Necrotic lesions were observed in the red superficial area of m. rectus femoris after 6 h and, in particular, after 9 h running. The results show that prolonged submaximal running also produces lethal and sublethal skeletal muscle fibre injuries, as well as exhaustive exercise or temporary ischaemia as reported earlier. It is suggested that sublethal injuries precede lethal ones and that acid proteolytic capacity increases especially in the sublethally injured muscle fibres.     

Effects of age and prolonged running on proteolytic capacity in mouse cardiac and skeletal muscles

Male NMRI-mice, aged 3, 6, 9, and 12 months, were made to run for a period of 4 h at a speed of 13.5 m/min on a motor-driven treadmill. 5 days after exertion, selected enzymatic estimates of acid and alkaline proteolytic as well as energy metabolic capacities were analyzed from the cardiac muscle and from the red and white parts of m. quadriceps femoris (MQF). The activities of alkaline and myofibrillar proteases increased most considerably in skeletal muscles with age. Cathepsin D and β-glucuronidase activities were less affected in both muscles. Prolonged running increased the activities of cathepsin D, dipeptidyl aminopeptidase I and β-glucuronidase in the white and, especially, in the red part of MQF. This stimulation of acid hydrolytic capacity was more prominent at the ages of 3 and 6 months than in the older animals. The estimates of alkaline proteolytic or energy metabolic capacities were not affected by prolonged running. In cardiac muscle, no significant changes were recorded in acid hydrolytic or energy metabolic capacity. Histological observation showed no necrosis or other pathological phenomena in the proximal part of m. rectus femoris after exertion. We suggest that the increased acid proteolytic capacity is involved in subcellular regenerative processes of skeletal muscle fibres. The smaller lysosomal response of older mice may indicate a reduced potential capacity for cellular repair.     

Human skeletal muscle mitochondrial capacity

Under aerobic work, the oxygen consumption and major ATP production occur in the mitochondria and it is therefore a relevant question whether the in vivo rates can be accounted for by mitochondrial capacities measured in vitro. Mitochondria were isolated from human quadriceps muscle biopsies in yields of approximately 45%. The tissue content of total creatine, mitochondrial protein and different cytochromes was estimated. A number of activities were measured in functional assays of the mitochondria: pyruvate, ketoglutarate, glutamate and succinate dehydrogenases, palmitoyl-carnitine respiration, cytochrome oxidase, the respiratory chain and the ATP synthesis. The activities involved in carbohydrate oxidation could account for in vivo oxygen uptakes of 15-16 mmol O2 min-1 kg-1 or slightly above the value measured at maximal work rates in the knee-extensor model of Saltin and co-workers, i.e. without limitation from the cardiac output. This probably indicates that the maximal oxygen consumption of the muscle is limited by the mitochondrial capacities. The in vitro activities of fatty acid oxidation corresponded to only 39% of those of carbohydrate oxidation. The maximal rate of free energy production from aerobic metabolism of glycogen was calculated from the mitochondrial activities and estimates of the DeltaG or ATP hydrolysis and the efficiency of the actin-myosin reaction. The resultant value was 20 W kg-1 or approximately 70% of the maximal in vivo work rates of which 10-20% probably are sustained by the anaerobic ATP production. The lack of aerobic in vitro ATP synthesis might reflect termination of some critical interplay between cytoplasm and mitochondria.     

Relationship between the size of the capillary bed and oxidative capacity in various cat skeletal muscles

The size of the capillary bed, assessed by capillary density (CD), capillary per muscle fibre ratio (C/F), total capillary length, surface area and volume was related to the oxidative capacity, assessed by the volume density of mitochondria and O₂max in cat muscles with a different composition of glycolytic and oxidative fibres: predominantly glycolytic gracilis, purely oxidative soleus and gracilis transformed towards oxidative by chronic low frequency (10 Hz) electrical stimulation. Maximal blood flow and lactate output were measured in the muscles during isometric contractions.When capillary supply was estimated by C/F ratio, there was a close correlation between various parameters only in stimulated gracilis. The combined data of all muscles showed a significant correlation between the total volume of mitochondria, O₂max and total capillary surface area. Capillary volume showed a tight correlation with maximal blood flow in both control and stimulated gracilis, but not in soleus. Maximal blood flow was correlated withVO₂max in oxidative muscles (stimulated gracilis and soleus) but not in control glycolytic gracilis. Moreover normal gracilis did not show any relationship between the volume density of mitochondria and the size of the capillary bed. The latter was inversely correlated with the output of lactate which was greater in muscles with a lower C/P ratio.The data on gracilis indicates that the capillary bed can adapt to the increased demand for oxygen and a greater oxidative capacity induced by long-term activity imposed on a glycolytic muscle, while it may be more important for the removal of lactate in the glycolytic muscles under their normal activity. The factors involved in the regulation of blood flow in control soleus — when the morphological size of the vascular bed is not related to blood flow — are discussed.     

OCTN2 is associated with carnitine transport capacity of rat skeletal muscles

Aim: Carnitine plays an essential role in fat oxidation in skeletal muscles; therefore carnitine influx could be crucial for muscle metabolism. OCTN2, a sodium-dependent solute carrier, is assumed to transport carnitine into various organs. However, OCTN2 protein expression and the functional importance of carnitine transport for muscle metabolism have not been studied. We tested the hypothesis that OCTN2 is expressed at higher levels in oxidative muscles than in other muscles, and that the carnitine uptake capacity of skeletal muscles depends on the amount of OCTN2. Methods: Rat hindlimb muscles (soleus, plantaris, and the surface and deep portions of gastrocnemius) were used for Western blotting to detect OCTN2. Tissue carnitine uptake was examined by an integration plot analysis using l -[³H]carnitine as a tracer. Tissue carnitine content was determined by enzymatic cycling methods. The percentage of type I fibres was determined by histochemical analysis. Results: OCTN2 was detected in all skeletal muscles although the amount was lower than that in the kidney. OCTN2 expression was significantly higher in soleus than in the other skeletal muscles. The amount of OCTN2 was positively correlated with the percentage of type I fibres in hindlimb muscles. The integration plot analysis revealed a positive correlation between the uptake clearance of l -[³H]carnitine and the amount of OCTN2 in skeletal muscles. However, the carnitine content in soleus was lower than that in other skeletal muscles. Conclusion: OCTN2 is functionally expressed in skeletal muscles and is involved in the import of carnitine for fatty acid oxidation, especially in highly oxidative muscles.     

Human skeletal muscle mitochondrial metabolism in youth and senescence: no signs of functional changes in ATP formation and mitochondrial oxidative capacity

The mitochondrial theory of ageing was tested. Isolated mitochondria from the quadriceps muscle from normal, healthy, young (age 20+ years, n=12) and elderly (70+ years, n=11) humans were studied in respiratory experiments and the data expressed as activities of the muscle. In each group, the subjects exhibited a variation of physical activity but, on average, the groups were representative for their age with maximum O(2) consumption rate of 50+/-9 and 34+/-13 ml min(-1) kg(-1) (mean+/-SD), respectively. Thirteen different activities were assayed. alpha-Glycerophosphate oxidation was lower in the 70+ group (38%, P~0.001), as was the respiratory capacity for fatty acids (19%, P~0.03). The remaining eleven activities, including those of the central bioenergetic reactions, were not lower in the 70+ group. Pyruvate and alpha-ketoglutarate dehydrogenase activities (i.e. the tricarboxylic acid cycle turnover) and the respiratory chain activity could all account for ~14 mmol O(2) min(-1) kg(-1) muscle (37 degrees C). The capacity for aerobic ATP synthesis was ~35 mmol ATP min(-1) kg(-1). The mitochondrial capacities were far in excess of whole-body performance. They were related to physical activity, but not to age. The mitochondrial theory of ageing, which attributes the age-related decline of muscle performance to decreased mitochondrial function, is incompatible with these results.     

Cigarette smoke-induced oxidative stress in skeletal muscles of mice

Cigarette smoke (CS)-induced oxidative stress may cause muscle alterations in chronic conditions such as chronic obstructive pulmonary disease (COPD). We sought to explore in AKR/J mice exposed to CS for 6 months and in control animals, levels of protein oxidation, oxidized proteins (immunoblotting, proteomics) and antioxidant mechanisms in both respiratory and limb muscles, body weight modifications, systemic inflammation, and lung structure. Compared to control mice, CS-exposed animals exhibited a reduction in body weight gain at 3 months and thereafter, showed lung emphysema, and exhibited increased oxidative stress levels in their diaphragms and gastrocnemius at 6 months. Proteins involved in glycolysis, ATP production and distribution, carbon dioxide hydration, and muscle contraction were carbonylated in respiratory and limb muscles. Blood tumor necrosis factor (TNF)-alpha levels were significantly greater in CS-exposed mice than in control animals. In AKR/J mice, chronic exposure to CS induces lung emphysema concomitantly with greater oxidative modifications on muscle proteins in both respiratory and limb muscles, and systemic inflammation.     

Localization of MyoD, myogenin and cell cycle regulatory factors in hypertrophying rat skeletal muscles

AIM: MyoD, myogenin, proliferating cell nuclear antigen (PCNA) and cyclin-dependent kinase inhibitor p21 (p21) proteins are key molecules in inducing the growth of myogenic cells in vitro. However, it has not been determined which cell types express these factors in hypertrophying skeletal muscles in vivo. METHODS: Using immunohistochemical techniques, we examined the spatial and temporal expression patterns of MyoD, myogenin, PCNA and p21 proteins in functionally overloaded rat plantaris muscles induced by ablation of the soleus and gastrocnemius muscles. RESULTS: MyoD and myogenin were detected in myonuclei located inside the dystrophin-positive plasma membrane of myofibres, m-cadherin-positive satellite cell nuclei and nuclei located in the interstitial spaces between myofibres on days 1, 3, 5 and 7 post-surgery. Entry of satellite cells into the cell cycle was indicated by the expression of PCNA on day 3 post-surgery, and withdrawal from the cell cycle was observed by the expression of p21 in satellite cell nuclei on day 5 post-surgery. However, the expression of both PCNA and p21 in satellite cell nuclei disappeared on day 7 post-surgery. CONCLUSION: These results indicate that proliferated satellite cell-derived myoblasts and undefined myogenic cells located in the interstitial spaces may contribute to an increase in myonuclear number and/or hyperplasia. Furthermore, we provide evidence that all of myonuclei, satellite cells and undefined myogenic cells express both MyoD and myogenin proteins. These results suggest that continual expression of MyoD and myogenin proteins in these cells is an essential molecular event which induces the successful hypertrophy of skeletal muscles.     

Excitation-contraction coupling of skeletal,cardiac and smooth muscles

Abstracts of the 1988 Annual Meeting on Muscle and Cell Motility Physiology

Excitation-contraction coupling of skeletal, cardiac and smooth muscles     

Age- and tissue-specific changes in mitochondrial and nuclear DNA base excision repair activity in mice: Susceptibility of skeletal muscles to oxidative injury

In this study, we investigated age- and tissue-dependent changes in the DNA base excision repair (BER) of oxidative lesions in mitochondrial and nuclear extracts by measuring single-nucleotide (SN)- and long-patch (LP)-BER activities in five tissues isolated from 4-, 10- and 20-month-old mice. Age-dependent SN-BER and LP-BER activity was increased in the mitochondria of liver, kidney and heart, but generally decreased in skeletal muscles. In contrast, no significant changes in repair activity were observed in nuclear extracts of the same tissues, except for quadriceps, where the SN-BER activity was higher in the old animals. Moreover, the BER activities in both the nucleus and the mitochondria were significantly lower in skeletal muscles compared to liver or kidney of the same mice. The protein level of three antioxidant enzymes, Mn and Cu/Zn superoxide dismutases (SOD) and catalase, was also significantly lower in skeletal muscle compared to liver or kidney. In addition, we found higher levels of protein carbonylation in the mitochondria of skeletal muscle relative to other tissues. Thus, it appears likely that mouse skeletal muscle is highly susceptible to oxidative stress due to deficiency in both repair of oxidative DNA damage and antioxidant enzymes, contributing to age-dependent muscle loss.     

Physical exercise increases mitochondrial function and reduces oxidative damage in skeletal muscle

The present study investigated mitochondrial adaptations and oxidative damage after 4 and 8 weeks of running training in skeletal muscle of mice. Twenty-one male mice (CF1, 30–35 g) were distributed into the following groups (n = 7): untrained (UT); trained—4 weeks (T4); trained—8 weeks (T8). Forty-eight hours after the last training session the animals were killed by decapitation and quadriceps (red portion) were removed and stored at −70°C. Succinate dehydrogenase (SDH), complexes I, II, II–III and IV, lipoperoxidation (TBARS), protein carbonyls (PC) and total thiol content were measured. Results show that endurance training (8-wk) increases the SDH activity and complexes (I, II, III, IV), decreases oxidative damage (TBARS, CP) and increases total thiol content in skeletal muscle when compared to untrained animals. In conclusion, eight weeks of running training are necessary for increases in mitochondrial respiratory chain enzyme activities to occur, in association with decreased oxidative damage.